prospect of stem cell conditioned medium in regenerative ......endothelialgrowth,homing,and aa [6]...
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Review ArticleProspect of Stem Cell Conditioned Medium inRegenerative Medicine
Jeanne Adiwinata Pawitan
Department of Histology Faculty of Medicine University of Indonesia Jalan Salemba 6 Jakarta 10430 Indonesia
Correspondence should be addressed to Jeanne Adiwinata Pawitan jeanneadiwipfkuiacid
Received 11 May 2014 Revised 22 July 2014 Accepted 25 July 2014 Published 28 August 2014
Academic Editor Antonio Salgado
Copyright copy 2014 Jeanne Adiwinata Pawitan This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Background Stem cell-derived conditioned medium has a promising prospect to be produced as pharmaceuticals for regenerativemedicine Objective To investigate various methods to obtain stem cell-derived conditioned medium (CM) to get an insight intotheir prospect of application in various diseasesMethods Systematic review using keywords ldquostem cellrdquo and ldquoconditionedmediumrdquoor ldquosecretomerdquo and ldquotherapyrdquo Data concerning treated conditionsdiseases type of cell that was cultured medium and supplementsto culture the cells culture condition CMprocessing growth factors and other secretions thatwere analyzedmethod of applicationand outcome were noted grouped tabulated and analyzed ResultsMost of CM using studies showed good results However thevarious CM even when they were derived from the same kind of cells were produced by different condition that is from differentpassage culturemedium and culture conditionThe growth factor yields of the various types of cells were available in some studiesand the cell number that was needed to produce CM for one application could be computed ConclusionVarious stem cell-derivedconditionedmedia were tested on various diseases andmostly showed good results However standardized methods of productionand validations of their use need to be conducted
1 Introduction
Data of the use of stem cells in various diseases are accu-mulating Some studies reported beneficial effects of stemcell therapy in degenerative diseases such as myocardialinfarction and revealed that stem cells cause tissue repair dueto their ability to secrete trophic factors that exert beneficialimpact on the damaged tissue rather than their capacity todifferentiate into the needed cells [1] Various studies on stemcell-derived secreted factors showed that the secreted factoralone without the stem cell itself may cause tissue repair invarious conditions that involved tissueorgan damage Thesecreted factors are referred to as secretome microvesiclesor exosome and can be found in the medium where the stemcells are cultured thus the medium is called conditionedmedium (CM) [2]
The use of secretome containing CM has several advan-tages compared to the use of stem cells as CM can bemanufactured freeze-dried packaged and transportedmoreeasily Moreover as it is devoid of cells there is no needto match the donor and the recipient to avoid rejection
problems Therefore stem cell-derived conditioned mediumhave a promising prospect to be produced as pharmaceuticalsfor regenerative medicine
To date no clinical trial that used CM for a certaindisease has been reported except two pilot studies on theuse of adipose derived mesenchymal stem cell CM forhair follicle regeneration [3] and fractional carbon dioxideresurfacing wound healing [4] in human which showedgood results The use of CM for therapy is very appealingand may be booming in the near future as studies on theuse of CM for various diseases are accumulating [1 3ndash33]Conditioned medium contains various growth factors andtissue regenerative agents which were secreted by the stemcells The fact that stem cells secrete various growth factorswas also shown by various proteomic studies which revealedthe presence of various growth factors and other cytokines inthe CM [5 7ndash9 13 17 20 22 28 34 35]
However various studies reported the use of variouskinds of stem cells and various methods to get the CM tocure various kinds of degenerative diseases in various animalmodelsTherefore this systematic review aimed to investigate
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 965849 14 pageshttpdxdoiorg1011552014965849
2 BioMed Research International
the various methods to get the CM and the various diseasesthat were treated to get an insight into the various kinds ofCM and their application benefit in various diseases
2 Materials and Methods
We performed ldquoall textrdquo searches without time restrictionon January 23 2014 in PubmedMedline using keywordsldquostem cellrdquo and ldquoconditioned mediumrdquo or ldquosecretomerdquo andldquotherapyrdquo ldquoall textrdquo searches in Cochrane library (trials) usingkeywords ldquosecretomerdquo or ldquoconditioned mediumrdquo and ldquoalltextrdquo searches in ClinicalTrialsgov using keywords ldquostemcellrdquo and ldquoconditionedmediumrdquo or ldquosecretomerdquo and ldquotherapyrdquoIn addition relevant existing articles in our library wereadded
Inclusion criteria are all studies that usedCM for a certaindisease Exclusion criteria are studies that did not containcomplete data concerning subject conditiondisease modelsource of CM and outcome of treatment with CM
Data collection is as follows treated conditionsdiseasestype of cell that was cultured detailed composition ofmedium and supplements that was used to culture the cellsculture condition (hypoxia or normoxia) to get the CM CMprocessing growth factors and other secretions that wereanalyzed method (mode) of application and outcome of CMapplication were noted grouped and tabulated
Data synthesis is as follows data were grouped accordingto treated disease and cell types that were used to producethe CM Further to know the growth factor yields of thevarious types of cells when available growth factor levelswere tabulated and grouped according to types of cells thatyielded the growth factor containing conditionedmedium inrelation to the number of cells type and duration of cultureand processing of the conditioned medium When the datawas available the number of cells thatwere needed to producethe CM for one application was computed
3 Results and Discussion
We got 39 articles that met the inclusion criteria and 7 wereexcluded due to incomplete data Various conditionsdiseaseswere treated by various cell-derived CM and mostly showedpromising results (Table 1)
The various conditioned media even when they werederived from same kind of cells were produced by differentcondition that is from different passage number of cellsculture medium and culture condition (Table 2)The growthfactor yields of the various types of cells can be seen inTable 3and the cell number that is needed to produce CM for oneapplication can be seen in Table 4
Various studies showed that conditioned medium havebeen tested in various kinds of diseasesconditions (Table 1)[1 3ndash33] that is alopecia [3 5] acute and chronic hind limbischemia [6ndash9] acute and chronic wound healing [4 10ndash14] myocardial infarct [1 15ndash18] acute liver injuryfailure[19ndash22] cerebral injuryischemiastroke [23ndash27] spinal cordinjury [28] lung injury [32] and bone defect [33] and
showed improvement of the conditions Moreover chronickidney disease that was treated using human embryonicstem cell-derived mesenchymal stem cell (huESC-MSC) CMshowed decreased systolic blood pressure and proteinuriaand improvement in tubular and glomerular damage renalblood flow and glomerular filtration rate [29] Howevernephropathy that was treated using CM from human umbili-cal cord blood unrestricted somatic stem cell (huUCB-USSC)or mouse bonemarrowmesenchymal stem cell (mBM-MSC)CM did not show improvement in serum urea and creati-nine level histopathological damage and physical activityscore [30] Moreover prevention of cancer using humanmesenchymal stem cell line CM showed increased tumor cellproliferation and vascularization [31]
In the two cases of kidney disease it can be concludedthat CM from hu-ESC-MSC can improve the condition andthe needed growth factor level is presumably enough asCM processing includes a 25-time concentration step [29]However for hu-UCB-USSC ormBM-MSC-CM lack of dataconcerning CMprocessing and growth factor level of the CM[30] prevent further analysis to conclude whether the failureto improve the condition is due to the lack of certain growthfactor or due to the level of growth factors that was too low togive an effect
The conditioned medium can be harvested from variouskinds of cells (Table 2) Moreover there are various methodsto get the conditioned medium which may interfere withthe growth factor types and levels that were harvested bythe methods Only some of the various studies using CMchecked the growth factor levels (Table 3) [5ndash10 13 17 2022 24 25 27 28 33 36] and the same type of cells yieldeddifferent growth factor levels when cell number culturemedium and condition and CM processing were different[6 24] Moreover growth factor measures also differed thatis pgmL or ngmL [6 8 10 13 17 24 27 28 33] pg120583g DNA[9 36] fgcell [25] spot density [5] and positivenegative[20] (Table 3) The measure of pg120583g DNA and fgcell can becomputed into pg or ngmL provided the DNA contentcelland cell number is known However in some studies theexact cell number that was used to produce the CM wasnot mentioned [7 8 10 13 28 33 36] In addition moststudies measured different sets of growth factors and othercytokinesfactors (Table 3)
31 Culture Medium and Supplement Some studies usedfetal bovine serum or other supplement containing completemedium while other studies used serum-free media More-over the basalmedia usedwere variable for example120572MEMDMEM DMEMF12 M199 EBM2 EGM-2 in vivo 15or chemically defined medium and the same type of cellmight be cultured in different kind of basalmedium (Table 2)Culture medium in in vitro culture represents microenvi-ronment in in vivo condition and may determine cell fateand thus cell secretion [37] Therefore the same type ofcells may secrete different level of growth factors when theywere cultured in different medium as can be seen in Table 3[25 27]
BioMed Research International 3
Table 1 Studies on various subjects conditions source of conditioned medium and outcome
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAlopeciamdashID Human Hu-AD-MSC Increased hair growth [3]BaldmdashSC C3HHeN nude mice Hu-AD-SC Hair growth [5]
Acute hind limbischemiamdashdirect IM
Female athymic mouse Hu-AD-SC
Decreased LL and FIncreased BF angiogenesisendothelial growth homing andAA
[6]
SCID mice Hu-ESCmdashendothelialcells
Vascularization and BF CMrestored defective diabetic PBderived PAC
[7]
Chronic hind limbischemiamdash7ndash10 days IM
Male nude athymic Hu-PB-MNC-EPCHu-UC-HUVEC Increased hind limb BF [8]
Male NOD-SCID mouse Hu-AFmdashSCmdashCkit (+)
Increased arteriogenesiscapillary density total perfusionarea and mobility and decreasedmuscular deg
[9]
Skin wound directmdashID[11] SC [10 12]topicalapplication [4 13]
Human Hu-AD-SC Enhanced wound healingReduced adverse effects [4]
BALBc nude mice
(i) Hu-UCB-MNC 997888rarrUCB-SC(endothelial + MSC)(ii) HUVEC
Faster wound healingUCB-SC was better thanHUVEC
[10]
Diabeticimmunodeficient mice Hu-UCB-CD34-EPC
Faster wound closureLess granulation tissue areaMore neovascularization
[11]
Male dbdb (diabetic)mice Hu-UC-MSC Faster wound closure
Increased capillary density [12]
BALBc-nude mouse(i) Hu-ESCmdashderivedEPC(ii) Hu-UCB-EPC
Faster wound healinggranulation and reepithelizationhuESC-EPC was better thanUCB-EPC
[13]
Skin woundmdash48 hourafter woundmdashSC Male NOD-SCID mice Hu-BM-MSC Faster wound healing [14]
MCImdashdirectmdashperi-infarctinjection
Male SCID or C57BL6mouse Hu-AD-SC
Improved cardiac functionReduced infarct sizeEffect of huAD-SC gt CM
[1]
MCImdashend of 2nd hourRmdashIC Female L pig Porcine PB-EPC
Reduced IZ-A and infarct sizeIncreased IZ angiogenesisIZ cardiomyocyte hypertrophyImproved LV contractility andrelaxation
[15]
MCImdash4 hoursmdashIV(jugular vein) DL pig Hu-ESC-MSC
Increased capillary densityReduced infarct sizePreserved S-D performance
[16]
MCImdash48 hours-IM yo Rat nude athymic Hu-BM-derived MPC
Improved LV functionReduced LV dilation myocyte Aand fibrosisIncreased neovascularization
[17]
MCImdash5min beforeRmdashIV -at RmdashIC Female DL pig Hu-ESC derived MSC (i) Reduced infarct size and A
(ii) Improved S-D performance [18]
MCImdash5min beforeRmdashIVmdash(tail) Mouse Hu-ESC derived MSC
Reduced infarct size(gt1000 kD100ndash220 nm) =10ndash220 nm lt 10ndash100 nm
RSLTmdashdirectmdashIVmdash(penile) Male SD rat Rat BM-MSC Reduced LIB and PIC
Increased survival [19]
4 BioMed Research International
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAcute hepaticfailuremdash24hoursmdashintrahepatic (leftliver lobe)
CCl4 injuredSCIDNODmice
1-Hu-AF MSC2-AF-MSC-hepaticprogenitor-like cells(HPL)
(i) AST ALT decreased(ii) Liver phenotypeimprovementHPL was better than MSC-CM
[20]
Fulminant hepaticfailuremdash24 hoursmdashIV(penile)
Male SD rat Hu-MSC
Reduced ALT AST TNF120572 IL6and IL1-rec-A level and HP ICIand AIncreased IL10 level liverregeneration and survival
[21]
Male SD rat Hu-BM-MSC
Reduced panlobular leucocyteinfiltrate hepatocellular deathand bile duct duplication andincreased survival
[22]
Focal cerebralischemiamdash72hoursmdashintranasal
Male SD rat (i) Hu-SC-EDT(ii) BM-MSC (Lonza)
Increasedmigration-diffmdashendogenousNPC vasculogenesis and motorfunction and reduced infarct size(Hu SC-EDT = BM-MSC)
[23]
Ischemic strokemdashafter 8daysmdashlateral ventricleinfusion
Male SD mice Hu-AD-MSC
Motor function maintainedreduced infarct volume neuralcell A and astrogliosis andincreased microvessel
[24]
Cerebral ischemiainfarctionmdash1daymdashICintracardiac(LV) injection
immunodeficient mice
(i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75(iv) Hu-fibro
Reduced cortical infarct volume(huBM-CD133-CM lthuBM-MSC-CM lt hufibroCM lthuBM-p75CM)
[25]
Fluidpercussion-TBImdashdirectIV jugular vein
Male SD rat Hu-BM-MSC
Reduced neuron loss A neuronA infarction volume and motordeficitIncreased VEGF(+) cells
[26]
Fluid percussionTBImdash12 hours aftermdashIV Male SD rat Hu-BM-MSC
Decreased brain damage volumebrain damage incidence andneuron A (hypoxia lt normoxia)Increased motorcognitivefunction and neurogenesis(hypoxia gt normoxia)
[27]
Contusion spinal cordinjurymdashdirect Female Wistar rat Rat-BM-MSC Increased motor recovery [28]
Chronic kidneydiseasemdashweek 5mdashIV(tail)
Male Le ratHu embryonicMSCmdashstablemdash80population doublings
Decreased systolic BPproteinuria and tubular +glomerular damageIncreased inulin and PAHclearance glomerularendothelium and DNA repair
[29]
Nephropathymdash24 hoursmdashIV (tail) Mouse BALBc (i) Hu-UCB-USSC
(ii) Mouse BM-MSC
No improvement in serum ureaand creatinine HP and physicalactivity score
[30]
Normalmdashcancer cell line+ CM xenograft BALB mice Hu-MSC (cell line)
Increased tumor cellproliferation (PCNA) andvascularization
[31]
VILImdashbeforeinductionmdashIVmdash(tail) Male C57BL6 mouse Mouse-iPSC
Reduced tidal volume andbronchial microstructurerestored
[32]
BioMed Research International 5
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberIntrabony periodontaldefect directmdashimplant Hybrid dog Hu-MSC (Lonza) Increased alveolar bone and
cementum regeneration [33]
ID intradermal IM intramuscular SC subcutaneous MCI myocardial infarct R reperfusion IC intracoronary artery IV intravenous Imyointramyocardial LV left ventricular RSLT 50 reduced size liver transplantation TBI traumatic brain injury VILI ventilator induced lung injury SCIDsevere combined immunodeficient NOD nonobese diabetic SD Sprague-Dawley DL Dalland Landrace L Landrace W Wistar Le Lewis hu humanAD adipose tissue derived MSC mesenchymal stem cells SC stem cell ESC embryonic stem cell PB peripheral blood MNC mononuclear cell UCumbilical cord UCB UC blood BM bonemarrow EPC endothelial progenitor cell HUVEC human umbilical vein endothelial cell AF amniotic fluid EDTexfoliated deciduous tooth MPC mesenchymal progenitor cell USSC unrestricted somatic stem cell iPSC induced pluripotent stem cell LL limb lost Ffibrosis BF blood flow AA antiapoptosis CM conditioned medium PAC proangiogenic cells deg degeneration IZ infarct zone A apoptosis ALT alanineamino transferase AST aspartate aminotransferase HP histopathology ICI immune cell infiltration S-D systolic-diastolic LIB liver injury biomarker PICproinflammatory cytokine Hu-SC- IL1-rec-A IL1 receptor antagonist NPC neural progenitor cell PAH para amino hippuric acid
32 Culture Duration Production of CM varies in cultureduration from sixteen hours to five days (Table 3) In casecomplete medium was used short culture duration mightleave certain serum derived growth factors that was notconsumed by the cells and might add to the growth factorlevel or on the contrary suppress growth factor secretionby the cells Possibility of the presence of residual growthfactor from themedium can be seen in a study which showedthat medium without cell contained a TGF-b1 level of 249 plusmn239 pgmL (Table 3) [24]
33 Culture Condition Some studies produce CM fromcell culture in normoxia (O
2level 20-21) and variable
oxygen deprived (hypoxia O2level 05 1 15 and
2) condition (Table 2) Some studies on various stem cellsshowed thatmost growth factors were upregulated in hypoxiacondition for example vascular endothelial derived growthfactor (VEGF) [5 8 27] hepatocyte growth factor (HGF)[8 27] platelet derived growth factor (PDGF) [5 8] placentagrowth factor (PlGF) [25] and insulin-like growth factorII (IGF-II) [5] except epidermal growth factor (EGF) thatwas downregulated [5] However another study showed thecontrary that is down regulation of VEGF and HGF inhypoxia condition [25]
Most studies produced CM in monolayer culture butseveral studies used spheroid cultures (Table 3) Spheroid cul-tures need a special handling and equipment (spinner flask)but yield more cells compared to conventional monolayercultures and thus more secreted factors [6 24] (Table 4) Inaddition cells located at the center of the spheroid may be inrelative hypoxic condition compared to cells on the surfacethus further increasing certain growth factor yield
34 Secreted Factorrsquos Role in Improvement of Diseases Var-ious cytokines were secreted by stem cells into the CMand they played a role in the improvement of variousdiseasesconditions Those cytokines can be grouped intogrowth factors proinflammatory and anti-inflammatorycytokines and other cytokines Various studies used variousmethods to assess various cytokines in the conditioned CMfrom the conventional ELISA assays [6 10 24 25 27 33 36]to proteomic profiling methods [5 7ndash9 13 17 20 22 28 3435]
341 Growth Factors Growth factors that are secreted byvarious kinds of stem cells are vascular endothelial derivedgrowth factor (VEGF) [5 6 8 10 13 17 24 25 27 3336] platelet derived growth factor (PDGF) [5 8 10 1324] epidermal growth factor (EGF) [5 10 13 20] insulin-like growth factor I (IGF-I) [33 36] insulin-like growthfactor II (IGF-II) [5] hepatocyte growth factor (HGF) [68 20 25 27 33] fibroblast growth factor 2basic fibroblastgrowth factor (FGF-2bFGF) [6 7 10 13] keratinocytegrowth factorfibroblast growth factor 7 (KGFFGF-7) [1020] platelet derived endothelial cell growth factor (PDEGF)[20] heparin binding epidermal growth factor (HEGF) [20]placenta growth factor (PlGF) [25] neural growth factor(NGF) [28] and brain derived neurotrophic factor (BDNF)[28]
Further studies that analyzed various growth factorsreported the presence of the various growth factors whichwere secreted by various stem cells into their conditionedmedium (Table 3) except for human MSC (Lonza) that didnot secrete FGF-2 PDGFBB BMP-2 and SDF-1 but secretedIGF-1 VEGF TGF 1205731 and HGF [33] Moreover differentculture condition and medium may yield different level ofgrowth factor secretions [6]
342 Pro- and Anti-Inflammatory Cytokines Anti-inflam-matory cytokines that are secreted by stem cells are TGF1205731[9 10 20 24 33] and some interleukins (IL) that is IL-6 [9 13 17 25 28] IL-10 IL-27 IL-17E IL-13 IL-12p70and IL-1 receptor antagonist (IL-1ra) [20] while the secretedproinflammatory cytokines are IL-8CXCL-8 [8 9 13] IL-9[13 35] and IL-1b [20]
343 Other Cytokines Other secreted factors are leptin [7]angiogenin [8] granulocyte colony stimulating factor (GCSF)[5 10] granulocyte macrophage CSF (GM-CSF) [5 10 13]macrophage CSF (MCSF) [5] fractalkine [13] monocytechemotactic protein (MCP-1) [9 13 17 20] serpin E-1 [20]endostatincollegen XVIII [20] UPA thrombospondins 1and 2 [20] tissue inhibitor of metalloproteinase-1 (TIMP-1)[20] IGF binding protein (IGFBP) [5 20] stem cell-derivedfactor 1 (SDF-1)CXCL-12 [6ndash9 20 25] adrenomedullin(ADM) [25] Dickkopf-1 (DKK-1) [25] and receptors that is
6 BioMed Research International
Table2Celltypemediumculture
cond
ition
celln
umberdu
ratio
npassageandprocessin
gof
cond
ition
edmedium
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[7]
Hu-ES
C-EC
HUVEC
EBM2
NA
NA
5days
NA
NA
[13]
(i)Hu-ES
C-CD
133KD
R-EP
C(ii)H
u-UCB
-EPC
EGM-2
(Lon
za)mdash
15mL
150m
mcultu
redish
NA
80
48ho
urs
P5ndash8
Con
c50x
10kD
[16]
[29]
Hu-ES
C-MSC
Hu-ES
C-MSC
DMEM
mdashinsulin
transferrin
seleno
protein
FGF2P
DGF-AB
glutam
ineand120573-M
E
NA
NA
NA
NA
3days
3days
ge80
PD80
PDCon
c25x10kD
mdash220n
mrarr
05m
gmLprotein
[18]
Hu-ES
C-MSC
Chem
icallydefin
edmedium
NA
NA
3days
80PD
Con
c25x
10kD119886
[9]
Hu-AFSC
mdashCk
it(+)
120572MEM
Six-wellplate
5CO
215000
070
16ho
urs
NA
from
50000
0cellsmdash1m
LmdashCon
crarr
80120583L
[20]
1-Hu-AF-MSC
2-hu
-AF-MSC
-HPL
DMEM
05
FBS
25cm
2TC
flask
5CO
215times10
6
80
24ho
urs
P5ndash13
Con
c25x
3kD
[8]
(i)Hu-PB
-MNC-
EPC
(ii)H
UVEC
EBM2(Lon
za)1FB
S15
O
25
CONA
72ho
urs
NA
NA
[15]
PorcineP
B-MNC-
EPC
Exvivo
15(Lon
za)mdash
VEG
F1n
gmLmdash
FCplate
5CO
2From
30ndash4
0mL
PB48
hours
P0Centr
600g
5min02120583mmdashice
[12]
Hu-UC-
MSC
M199
5CO
2NA
24ho
urs
P3Con
c-02120583
m
[10]
(i)Hu-UCB
-MNC-
SC(end
othelial+
MSC
)(ii)H
UVEC
EGM-2ndash15m
L150m
mcultu
redish
5CO
280
48ho
urs
P5ndash8
Con
c50xmdash
10kD
[11]
Hu-UCB
-CD34-EPC
M199basalm
edium
5CO
21times
106
24ho
urs
NA
Con
c
[30]
(i)Hu-UCB
-USSC
(ii)m
BM-M
SCUltraC
ULT
URE
medium
75
BABL
cserum
5CO
260
48ho
urs
NA
NA
[14]
Hu-BM
-MSC
120572MEM
10FB
S5
CO2
2times10
7 flask
60ndash70
Till60ndash70
P5Con
c50xmdash
5kD
5flaskrarr
100120583
L[22]
Hu-BM
-MSC
NA-
005BS
ANA
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[26]
Hu-BM
-MSC
DMEM
-005
BSA
norm
oxia
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[27]
Hu-BM
-MSC
DMEM
-005
BSA
5CO
205
O2
2times10
6rarr
split
12
rarrconfl
24ho
urs
P3ndash7
Con
c25x
3kD
[17]
Hu-BM
-MNC-
stro-3-MPC
120572MEM
NA
1times10
6MPC
NA
P5Con
c
[25]
(i)Hu-BM
-MSC
(ii)H
u-BM
-CD133
(iii)Hu-BM
-p75
(iv)H
u-fib
ro
120572MEM
5CO
2(i)
1O
2(ii)2
1O
2
1times10
6
90
48ho
urs
P4-5
02120583
mmdash80∘C
conc40xmdash5k
D
[28]
Rat-B
M-M
SCDMEM
T75flask
NA
90
48ho
urs
P2ndash4
Con
c40xmdash
10kD
10m
Lrarr
250120583
L022120583mmdash80∘C
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
2 BioMed Research International
the various methods to get the CM and the various diseasesthat were treated to get an insight into the various kinds ofCM and their application benefit in various diseases
2 Materials and Methods
We performed ldquoall textrdquo searches without time restrictionon January 23 2014 in PubmedMedline using keywordsldquostem cellrdquo and ldquoconditioned mediumrdquo or ldquosecretomerdquo andldquotherapyrdquo ldquoall textrdquo searches in Cochrane library (trials) usingkeywords ldquosecretomerdquo or ldquoconditioned mediumrdquo and ldquoalltextrdquo searches in ClinicalTrialsgov using keywords ldquostemcellrdquo and ldquoconditionedmediumrdquo or ldquosecretomerdquo and ldquotherapyrdquoIn addition relevant existing articles in our library wereadded
Inclusion criteria are all studies that usedCM for a certaindisease Exclusion criteria are studies that did not containcomplete data concerning subject conditiondisease modelsource of CM and outcome of treatment with CM
Data collection is as follows treated conditionsdiseasestype of cell that was cultured detailed composition ofmedium and supplements that was used to culture the cellsculture condition (hypoxia or normoxia) to get the CM CMprocessing growth factors and other secretions that wereanalyzed method (mode) of application and outcome of CMapplication were noted grouped and tabulated
Data synthesis is as follows data were grouped accordingto treated disease and cell types that were used to producethe CM Further to know the growth factor yields of thevarious types of cells when available growth factor levelswere tabulated and grouped according to types of cells thatyielded the growth factor containing conditionedmedium inrelation to the number of cells type and duration of cultureand processing of the conditioned medium When the datawas available the number of cells thatwere needed to producethe CM for one application was computed
3 Results and Discussion
We got 39 articles that met the inclusion criteria and 7 wereexcluded due to incomplete data Various conditionsdiseaseswere treated by various cell-derived CM and mostly showedpromising results (Table 1)
The various conditioned media even when they werederived from same kind of cells were produced by differentcondition that is from different passage number of cellsculture medium and culture condition (Table 2)The growthfactor yields of the various types of cells can be seen inTable 3and the cell number that is needed to produce CM for oneapplication can be seen in Table 4
Various studies showed that conditioned medium havebeen tested in various kinds of diseasesconditions (Table 1)[1 3ndash33] that is alopecia [3 5] acute and chronic hind limbischemia [6ndash9] acute and chronic wound healing [4 10ndash14] myocardial infarct [1 15ndash18] acute liver injuryfailure[19ndash22] cerebral injuryischemiastroke [23ndash27] spinal cordinjury [28] lung injury [32] and bone defect [33] and
showed improvement of the conditions Moreover chronickidney disease that was treated using human embryonicstem cell-derived mesenchymal stem cell (huESC-MSC) CMshowed decreased systolic blood pressure and proteinuriaand improvement in tubular and glomerular damage renalblood flow and glomerular filtration rate [29] Howevernephropathy that was treated using CM from human umbili-cal cord blood unrestricted somatic stem cell (huUCB-USSC)or mouse bonemarrowmesenchymal stem cell (mBM-MSC)CM did not show improvement in serum urea and creati-nine level histopathological damage and physical activityscore [30] Moreover prevention of cancer using humanmesenchymal stem cell line CM showed increased tumor cellproliferation and vascularization [31]
In the two cases of kidney disease it can be concludedthat CM from hu-ESC-MSC can improve the condition andthe needed growth factor level is presumably enough asCM processing includes a 25-time concentration step [29]However for hu-UCB-USSC ormBM-MSC-CM lack of dataconcerning CMprocessing and growth factor level of the CM[30] prevent further analysis to conclude whether the failureto improve the condition is due to the lack of certain growthfactor or due to the level of growth factors that was too low togive an effect
The conditioned medium can be harvested from variouskinds of cells (Table 2) Moreover there are various methodsto get the conditioned medium which may interfere withthe growth factor types and levels that were harvested bythe methods Only some of the various studies using CMchecked the growth factor levels (Table 3) [5ndash10 13 17 2022 24 25 27 28 33 36] and the same type of cells yieldeddifferent growth factor levels when cell number culturemedium and condition and CM processing were different[6 24] Moreover growth factor measures also differed thatis pgmL or ngmL [6 8 10 13 17 24 27 28 33] pg120583g DNA[9 36] fgcell [25] spot density [5] and positivenegative[20] (Table 3) The measure of pg120583g DNA and fgcell can becomputed into pg or ngmL provided the DNA contentcelland cell number is known However in some studies theexact cell number that was used to produce the CM wasnot mentioned [7 8 10 13 28 33 36] In addition moststudies measured different sets of growth factors and othercytokinesfactors (Table 3)
31 Culture Medium and Supplement Some studies usedfetal bovine serum or other supplement containing completemedium while other studies used serum-free media More-over the basalmedia usedwere variable for example120572MEMDMEM DMEMF12 M199 EBM2 EGM-2 in vivo 15or chemically defined medium and the same type of cellmight be cultured in different kind of basalmedium (Table 2)Culture medium in in vitro culture represents microenvi-ronment in in vivo condition and may determine cell fateand thus cell secretion [37] Therefore the same type ofcells may secrete different level of growth factors when theywere cultured in different medium as can be seen in Table 3[25 27]
BioMed Research International 3
Table 1 Studies on various subjects conditions source of conditioned medium and outcome
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAlopeciamdashID Human Hu-AD-MSC Increased hair growth [3]BaldmdashSC C3HHeN nude mice Hu-AD-SC Hair growth [5]
Acute hind limbischemiamdashdirect IM
Female athymic mouse Hu-AD-SC
Decreased LL and FIncreased BF angiogenesisendothelial growth homing andAA
[6]
SCID mice Hu-ESCmdashendothelialcells
Vascularization and BF CMrestored defective diabetic PBderived PAC
[7]
Chronic hind limbischemiamdash7ndash10 days IM
Male nude athymic Hu-PB-MNC-EPCHu-UC-HUVEC Increased hind limb BF [8]
Male NOD-SCID mouse Hu-AFmdashSCmdashCkit (+)
Increased arteriogenesiscapillary density total perfusionarea and mobility and decreasedmuscular deg
[9]
Skin wound directmdashID[11] SC [10 12]topicalapplication [4 13]
Human Hu-AD-SC Enhanced wound healingReduced adverse effects [4]
BALBc nude mice
(i) Hu-UCB-MNC 997888rarrUCB-SC(endothelial + MSC)(ii) HUVEC
Faster wound healingUCB-SC was better thanHUVEC
[10]
Diabeticimmunodeficient mice Hu-UCB-CD34-EPC
Faster wound closureLess granulation tissue areaMore neovascularization
[11]
Male dbdb (diabetic)mice Hu-UC-MSC Faster wound closure
Increased capillary density [12]
BALBc-nude mouse(i) Hu-ESCmdashderivedEPC(ii) Hu-UCB-EPC
Faster wound healinggranulation and reepithelizationhuESC-EPC was better thanUCB-EPC
[13]
Skin woundmdash48 hourafter woundmdashSC Male NOD-SCID mice Hu-BM-MSC Faster wound healing [14]
MCImdashdirectmdashperi-infarctinjection
Male SCID or C57BL6mouse Hu-AD-SC
Improved cardiac functionReduced infarct sizeEffect of huAD-SC gt CM
[1]
MCImdashend of 2nd hourRmdashIC Female L pig Porcine PB-EPC
Reduced IZ-A and infarct sizeIncreased IZ angiogenesisIZ cardiomyocyte hypertrophyImproved LV contractility andrelaxation
[15]
MCImdash4 hoursmdashIV(jugular vein) DL pig Hu-ESC-MSC
Increased capillary densityReduced infarct sizePreserved S-D performance
[16]
MCImdash48 hours-IM yo Rat nude athymic Hu-BM-derived MPC
Improved LV functionReduced LV dilation myocyte Aand fibrosisIncreased neovascularization
[17]
MCImdash5min beforeRmdashIV -at RmdashIC Female DL pig Hu-ESC derived MSC (i) Reduced infarct size and A
(ii) Improved S-D performance [18]
MCImdash5min beforeRmdashIVmdash(tail) Mouse Hu-ESC derived MSC
Reduced infarct size(gt1000 kD100ndash220 nm) =10ndash220 nm lt 10ndash100 nm
RSLTmdashdirectmdashIVmdash(penile) Male SD rat Rat BM-MSC Reduced LIB and PIC
Increased survival [19]
4 BioMed Research International
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAcute hepaticfailuremdash24hoursmdashintrahepatic (leftliver lobe)
CCl4 injuredSCIDNODmice
1-Hu-AF MSC2-AF-MSC-hepaticprogenitor-like cells(HPL)
(i) AST ALT decreased(ii) Liver phenotypeimprovementHPL was better than MSC-CM
[20]
Fulminant hepaticfailuremdash24 hoursmdashIV(penile)
Male SD rat Hu-MSC
Reduced ALT AST TNF120572 IL6and IL1-rec-A level and HP ICIand AIncreased IL10 level liverregeneration and survival
[21]
Male SD rat Hu-BM-MSC
Reduced panlobular leucocyteinfiltrate hepatocellular deathand bile duct duplication andincreased survival
[22]
Focal cerebralischemiamdash72hoursmdashintranasal
Male SD rat (i) Hu-SC-EDT(ii) BM-MSC (Lonza)
Increasedmigration-diffmdashendogenousNPC vasculogenesis and motorfunction and reduced infarct size(Hu SC-EDT = BM-MSC)
[23]
Ischemic strokemdashafter 8daysmdashlateral ventricleinfusion
Male SD mice Hu-AD-MSC
Motor function maintainedreduced infarct volume neuralcell A and astrogliosis andincreased microvessel
[24]
Cerebral ischemiainfarctionmdash1daymdashICintracardiac(LV) injection
immunodeficient mice
(i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75(iv) Hu-fibro
Reduced cortical infarct volume(huBM-CD133-CM lthuBM-MSC-CM lt hufibroCM lthuBM-p75CM)
[25]
Fluidpercussion-TBImdashdirectIV jugular vein
Male SD rat Hu-BM-MSC
Reduced neuron loss A neuronA infarction volume and motordeficitIncreased VEGF(+) cells
[26]
Fluid percussionTBImdash12 hours aftermdashIV Male SD rat Hu-BM-MSC
Decreased brain damage volumebrain damage incidence andneuron A (hypoxia lt normoxia)Increased motorcognitivefunction and neurogenesis(hypoxia gt normoxia)
[27]
Contusion spinal cordinjurymdashdirect Female Wistar rat Rat-BM-MSC Increased motor recovery [28]
Chronic kidneydiseasemdashweek 5mdashIV(tail)
Male Le ratHu embryonicMSCmdashstablemdash80population doublings
Decreased systolic BPproteinuria and tubular +glomerular damageIncreased inulin and PAHclearance glomerularendothelium and DNA repair
[29]
Nephropathymdash24 hoursmdashIV (tail) Mouse BALBc (i) Hu-UCB-USSC
(ii) Mouse BM-MSC
No improvement in serum ureaand creatinine HP and physicalactivity score
[30]
Normalmdashcancer cell line+ CM xenograft BALB mice Hu-MSC (cell line)
Increased tumor cellproliferation (PCNA) andvascularization
[31]
VILImdashbeforeinductionmdashIVmdash(tail) Male C57BL6 mouse Mouse-iPSC
Reduced tidal volume andbronchial microstructurerestored
[32]
BioMed Research International 5
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberIntrabony periodontaldefect directmdashimplant Hybrid dog Hu-MSC (Lonza) Increased alveolar bone and
cementum regeneration [33]
ID intradermal IM intramuscular SC subcutaneous MCI myocardial infarct R reperfusion IC intracoronary artery IV intravenous Imyointramyocardial LV left ventricular RSLT 50 reduced size liver transplantation TBI traumatic brain injury VILI ventilator induced lung injury SCIDsevere combined immunodeficient NOD nonobese diabetic SD Sprague-Dawley DL Dalland Landrace L Landrace W Wistar Le Lewis hu humanAD adipose tissue derived MSC mesenchymal stem cells SC stem cell ESC embryonic stem cell PB peripheral blood MNC mononuclear cell UCumbilical cord UCB UC blood BM bonemarrow EPC endothelial progenitor cell HUVEC human umbilical vein endothelial cell AF amniotic fluid EDTexfoliated deciduous tooth MPC mesenchymal progenitor cell USSC unrestricted somatic stem cell iPSC induced pluripotent stem cell LL limb lost Ffibrosis BF blood flow AA antiapoptosis CM conditioned medium PAC proangiogenic cells deg degeneration IZ infarct zone A apoptosis ALT alanineamino transferase AST aspartate aminotransferase HP histopathology ICI immune cell infiltration S-D systolic-diastolic LIB liver injury biomarker PICproinflammatory cytokine Hu-SC- IL1-rec-A IL1 receptor antagonist NPC neural progenitor cell PAH para amino hippuric acid
32 Culture Duration Production of CM varies in cultureduration from sixteen hours to five days (Table 3) In casecomplete medium was used short culture duration mightleave certain serum derived growth factors that was notconsumed by the cells and might add to the growth factorlevel or on the contrary suppress growth factor secretionby the cells Possibility of the presence of residual growthfactor from themedium can be seen in a study which showedthat medium without cell contained a TGF-b1 level of 249 plusmn239 pgmL (Table 3) [24]
33 Culture Condition Some studies produce CM fromcell culture in normoxia (O
2level 20-21) and variable
oxygen deprived (hypoxia O2level 05 1 15 and
2) condition (Table 2) Some studies on various stem cellsshowed thatmost growth factors were upregulated in hypoxiacondition for example vascular endothelial derived growthfactor (VEGF) [5 8 27] hepatocyte growth factor (HGF)[8 27] platelet derived growth factor (PDGF) [5 8] placentagrowth factor (PlGF) [25] and insulin-like growth factorII (IGF-II) [5] except epidermal growth factor (EGF) thatwas downregulated [5] However another study showed thecontrary that is down regulation of VEGF and HGF inhypoxia condition [25]
Most studies produced CM in monolayer culture butseveral studies used spheroid cultures (Table 3) Spheroid cul-tures need a special handling and equipment (spinner flask)but yield more cells compared to conventional monolayercultures and thus more secreted factors [6 24] (Table 4) Inaddition cells located at the center of the spheroid may be inrelative hypoxic condition compared to cells on the surfacethus further increasing certain growth factor yield
34 Secreted Factorrsquos Role in Improvement of Diseases Var-ious cytokines were secreted by stem cells into the CMand they played a role in the improvement of variousdiseasesconditions Those cytokines can be grouped intogrowth factors proinflammatory and anti-inflammatorycytokines and other cytokines Various studies used variousmethods to assess various cytokines in the conditioned CMfrom the conventional ELISA assays [6 10 24 25 27 33 36]to proteomic profiling methods [5 7ndash9 13 17 20 22 28 3435]
341 Growth Factors Growth factors that are secreted byvarious kinds of stem cells are vascular endothelial derivedgrowth factor (VEGF) [5 6 8 10 13 17 24 25 27 3336] platelet derived growth factor (PDGF) [5 8 10 1324] epidermal growth factor (EGF) [5 10 13 20] insulin-like growth factor I (IGF-I) [33 36] insulin-like growthfactor II (IGF-II) [5] hepatocyte growth factor (HGF) [68 20 25 27 33] fibroblast growth factor 2basic fibroblastgrowth factor (FGF-2bFGF) [6 7 10 13] keratinocytegrowth factorfibroblast growth factor 7 (KGFFGF-7) [1020] platelet derived endothelial cell growth factor (PDEGF)[20] heparin binding epidermal growth factor (HEGF) [20]placenta growth factor (PlGF) [25] neural growth factor(NGF) [28] and brain derived neurotrophic factor (BDNF)[28]
Further studies that analyzed various growth factorsreported the presence of the various growth factors whichwere secreted by various stem cells into their conditionedmedium (Table 3) except for human MSC (Lonza) that didnot secrete FGF-2 PDGFBB BMP-2 and SDF-1 but secretedIGF-1 VEGF TGF 1205731 and HGF [33] Moreover differentculture condition and medium may yield different level ofgrowth factor secretions [6]
342 Pro- and Anti-Inflammatory Cytokines Anti-inflam-matory cytokines that are secreted by stem cells are TGF1205731[9 10 20 24 33] and some interleukins (IL) that is IL-6 [9 13 17 25 28] IL-10 IL-27 IL-17E IL-13 IL-12p70and IL-1 receptor antagonist (IL-1ra) [20] while the secretedproinflammatory cytokines are IL-8CXCL-8 [8 9 13] IL-9[13 35] and IL-1b [20]
343 Other Cytokines Other secreted factors are leptin [7]angiogenin [8] granulocyte colony stimulating factor (GCSF)[5 10] granulocyte macrophage CSF (GM-CSF) [5 10 13]macrophage CSF (MCSF) [5] fractalkine [13] monocytechemotactic protein (MCP-1) [9 13 17 20] serpin E-1 [20]endostatincollegen XVIII [20] UPA thrombospondins 1and 2 [20] tissue inhibitor of metalloproteinase-1 (TIMP-1)[20] IGF binding protein (IGFBP) [5 20] stem cell-derivedfactor 1 (SDF-1)CXCL-12 [6ndash9 20 25] adrenomedullin(ADM) [25] Dickkopf-1 (DKK-1) [25] and receptors that is
6 BioMed Research International
Table2Celltypemediumculture
cond
ition
celln
umberdu
ratio
npassageandprocessin
gof
cond
ition
edmedium
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[7]
Hu-ES
C-EC
HUVEC
EBM2
NA
NA
5days
NA
NA
[13]
(i)Hu-ES
C-CD
133KD
R-EP
C(ii)H
u-UCB
-EPC
EGM-2
(Lon
za)mdash
15mL
150m
mcultu
redish
NA
80
48ho
urs
P5ndash8
Con
c50x
10kD
[16]
[29]
Hu-ES
C-MSC
Hu-ES
C-MSC
DMEM
mdashinsulin
transferrin
seleno
protein
FGF2P
DGF-AB
glutam
ineand120573-M
E
NA
NA
NA
NA
3days
3days
ge80
PD80
PDCon
c25x10kD
mdash220n
mrarr
05m
gmLprotein
[18]
Hu-ES
C-MSC
Chem
icallydefin
edmedium
NA
NA
3days
80PD
Con
c25x
10kD119886
[9]
Hu-AFSC
mdashCk
it(+)
120572MEM
Six-wellplate
5CO
215000
070
16ho
urs
NA
from
50000
0cellsmdash1m
LmdashCon
crarr
80120583L
[20]
1-Hu-AF-MSC
2-hu
-AF-MSC
-HPL
DMEM
05
FBS
25cm
2TC
flask
5CO
215times10
6
80
24ho
urs
P5ndash13
Con
c25x
3kD
[8]
(i)Hu-PB
-MNC-
EPC
(ii)H
UVEC
EBM2(Lon
za)1FB
S15
O
25
CONA
72ho
urs
NA
NA
[15]
PorcineP
B-MNC-
EPC
Exvivo
15(Lon
za)mdash
VEG
F1n
gmLmdash
FCplate
5CO
2From
30ndash4
0mL
PB48
hours
P0Centr
600g
5min02120583mmdashice
[12]
Hu-UC-
MSC
M199
5CO
2NA
24ho
urs
P3Con
c-02120583
m
[10]
(i)Hu-UCB
-MNC-
SC(end
othelial+
MSC
)(ii)H
UVEC
EGM-2ndash15m
L150m
mcultu
redish
5CO
280
48ho
urs
P5ndash8
Con
c50xmdash
10kD
[11]
Hu-UCB
-CD34-EPC
M199basalm
edium
5CO
21times
106
24ho
urs
NA
Con
c
[30]
(i)Hu-UCB
-USSC
(ii)m
BM-M
SCUltraC
ULT
URE
medium
75
BABL
cserum
5CO
260
48ho
urs
NA
NA
[14]
Hu-BM
-MSC
120572MEM
10FB
S5
CO2
2times10
7 flask
60ndash70
Till60ndash70
P5Con
c50xmdash
5kD
5flaskrarr
100120583
L[22]
Hu-BM
-MSC
NA-
005BS
ANA
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[26]
Hu-BM
-MSC
DMEM
-005
BSA
norm
oxia
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[27]
Hu-BM
-MSC
DMEM
-005
BSA
5CO
205
O2
2times10
6rarr
split
12
rarrconfl
24ho
urs
P3ndash7
Con
c25x
3kD
[17]
Hu-BM
-MNC-
stro-3-MPC
120572MEM
NA
1times10
6MPC
NA
P5Con
c
[25]
(i)Hu-BM
-MSC
(ii)H
u-BM
-CD133
(iii)Hu-BM
-p75
(iv)H
u-fib
ro
120572MEM
5CO
2(i)
1O
2(ii)2
1O
2
1times10
6
90
48ho
urs
P4-5
02120583
mmdash80∘C
conc40xmdash5k
D
[28]
Rat-B
M-M
SCDMEM
T75flask
NA
90
48ho
urs
P2ndash4
Con
c40xmdash
10kD
10m
Lrarr
250120583
L022120583mmdash80∘C
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
BioMed Research International 3
Table 1 Studies on various subjects conditions source of conditioned medium and outcome
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAlopeciamdashID Human Hu-AD-MSC Increased hair growth [3]BaldmdashSC C3HHeN nude mice Hu-AD-SC Hair growth [5]
Acute hind limbischemiamdashdirect IM
Female athymic mouse Hu-AD-SC
Decreased LL and FIncreased BF angiogenesisendothelial growth homing andAA
[6]
SCID mice Hu-ESCmdashendothelialcells
Vascularization and BF CMrestored defective diabetic PBderived PAC
[7]
Chronic hind limbischemiamdash7ndash10 days IM
Male nude athymic Hu-PB-MNC-EPCHu-UC-HUVEC Increased hind limb BF [8]
Male NOD-SCID mouse Hu-AFmdashSCmdashCkit (+)
Increased arteriogenesiscapillary density total perfusionarea and mobility and decreasedmuscular deg
[9]
Skin wound directmdashID[11] SC [10 12]topicalapplication [4 13]
Human Hu-AD-SC Enhanced wound healingReduced adverse effects [4]
BALBc nude mice
(i) Hu-UCB-MNC 997888rarrUCB-SC(endothelial + MSC)(ii) HUVEC
Faster wound healingUCB-SC was better thanHUVEC
[10]
Diabeticimmunodeficient mice Hu-UCB-CD34-EPC
Faster wound closureLess granulation tissue areaMore neovascularization
[11]
Male dbdb (diabetic)mice Hu-UC-MSC Faster wound closure
Increased capillary density [12]
BALBc-nude mouse(i) Hu-ESCmdashderivedEPC(ii) Hu-UCB-EPC
Faster wound healinggranulation and reepithelizationhuESC-EPC was better thanUCB-EPC
[13]
Skin woundmdash48 hourafter woundmdashSC Male NOD-SCID mice Hu-BM-MSC Faster wound healing [14]
MCImdashdirectmdashperi-infarctinjection
Male SCID or C57BL6mouse Hu-AD-SC
Improved cardiac functionReduced infarct sizeEffect of huAD-SC gt CM
[1]
MCImdashend of 2nd hourRmdashIC Female L pig Porcine PB-EPC
Reduced IZ-A and infarct sizeIncreased IZ angiogenesisIZ cardiomyocyte hypertrophyImproved LV contractility andrelaxation
[15]
MCImdash4 hoursmdashIV(jugular vein) DL pig Hu-ESC-MSC
Increased capillary densityReduced infarct sizePreserved S-D performance
[16]
MCImdash48 hours-IM yo Rat nude athymic Hu-BM-derived MPC
Improved LV functionReduced LV dilation myocyte Aand fibrosisIncreased neovascularization
[17]
MCImdash5min beforeRmdashIV -at RmdashIC Female DL pig Hu-ESC derived MSC (i) Reduced infarct size and A
(ii) Improved S-D performance [18]
MCImdash5min beforeRmdashIVmdash(tail) Mouse Hu-ESC derived MSC
Reduced infarct size(gt1000 kD100ndash220 nm) =10ndash220 nm lt 10ndash100 nm
RSLTmdashdirectmdashIVmdash(penile) Male SD rat Rat BM-MSC Reduced LIB and PIC
Increased survival [19]
4 BioMed Research International
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAcute hepaticfailuremdash24hoursmdashintrahepatic (leftliver lobe)
CCl4 injuredSCIDNODmice
1-Hu-AF MSC2-AF-MSC-hepaticprogenitor-like cells(HPL)
(i) AST ALT decreased(ii) Liver phenotypeimprovementHPL was better than MSC-CM
[20]
Fulminant hepaticfailuremdash24 hoursmdashIV(penile)
Male SD rat Hu-MSC
Reduced ALT AST TNF120572 IL6and IL1-rec-A level and HP ICIand AIncreased IL10 level liverregeneration and survival
[21]
Male SD rat Hu-BM-MSC
Reduced panlobular leucocyteinfiltrate hepatocellular deathand bile duct duplication andincreased survival
[22]
Focal cerebralischemiamdash72hoursmdashintranasal
Male SD rat (i) Hu-SC-EDT(ii) BM-MSC (Lonza)
Increasedmigration-diffmdashendogenousNPC vasculogenesis and motorfunction and reduced infarct size(Hu SC-EDT = BM-MSC)
[23]
Ischemic strokemdashafter 8daysmdashlateral ventricleinfusion
Male SD mice Hu-AD-MSC
Motor function maintainedreduced infarct volume neuralcell A and astrogliosis andincreased microvessel
[24]
Cerebral ischemiainfarctionmdash1daymdashICintracardiac(LV) injection
immunodeficient mice
(i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75(iv) Hu-fibro
Reduced cortical infarct volume(huBM-CD133-CM lthuBM-MSC-CM lt hufibroCM lthuBM-p75CM)
[25]
Fluidpercussion-TBImdashdirectIV jugular vein
Male SD rat Hu-BM-MSC
Reduced neuron loss A neuronA infarction volume and motordeficitIncreased VEGF(+) cells
[26]
Fluid percussionTBImdash12 hours aftermdashIV Male SD rat Hu-BM-MSC
Decreased brain damage volumebrain damage incidence andneuron A (hypoxia lt normoxia)Increased motorcognitivefunction and neurogenesis(hypoxia gt normoxia)
[27]
Contusion spinal cordinjurymdashdirect Female Wistar rat Rat-BM-MSC Increased motor recovery [28]
Chronic kidneydiseasemdashweek 5mdashIV(tail)
Male Le ratHu embryonicMSCmdashstablemdash80population doublings
Decreased systolic BPproteinuria and tubular +glomerular damageIncreased inulin and PAHclearance glomerularendothelium and DNA repair
[29]
Nephropathymdash24 hoursmdashIV (tail) Mouse BALBc (i) Hu-UCB-USSC
(ii) Mouse BM-MSC
No improvement in serum ureaand creatinine HP and physicalactivity score
[30]
Normalmdashcancer cell line+ CM xenograft BALB mice Hu-MSC (cell line)
Increased tumor cellproliferation (PCNA) andvascularization
[31]
VILImdashbeforeinductionmdashIVmdash(tail) Male C57BL6 mouse Mouse-iPSC
Reduced tidal volume andbronchial microstructurerestored
[32]
BioMed Research International 5
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberIntrabony periodontaldefect directmdashimplant Hybrid dog Hu-MSC (Lonza) Increased alveolar bone and
cementum regeneration [33]
ID intradermal IM intramuscular SC subcutaneous MCI myocardial infarct R reperfusion IC intracoronary artery IV intravenous Imyointramyocardial LV left ventricular RSLT 50 reduced size liver transplantation TBI traumatic brain injury VILI ventilator induced lung injury SCIDsevere combined immunodeficient NOD nonobese diabetic SD Sprague-Dawley DL Dalland Landrace L Landrace W Wistar Le Lewis hu humanAD adipose tissue derived MSC mesenchymal stem cells SC stem cell ESC embryonic stem cell PB peripheral blood MNC mononuclear cell UCumbilical cord UCB UC blood BM bonemarrow EPC endothelial progenitor cell HUVEC human umbilical vein endothelial cell AF amniotic fluid EDTexfoliated deciduous tooth MPC mesenchymal progenitor cell USSC unrestricted somatic stem cell iPSC induced pluripotent stem cell LL limb lost Ffibrosis BF blood flow AA antiapoptosis CM conditioned medium PAC proangiogenic cells deg degeneration IZ infarct zone A apoptosis ALT alanineamino transferase AST aspartate aminotransferase HP histopathology ICI immune cell infiltration S-D systolic-diastolic LIB liver injury biomarker PICproinflammatory cytokine Hu-SC- IL1-rec-A IL1 receptor antagonist NPC neural progenitor cell PAH para amino hippuric acid
32 Culture Duration Production of CM varies in cultureduration from sixteen hours to five days (Table 3) In casecomplete medium was used short culture duration mightleave certain serum derived growth factors that was notconsumed by the cells and might add to the growth factorlevel or on the contrary suppress growth factor secretionby the cells Possibility of the presence of residual growthfactor from themedium can be seen in a study which showedthat medium without cell contained a TGF-b1 level of 249 plusmn239 pgmL (Table 3) [24]
33 Culture Condition Some studies produce CM fromcell culture in normoxia (O
2level 20-21) and variable
oxygen deprived (hypoxia O2level 05 1 15 and
2) condition (Table 2) Some studies on various stem cellsshowed thatmost growth factors were upregulated in hypoxiacondition for example vascular endothelial derived growthfactor (VEGF) [5 8 27] hepatocyte growth factor (HGF)[8 27] platelet derived growth factor (PDGF) [5 8] placentagrowth factor (PlGF) [25] and insulin-like growth factorII (IGF-II) [5] except epidermal growth factor (EGF) thatwas downregulated [5] However another study showed thecontrary that is down regulation of VEGF and HGF inhypoxia condition [25]
Most studies produced CM in monolayer culture butseveral studies used spheroid cultures (Table 3) Spheroid cul-tures need a special handling and equipment (spinner flask)but yield more cells compared to conventional monolayercultures and thus more secreted factors [6 24] (Table 4) Inaddition cells located at the center of the spheroid may be inrelative hypoxic condition compared to cells on the surfacethus further increasing certain growth factor yield
34 Secreted Factorrsquos Role in Improvement of Diseases Var-ious cytokines were secreted by stem cells into the CMand they played a role in the improvement of variousdiseasesconditions Those cytokines can be grouped intogrowth factors proinflammatory and anti-inflammatorycytokines and other cytokines Various studies used variousmethods to assess various cytokines in the conditioned CMfrom the conventional ELISA assays [6 10 24 25 27 33 36]to proteomic profiling methods [5 7ndash9 13 17 20 22 28 3435]
341 Growth Factors Growth factors that are secreted byvarious kinds of stem cells are vascular endothelial derivedgrowth factor (VEGF) [5 6 8 10 13 17 24 25 27 3336] platelet derived growth factor (PDGF) [5 8 10 1324] epidermal growth factor (EGF) [5 10 13 20] insulin-like growth factor I (IGF-I) [33 36] insulin-like growthfactor II (IGF-II) [5] hepatocyte growth factor (HGF) [68 20 25 27 33] fibroblast growth factor 2basic fibroblastgrowth factor (FGF-2bFGF) [6 7 10 13] keratinocytegrowth factorfibroblast growth factor 7 (KGFFGF-7) [1020] platelet derived endothelial cell growth factor (PDEGF)[20] heparin binding epidermal growth factor (HEGF) [20]placenta growth factor (PlGF) [25] neural growth factor(NGF) [28] and brain derived neurotrophic factor (BDNF)[28]
Further studies that analyzed various growth factorsreported the presence of the various growth factors whichwere secreted by various stem cells into their conditionedmedium (Table 3) except for human MSC (Lonza) that didnot secrete FGF-2 PDGFBB BMP-2 and SDF-1 but secretedIGF-1 VEGF TGF 1205731 and HGF [33] Moreover differentculture condition and medium may yield different level ofgrowth factor secretions [6]
342 Pro- and Anti-Inflammatory Cytokines Anti-inflam-matory cytokines that are secreted by stem cells are TGF1205731[9 10 20 24 33] and some interleukins (IL) that is IL-6 [9 13 17 25 28] IL-10 IL-27 IL-17E IL-13 IL-12p70and IL-1 receptor antagonist (IL-1ra) [20] while the secretedproinflammatory cytokines are IL-8CXCL-8 [8 9 13] IL-9[13 35] and IL-1b [20]
343 Other Cytokines Other secreted factors are leptin [7]angiogenin [8] granulocyte colony stimulating factor (GCSF)[5 10] granulocyte macrophage CSF (GM-CSF) [5 10 13]macrophage CSF (MCSF) [5] fractalkine [13] monocytechemotactic protein (MCP-1) [9 13 17 20] serpin E-1 [20]endostatincollegen XVIII [20] UPA thrombospondins 1and 2 [20] tissue inhibitor of metalloproteinase-1 (TIMP-1)[20] IGF binding protein (IGFBP) [5 20] stem cell-derivedfactor 1 (SDF-1)CXCL-12 [6ndash9 20 25] adrenomedullin(ADM) [25] Dickkopf-1 (DKK-1) [25] and receptors that is
6 BioMed Research International
Table2Celltypemediumculture
cond
ition
celln
umberdu
ratio
npassageandprocessin
gof
cond
ition
edmedium
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[7]
Hu-ES
C-EC
HUVEC
EBM2
NA
NA
5days
NA
NA
[13]
(i)Hu-ES
C-CD
133KD
R-EP
C(ii)H
u-UCB
-EPC
EGM-2
(Lon
za)mdash
15mL
150m
mcultu
redish
NA
80
48ho
urs
P5ndash8
Con
c50x
10kD
[16]
[29]
Hu-ES
C-MSC
Hu-ES
C-MSC
DMEM
mdashinsulin
transferrin
seleno
protein
FGF2P
DGF-AB
glutam
ineand120573-M
E
NA
NA
NA
NA
3days
3days
ge80
PD80
PDCon
c25x10kD
mdash220n
mrarr
05m
gmLprotein
[18]
Hu-ES
C-MSC
Chem
icallydefin
edmedium
NA
NA
3days
80PD
Con
c25x
10kD119886
[9]
Hu-AFSC
mdashCk
it(+)
120572MEM
Six-wellplate
5CO
215000
070
16ho
urs
NA
from
50000
0cellsmdash1m
LmdashCon
crarr
80120583L
[20]
1-Hu-AF-MSC
2-hu
-AF-MSC
-HPL
DMEM
05
FBS
25cm
2TC
flask
5CO
215times10
6
80
24ho
urs
P5ndash13
Con
c25x
3kD
[8]
(i)Hu-PB
-MNC-
EPC
(ii)H
UVEC
EBM2(Lon
za)1FB
S15
O
25
CONA
72ho
urs
NA
NA
[15]
PorcineP
B-MNC-
EPC
Exvivo
15(Lon
za)mdash
VEG
F1n
gmLmdash
FCplate
5CO
2From
30ndash4
0mL
PB48
hours
P0Centr
600g
5min02120583mmdashice
[12]
Hu-UC-
MSC
M199
5CO
2NA
24ho
urs
P3Con
c-02120583
m
[10]
(i)Hu-UCB
-MNC-
SC(end
othelial+
MSC
)(ii)H
UVEC
EGM-2ndash15m
L150m
mcultu
redish
5CO
280
48ho
urs
P5ndash8
Con
c50xmdash
10kD
[11]
Hu-UCB
-CD34-EPC
M199basalm
edium
5CO
21times
106
24ho
urs
NA
Con
c
[30]
(i)Hu-UCB
-USSC
(ii)m
BM-M
SCUltraC
ULT
URE
medium
75
BABL
cserum
5CO
260
48ho
urs
NA
NA
[14]
Hu-BM
-MSC
120572MEM
10FB
S5
CO2
2times10
7 flask
60ndash70
Till60ndash70
P5Con
c50xmdash
5kD
5flaskrarr
100120583
L[22]
Hu-BM
-MSC
NA-
005BS
ANA
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[26]
Hu-BM
-MSC
DMEM
-005
BSA
norm
oxia
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[27]
Hu-BM
-MSC
DMEM
-005
BSA
5CO
205
O2
2times10
6rarr
split
12
rarrconfl
24ho
urs
P3ndash7
Con
c25x
3kD
[17]
Hu-BM
-MNC-
stro-3-MPC
120572MEM
NA
1times10
6MPC
NA
P5Con
c
[25]
(i)Hu-BM
-MSC
(ii)H
u-BM
-CD133
(iii)Hu-BM
-p75
(iv)H
u-fib
ro
120572MEM
5CO
2(i)
1O
2(ii)2
1O
2
1times10
6
90
48ho
urs
P4-5
02120583
mmdash80∘C
conc40xmdash5k
D
[28]
Rat-B
M-M
SCDMEM
T75flask
NA
90
48ho
urs
P2ndash4
Con
c40xmdash
10kD
10m
Lrarr
250120583
L022120583mmdash80∘C
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
4 BioMed Research International
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberAcute hepaticfailuremdash24hoursmdashintrahepatic (leftliver lobe)
CCl4 injuredSCIDNODmice
1-Hu-AF MSC2-AF-MSC-hepaticprogenitor-like cells(HPL)
(i) AST ALT decreased(ii) Liver phenotypeimprovementHPL was better than MSC-CM
[20]
Fulminant hepaticfailuremdash24 hoursmdashIV(penile)
Male SD rat Hu-MSC
Reduced ALT AST TNF120572 IL6and IL1-rec-A level and HP ICIand AIncreased IL10 level liverregeneration and survival
[21]
Male SD rat Hu-BM-MSC
Reduced panlobular leucocyteinfiltrate hepatocellular deathand bile duct duplication andincreased survival
[22]
Focal cerebralischemiamdash72hoursmdashintranasal
Male SD rat (i) Hu-SC-EDT(ii) BM-MSC (Lonza)
Increasedmigration-diffmdashendogenousNPC vasculogenesis and motorfunction and reduced infarct size(Hu SC-EDT = BM-MSC)
[23]
Ischemic strokemdashafter 8daysmdashlateral ventricleinfusion
Male SD mice Hu-AD-MSC
Motor function maintainedreduced infarct volume neuralcell A and astrogliosis andincreased microvessel
[24]
Cerebral ischemiainfarctionmdash1daymdashICintracardiac(LV) injection
immunodeficient mice
(i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75(iv) Hu-fibro
Reduced cortical infarct volume(huBM-CD133-CM lthuBM-MSC-CM lt hufibroCM lthuBM-p75CM)
[25]
Fluidpercussion-TBImdashdirectIV jugular vein
Male SD rat Hu-BM-MSC
Reduced neuron loss A neuronA infarction volume and motordeficitIncreased VEGF(+) cells
[26]
Fluid percussionTBImdash12 hours aftermdashIV Male SD rat Hu-BM-MSC
Decreased brain damage volumebrain damage incidence andneuron A (hypoxia lt normoxia)Increased motorcognitivefunction and neurogenesis(hypoxia gt normoxia)
[27]
Contusion spinal cordinjurymdashdirect Female Wistar rat Rat-BM-MSC Increased motor recovery [28]
Chronic kidneydiseasemdashweek 5mdashIV(tail)
Male Le ratHu embryonicMSCmdashstablemdash80population doublings
Decreased systolic BPproteinuria and tubular +glomerular damageIncreased inulin and PAHclearance glomerularendothelium and DNA repair
[29]
Nephropathymdash24 hoursmdashIV (tail) Mouse BALBc (i) Hu-UCB-USSC
(ii) Mouse BM-MSC
No improvement in serum ureaand creatinine HP and physicalactivity score
[30]
Normalmdashcancer cell line+ CM xenograft BALB mice Hu-MSC (cell line)
Increased tumor cellproliferation (PCNA) andvascularization
[31]
VILImdashbeforeinductionmdashIVmdash(tail) Male C57BL6 mouse Mouse-iPSC
Reduced tidal volume andbronchial microstructurerestored
[32]
BioMed Research International 5
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberIntrabony periodontaldefect directmdashimplant Hybrid dog Hu-MSC (Lonza) Increased alveolar bone and
cementum regeneration [33]
ID intradermal IM intramuscular SC subcutaneous MCI myocardial infarct R reperfusion IC intracoronary artery IV intravenous Imyointramyocardial LV left ventricular RSLT 50 reduced size liver transplantation TBI traumatic brain injury VILI ventilator induced lung injury SCIDsevere combined immunodeficient NOD nonobese diabetic SD Sprague-Dawley DL Dalland Landrace L Landrace W Wistar Le Lewis hu humanAD adipose tissue derived MSC mesenchymal stem cells SC stem cell ESC embryonic stem cell PB peripheral blood MNC mononuclear cell UCumbilical cord UCB UC blood BM bonemarrow EPC endothelial progenitor cell HUVEC human umbilical vein endothelial cell AF amniotic fluid EDTexfoliated deciduous tooth MPC mesenchymal progenitor cell USSC unrestricted somatic stem cell iPSC induced pluripotent stem cell LL limb lost Ffibrosis BF blood flow AA antiapoptosis CM conditioned medium PAC proangiogenic cells deg degeneration IZ infarct zone A apoptosis ALT alanineamino transferase AST aspartate aminotransferase HP histopathology ICI immune cell infiltration S-D systolic-diastolic LIB liver injury biomarker PICproinflammatory cytokine Hu-SC- IL1-rec-A IL1 receptor antagonist NPC neural progenitor cell PAH para amino hippuric acid
32 Culture Duration Production of CM varies in cultureduration from sixteen hours to five days (Table 3) In casecomplete medium was used short culture duration mightleave certain serum derived growth factors that was notconsumed by the cells and might add to the growth factorlevel or on the contrary suppress growth factor secretionby the cells Possibility of the presence of residual growthfactor from themedium can be seen in a study which showedthat medium without cell contained a TGF-b1 level of 249 plusmn239 pgmL (Table 3) [24]
33 Culture Condition Some studies produce CM fromcell culture in normoxia (O
2level 20-21) and variable
oxygen deprived (hypoxia O2level 05 1 15 and
2) condition (Table 2) Some studies on various stem cellsshowed thatmost growth factors were upregulated in hypoxiacondition for example vascular endothelial derived growthfactor (VEGF) [5 8 27] hepatocyte growth factor (HGF)[8 27] platelet derived growth factor (PDGF) [5 8] placentagrowth factor (PlGF) [25] and insulin-like growth factorII (IGF-II) [5] except epidermal growth factor (EGF) thatwas downregulated [5] However another study showed thecontrary that is down regulation of VEGF and HGF inhypoxia condition [25]
Most studies produced CM in monolayer culture butseveral studies used spheroid cultures (Table 3) Spheroid cul-tures need a special handling and equipment (spinner flask)but yield more cells compared to conventional monolayercultures and thus more secreted factors [6 24] (Table 4) Inaddition cells located at the center of the spheroid may be inrelative hypoxic condition compared to cells on the surfacethus further increasing certain growth factor yield
34 Secreted Factorrsquos Role in Improvement of Diseases Var-ious cytokines were secreted by stem cells into the CMand they played a role in the improvement of variousdiseasesconditions Those cytokines can be grouped intogrowth factors proinflammatory and anti-inflammatorycytokines and other cytokines Various studies used variousmethods to assess various cytokines in the conditioned CMfrom the conventional ELISA assays [6 10 24 25 27 33 36]to proteomic profiling methods [5 7ndash9 13 17 20 22 28 3435]
341 Growth Factors Growth factors that are secreted byvarious kinds of stem cells are vascular endothelial derivedgrowth factor (VEGF) [5 6 8 10 13 17 24 25 27 3336] platelet derived growth factor (PDGF) [5 8 10 1324] epidermal growth factor (EGF) [5 10 13 20] insulin-like growth factor I (IGF-I) [33 36] insulin-like growthfactor II (IGF-II) [5] hepatocyte growth factor (HGF) [68 20 25 27 33] fibroblast growth factor 2basic fibroblastgrowth factor (FGF-2bFGF) [6 7 10 13] keratinocytegrowth factorfibroblast growth factor 7 (KGFFGF-7) [1020] platelet derived endothelial cell growth factor (PDEGF)[20] heparin binding epidermal growth factor (HEGF) [20]placenta growth factor (PlGF) [25] neural growth factor(NGF) [28] and brain derived neurotrophic factor (BDNF)[28]
Further studies that analyzed various growth factorsreported the presence of the various growth factors whichwere secreted by various stem cells into their conditionedmedium (Table 3) except for human MSC (Lonza) that didnot secrete FGF-2 PDGFBB BMP-2 and SDF-1 but secretedIGF-1 VEGF TGF 1205731 and HGF [33] Moreover differentculture condition and medium may yield different level ofgrowth factor secretions [6]
342 Pro- and Anti-Inflammatory Cytokines Anti-inflam-matory cytokines that are secreted by stem cells are TGF1205731[9 10 20 24 33] and some interleukins (IL) that is IL-6 [9 13 17 25 28] IL-10 IL-27 IL-17E IL-13 IL-12p70and IL-1 receptor antagonist (IL-1ra) [20] while the secretedproinflammatory cytokines are IL-8CXCL-8 [8 9 13] IL-9[13 35] and IL-1b [20]
343 Other Cytokines Other secreted factors are leptin [7]angiogenin [8] granulocyte colony stimulating factor (GCSF)[5 10] granulocyte macrophage CSF (GM-CSF) [5 10 13]macrophage CSF (MCSF) [5] fractalkine [13] monocytechemotactic protein (MCP-1) [9 13 17 20] serpin E-1 [20]endostatincollegen XVIII [20] UPA thrombospondins 1and 2 [20] tissue inhibitor of metalloproteinase-1 (TIMP-1)[20] IGF binding protein (IGFBP) [5 20] stem cell-derivedfactor 1 (SDF-1)CXCL-12 [6ndash9 20 25] adrenomedullin(ADM) [25] Dickkopf-1 (DKK-1) [25] and receptors that is
6 BioMed Research International
Table2Celltypemediumculture
cond
ition
celln
umberdu
ratio
npassageandprocessin
gof
cond
ition
edmedium
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[7]
Hu-ES
C-EC
HUVEC
EBM2
NA
NA
5days
NA
NA
[13]
(i)Hu-ES
C-CD
133KD
R-EP
C(ii)H
u-UCB
-EPC
EGM-2
(Lon
za)mdash
15mL
150m
mcultu
redish
NA
80
48ho
urs
P5ndash8
Con
c50x
10kD
[16]
[29]
Hu-ES
C-MSC
Hu-ES
C-MSC
DMEM
mdashinsulin
transferrin
seleno
protein
FGF2P
DGF-AB
glutam
ineand120573-M
E
NA
NA
NA
NA
3days
3days
ge80
PD80
PDCon
c25x10kD
mdash220n
mrarr
05m
gmLprotein
[18]
Hu-ES
C-MSC
Chem
icallydefin
edmedium
NA
NA
3days
80PD
Con
c25x
10kD119886
[9]
Hu-AFSC
mdashCk
it(+)
120572MEM
Six-wellplate
5CO
215000
070
16ho
urs
NA
from
50000
0cellsmdash1m
LmdashCon
crarr
80120583L
[20]
1-Hu-AF-MSC
2-hu
-AF-MSC
-HPL
DMEM
05
FBS
25cm
2TC
flask
5CO
215times10
6
80
24ho
urs
P5ndash13
Con
c25x
3kD
[8]
(i)Hu-PB
-MNC-
EPC
(ii)H
UVEC
EBM2(Lon
za)1FB
S15
O
25
CONA
72ho
urs
NA
NA
[15]
PorcineP
B-MNC-
EPC
Exvivo
15(Lon
za)mdash
VEG
F1n
gmLmdash
FCplate
5CO
2From
30ndash4
0mL
PB48
hours
P0Centr
600g
5min02120583mmdashice
[12]
Hu-UC-
MSC
M199
5CO
2NA
24ho
urs
P3Con
c-02120583
m
[10]
(i)Hu-UCB
-MNC-
SC(end
othelial+
MSC
)(ii)H
UVEC
EGM-2ndash15m
L150m
mcultu
redish
5CO
280
48ho
urs
P5ndash8
Con
c50xmdash
10kD
[11]
Hu-UCB
-CD34-EPC
M199basalm
edium
5CO
21times
106
24ho
urs
NA
Con
c
[30]
(i)Hu-UCB
-USSC
(ii)m
BM-M
SCUltraC
ULT
URE
medium
75
BABL
cserum
5CO
260
48ho
urs
NA
NA
[14]
Hu-BM
-MSC
120572MEM
10FB
S5
CO2
2times10
7 flask
60ndash70
Till60ndash70
P5Con
c50xmdash
5kD
5flaskrarr
100120583
L[22]
Hu-BM
-MSC
NA-
005BS
ANA
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[26]
Hu-BM
-MSC
DMEM
-005
BSA
norm
oxia
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[27]
Hu-BM
-MSC
DMEM
-005
BSA
5CO
205
O2
2times10
6rarr
split
12
rarrconfl
24ho
urs
P3ndash7
Con
c25x
3kD
[17]
Hu-BM
-MNC-
stro-3-MPC
120572MEM
NA
1times10
6MPC
NA
P5Con
c
[25]
(i)Hu-BM
-MSC
(ii)H
u-BM
-CD133
(iii)Hu-BM
-p75
(iv)H
u-fib
ro
120572MEM
5CO
2(i)
1O
2(ii)2
1O
2
1times10
6
90
48ho
urs
P4-5
02120583
mmdash80∘C
conc40xmdash5k
D
[28]
Rat-B
M-M
SCDMEM
T75flask
NA
90
48ho
urs
P2ndash4
Con
c40xmdash
10kD
10m
Lrarr
250120583
L022120583mmdash80∘C
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
BioMed Research International 5
Table 1 Continued
Conditiondisease Subject Source of conditionedmedium Outcome Reference
numberIntrabony periodontaldefect directmdashimplant Hybrid dog Hu-MSC (Lonza) Increased alveolar bone and
cementum regeneration [33]
ID intradermal IM intramuscular SC subcutaneous MCI myocardial infarct R reperfusion IC intracoronary artery IV intravenous Imyointramyocardial LV left ventricular RSLT 50 reduced size liver transplantation TBI traumatic brain injury VILI ventilator induced lung injury SCIDsevere combined immunodeficient NOD nonobese diabetic SD Sprague-Dawley DL Dalland Landrace L Landrace W Wistar Le Lewis hu humanAD adipose tissue derived MSC mesenchymal stem cells SC stem cell ESC embryonic stem cell PB peripheral blood MNC mononuclear cell UCumbilical cord UCB UC blood BM bonemarrow EPC endothelial progenitor cell HUVEC human umbilical vein endothelial cell AF amniotic fluid EDTexfoliated deciduous tooth MPC mesenchymal progenitor cell USSC unrestricted somatic stem cell iPSC induced pluripotent stem cell LL limb lost Ffibrosis BF blood flow AA antiapoptosis CM conditioned medium PAC proangiogenic cells deg degeneration IZ infarct zone A apoptosis ALT alanineamino transferase AST aspartate aminotransferase HP histopathology ICI immune cell infiltration S-D systolic-diastolic LIB liver injury biomarker PICproinflammatory cytokine Hu-SC- IL1-rec-A IL1 receptor antagonist NPC neural progenitor cell PAH para amino hippuric acid
32 Culture Duration Production of CM varies in cultureduration from sixteen hours to five days (Table 3) In casecomplete medium was used short culture duration mightleave certain serum derived growth factors that was notconsumed by the cells and might add to the growth factorlevel or on the contrary suppress growth factor secretionby the cells Possibility of the presence of residual growthfactor from themedium can be seen in a study which showedthat medium without cell contained a TGF-b1 level of 249 plusmn239 pgmL (Table 3) [24]
33 Culture Condition Some studies produce CM fromcell culture in normoxia (O
2level 20-21) and variable
oxygen deprived (hypoxia O2level 05 1 15 and
2) condition (Table 2) Some studies on various stem cellsshowed thatmost growth factors were upregulated in hypoxiacondition for example vascular endothelial derived growthfactor (VEGF) [5 8 27] hepatocyte growth factor (HGF)[8 27] platelet derived growth factor (PDGF) [5 8] placentagrowth factor (PlGF) [25] and insulin-like growth factorII (IGF-II) [5] except epidermal growth factor (EGF) thatwas downregulated [5] However another study showed thecontrary that is down regulation of VEGF and HGF inhypoxia condition [25]
Most studies produced CM in monolayer culture butseveral studies used spheroid cultures (Table 3) Spheroid cul-tures need a special handling and equipment (spinner flask)but yield more cells compared to conventional monolayercultures and thus more secreted factors [6 24] (Table 4) Inaddition cells located at the center of the spheroid may be inrelative hypoxic condition compared to cells on the surfacethus further increasing certain growth factor yield
34 Secreted Factorrsquos Role in Improvement of Diseases Var-ious cytokines were secreted by stem cells into the CMand they played a role in the improvement of variousdiseasesconditions Those cytokines can be grouped intogrowth factors proinflammatory and anti-inflammatorycytokines and other cytokines Various studies used variousmethods to assess various cytokines in the conditioned CMfrom the conventional ELISA assays [6 10 24 25 27 33 36]to proteomic profiling methods [5 7ndash9 13 17 20 22 28 3435]
341 Growth Factors Growth factors that are secreted byvarious kinds of stem cells are vascular endothelial derivedgrowth factor (VEGF) [5 6 8 10 13 17 24 25 27 3336] platelet derived growth factor (PDGF) [5 8 10 1324] epidermal growth factor (EGF) [5 10 13 20] insulin-like growth factor I (IGF-I) [33 36] insulin-like growthfactor II (IGF-II) [5] hepatocyte growth factor (HGF) [68 20 25 27 33] fibroblast growth factor 2basic fibroblastgrowth factor (FGF-2bFGF) [6 7 10 13] keratinocytegrowth factorfibroblast growth factor 7 (KGFFGF-7) [1020] platelet derived endothelial cell growth factor (PDEGF)[20] heparin binding epidermal growth factor (HEGF) [20]placenta growth factor (PlGF) [25] neural growth factor(NGF) [28] and brain derived neurotrophic factor (BDNF)[28]
Further studies that analyzed various growth factorsreported the presence of the various growth factors whichwere secreted by various stem cells into their conditionedmedium (Table 3) except for human MSC (Lonza) that didnot secrete FGF-2 PDGFBB BMP-2 and SDF-1 but secretedIGF-1 VEGF TGF 1205731 and HGF [33] Moreover differentculture condition and medium may yield different level ofgrowth factor secretions [6]
342 Pro- and Anti-Inflammatory Cytokines Anti-inflam-matory cytokines that are secreted by stem cells are TGF1205731[9 10 20 24 33] and some interleukins (IL) that is IL-6 [9 13 17 25 28] IL-10 IL-27 IL-17E IL-13 IL-12p70and IL-1 receptor antagonist (IL-1ra) [20] while the secretedproinflammatory cytokines are IL-8CXCL-8 [8 9 13] IL-9[13 35] and IL-1b [20]
343 Other Cytokines Other secreted factors are leptin [7]angiogenin [8] granulocyte colony stimulating factor (GCSF)[5 10] granulocyte macrophage CSF (GM-CSF) [5 10 13]macrophage CSF (MCSF) [5] fractalkine [13] monocytechemotactic protein (MCP-1) [9 13 17 20] serpin E-1 [20]endostatincollegen XVIII [20] UPA thrombospondins 1and 2 [20] tissue inhibitor of metalloproteinase-1 (TIMP-1)[20] IGF binding protein (IGFBP) [5 20] stem cell-derivedfactor 1 (SDF-1)CXCL-12 [6ndash9 20 25] adrenomedullin(ADM) [25] Dickkopf-1 (DKK-1) [25] and receptors that is
6 BioMed Research International
Table2Celltypemediumculture
cond
ition
celln
umberdu
ratio
npassageandprocessin
gof
cond
ition
edmedium
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[7]
Hu-ES
C-EC
HUVEC
EBM2
NA
NA
5days
NA
NA
[13]
(i)Hu-ES
C-CD
133KD
R-EP
C(ii)H
u-UCB
-EPC
EGM-2
(Lon
za)mdash
15mL
150m
mcultu
redish
NA
80
48ho
urs
P5ndash8
Con
c50x
10kD
[16]
[29]
Hu-ES
C-MSC
Hu-ES
C-MSC
DMEM
mdashinsulin
transferrin
seleno
protein
FGF2P
DGF-AB
glutam
ineand120573-M
E
NA
NA
NA
NA
3days
3days
ge80
PD80
PDCon
c25x10kD
mdash220n
mrarr
05m
gmLprotein
[18]
Hu-ES
C-MSC
Chem
icallydefin
edmedium
NA
NA
3days
80PD
Con
c25x
10kD119886
[9]
Hu-AFSC
mdashCk
it(+)
120572MEM
Six-wellplate
5CO
215000
070
16ho
urs
NA
from
50000
0cellsmdash1m
LmdashCon
crarr
80120583L
[20]
1-Hu-AF-MSC
2-hu
-AF-MSC
-HPL
DMEM
05
FBS
25cm
2TC
flask
5CO
215times10
6
80
24ho
urs
P5ndash13
Con
c25x
3kD
[8]
(i)Hu-PB
-MNC-
EPC
(ii)H
UVEC
EBM2(Lon
za)1FB
S15
O
25
CONA
72ho
urs
NA
NA
[15]
PorcineP
B-MNC-
EPC
Exvivo
15(Lon
za)mdash
VEG
F1n
gmLmdash
FCplate
5CO
2From
30ndash4
0mL
PB48
hours
P0Centr
600g
5min02120583mmdashice
[12]
Hu-UC-
MSC
M199
5CO
2NA
24ho
urs
P3Con
c-02120583
m
[10]
(i)Hu-UCB
-MNC-
SC(end
othelial+
MSC
)(ii)H
UVEC
EGM-2ndash15m
L150m
mcultu
redish
5CO
280
48ho
urs
P5ndash8
Con
c50xmdash
10kD
[11]
Hu-UCB
-CD34-EPC
M199basalm
edium
5CO
21times
106
24ho
urs
NA
Con
c
[30]
(i)Hu-UCB
-USSC
(ii)m
BM-M
SCUltraC
ULT
URE
medium
75
BABL
cserum
5CO
260
48ho
urs
NA
NA
[14]
Hu-BM
-MSC
120572MEM
10FB
S5
CO2
2times10
7 flask
60ndash70
Till60ndash70
P5Con
c50xmdash
5kD
5flaskrarr
100120583
L[22]
Hu-BM
-MSC
NA-
005BS
ANA
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[26]
Hu-BM
-MSC
DMEM
-005
BSA
norm
oxia
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[27]
Hu-BM
-MSC
DMEM
-005
BSA
5CO
205
O2
2times10
6rarr
split
12
rarrconfl
24ho
urs
P3ndash7
Con
c25x
3kD
[17]
Hu-BM
-MNC-
stro-3-MPC
120572MEM
NA
1times10
6MPC
NA
P5Con
c
[25]
(i)Hu-BM
-MSC
(ii)H
u-BM
-CD133
(iii)Hu-BM
-p75
(iv)H
u-fib
ro
120572MEM
5CO
2(i)
1O
2(ii)2
1O
2
1times10
6
90
48ho
urs
P4-5
02120583
mmdash80∘C
conc40xmdash5k
D
[28]
Rat-B
M-M
SCDMEM
T75flask
NA
90
48ho
urs
P2ndash4
Con
c40xmdash
10kD
10m
Lrarr
250120583
L022120583mmdash80∘C
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
6 BioMed Research International
Table2Celltypemediumculture
cond
ition
celln
umberdu
ratio
npassageandprocessin
gof
cond
ition
edmedium
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[7]
Hu-ES
C-EC
HUVEC
EBM2
NA
NA
5days
NA
NA
[13]
(i)Hu-ES
C-CD
133KD
R-EP
C(ii)H
u-UCB
-EPC
EGM-2
(Lon
za)mdash
15mL
150m
mcultu
redish
NA
80
48ho
urs
P5ndash8
Con
c50x
10kD
[16]
[29]
Hu-ES
C-MSC
Hu-ES
C-MSC
DMEM
mdashinsulin
transferrin
seleno
protein
FGF2P
DGF-AB
glutam
ineand120573-M
E
NA
NA
NA
NA
3days
3days
ge80
PD80
PDCon
c25x10kD
mdash220n
mrarr
05m
gmLprotein
[18]
Hu-ES
C-MSC
Chem
icallydefin
edmedium
NA
NA
3days
80PD
Con
c25x
10kD119886
[9]
Hu-AFSC
mdashCk
it(+)
120572MEM
Six-wellplate
5CO
215000
070
16ho
urs
NA
from
50000
0cellsmdash1m
LmdashCon
crarr
80120583L
[20]
1-Hu-AF-MSC
2-hu
-AF-MSC
-HPL
DMEM
05
FBS
25cm
2TC
flask
5CO
215times10
6
80
24ho
urs
P5ndash13
Con
c25x
3kD
[8]
(i)Hu-PB
-MNC-
EPC
(ii)H
UVEC
EBM2(Lon
za)1FB
S15
O
25
CONA
72ho
urs
NA
NA
[15]
PorcineP
B-MNC-
EPC
Exvivo
15(Lon
za)mdash
VEG
F1n
gmLmdash
FCplate
5CO
2From
30ndash4
0mL
PB48
hours
P0Centr
600g
5min02120583mmdashice
[12]
Hu-UC-
MSC
M199
5CO
2NA
24ho
urs
P3Con
c-02120583
m
[10]
(i)Hu-UCB
-MNC-
SC(end
othelial+
MSC
)(ii)H
UVEC
EGM-2ndash15m
L150m
mcultu
redish
5CO
280
48ho
urs
P5ndash8
Con
c50xmdash
10kD
[11]
Hu-UCB
-CD34-EPC
M199basalm
edium
5CO
21times
106
24ho
urs
NA
Con
c
[30]
(i)Hu-UCB
-USSC
(ii)m
BM-M
SCUltraC
ULT
URE
medium
75
BABL
cserum
5CO
260
48ho
urs
NA
NA
[14]
Hu-BM
-MSC
120572MEM
10FB
S5
CO2
2times10
7 flask
60ndash70
Till60ndash70
P5Con
c50xmdash
5kD
5flaskrarr
100120583
L[22]
Hu-BM
-MSC
NA-
005BS
ANA
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[26]
Hu-BM
-MSC
DMEM
-005
BSA
norm
oxia
2times10
624
hours
P3ndash7
Con
c25xmdash
3kD
[27]
Hu-BM
-MSC
DMEM
-005
BSA
5CO
205
O2
2times10
6rarr
split
12
rarrconfl
24ho
urs
P3ndash7
Con
c25x
3kD
[17]
Hu-BM
-MNC-
stro-3-MPC
120572MEM
NA
1times10
6MPC
NA
P5Con
c
[25]
(i)Hu-BM
-MSC
(ii)H
u-BM
-CD133
(iii)Hu-BM
-p75
(iv)H
u-fib
ro
120572MEM
5CO
2(i)
1O
2(ii)2
1O
2
1times10
6
90
48ho
urs
P4-5
02120583
mmdash80∘C
conc40xmdash5k
D
[28]
Rat-B
M-M
SCDMEM
T75flask
NA
90
48ho
urs
P2ndash4
Con
c40xmdash
10kD
10m
Lrarr
250120583
L022120583mmdash80∘C
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
BioMed Research International 7
Table2Con
tinued
Reference
number
Cell
type
Mediumvessel
Cultu
recond
ition
Cell
number
Duration
Passage
CMprocessin
g
[19]
RatB
M-M
SCDMEM
-005
BSA
10mL
NA
80ndash9
012
hours
P3-4
Con
c25x
3kD
[23]
(i)Hu-SC
-EDT
(ii)B
M-M
SC(Lon
za)
DMEM
5CO
24times10
548
hours
P3ndash5
3000
rpmmdash3m
inrarr
supernatant
[3]
Hu-AD-M
SCNA
Hypoxia
NA
NA
NA
Con
c-fre
ezed
ried
[24]
Hu-AD-M
SC120572MEM
70mL
Spheroid
1O
2mdash5
CO2
42times107
2days
NA
Centr
[6]
Hu-AD-SC
CRM120572MEM
CRMmdashhu
allo10
120572MEM
mdashFB
S10
1O
2mdash5
CO2
25ndash3times
105 mLmdash
24mLmdash
150c
mdish
2days
Upto
P5Centr
CRM120572MEM
CRMmdashhu
allo10
120572MEM
FBS10
Spheroid
1O
2mdash5
CO2
6ndash12times
105 mLmdash
30120583L
rarr70
mLflask
[1]
Hu-AD-SC
NA
NA
Reference[1314]
1times10
5rarr
80
24ho
urs
NA
Centr
300g
5minmdash220n
m
[5]
Hu-AD-SC
DMEM
F12
2O
25
CO2
4times10
572
hours
P4-5
Centr
300g
5rsquo022120583m3
kD
[23]
Hu-MSC
DMEM
-005
BSA
15mLmdash
175c
m2flask
NA
1times10
6
70ndash80
24ho
urs
NA
Con
c25x3k
D
[31]
Hu-MSC
(cellline)
DMEM
-10
FBS
5mL
5CO
270
48ho
urs
NA
10000
0gmdash
1hou
rrarr
supernatant
022120583m
[33]
Hu-MSC
(Lon
za)
DMEM
5CO
270
48ho
urs
P3ndash9
4∘Corminus80∘C
[32]
Mou
se-iP
SCNA
NA
NA
NA
NA
NA
Hu
humanE
SCembryonicste
mcell
ECend
othelialcellHUVEC
hum
anum
bilical
vein
endo
thelialcellEP
Cendo
thelialp
rogenitorcell
AF
amniotic
fluidS
Cste
mcell
MSC
mesenchym
alSC
HPL
hepatic
progenito
r-lik
ecell
ADadipo
setissuederiv
edP
Bperip
heralb
lood
MNC
mon
onuclear
cell
UC
umbilicalcordU
CBU
Cbloo
dmm
ouseB
Mb
onemarrow
MPC
mesenchym
alprogenito
rcell
fibrofibrob
last
EDT
exfoliateddecidu
oustoo
thiPS
Cindu
cedpluripotentS
CTC
tissue
cultu
reN
Anot
availableFB
Sfetalbovines
erum
allo
allo
genics
erum
BSA
bovines
erum
albu
minlowastFiltered220n
mrarr
10nmrarr
100n
myield
ed10ndash220
nmversus
10ndash100
nm(lt1000
kD)v
ersus100ndash220
nm(gt1000
kD)FC
fibron
ectin
coatedP
passageP
Dpop
ulationdo
ublin
gCM
con
ditio
nedmediumcon
cconcentrated
centrcentrifugation
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
8 BioMed Research International
Table 3 Growth factor level from various cell sources culture duration cell number and processing of conditioned medium
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[7] Hu-ESC-EC Monolayer mdash5 days NA
Angiogenic cytokine VEGF SDF-1 PlGFleptin EGF bFGF and HGFCM DM-PAC lt CM cPACAngiogenic cytokine VEGF PDGFICAM-1 EGF and bFGF CM ESC-EC gtCM c-PAC = CM D-PAC
[13] (i) Hu-ESC-CD133KDR-EPC(ii) Hu-UCB-EPC
Monolayermdash48hours
80 in 150mmculture dishConc50x
EGF
ESC-EPC versus CB-EPC versusEGM-212584 versus 12654 versus9 pgmL
FGF-2 383 versus 652 versus 61Fractalkine 1605 versus 150 versus 133GM-CSF 755 versus 323 versus 313IL-6 4332 versus 1961 versus 2463IL-8 239030 versus 13629 versus 7IL-9 345 versus 42 versus 9IP-10 458 versus 513 versus 511MCP-1 63 versus 3201 versus 1902PDGF-AA 6667 versus 5568 versus 41PDGF-ABBB 17 versus 1884 versus 75VEGF 4265 versus 538 versus 42
[9] Hu-AF SCmdashCkit (+) Monolayermdash16hours
15 times104mdash70from 5 times105 cells Conc125x
VEGF IL-8SDF-1 1 ng170000 cell (1 120583g DNA)
IL-6 MCP-1 05 ng170000 cellTGF-b 02 ng170000 cellIFNg mdashIP-10 CXCL10 mdashIL-1a mdash
[20] (i) Hu-AF-MSC(ii) Hu-AF-MSC-HPL
Monolayermdash24hours
15 times 10680Conc 25x
Proteome analysisHu-AF-MSC-HPL-CMAnti-inflammatory cytokine IL-10 IL-27IL-17E IL-13 IL-12p70 and IL-1raLiver protection MCP- 1 IL-1bHu-AF-MSC-HPL-CM AF-MSC-CMAnti-inflammatory cytokine TGF1205731Tissue repair serpin E1 SDF-1Angiogenesis VEGF PDEGF andendostatincollagen XVIIILiver regeneration UPA thrombospondin 1and 2 HEGF FGF-7 EGF and HGFAnti-apoptotic markers TIMP-1 IGFBP
[8](i) Hu-PB-MNC-EPC(ii) HUVEC
Monolayermdash72hours NA
IL-8CXCL8Hypox versusnorm
290907 plusmn 122794 pgmLversus 22821 plusmn 4063 pgmL
SDF-1CXCL12 60599 plusmn 6546 pgmLversus 31799 plusmn 4880 pgmL
HGF 5395 plusmn 1417 pgmLversus 3434 plusmn 748 pgmL
Angiogenin 1446 plusmn 682 pgmLversus 725 plusmn 158 pgmL
PDGF-BB 1116 plusmn 2702 pgmLversus 199 plusmn 22 pgmL
VEGF-A 255 plusmn 48 pgmLversus 114 plusmn 52 pgmL
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
BioMed Research International 9
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[10]
Hu-UCB SC(endothelial + MSC)
Monolayermdash48hours 80Conc 50x
EGF 3286 plusmn 419 pgmLVEGF 2463 plusmn 151 pgmLG-CSF 3615 plusmn 173 pgmLGM-CSF 3623 plusmn 345 pgmLTGF-1205731 PDGFbFGF and KGF =HUVEC
HUVEC Monolayermdash48hours 80Conc 50x
EGF UCB-SC-48XVEGF UCB-SC-42xG-CSF UCB-SC-37xGM-CSF UCB-SC-24x
[22] Hu-BM-MSC Monolayermdash24hours 2 times 106Conc 25x 69 from 174 prot tested (+)
(concentration NA)
[27] Hu-BM-MSC Monolayermdash24hours 4 times 106Conc 25x
VEGF
HGF
Normoxia 230 pgmLHypoxia 450 pgmLNormoxia 600 pgmLHypoxia 750 pgmL
[17] Hu-BM-MNC-stro-3-MPC NA 1 times 106Conc
IL-6 = 2x C 11804 plusmn 027 pgmLMCP-1 = 2x C 52189 plusmn 148 pgmLVEGF = 2x C 3395 plusmn 298 pgmL
[25](i) Hu-BM-MSC(ii) Hu-BM-CD133(iii) Hu-BM-p75
Monolayermdash48hours
1 times 10690Conc 40x
Secretioncell P75 versus CD133 versusBMMSC
IL6mdashnorm 38 versus 08 versus 06 fgIL6mdashhypox 025 = 025 versus 01 fgPlGFmdashnorm 0045 versus 001 versus 0 fgPlGFmdashhypox 0043 versus 0025 versus 015 fgADMmdashnorm 01 versus 005 versus 02 fgADMmdashhypox 58 versus 54 versus 115 fgVEGFmdashnorm 15 versus 10 versus 135 fgVEGFmdashhypox 07 versus 09 versus 095 fgSDF-1mdashnorm 135 versus 075 versus 015 fgSDF-1mdashhypox 04 versus 07 versus 10 fgHGFmdashnorm 084 versus 07 versus 025 fgHGFmdashhypox 001 versus 025 versus 001 fgDKK-Imdashnorm 4 versus 4 versus 45 fgDKK-1mdashhypox 68 versus 65 versus 105 fg
[28] Rat-BM-MSC Monolayermdash48hours
90T75Conc 40x
23 from 90 prot tested (+)NGF 356 plusmn 117 pgmLBDNF 208 plusmn 57 pgmLIL-6 427plusmn 168 pgmL
[24] Hu-AD-MSC Spheroidmdash2 days 42 times 107Centr
CM versus120572MEMhTGF-b1
1433 plusmn 671 versus 249 plusmn239 pgmL
hVEGF 101517 plusmn 17097 pgmL versusND
hPDGF-AA Both ND
[6]
Hu-AD-SCIn 120572MEMmdashFBS Spheroidmdash
2 days 105Centr
VEGF 144 plusmn 04 ngmLFGF2 132 plusmn 22 ngmLHGF 133 plusmn 23 ngmLCXCL12 166 plusmn 29 ngmL
In CRM-hu allo No diff gtlt 120572MEM-FBSIn CRM-serum (minus) GF lt
In 120572MEMmdashFBS Monolayermdash2days 105Centr GFltltlt
In CRM-hu allo GFltltlt
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
10 BioMed Research International
Table 3 Continued
Referencenumber Cell source Cultureduration Cell
numberprocessing Growth factor level
[5] Hu-AD-SC Monolayermdash72 hours 4 times 105Conc
Spot density array hypox versus nomoxiaGCSF 1407 plusmn 384 versus 1013 plusmn 421GM-CSF 1353 plusmn 126 versus 1021 plusmn 144IGFBP-1 948 plusmn 044 versus 556 plusmn 044IGFBP-2 891 plusmn 002 versus 673 plusmn 031IGF-II 1062 plusmn 085 versus 461 plusmn 093M-CSF 1406 plusmn 013 versus 746 plusmn 169M-CSF R 909 plusmn 020 versus 331 plusmn 175PDGF R120573 1767 plusmn 132 versus 1147 plusmn 140PDGF-AA 1663 plusmn 133 versus 1214 plusmn 212VEGF 1347 plusmn 126 versus 559 plusmn 122EGF 1106 plusmn 245 versus 3414 plusmn 675
[36](i) AD-SC(ii) Hu dermalfibroblast
NA NA
AD-SCVEGF 81065 plusmn 5692 pg120583g DNAIGF-I 32833 plusmn 227 pg120583g DNA
Hu dermal fibroblastVEGF 284 plusmn 225 pg120583g DNAIGFI Undetectable
[33] Hu-MSC (Lonza) Monolayermdash48hours 70(mdash)
IGF-1 15156 plusmn 2118 pgmLVEGF 4658 plusmn 1088 pgmLTGF-b1 3398 plusmn 144 pgmLHGF 203 plusmn 79 pgmL
FGF-2 PDGFBB BMP-2 and SDF-1 (mdash)CRM clinically relevant med hu allo human allogenic serum MP microparticle ND not detected SDF-1 stromal derived factor-1 PlGF placental GFbFGF basic FGF HGF hepatocyte GF PAC peripheral blood angiogenic cells (from PBMN cells-floating) cPAC healthy control PAC ESC-EC ESC derivedendothelial cell MCP-1 monocyte chemotactic protein-1 PDEGF platelet derived endothelial cell GF UPA urokinase plasminogen activator HEGF heparinbinding epidermal GF TIMP-1 tissue inhibitor of metalloproteinase-1 IGFBP insulin-like GF binding protein IP-10 interferon inducible protein-1 ADMadrenomedullin DKK-1 Dickkopf-1 norm normoxic hypox = hypoxic fg = fentogram
MCSF receptor (MCSFR) [5] and PDGF receptor (PDGFR)[5]
35 Translation of Conditioned Medium Usage in PatientsIn conditioned medium various factors may be presentas a cocktail and act in concert to promote regenerationTherefore it is important to analyze a complete set of growthfactor and cytokine levels for every kind of stem cell-derivedconditionedmedium and to know the culture condition con-ditioned medium processing and diseasesconditions thatare responsive to a certain conditioned medium treatmentWhen the content of the various cytokines in a certainconditioned medium is known the result of the conditionedmedium on a certain diseasecondition can be determinedand the way to translation into patients is open
From studies that analyzed VEGF level we can concludethat most stem cells secrete VEGF As VEGF plays a roleon angiogenesis [36] that is important in regeneration ofinjureddamaged tissuesorgans various stem cell-derivedconditioned media are able to cure various diseases and willhave more impact on diseases with ischemia In additionVEGF may prevent apoptosis in hypoxic condition thuspreventing further damage [6]
Concerning angiogenesis other thanVEGF other growthfactors that may play a role in angiogenesis are FGF2 [7 38]EGF [7] HGF [7 8] PlGF [7] SDF-1 [7] PDGF [7 38]TGF1205731 [38] and PDEGF [39] In addition various cytokinesthat is interleukin [38] IL-8 [8 9 13] chemokines [38]monocyte chemotactic protein (MCP-1) [9 13 17 20] leptin[7] angiogenin [8] and endostatincollagen XVIII [20] alsoplay a role in angiogenesis
Moreover FGF2 is a more potent angiogenic factorcompared to VEGF with additional effect on proliferationof fibroblasts preadipocytes and endothelial epithelial andneural stem cells on migration of neural crest derived glialand myogenic cells and on differentiation of neuroepithelialcells into mature neurons and glial cells [38]
Other growth factors contribute in the regeneration ofinjureddamaged tissue organs with special emphasis on pro-liferation that is PDGF for connective tissue glial and othercells EGF for mesenchymal glial and epithelial cells andIGF-I and IGF-II for various kinds of cells [40] In additionPlGF that is a member of VEGF family increases the activityofVEGF in vitro and in vivo [41] KGF inhibits oxidative stressinduced epithelial cell death [42] NGF promotes neuriteoutgrowth and neural cell survival [40] BDNF is neuropro-tective promotes cell survival and reduces astroglial scar
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
BioMed Research International 11
Table4Celln
umbertoprod
uceC
Mpera
pplication
volumeandmod
eofd
eliveryof
vario
uscellsourcesfor
vario
uscond
ition
sand
theo
utcome
Reference
number
Con
ditio
ndisease
Species
Cellsou
rceo
fCM
Cultu
remediumculture
typemdashcond
ition
Cell
numberapplication
Volumea
ndmod
eof
delivery
Outcome
[6]
Hindlim
bisc
hemiamdashdirect
Femalea
thym
icmicemdash
20ndash25g
rHu-AD-SC
120572MEM
mdashFB
S10m
onolayermdashhypo
x1
12000
40120583Lmdash
IMmdash7x
Goo
dresult
CRMmdashHu
allo10sph
eroidmdash
hypo
x1
48000
Bette
rresult
120572MEM
mdashFB
S10sph
eroidmdash
hypo
x1
Bette
rresult
[9]
Hindlim
bisc
hemiamdash10
days
MaleN
OD-SCI
Dmicemdash
10ndash12weeks
Hu-AF-SC
ndashCkit
(+)
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
50000
080120583Lmdash
IMmdash4x
Goo
dresult
[11]
Fullthickn
ess
wou
ndmdash5m
mdirect
Diabetic
-immun
odef
micemdash
17ndash23g
Hu-UCB
-CD
34-EPC
M199basalm
edium
(minus)m
onolayermdashno
rmoxia
1times10
6100120583
Lmdashintradermal
injection
Goo
dresult
[14]
Wou
nd30ndash50m
m2
120ndash
140m
m2 mdash
48ho
urs
MaleN
OD-SCI
Dmicemdash
4-5w
eeks
Hu-BM
-MSC
120572MEM
mdash10
FBSmon
olayermdashno
rmoxia
1times10
8100120583
LmdashSC
mdashperip
hery
wou
ndGoo
dresult
[17]
MCI
48ho
urs
Nud
e-athymicratmdash
6ndash8weeks
Hu-BM
-MNC-
stro-3-MPC
120572MEM
mdash(minus)m
onolayermdash
norm
oxia
1times10
6250120583
LIntram
yocardial
Goo
dresult
[20]
CCl4injuredacute
hepatic
failu
remdash24
hours
SCID
-NOD
micemdash
6ndash8weeks
Hu-AF-MSC
DMEM
mdash05
FBSmon
olayermdashno
rmoxia
15times10
6200120583
Lmdashintrahepatic
(leftliver
lobe)
Goo
dresult
Hu-AF-MSC
-HPL
Bette
rresult
[21]
Fulm
inanth
epatic
failu
remdash24
hours
MaleS
Dratmdash
250ndash
300g
Hu-MSC
DMEM
mdash005bo
vine
serum
albu
minm
onolayermdash
norm
oxia
15times10
6900120583
Lpenilevein
Goo
dresult
Increasedsurvival
[22]
MaleS
Dratmdash
280ndash
370g
Hu-BM
-MSC
NAmdash
005
BSAm
onolayermdash
norm
oxia
2times10
6900120583
LCM
Penilevein
Goo
dresult
Increasedsurvival
[23]
Focalcerebral
ischemiamdash72
hours
MaleS
Dratmdash
350ndash
400g
Hu-ED
T-SC
DMEM
(minus)m
onolayermdashno
rmoxia
40000
010x10120583
Lmdashintranasal
(left-rig
ht)
EverydayD3-D15
Goo
dresult
BM-M
SC(Lon
za)
Goo
dresult
[24]
Ischem
icStrokemdash8days
MaleS
Dmou
semdash8
weeks
Hu-AD-M
SC120572MEM
mdash(minus)s
pheroidmdash
hypo
xia
150400
Infusio
n05120583
Lho
ur-7
daysmdashlateralventricle
Goo
dresult
SCID
severec
ombinedim
mun
odeficiencyN
ODnon
obesed
iabeticSDSprague-D
awleyHuhu
manA
Dadipo
setissueSC
stem
cellAFam
nioticflu
idU
CBumbilicalcord
bloo
dEP
Cendo
thelialprogenitor
cellBM
bon
emarrow
MSC
mesenchym
alSC
MNC
mon
onuclear
cellMPC
mesenchym
alprogenito
rcellHPL
hepaticprogenito
r-lik
ecellandED
Texfoliateddecidu
oustoo
th
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
12 BioMed Research International
Adipose tissue
Bone marrow
Amniotic fluid
Umbilical cord
MSCCM
EPCCM
PB
UCB
AlopeciaSkin regenerationWound Cerebral ischemiastrokeBrainspinal cord injuryBonecartilage defectLiver failure
Myocardial infarct
Limb ischemia
MSC = mesenchymal stem cellCM = conditioned mediumPB = peripheral blood
UCB = umbilical cord bloodEPC = endothelial progenitor cell
Figure 1 Various possible applications of CM for various conditions
formation [28] and some growth factors including HEGFFGF-7 EGF and HGF promote liver regeneration [20]
Proinflammatory cytokines that play a role in regen-eration are IL-1b due to its liver protective role [20] IL-8 due to its angiogenic activity [8 9 13] and IL-9 due towound healing promotion activity [13 43] In addition anti-inflammatory cytokines prevent inflammation and promoteliver regeneration [20]
Other cytokines that is UPA and thrombospondins 1 and2 promote liver regeneration [20] serpin E-1 [20] and SDF-1[6ndash9 20 25] promote tissue repair [20] TIMP-1 and IGFBP[5 20] prevent apoptosis [20] ADM causes vasodilatationand reduces cellular oxidative stress and apoptosis [25]DKK-1 initiates bone marrow stem cell proliferation [25] andfractalkine prevents apoptosis [13 44]
Various colony stimulating factors that is granulocytecolony stimulating factor (GCSF) [5 10] granulocyte macro-phage CSF (GM-CSF) [5 10 13] and macrophage CSF(MCSF) [5] may recruit various resident stem cellspro-genitor cells including endothelial progenitors to site ofinjurydamage and promote wound healing process [10 13]or hair growth [5]
MCSF receptor (MCSFR) [5] promotes myeloid pro-genitor mononuclear phagocyte and placental trophoblastgrowth and development [45] and PDGFR [5] may interactwith various signalingmolecules or integrin to cause cell pro-liferation motility differentiation or survival by apoptosisinhibition [46]
Moreover one factor may contribute to more than onemode of regenerative action such as MCP-1 that is involvedin angiogenesis [9 13 17 20] and liver protection activity [20]Further for production of CM to be applied in various human
diseases data from animal studies that showed promisingoutcome are very valuable
351 Production of CM for Translation into Various HumanDiseases To use CM for various human diseases productionmethod of the CM needs to be standardized in terms of thetype andnumber of cells thatwere needed to produce theCMculture medium and condition and conditioned mediumprocessing In addition the volume and mode of deliveryare also important As various studies used various numbersand type of cells and various doses of CM it is importantto know the number of cells that yielded the CM for oneapplication which may be interpolated for human studiesTherefore in Table 4 we summarized all data that may beneeded for interpolation into human studies that is diseasesthat were treated species and age or body weight of theanimal type of cell culture medium and condition numberof cells to produceCM for one application volume andmodeof applicationMoreover various possible applications of CMfor various conditions are summarized in Figure 1
In addition for translation into patients it is very impor-tant to analyze and to note the various cytokine contentsof the various conditioned media Further for every condi-tioned medium with known cytokine content validation ofits use on various diseases needs to be conducted Finally thepossibility of promotion of existing cancer should be testedfor everyCM and caution should be taken beforeCM therapyto ensure that the recipient is free from cancer
Advantages of production of various CM for patientslie in the possibility of mass production by pharmaceuticalcompanies when production methods have been standard-ized Conditioned media are not like stem cells that need agood manufacturing practice (GMP) facility to be applied
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
BioMed Research International 13
to patients [47] When CM has been packaged properlyit can be transported easily as drugs and does not needcryopreservation such as that the stem cells need Howevercompared to stem cells that may survive for a rather longperiod CM needs to be given more frequently as cytokinesrsquoand growth factorsrsquo half-lives are mostly shorter [48 49]which is a disadvantage for the patients but will give moreprofit to pharmaceutical companies
4 Conclusion
Various stem cell-derived conditioned media were producedby various methods and processing and tested on variousdiseases and mostly showed good results However stan-dardized methods for various conditioned media productionand validations of their use on various diseases need to beconducted
Conflict of Interests
The author declares that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was funded by the research grant from IndonesianMinistry of Education and Culture (Pusnas 2014) Contractno 2218H2R12HKP05002014
References
[1] D Yang W Wang L Li et al ldquoThe relative contribution ofparacine effect versus direct differentiation on adipose-derivedstem cell transplantation mediated cardiac repairrdquo PLoS ONEvol 8 no 3 Article ID e59020 2013
[2] H O Kim and S Choi ldquoMesenchymal stem cell-derivedsecretome and microvesicles as a cell-free therapeutics for neu-rodegenerative disordersrdquo Tissue Engineering and RegenerativeMedicine vol 10 no 3 pp 93ndash101 2013
[3] H Fukuoka H Suga K Narita R Watanabe and S ShintanildquoThe latest advance in hair regeneration therapy using proteinssecreted by adipose-derived stem cellsrdquo American Journal ofCosmetic Surgery vol 29 no 4 pp 273ndash282 2012
[4] B R Zhou Y Xu S L Guo et al ldquoThe effect of conditionedmedia of adipose-derived stem cells on wound healing afterablative fractional carbon dioxide laser resurfacingrdquo BioMedResearch International vol 2013 Article ID 519126 9 pages2013
[5] B S ParkW S Kim J S Choi et al ldquoHair growth stimulated byconditioned medium of adipose-derived stem cells is enhancedby hypoxia evidence of increased growth factor secretionrdquoBiomedical Research vol 31 no 1 pp 27ndash34 2010
[6] S H Bhang S Lee J Y Shin T J Lee H K Jang andB S Kim ldquoEfficacious and clinically relevant conditioned-medium of human adipose-derived stem cells for therapeuticangiogenesisrdquoMolecular Therapy vol 22 no 4 p 862 2014
[7] J C Y Ho W Lai M Li et al ldquoReversal of endothelialprogenitor cell dysfunction in patients with type 2 diabetes
using a conditioned medium of human embryonic stem cell-derived endothelial cellsrdquo DiabetesMetabolism Research andReviews vol 28 no 5 pp 462ndash473 2012
[8] S di Santo Z Yang M W von Ballmoos et al ldquoNovel cell-freestrategy for therapeutic angiogenesis in vitro generated con-ditioned medium can replace progenitor cell transplantationrdquoPLoS ONE vol 4 no 5 Article ID e5643 2009
[9] T Mirabella M Cilli S Carlone R Cancedda and C GentilildquoAmniotic liquid derived stem cells as reservoir of secretedangiogenic factors capable of stimulating neo-arteriogenesis inan ischemicmodelrdquo Biomaterials vol 32 no 15 pp 3689ndash36992011
[10] J Kim J H Lee S M Yeo H M Chung and J I Chae ldquoStemcell recruitment factors secreted from cord blood-derived stemcells that are not secreted frommature endothelial cells enhancewound healingrdquo In Vitro Cellular amp Developmental BiologyAnimal vol 50 no 2 pp 146ndash154 2014
[11] J Y Kim S H Song K L Kim et al ldquoHuman cord blood-derived endothelial progenitor cells and their conditionedmedia exhibit therapeutic equivalence for diabetic woundhealingrdquo Cell Transplantation vol 19 no 12 pp 1635ndash16442010
[12] C Shrestha L Zhao K Chen H He and Z Mo ldquoEnhancedhealing of diabetic wounds by subcutaneous administration ofhuman umbilical cord derived stem cells and their conditionedmediardquo International Journal of Endocrinology vol 2013 ArticleID 592454 10 pages 2013
[13] M J Lee J Kim K I Lee J M Shin J I Chae and H MChung ldquoEnhancement of wound healing by secretory factorsof endothelial precursor cells derived from human embryonicstem cellsrdquo Cytotherapy vol 13 no 2 pp 165ndash178 2011
[14] P J Mishra and D Banerjee ldquoCell-free derivatives from mes-enchymal stem cells are effective in wound therapyrdquo WorldJournal of Stem Cells vol 4 no 5 pp 35ndash43 2012
[15] B Hynes A H S Kumar J OrsquoSullivan et al ldquoPotent endothelialprogenitor cell-conditioned media-related anti-apoptotic car-diotrophic and pro-angiogenic effects post-myocardial infarc-tion are mediated by insulin-like growth factor-1rdquo EuropeanHeart Journal vol 34 no 10 pp 782ndash789 2013
[16] L Timmers S K Lim I E Hoefer et al ldquoHuman mesenchy-mal stem cell-conditioned medium improves cardiac functionfollowing myocardial infarctionrdquo Stem Cell Research vol 6 no3 pp 206ndash214 2011
[17] F See T Seki P J Psaltis et al ldquoTherapeutic effects ofhumanSTRO-3-selectedmesenchymal precursor cells and theirsoluble factors in experimental myocardial ischemiardquo Journal ofCellular and Molecular Medicine vol 15 no 10 pp 2117ndash21292011
[18] L Timmers S K Lim F Arslan et al ldquoReduction ofmyocardialinfarct size by human mesenchymal stem cell conditionedmediumrdquo Stem Cell Research vol 1 no 2 pp 129ndash137 2008
[19] Z Du C Wei K Cheng et al ldquoMesenchymal stem cell-conditioned medium reduces liver injury and enhances regen-eration in reduced-size rat liver transplantationrdquo Journal ofSurgical Research vol 183 no 2 pp 907ndash915 2013
[20] D S Zagoura M G Roubelakis V Bitsika et al ldquoTherapeuticpotential of a distinct population of human amniotic fluidmesenchymal stem cells and their secreted molecules in micewith acute hepatic failurerdquoGut vol 61 no 6 pp 894ndash906 2012
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007
14 BioMed Research International
[21] D van Poll B Parekkadan CH Cho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008
[22] B Parekkadan D Van Poll K Suganuma et al ldquoMesenchymalstem cell-derived molecules reverse fulminant hepatic failurerdquoPLoS ONE vol 2 no 9 article e941 2007
[23] T Inoue M Sugiyama H Hattori H Wakita T Wakabayashiand M Ueda ldquoStem cells from human exfoliated deciduoustooth-derived conditioned medium enhance recovery of focalcerebral ischemia in ratsrdquo Tissue Engineering A vol 19 no 1-2pp 24ndash29 2013
[24] Y J Cho H S Song S Bhang et al ldquoTherapeutic effectsof human adipose stem cell-conditioned medium on strokerdquoJournal of Neuroscience Research vol 90 no 9 pp 1794ndash18022012
[25] B Bakondi I S Shimada A Perry et al ldquoCD133 identifiesa human bone marrow stemprogenitor cell sub-populationwith a repertoire of secreted factors that protect against strokerdquoMolecular Therapy vol 17 no 11 pp 1938ndash1947 2009
[26] T J Chuang K C Lin C C Chio C C Wang C P Changand J R Kuo ldquoEffects of secretome obtained from normoxia-preconditioned human mesenchymal stem cells in traumaticbrain injury ratsrdquo Journal of Trauma and Acute Care Surgeryvol 73 no 5 pp 1161ndash1167 2012
[27] C Chang C Chio C Cheong C Chao B Cheng and M LinldquoHypoxic preconditioning enhances the therapeutic potential ofthe secretome from cultured humanmesenchymal stem cells inexperimental traumatic brain injuryrdquo Clinical Science vol 124no 3 pp 165ndash176 2013
[28] D Cantinieaux R Quertainmont S Blacher et al ldquoCondi-tionedmedium from bonemarrow-derived mesenchymal stemcells improves recovery after spinal cord injury in rats anoriginal strategy to avoid cell transplantationrdquo PLoS ONE vol8 no 8 Article ID e69515 2013
[29] A van Koppen J A Joles B W M van Balkom et alldquoHuman embryonic mesenchymal stem cell-derived condi-tionedmedium rescues kidney function in rats with establishedchronic kidney diseaserdquo PLoS ONE vol 7 no 6 Article IDe38746 2012
[30] Y Gheisari N Ahmadbeigi M Naderi S M Nassiri S Nadriand M Soleimani ldquoStem cell-conditioned medium does notprotect against kidney failurerdquo Cell Biology International vol35 no 3 pp 209ndash213 2011
[31] W Zhu L Huang Y Li et al ldquoMesenchymal stem cell-secretedsoluble signaling molecules potentiate tumor growthrdquo CellCycle vol 10 no 18 pp 3198ndash3207 2011
[32] L Li Y Liu C Yang et al ldquoImprovement of ventilator-inducedlung injury by IPS cell-derived conditioned medium via inhi-bition of PI3KAkt pathway and IP-10-dependent paracrineregulationrdquo Biomaterials vol 34 no 1 pp 78ndash91 2013
[33] T Inukai W Katagiri R Yoshimi et al ldquoNovel applicationof stem cell-derived factors for periodontal regenerationrdquo Bio-chemical and Biophysical Research Communications vol 430no 2 pp 763ndash768 2013
[34] E Ivanova-Todorova I Bochev R Dimitrov et al ldquoCondi-tionedmedium from adipose tissue-derivedmesenchymal stemcells induces CD4+FOXP3+ cells and increases IL-10 secretionrdquoJournal of Biomedicine and Biotechnology vol 2012 Article ID295167 8 pages 2012
[35] S K Sze D P V de Kleijn R C Lai et al ldquoElucidatingthe secretion proteome of human embryonic stem cell-derivedmesenchymal stem cellsrdquo Molecular and Cellular Proteomicsvol 6 no 10 pp 1680ndash1689 2007
[36] S Sadat S Gehmert Y Song et al ldquoThe cardioprotective effectof mesenchymal stem cells is mediated by IGF-I and VEGFrdquoBiochemical and Biophysical Research Communications vol 363no 3 pp 674ndash679 2007
[37] R Goswami and M H Kaplan ldquoA brief history of IL-9rdquo TheJournal of Immunology vol 186 no 6 pp 3283ndash3288 2011
[38] Y R Yun J E Won E Jeon et al ldquoFibroblast growth factorsbiology function and application for tissue regenerationrdquoJournal of Tissue Engineering vol 2010 Article ID 218142 2010
[39] T Saeki M Tanada S Takashima et al ldquoCorrelation betweenexpression of platelet-derived endothelial cell growth factor(thymidine phosphorylase ) and microvessel density in early-stage human colon carcinomasrdquo Japanese Journal of ClinicalOncology vol 27 no 4 pp 227ndash230 1997
[40] G Litwack ldquoGrowth factors and cytokinesrdquo in Human Bio-chemistry and Disease G Litwack Ed pp 587ndash683 ElsevierAcademic Press 2008
[41] J E Park H H Chen J Winer K A Houck and N FerraraldquoPlacenta growth factor potentiation of vascular endothelialgrowth factor bioactivity in vitro and in vivo and high affinitybinding to Flt-1 but not to Flk-1KDRrdquoThe Journal of BiologicalChemistry vol 269 no 41 pp 25646ndash25654 1994
[42] P Ray Y Devaux D B Stolz et al ldquoInducible expressionof keratinocyte growth factor (KGF) in mice inhibits lungepithelial cell death induced by hyperoxiardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 10 pp 6098ndash6103 2003
[43] J E Turner P J Morrison C Wilhelm et al ldquoIL-9-mediatedsurvival of type 2 innate lymphoid cells promotes damagecontrol in helminthmdashinduced lung inflammationrdquoThe Journalof Experimental Medicine vol 210 no 13 pp 2951ndash2965 2013
[44] G E White and D R Greaves ldquoFractalkine a survivorrsquosguide chemokines as antiapoptotic mediatorsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 3 pp 589ndash5942012
[45] A Mancini A Koch A D Whetton and T Tamura ldquoTheM-CSF receptor substrate and interacting protein FMIP isgoverned in its subcellular localization by protein kinase C-mediated phosphorylation and thereby potentiates M-CSF-mediated differentiationrdquo Oncogene vol 23 no 39 pp 6581ndash6589 2004
[46] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008
[47] P Wuchter K Bieback H Schrezenmeier et al ldquoStandardiza-tion of GoodManufacturing Practicendashcompliant production ofbone marrowndashderived human mesenchymal stromal cells forimmunotherapeutic applicationsrdquo Cytotherapy 2014
[48] P Yde B Mengel M H Jensen S Krishna and A TrusinaldquoModeling the NF-120581B mediated inflammatory response pre-dicts cytokine waves in tissuerdquo BMC Systems Biology vol 5article 115 2011
[49] A Khosravi C M Cutler M H Kelly et al ldquoDeterminationof the elimination half-life of fibroblast growth factor-23rdquo TheJournal of Clinical Endocrinology amp Metabolism vol 92 no 6pp 2374ndash2377 2007