Hindawi Publishing CorporationStem Cells InternationalVolume 2013 Article ID 246134 12 pageshttpdxdoiorg1011552013246134

Research ArticleComparative Evaluation of Human Mesenchymal Stem Cells ofFetal (Whartonrsquos Jelly) and Adult (Adipose Tissue) Origin duringProlonged In Vitro Expansion Considerations for Cytotherapy

I Christodoulou1 F N Kolisis2 D Papaevangeliou3 and V Zoumpourlis1

1 Institute of Biology Medicinal Chemistry amp Biotechnology National Hellenic Research Foundation (NHRF)48 Vasileos Konstantinou Avenue 11635 Athens Greece

2 School of Chemical Engineering National Technical University of Athens 15780 Athens Greece3 TAK-EIE Stem Cell Bank SA NHRF 11635 Athens Greece

Correspondence should be addressed to V Zoumpourlis vzubeiegr

Received 5 October 2012 Revised 28 November 2012 Accepted 28 November 2012

Academic Editor Pranela Rameshwar

Copyright copy 2013 I Christodoulou et al 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

Mesenchymal stem cells (MSCs) are somatic cells with a dual capacity for self-renewal and differentiation and diverse therapeuticapplicability both experimentally and in the clinic These cells can be isolated from various human tissues that may differanatomically or developmentally with relative ease Heterogeneity due to biological origin or in vitromanipulation is neverthelessconsiderable and may equate to differences in qualitative and quantitative characteristics which can prove crucial for successfultherapeutic use With this in mind in the present study we have evaluated the proliferation kinetics and phenotypic characteristicsof MSCs derived from two abundant sources that is fetal umbilical cord matrix (Whartonrsquos jelly) and adult adipose tissue(termed WJSC and ADSC resp) during prolonged in vitro expansion a process necessary for obtaining cell numbers sufficientfor clinical application Our results show that WJSC are derived with relatively high efficiency and bear a substantially increasedproliferation capacity whilst largely sustaining the expression of typical immunophenotypic markers whereas ADSC exhibit areduced proliferation potential showing typical signs of senescence at an early stage By combining kinetic with phenotypic data weidentify culture thresholds up to which both cell types maintain their stem properties and we discuss the practical implications oftheir differences

1 Introduction

Mesenchymal stem cells (MSCs) are somatic cells with anability to self-renew and to differentiate towards a variety ofspecialized cell types through a combination of symmetricand asymmetric divisions Populations of MSCs can berelatively easily isolated from a number of tissues that maydiffer both developmentally (eg fetal versus adult) andanatomically (eg bone marrow versus fat) Due to theirunique properties of self-renewal and multipotency as wellas their immunogenic profile [1ndash3] (in many cases theyhave been shown to be hypoimmunogenic andor can betransplanted autologously following ex vivo expansion) theyhave been utilized in several therapeutic applications such as

tissue repair and regeneration and autoimmune disease overthe last decade [4]

Despite the broad commonalities in defining characteris-tics and clinical applicability potential there are unquestion-able qualitative and quantitative differences in terms of theirisolation efficiency and in vitro manipulation performanceas well as their efficacy in animal models and clinical studieswhich have been well highlighted in a number of publications[5ndash8] Indeed the process of getting MSCs from the tissuesource to the patient is far from streamlined and includesseveral factors of heterogeneity the main ones relating toisolation and expansion of these cell populations [9] Inparticular lack of consistencyconsensus in cell manipulation(ie diversity in the isolation and culture protocols applied)

2 Stem Cells International

in addition to inherent heterogeneity in the samples (relatedto donor tissue origin age sex and underlying pathology)may have an impact on the quality and quantity of isolatedcells Moreover MSCs (in contrast to embryonic stem cells)are bound to have to a lesser or greater extent a limitedreplication potential in an artificial in vitro culture environ-ment Indeed Hayflickrsquos theory states that most somatic celltypes may undergo a maximum of 70 population doublingsbefore reaching replicative senescence [10 11] and MSCsare bound to be constrained by this limitation Neverthelessin the case of MSCs-based cytotherapy it is estimated thatapproximately 1ndash5 million cellskg body are required forsuccessful outcome therefore a degree of ex vivo expansionis necessary and this holds true in over 60 of casesof cellular transplants [12 13] A question therefore ariseswhether MSCs can sustain their expansion potential andphenotypic stability over prolonged culture periods for howlong and whether possible disparities exist between diversepopulations rendering some of them more competent andsuitable for consideration in cytotherapy protocols

In the present study we have focused on the charac-terization of postnatal human MSC populations originatingfrom two sources that differ both developmentally as well asanatomically that is cells isolated from the matrix (Whar-tonrsquos jelly) of the fetal umbilical cord (UC) tissue (termedWJSC) [14] and from the abdominal adipose tissue of adults(adipose-derived ADSC) [15] The main advantages of theseMSC sources compared to other stem cell origins are (a)abundant cell availability (b) relative ease of access for stemcell isolation with minimal or no tissue morbidity and (c)an immunogenic profile of isolated cells that is favorable forcellular transplantation [16 17]

Themain hypothesis behind this work has been that thereare differences in qualitative and quantitative characteristicsof these MSC populations in terms of their adaptationto extended culture which ultimately relate to practicalconsiderations regarding their clinical use This study whichto our knowledge is the first to address this issue describesa culture regime that sustains the MSC phenotype whilehighlighting a threshold for optimal cell expansion

2 Materials and Methods

21 MSC Isolation

211 WJSC WJSC were isolated from donated UC tissuesamples from normal full-term deliveries following approvalby the institutional bioethics review board Umbilical cordswere transported to the lab in Hanks Balanced Salt Solution(HBSS) containing 1 penicillinstreptomycin solution (bothfrom Invitrogen) and cell isolation was carried out withina maximum of 48 hrs from tissue collection according to amodified protocol based on that described by Seshareddyand colleagues [18] using reagents free of animal productsBriefly the blood vessels were carefully removed and the UCwas minced into 1-2mm3 fragments and then incubated witha mixture of 026 collagenase type I (Biochrom) and 007hyaluronidase (Applichem) for 2 hrs followed by 0125

trypsin (Biochrom) for 30min with gentle agitation at 37∘CThe digested mixture was then passed through a 100 120583mcell strainer (BD) and washed in PBS (Biochrom) to obtaincell suspensions that were stained with 04 trypan blue(Biochrom) counted on a hemocytometer aliquoted andfrozen down in liquid N

2

212 ADSC ADSC were isolated from liposuction aspiratesdonated for research purposes which were obtained fromsubcutaneous adipose tissue sites of subjects (age 44plusmn11 yrssex 7129 malefemale) undergoing elective procedures inlocal plastic surgery offices The research protocol used hadbeen approved by the institutional bioethics review boardCell isolation was carried out under xeno-free conditionswithin 48 hrs from collection following a modified protocolbased on that described by Zuk et al [19] Briefly lipoaspiratesamples were first washed in the collection bag using 250mLnormal saline and after removal of the blood fraction adi-pose tissue was dispersed by digestion with 02 collagenasetype I (Biochrom) at 37∘C for 1 hr with agitation After onemore wash the layer below the floating lipid-filled adipocyteswas dispensed into 50mL tubes and centrifuged at 900 g for45min Subsequently the supernatant was aspirated and thecell pellets (stromal vascular fraction (SVF)) frozen down inliquid N

2 after determining cell numbers with a hematology

blood analyzer (Beckman-Coulter)

22 MSC Culture

221 WJSC The five best samples (ie with the highestnumber of nucleated cells) out of a pool of 34 samples wereresuscitated plated on noncoated 75-cm2 flasks (Corning)and cultured in growth medium (GM) which consisted ofDMEMF12 (with 35 gL glucose ultraglutamine I and Napyruvate Lonza) supplemented with 10 batch-tested fetalbovine serum (FBS) 15mM HEPES 1x nonessential aminoacids and 1 penicillinstreptomycin (all from Invitrogen)Preliminary experiments testing DMEM and DMEMF12-based media (both used in the literature for maintenance ofWJSC as well as ADSC cultures) had established DMEMF12containing stable L-glutamine dipeptide as the optimal basalmedium for propagation of both MSC types (see Section 3)Three days after initial plating nonadherent cells wereremoved and the medium was replaced with fresh GM Cellswere maintained in a humidified atmosphere with 5 CO

2

in air at 37∘C with media changes (complete) every 3-4days until 70ndash80 confluence Subcultivation (passaging)was carried out by trypsinization using 005 trypsin-EDTAsolution (TE Invitrogen) and replating the cells at a densityof 4000 cellscm2 in 75 cm2 flasks Frozen stocks of 05ndash2million cells were maintained in 10 DMSO in FBS in 2mLcryovials (Nunc) stored in liquid N

2

222 ADSC The eight best samples (ie with the highestnumber of nucleated cells) out of a pool of 29 samples wereresuscitated plated on noncoated 25 cm2 flasks (Greiner) andcultured in GM as described previously After three days ofculture nonadherent cells were removed and the medium

Stem Cells International 3

was replaced Adherent cells were grown at 37∘C in 5 CO2

at saturating humidity with medium changes twice weeklyOnce 70ndash80 confluence was reached cells were detachedwith 005 trypsin-EDTA and replated (passaged) in 75 cm2flasks at a seeding density of 6000 cellscm2 Frozen stocks of05ndash2 million cellscryovial were maintained in liquid N

2 as

described previously

23 Determination of Proliferation Kinetics and Cell Size Thenumber of doublings in cell populations (PD) and the timeperiod (in hrs) in which they occur (PDT) were calculatedaccording to the following formulas

PD = ( 1log102) times log

10(NtNo)

PDT = 119905 times [log102

(log10Nt minus log

10No)]

(1)

where No is number of viable (as determined by trypan blueexclusion) cells at seeding Nt is number of viable cells atharvest and t is time (in hrs) between seeding and harvest Aproliferation index (PI) was used as a standardized measureof the proliferative capacity of the cells PI was defined as theratio of the number of population doublings (PD) in a specificpassage over the respective time period (PDT) in which theformer take place

To determine cell size (surface area in 120583m2) photos of sixrandom nonoverlapping fields were captured at 10x magnifi-cation using an Olympus E520 digital camera mounted on anAxiovert 40 CFL (Zeiss) inverted microscope The area of atleast five different cells per field was measured using AdobePhotoshop CS5 Extended Cells with sizes in the top 25 ofeach size range were considered as ldquogiantrdquo cells (gt4600 120583m2and gt3880120583m2 for ADSC and WJSC resp)

24 Characterization of Isolated Stem Cell Populations

241 Immunophenotyping Cultured WJSC and ADSC werecharacterized for the expression of the following mark-ers CD29 (120573

1-integrin) CD44 (H-CAM) CD73 (ECTO-

51015840 nucleaseSH3) CD90 (THY-1) CD105 (endoglinSH2)CD14 (LeuM3MY4) CD34 (HPCA1gp105ndash120) and CD45(LCA) Cells were detached using StemPro Accutase (Invit-rogen) washed in PBS and stained using specific FITC- PE-ECD- or PC5-conjugated monoclonal antibodies or isotype-matched (IgG1aIgG2a) controls (all by Beckman-Coulterexcept CD105-PE which was purchased from BD) 7-AAD(Beckman-Coulter) was used as a viability stain Analysiswas performed on a Beckman-Coulter EPICS XL-MCL flowcytometer At least 20000 live events were acquired

242 CFU-F Assay The capacity of ADSC and WJSC forself-renewal was evaluated by seeding cells at clonal densities(20ndash80 cellscm2) in 10 cm dishes and fixing and stainingthe resulting CFU-F two weeks later with 05 crystal vio-letmethanol solution (Digirolamo et al 1999) [20] Coloniesgt 2mm in diameter were counted manually

243 ALP Assay Alkaline-phosphatase-(ALP-)-positiveCFU which represent clonal populations of cells with os-teoprtogenitor properties were detected in duplicate culturesof ADSCWJSC (set up as described for CFU-F assaypreviously mentioned) using the Sigma leukocyte ALP histo-chemistry staining kit (Kaplow 1968) [21] following themanufacturerrsquos recommendations Colonies counterstainedwith hematoxylin and those gt 2mm in diameter werecounted manually

244 Osteogenic Differentiation The potency of WJSC andADSC for directed differentiation towards osteoblast lineagewas tested by alizarin red staining of mineralized bonenodules [22] Cells were grown in triplicate in 6-well plate for21 days in the presence of osteogenic supplements (50120583gmL120573-glycerophosphate 10mML-ascorbic acid 2-phosphate and5 times 10

minus7M dexamethasone all from Sigma) and then fixedin 10 formol saline and stained with 2 alizarin red Bonenodules gt 1mm were manually counted and scored in threedifferent categories + (lt 20) ++ (20ndash50) and +++ (gt 50)

245 Calcium Assay The Ca content of mineralized culturelysates was measured by a colorimetric assay (Cayman) [23]following the manufacturerrsquos recommendations Cells wereseeded at a density of 500cm2 in duplicate in 10 cm dishesand grown in the presence of osteogenic supplements (seeaforementioned) for 21 days Following a wash in CaMg-free PBS they were lysed in 100mM ice-cold Tris buffer spunat 10000 g and the supernatants stored at minus80∘C until usedSample absorbances were measured at 570 nm using a Tecanmicroplate reader and compared across a reference curveconstructed using calcium standards The assay detectionlimit was 05mgdL

246 Senescence Assay Cellular senescence was determinedby 120573-gal staining of subconfluent day 4-5 cultures set up in6-well plates using the Sigma cell senescence histochemicalkit [24] according to the manufacturerrsquos instructions Brieflycells were fixed and incubated with the staining mix contain-ing X-gal overnight at 37∘C in a low CO

2environment The

occurrence of 120573-gal positive blue-green cells was determinedby counting a minimum of 50 cells in at least five randomnonoverlapping fields under a microscope Each assay wasperformed in duplicate

247 Adherence Assay Cell detachment was measured intime series experiments set up in 6-well plates in triplicateCells were seeded at 4000ndash6000cm2 and left to grow to70ndash80 confluence upon which they were washed in PBSand incubated with 600 120583L warm 005 trypsin-EDTA (TE)per well for up to 30min without agitation Every 5min thedigested cell suspensions were drawn out into 15mL tubesand an aliquot was mixed with trypan blue and live cellscounted on a Neubauer plate while 600120583L of fresh warm TEwas added per well for the next measurement Control cellswere incubated with TE at 37∘C with frequent tapping until100 detached from culture plastic

4 Stem Cells International

25 Statistical Analysis Parametric and nonparametric tests(Spearman correlation analysis Mann-Whitney pairwisedata comparisons and ANOVA) were conducted usingGraphPad Prism 504 statistical analysis software Data aredepicted as means plusmn SD unless otherwise stated A P valuelt 005 was considered as statistically significant

3 Results

31 Primary Cell Isolation and Culture Initiation Efficiency

311 WJSC Mean nucleated cell yield following UC tissueprocessing was about 23 times 106 and was found to positivelycorrelate (Spearman 119903 = 0722119875 lt 0001) with tissue proper-ties (ie UC length) (Table 1 and see in SupplementaryMate-rial available online at httpdxdoiorg1011552013246134Figure 1) The isolation protocol involved removal of thevessels that due to the supercoiled anatomy of the UCincreases tissue processing time and hence results in some cellloss compared to protocols based on digestion of whole UCNevertheless our protocol produces a more homogeneouspopulation than the latter which results in the isolation ofa mixed population of at least four stem cell types [25 26]Five out of five (100) cultures of adherent cells derived fromplated UC nucleated cell samples led to the establishment ofcultures that grew successfully beyond passage 3 (Table 1)The three of these cultures with the shortest PDT (see thefollowing) were further subcultured and used for the exper-iments described in this study The percentage of adherentcells in the primary cultures was estimated at 12

312 ADSC ForADSCmeannucleated cell yield at isolationwas 312 times 106 cells (Table 1) Regression analysis of samplesshowed that this yield was not significantly associated withinitial tissue quantity (donor adipose weight) (see Supple-mentary Figure 1) or other donor characteristics such asage or sex Nevertheless there was a strong linear correlationof the number of isolated cells with adipose tissue weightfor samples up to 200 g (Spearman 119903 = 0952 119875 lt0001) highlighting the increased efficiency of the protocolfor samples in that range The eight adipose tissue sampleswith the highest mononuclear cell yields were plated out andused to establish cultures of ADSC Five of these samplestested proliferated very slowly (less than 20 increase incell yield between passages) and failed to grow beyondpassage 3 whereas 38 (375) proliferated further and wereused for the experiments ADSC yield was approximately18 times 10

6 cellsg lipoaspirate with the percentage of adherentcells in the primary cultures being much lower than thatof WJSC at 093 This yield is slightly higher than thatreported elsewhere (1 times 106 cellsg) [27] Viability of isolatedcells and resuscitated cells following initial cryopreservationwas consistently high (gt92) and similar to that of WJSC(Table 1)

32 Proliferation Kinetics of Long-Term Cultures Figures1(a)ndash1(c) depict the proliferation dynamics of the two MSCpopulations during prolonged in vitro subcultivation while

Table 2 summarises the main findings The mean cell yieldshown in Table 2 is the one that arises from the serial prop-agation of cells following the subculture protocol describedin Section 2 This was higher (by more than twofold) forWJSC and related to a constant number of about three PD perpassage (depicted by the linearity of the graph in Figure 1(a))accumulating to a total of 58 PD in the 100-day cultureperiod Mean PDT was 384 plusmn 86 hrs over that period Peakproliferative rate was observed at passage 6 (considering PIand PDT Figures 1(b) and 1(c) resp) which equated tonearly 19 PD over a month in culture Maximum PI of WJSC(observed at passage 6) was over 15-fold higher than that ofADSC and coincided with a maximum number of 372 plusmn032 PD WJSC showed no signs of slowdown even after 15passages (45 PD) (119875 gt 005 ANOVA on PDT of passages 2ndash16)There was nevertheless a significant increase in PDT afterpassage 17 which exceeded 50 hrs by passage 20

ADSC proliferation capacity was maximum at passage 2(see Figures 1(b) and 1(c)) and remained above average untilpassage 5 although related to a poor number of cumulativePD (just over five) Signs of senescence and essentially ces-sation of proliferation appeared after passage 6 Thencefortha plateau in PD accumulation a consistently high PDT (ofnearly 37 days) and low expansion rate (lt20 per passage)were observed Mean cell yield per passage in our cultureprotocol was two-three times lower for ADSC (Table 2)

The growth curves (Figure 1(d)) depict the kinetics ofproliferation within a single passage Both cell types followa typical sigmoid growth pattern but with different phasedurations WJSC exhibit a lag phase similar to that of ADSC(about 25 days) but a much shorter log phase (35 as opposedto 75 days) during which they expand more promptly(about fivefold expansion of cell population) WJSC becomeconfluent by day 7 while they continue to grow by 15-20 even when superconflluent forming dense cultures ofcompacted cells whereas ADSC undergo contact inhibitionafter day 11 not exceeding 90 confluency GM containingthe dipeptide form of glutamine increases cell yield duringlog phase by up to 200 (especially for ADSC 119875 lt 001 day9) while it enhances survival of postconfluentWJSC cultures(119875 lt 005 day 11 see online Supplementary Figure 2) Thisis in concordance with the effects of glutamine dipeptide onbone-marrow-derived MSC survival and growth [28]

33 Phenotypic Characteristics of Long-Term CulturesFigure 2(a) illustrates the change in cell size as a phenotypiccharacteristic during prolonged subculture A markedgreather than 300 change in cell size over culture timewas observed for both MSC populations ADSC mean sizewas 3876 120583m2 with minimum (1648) and maximum (6130)values observed at passages 1 and 8 respectively (Figure 2(a))WJSC had an average size of 2926120583m2 (min 1437 max 5183at passages 1 and 11 resp Figure 2(a)) WJSC exhibited aperiodicity in cell size that might be attributed to smallrapidly proliferating cells undergoing rounds of symmetricand asymmetric divisions which on one hand give rise todifferentiating daughter cells that gradually increase in sizeand probably become senescent (see also Figures 2(c) and 3)

Stem Cells International 5

Table 1 Cell isolation and culture establishment efficiency

WJSC ADSC 119875 value

Mean tissue length-weight 133 cm plusmn 2lowast 1738 g plusmn 1357 mdashMean nucleated cell yield (times106)

(per unit length-weight)228 plusmn 155(017cm)

312 plusmn 198(18g) mdash

Mean viability at isolation () 943 plusmn 22 966 plusmn 16 gt005Mean viability at resuscitation () 929 plusmn 43 927 plusmn 49 gt005 of adherent cells in starting culture 173 plusmn 46 093 plusmn 045 lt0001Adherent cultures beyond p3 100 (55) 375 (38) lt005Mean passage-to-passage expansion rate (p1ndash5) 974 plusmn 317 175 plusmn 56 lt001Mean subculture viability (p1ndash5) 976 plusmn 05 964 plusmn 19 gt005119875 value WJSC versus ADSC lowastA statistically significant association was found between UC tissue sample length and the yield of isolated nucleated cellsfollowing tissue processing (see text for details)

while at the same time replenishing the culture with moresubsets of small rapidly proliferating stem cells In support ofthis is the association of kinetics (PDT) with cell size (Figures2(b) and 2(c)) Spearman analysis showed that small cell sizecorrelates better with low PDT while cells that take longerto undergo rounds of division (senescent) tend to be largerSpecifically the cells with sizes in the top 25 (arbitrarilydenoted as giant cells) have also the highest PDT and thelatter correspond to mid to late passages (p7 or greater forADSC and p9 or greater for WJSC)

Besides cell size a marked change was observed in otherphenotypic properties of MSCs over in vitro expansionFigure 3 outlines these differences between early cultures andculture endpoints In terms of morphological characteristicsthree main morphologies were observed (a) spindle-shapedelongated (type I) (b) polygonal with few small membraneprotrusions (type II) (c) flattened with irregular shape andlarge (frequently multiple) nuclei (type III) Early-passage(p2) WJSC and ADSC have mainly type II and type Imorphologies respectively (see Table 3 and Figure 3(a))Serial passaging of both MSC populations results in gradualacquisition of type III morphology which especially inthe case of WJSC increases by 28-fold in occurrence andbecomes predominant by passage 20 In these culture pointscells with either type I or type III morphologies accountedfor over 70 of the cultures (see Table 3) with at least one-in-three cells being a giant cell (see Methods section for sizedefinition) bearing enlarged often multiple nuclei and beinglargely senescent staining positive for 120573-gal (Figure 3(b))The occurrence of senescent cells in ADSC cultures was fairlyfrequent with roughly one cell out of five staining positiveirrespective of the culture point (Table 3) On the contrarythe senescence rate of early WJSC culture which is very lowincreases 5-6 fold in later passages albeit with considerableinterculture variability An interesting observation was alsothat cytoplasmic granularity ofWJSC decreased by 35ndash40with passaging probably relating to rearrangements in thecytoskeleton and protein synthesis machinery (Table 3 andonline Supplementary Figure 3)

In terms of stemness properties both cell types have acapacity for clonogenicityself-renewal and lineage-specific

differentiation and express a typical immunophenotypic pro-file with high MSC marker (gt97 positive except CD44of late-passage ADSC which was slightly lower at 92 plusmn23) and low HSC marker expression (lt3 positive withthe exception of 8 plusmn 02 of early-passage ADSC whichexpressed monocyte-related CD14) (Figure 4) The mostavidly expressed markers (high signal intensity) were CD44(H-CAM) and CD105 (endoglin) for WJSC and ADSCrespectively But although expression levels of CD surfacemarkers remained constant over time in culture clonogenic-ity was reduced ForWJSC about 1 in 45 cells was clonogenicand this ability diminished by less than twofold over the first10 passages (+24 PD) but almost by a factor of five in thenext 10 passages (+28 PD) (Figure 3(c) and Table 3) ADSCformed robustly clones at all densities tested however theof CFU-F ratios were lower than those of WJSC that hadundergone many more PD Differentiation capacityinducedlineage specific differentiation (osteoblasts) also diminishedwith ageing in culture (Figures 3(c) and 3(d) Table 3) ForWJSC again this decline was more acute for the last 10passages although spontaneous differentiation to CFU-ALPincreased as a percentage relative to CFU-F probably asign of relative lineage commitmentrestriction In passage-2 cultures calcium concentration was marginally higher forADSC probably owing to the presence of bigger and bettermineralized nodules but was undetected in later passages

Last it is worth noting an increased resistance to trypsindigestion for ADSC The time period required for 50detachment at passage 4 was 10min (60) longer forADSC as compared to WJSC passaging did not significantlyincrease trypsinization time (see online Supplementary Fig-ure 4) Since the cells have similar sizes (at respectivepassages) it is suggested that the increased resistance totrypsinization may be due to differential adhesion moleculeexpression

4 Discussion

MSC populations share common characteristics howeverphenotypic diversification is bound to exist especially inan in vitro environment Indeed microarray studies show

6 Stem Cells International

Table 2 Synopsis of proliferation kinetics data of WJSC and ADSC during long-term in vitro expansion

Mean PDT(hrs)

Mean PDper passage

Mean cell yield perpassage (times106)

Theoretical total yield(times108) after five passages

Average time (days)required for obtaining

80 times 106 cells

WJSC 319 plusmn 68 384 plusmn 52lowast

29 plusmn 063 28 plusmn 059

30 plusmn 082 225 plusmn 08 1205 178

ADSC 4023 plusmn 2151 077 plusmn 024 103 plusmn 035 103 348WJSC data pairs in the first three columns correspond to cultures in the first 10 passages and in passages 11 to 20 respectively lowast119875 lt 0001 Values in the lasttwo columns have been calculated on the basis of the assumption that all isolated cells are serially subcultivated with no intermediate freezing of cell stocksbetween successive passages

Table 3 Overview of phenotypic characteristics of ADSC and WJSC at early (p2) and late (p10 and p1020 resp) passages

Passage (cumPD)WJSC ADSC

2 (45) 10 (289) 20 (567) 2 (24) 10 (81)Mean cell size (120583m2

plusmn SEM) 2340 plusmn 180 4882 plusmn 646 5003 plusmn 712 2407 plusmn 183 4907 plusmn 653 giant cellslowast 0 44 plusmn 4 30 plusmn 7 20 plusmn 5 31 plusmn 8Predominant morphology types () III (4638) III (3832) IIII (4436) IIII (5726) IIII (4233)Intracellular complexity (SSC) 130 85 77 132 115Senescence (120573-gal positive) 21 plusmn 09 113 plusmn 83 129 plusmn 110 17 plusmn 62 246 plusmn 97Self-renewal (CFU-F cells seeded) 1 46 1 73 1 366 1 68 1 140Osteogenic differentiation

CFU-ALP cells seeded( of CFU-F)

1 7860 plusmn 63

1 9876 plusmn 26

1 45083 plusmn 12

1 14946 plusmn 62

1 44831 plusmn 15

Bone nodule formation +++ ++ + +++ +(Ca2+) (mgdL) 108 plusmn 004 ND ND 156 plusmn 038 ND

lowastGiant cells gt3880120583m2 (WJSC) gt4600 120583m2 (ADSC)Bone nodule count scoring + (lt20 nodules) ++ (20ndash50) and +++ (gt50)ND not detected

that phenotypic diversification can actually in many cases beattributed more to differential adaptation to in vitro cultureconditions than to inherent donor sample heterogeneity dueto age sex pathology and so forth [29] In the present studywe have focused on the in vitro characterization of postnatalhumanMSC populations originating from sources that differboth developmentally as well as anatomically that is cellsisolated from the matrix (Whartonrsquos jelly) of the fetal UCtissue (WJSC) [14] and from the abdominal adipose tissue ofadults (ADSC) [15] We have chosen these two diverse popu-lations not only because they are representative of the adultand fetal human stem cell phenotype allowing comparisonsto be made but also for practical reasons sincemdashin contrastto other MSC populationsmdashhigh numbers can be relativelyeasily isolated from the respective tissues with no (as in thecase of WJSC) or minimal donor site morbidity We thenserially propagated these two MSC types over an extendedperiod under standardized culture conditions in an effortto investigate whether and to what extent they sustain theirproliferative capacity and phenotypic stability

With regard to ADSC their isolation is uncomplicatedand efficient especially for smaller tissue samples providingon average about two million cells per gram lipoaspirateEstablishment of primary cultures was however not 100efficient and propagation was slow Culture threshold waspinpointed at passage 5 up to which the cells divide on

average every eight days giving a total of 52 PD in a 40-dayperiod This equates following serial passaging to 103 times 108cells enough for a single administration to an 80 kg patientThis yield is comparable to that reported by other studiesfocusing on ADSC [27] as well as to the yield obtained bythe expansion of other adult MSC populations (eg BM-MSCs) [30] Naturally as in the case of other adult MSCssuch as peripheral blood- and BM-MSCs ADSC can beused autologously following ex vivo expansion Moreoverthey bear the additional advantage of probably being themost abundantly available and readily obtainable MSCscompared to the aforementioned cell types A comparativecharacterization of ADSC and various fetal and adult stemcell populations has been accounted by a number of studieshowever a comprehensive comparison to WJSC especiallywith respect to long-term culture is lacking

As highlighted earlier an accumulating number of pub-lications show that fetal (prenatal as well as perinatal) stemcells bear advantages in comparison to their adult counter-parts These concern increased proliferation rates prolongedcapacity for differentiation enhanced sensitivity to chemicalstimuli in the extracellular milieu and adaptation to cultureconditions [8 31ndash33] Moreover there is strong evidence thatfetal tissue MSCs are hypoimmunogenic and can be well tol-erated in allogeneic transplantation [1 34] Possible sourcesof fetal MSCs that can be easily harvested perinatally are

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

150

200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Stem Cells International

in addition to inherent heterogeneity in the samples (relatedto donor tissue origin age sex and underlying pathology)may have an impact on the quality and quantity of isolatedcells Moreover MSCs (in contrast to embryonic stem cells)are bound to have to a lesser or greater extent a limitedreplication potential in an artificial in vitro culture environ-ment Indeed Hayflickrsquos theory states that most somatic celltypes may undergo a maximum of 70 population doublingsbefore reaching replicative senescence [10 11] and MSCsare bound to be constrained by this limitation Neverthelessin the case of MSCs-based cytotherapy it is estimated thatapproximately 1ndash5 million cellskg body are required forsuccessful outcome therefore a degree of ex vivo expansionis necessary and this holds true in over 60 of casesof cellular transplants [12 13] A question therefore ariseswhether MSCs can sustain their expansion potential andphenotypic stability over prolonged culture periods for howlong and whether possible disparities exist between diversepopulations rendering some of them more competent andsuitable for consideration in cytotherapy protocols

In the present study we have focused on the charac-terization of postnatal human MSC populations originatingfrom two sources that differ both developmentally as well asanatomically that is cells isolated from the matrix (Whar-tonrsquos jelly) of the fetal umbilical cord (UC) tissue (termedWJSC) [14] and from the abdominal adipose tissue of adults(adipose-derived ADSC) [15] The main advantages of theseMSC sources compared to other stem cell origins are (a)abundant cell availability (b) relative ease of access for stemcell isolation with minimal or no tissue morbidity and (c)an immunogenic profile of isolated cells that is favorable forcellular transplantation [16 17]

Themain hypothesis behind this work has been that thereare differences in qualitative and quantitative characteristicsof these MSC populations in terms of their adaptationto extended culture which ultimately relate to practicalconsiderations regarding their clinical use This study whichto our knowledge is the first to address this issue describesa culture regime that sustains the MSC phenotype whilehighlighting a threshold for optimal cell expansion

2 Materials and Methods

21 MSC Isolation

211 WJSC WJSC were isolated from donated UC tissuesamples from normal full-term deliveries following approvalby the institutional bioethics review board Umbilical cordswere transported to the lab in Hanks Balanced Salt Solution(HBSS) containing 1 penicillinstreptomycin solution (bothfrom Invitrogen) and cell isolation was carried out withina maximum of 48 hrs from tissue collection according to amodified protocol based on that described by Seshareddyand colleagues [18] using reagents free of animal productsBriefly the blood vessels were carefully removed and the UCwas minced into 1-2mm3 fragments and then incubated witha mixture of 026 collagenase type I (Biochrom) and 007hyaluronidase (Applichem) for 2 hrs followed by 0125

trypsin (Biochrom) for 30min with gentle agitation at 37∘CThe digested mixture was then passed through a 100 120583mcell strainer (BD) and washed in PBS (Biochrom) to obtaincell suspensions that were stained with 04 trypan blue(Biochrom) counted on a hemocytometer aliquoted andfrozen down in liquid N

2

212 ADSC ADSC were isolated from liposuction aspiratesdonated for research purposes which were obtained fromsubcutaneous adipose tissue sites of subjects (age 44plusmn11 yrssex 7129 malefemale) undergoing elective procedures inlocal plastic surgery offices The research protocol used hadbeen approved by the institutional bioethics review boardCell isolation was carried out under xeno-free conditionswithin 48 hrs from collection following a modified protocolbased on that described by Zuk et al [19] Briefly lipoaspiratesamples were first washed in the collection bag using 250mLnormal saline and after removal of the blood fraction adi-pose tissue was dispersed by digestion with 02 collagenasetype I (Biochrom) at 37∘C for 1 hr with agitation After onemore wash the layer below the floating lipid-filled adipocyteswas dispensed into 50mL tubes and centrifuged at 900 g for45min Subsequently the supernatant was aspirated and thecell pellets (stromal vascular fraction (SVF)) frozen down inliquid N

2 after determining cell numbers with a hematology

blood analyzer (Beckman-Coulter)

22 MSC Culture

221 WJSC The five best samples (ie with the highestnumber of nucleated cells) out of a pool of 34 samples wereresuscitated plated on noncoated 75-cm2 flasks (Corning)and cultured in growth medium (GM) which consisted ofDMEMF12 (with 35 gL glucose ultraglutamine I and Napyruvate Lonza) supplemented with 10 batch-tested fetalbovine serum (FBS) 15mM HEPES 1x nonessential aminoacids and 1 penicillinstreptomycin (all from Invitrogen)Preliminary experiments testing DMEM and DMEMF12-based media (both used in the literature for maintenance ofWJSC as well as ADSC cultures) had established DMEMF12containing stable L-glutamine dipeptide as the optimal basalmedium for propagation of both MSC types (see Section 3)Three days after initial plating nonadherent cells wereremoved and the medium was replaced with fresh GM Cellswere maintained in a humidified atmosphere with 5 CO

2

in air at 37∘C with media changes (complete) every 3-4days until 70ndash80 confluence Subcultivation (passaging)was carried out by trypsinization using 005 trypsin-EDTAsolution (TE Invitrogen) and replating the cells at a densityof 4000 cellscm2 in 75 cm2 flasks Frozen stocks of 05ndash2million cells were maintained in 10 DMSO in FBS in 2mLcryovials (Nunc) stored in liquid N

2

222 ADSC The eight best samples (ie with the highestnumber of nucleated cells) out of a pool of 29 samples wereresuscitated plated on noncoated 25 cm2 flasks (Greiner) andcultured in GM as described previously After three days ofculture nonadherent cells were removed and the medium

Stem Cells International 3

was replaced Adherent cells were grown at 37∘C in 5 CO2

at saturating humidity with medium changes twice weeklyOnce 70ndash80 confluence was reached cells were detachedwith 005 trypsin-EDTA and replated (passaged) in 75 cm2flasks at a seeding density of 6000 cellscm2 Frozen stocks of05ndash2 million cellscryovial were maintained in liquid N

2 as

described previously

23 Determination of Proliferation Kinetics and Cell Size Thenumber of doublings in cell populations (PD) and the timeperiod (in hrs) in which they occur (PDT) were calculatedaccording to the following formulas

PD = ( 1log102) times log

10(NtNo)

PDT = 119905 times [log102

(log10Nt minus log

10No)]

(1)

where No is number of viable (as determined by trypan blueexclusion) cells at seeding Nt is number of viable cells atharvest and t is time (in hrs) between seeding and harvest Aproliferation index (PI) was used as a standardized measureof the proliferative capacity of the cells PI was defined as theratio of the number of population doublings (PD) in a specificpassage over the respective time period (PDT) in which theformer take place

To determine cell size (surface area in 120583m2) photos of sixrandom nonoverlapping fields were captured at 10x magnifi-cation using an Olympus E520 digital camera mounted on anAxiovert 40 CFL (Zeiss) inverted microscope The area of atleast five different cells per field was measured using AdobePhotoshop CS5 Extended Cells with sizes in the top 25 ofeach size range were considered as ldquogiantrdquo cells (gt4600 120583m2and gt3880120583m2 for ADSC and WJSC resp)

24 Characterization of Isolated Stem Cell Populations

241 Immunophenotyping Cultured WJSC and ADSC werecharacterized for the expression of the following mark-ers CD29 (120573

1-integrin) CD44 (H-CAM) CD73 (ECTO-

51015840 nucleaseSH3) CD90 (THY-1) CD105 (endoglinSH2)CD14 (LeuM3MY4) CD34 (HPCA1gp105ndash120) and CD45(LCA) Cells were detached using StemPro Accutase (Invit-rogen) washed in PBS and stained using specific FITC- PE-ECD- or PC5-conjugated monoclonal antibodies or isotype-matched (IgG1aIgG2a) controls (all by Beckman-Coulterexcept CD105-PE which was purchased from BD) 7-AAD(Beckman-Coulter) was used as a viability stain Analysiswas performed on a Beckman-Coulter EPICS XL-MCL flowcytometer At least 20000 live events were acquired

242 CFU-F Assay The capacity of ADSC and WJSC forself-renewal was evaluated by seeding cells at clonal densities(20ndash80 cellscm2) in 10 cm dishes and fixing and stainingthe resulting CFU-F two weeks later with 05 crystal vio-letmethanol solution (Digirolamo et al 1999) [20] Coloniesgt 2mm in diameter were counted manually

243 ALP Assay Alkaline-phosphatase-(ALP-)-positiveCFU which represent clonal populations of cells with os-teoprtogenitor properties were detected in duplicate culturesof ADSCWJSC (set up as described for CFU-F assaypreviously mentioned) using the Sigma leukocyte ALP histo-chemistry staining kit (Kaplow 1968) [21] following themanufacturerrsquos recommendations Colonies counterstainedwith hematoxylin and those gt 2mm in diameter werecounted manually

244 Osteogenic Differentiation The potency of WJSC andADSC for directed differentiation towards osteoblast lineagewas tested by alizarin red staining of mineralized bonenodules [22] Cells were grown in triplicate in 6-well plate for21 days in the presence of osteogenic supplements (50120583gmL120573-glycerophosphate 10mML-ascorbic acid 2-phosphate and5 times 10

minus7M dexamethasone all from Sigma) and then fixedin 10 formol saline and stained with 2 alizarin red Bonenodules gt 1mm were manually counted and scored in threedifferent categories + (lt 20) ++ (20ndash50) and +++ (gt 50)

245 Calcium Assay The Ca content of mineralized culturelysates was measured by a colorimetric assay (Cayman) [23]following the manufacturerrsquos recommendations Cells wereseeded at a density of 500cm2 in duplicate in 10 cm dishesand grown in the presence of osteogenic supplements (seeaforementioned) for 21 days Following a wash in CaMg-free PBS they were lysed in 100mM ice-cold Tris buffer spunat 10000 g and the supernatants stored at minus80∘C until usedSample absorbances were measured at 570 nm using a Tecanmicroplate reader and compared across a reference curveconstructed using calcium standards The assay detectionlimit was 05mgdL

246 Senescence Assay Cellular senescence was determinedby 120573-gal staining of subconfluent day 4-5 cultures set up in6-well plates using the Sigma cell senescence histochemicalkit [24] according to the manufacturerrsquos instructions Brieflycells were fixed and incubated with the staining mix contain-ing X-gal overnight at 37∘C in a low CO

2environment The

occurrence of 120573-gal positive blue-green cells was determinedby counting a minimum of 50 cells in at least five randomnonoverlapping fields under a microscope Each assay wasperformed in duplicate

247 Adherence Assay Cell detachment was measured intime series experiments set up in 6-well plates in triplicateCells were seeded at 4000ndash6000cm2 and left to grow to70ndash80 confluence upon which they were washed in PBSand incubated with 600 120583L warm 005 trypsin-EDTA (TE)per well for up to 30min without agitation Every 5min thedigested cell suspensions were drawn out into 15mL tubesand an aliquot was mixed with trypan blue and live cellscounted on a Neubauer plate while 600120583L of fresh warm TEwas added per well for the next measurement Control cellswere incubated with TE at 37∘C with frequent tapping until100 detached from culture plastic

4 Stem Cells International

25 Statistical Analysis Parametric and nonparametric tests(Spearman correlation analysis Mann-Whitney pairwisedata comparisons and ANOVA) were conducted usingGraphPad Prism 504 statistical analysis software Data aredepicted as means plusmn SD unless otherwise stated A P valuelt 005 was considered as statistically significant

3 Results

31 Primary Cell Isolation and Culture Initiation Efficiency

311 WJSC Mean nucleated cell yield following UC tissueprocessing was about 23 times 106 and was found to positivelycorrelate (Spearman 119903 = 0722119875 lt 0001) with tissue proper-ties (ie UC length) (Table 1 and see in SupplementaryMate-rial available online at httpdxdoiorg1011552013246134Figure 1) The isolation protocol involved removal of thevessels that due to the supercoiled anatomy of the UCincreases tissue processing time and hence results in some cellloss compared to protocols based on digestion of whole UCNevertheless our protocol produces a more homogeneouspopulation than the latter which results in the isolation ofa mixed population of at least four stem cell types [25 26]Five out of five (100) cultures of adherent cells derived fromplated UC nucleated cell samples led to the establishment ofcultures that grew successfully beyond passage 3 (Table 1)The three of these cultures with the shortest PDT (see thefollowing) were further subcultured and used for the exper-iments described in this study The percentage of adherentcells in the primary cultures was estimated at 12

312 ADSC ForADSCmeannucleated cell yield at isolationwas 312 times 106 cells (Table 1) Regression analysis of samplesshowed that this yield was not significantly associated withinitial tissue quantity (donor adipose weight) (see Supple-mentary Figure 1) or other donor characteristics such asage or sex Nevertheless there was a strong linear correlationof the number of isolated cells with adipose tissue weightfor samples up to 200 g (Spearman 119903 = 0952 119875 lt0001) highlighting the increased efficiency of the protocolfor samples in that range The eight adipose tissue sampleswith the highest mononuclear cell yields were plated out andused to establish cultures of ADSC Five of these samplestested proliferated very slowly (less than 20 increase incell yield between passages) and failed to grow beyondpassage 3 whereas 38 (375) proliferated further and wereused for the experiments ADSC yield was approximately18 times 10

6 cellsg lipoaspirate with the percentage of adherentcells in the primary cultures being much lower than thatof WJSC at 093 This yield is slightly higher than thatreported elsewhere (1 times 106 cellsg) [27] Viability of isolatedcells and resuscitated cells following initial cryopreservationwas consistently high (gt92) and similar to that of WJSC(Table 1)

32 Proliferation Kinetics of Long-Term Cultures Figures1(a)ndash1(c) depict the proliferation dynamics of the two MSCpopulations during prolonged in vitro subcultivation while

Table 2 summarises the main findings The mean cell yieldshown in Table 2 is the one that arises from the serial prop-agation of cells following the subculture protocol describedin Section 2 This was higher (by more than twofold) forWJSC and related to a constant number of about three PD perpassage (depicted by the linearity of the graph in Figure 1(a))accumulating to a total of 58 PD in the 100-day cultureperiod Mean PDT was 384 plusmn 86 hrs over that period Peakproliferative rate was observed at passage 6 (considering PIand PDT Figures 1(b) and 1(c) resp) which equated tonearly 19 PD over a month in culture Maximum PI of WJSC(observed at passage 6) was over 15-fold higher than that ofADSC and coincided with a maximum number of 372 plusmn032 PD WJSC showed no signs of slowdown even after 15passages (45 PD) (119875 gt 005 ANOVA on PDT of passages 2ndash16)There was nevertheless a significant increase in PDT afterpassage 17 which exceeded 50 hrs by passage 20

ADSC proliferation capacity was maximum at passage 2(see Figures 1(b) and 1(c)) and remained above average untilpassage 5 although related to a poor number of cumulativePD (just over five) Signs of senescence and essentially ces-sation of proliferation appeared after passage 6 Thencefortha plateau in PD accumulation a consistently high PDT (ofnearly 37 days) and low expansion rate (lt20 per passage)were observed Mean cell yield per passage in our cultureprotocol was two-three times lower for ADSC (Table 2)

The growth curves (Figure 1(d)) depict the kinetics ofproliferation within a single passage Both cell types followa typical sigmoid growth pattern but with different phasedurations WJSC exhibit a lag phase similar to that of ADSC(about 25 days) but a much shorter log phase (35 as opposedto 75 days) during which they expand more promptly(about fivefold expansion of cell population) WJSC becomeconfluent by day 7 while they continue to grow by 15-20 even when superconflluent forming dense cultures ofcompacted cells whereas ADSC undergo contact inhibitionafter day 11 not exceeding 90 confluency GM containingthe dipeptide form of glutamine increases cell yield duringlog phase by up to 200 (especially for ADSC 119875 lt 001 day9) while it enhances survival of postconfluentWJSC cultures(119875 lt 005 day 11 see online Supplementary Figure 2) Thisis in concordance with the effects of glutamine dipeptide onbone-marrow-derived MSC survival and growth [28]

33 Phenotypic Characteristics of Long-Term CulturesFigure 2(a) illustrates the change in cell size as a phenotypiccharacteristic during prolonged subculture A markedgreather than 300 change in cell size over culture timewas observed for both MSC populations ADSC mean sizewas 3876 120583m2 with minimum (1648) and maximum (6130)values observed at passages 1 and 8 respectively (Figure 2(a))WJSC had an average size of 2926120583m2 (min 1437 max 5183at passages 1 and 11 resp Figure 2(a)) WJSC exhibited aperiodicity in cell size that might be attributed to smallrapidly proliferating cells undergoing rounds of symmetricand asymmetric divisions which on one hand give rise todifferentiating daughter cells that gradually increase in sizeand probably become senescent (see also Figures 2(c) and 3)

Stem Cells International 5

Table 1 Cell isolation and culture establishment efficiency

WJSC ADSC 119875 value

Mean tissue length-weight 133 cm plusmn 2lowast 1738 g plusmn 1357 mdashMean nucleated cell yield (times106)

(per unit length-weight)228 plusmn 155(017cm)

312 plusmn 198(18g) mdash

Mean viability at isolation () 943 plusmn 22 966 plusmn 16 gt005Mean viability at resuscitation () 929 plusmn 43 927 plusmn 49 gt005 of adherent cells in starting culture 173 plusmn 46 093 plusmn 045 lt0001Adherent cultures beyond p3 100 (55) 375 (38) lt005Mean passage-to-passage expansion rate (p1ndash5) 974 plusmn 317 175 plusmn 56 lt001Mean subculture viability (p1ndash5) 976 plusmn 05 964 plusmn 19 gt005119875 value WJSC versus ADSC lowastA statistically significant association was found between UC tissue sample length and the yield of isolated nucleated cellsfollowing tissue processing (see text for details)

while at the same time replenishing the culture with moresubsets of small rapidly proliferating stem cells In support ofthis is the association of kinetics (PDT) with cell size (Figures2(b) and 2(c)) Spearman analysis showed that small cell sizecorrelates better with low PDT while cells that take longerto undergo rounds of division (senescent) tend to be largerSpecifically the cells with sizes in the top 25 (arbitrarilydenoted as giant cells) have also the highest PDT and thelatter correspond to mid to late passages (p7 or greater forADSC and p9 or greater for WJSC)

Besides cell size a marked change was observed in otherphenotypic properties of MSCs over in vitro expansionFigure 3 outlines these differences between early cultures andculture endpoints In terms of morphological characteristicsthree main morphologies were observed (a) spindle-shapedelongated (type I) (b) polygonal with few small membraneprotrusions (type II) (c) flattened with irregular shape andlarge (frequently multiple) nuclei (type III) Early-passage(p2) WJSC and ADSC have mainly type II and type Imorphologies respectively (see Table 3 and Figure 3(a))Serial passaging of both MSC populations results in gradualacquisition of type III morphology which especially inthe case of WJSC increases by 28-fold in occurrence andbecomes predominant by passage 20 In these culture pointscells with either type I or type III morphologies accountedfor over 70 of the cultures (see Table 3) with at least one-in-three cells being a giant cell (see Methods section for sizedefinition) bearing enlarged often multiple nuclei and beinglargely senescent staining positive for 120573-gal (Figure 3(b))The occurrence of senescent cells in ADSC cultures was fairlyfrequent with roughly one cell out of five staining positiveirrespective of the culture point (Table 3) On the contrarythe senescence rate of early WJSC culture which is very lowincreases 5-6 fold in later passages albeit with considerableinterculture variability An interesting observation was alsothat cytoplasmic granularity ofWJSC decreased by 35ndash40with passaging probably relating to rearrangements in thecytoskeleton and protein synthesis machinery (Table 3 andonline Supplementary Figure 3)

In terms of stemness properties both cell types have acapacity for clonogenicityself-renewal and lineage-specific

differentiation and express a typical immunophenotypic pro-file with high MSC marker (gt97 positive except CD44of late-passage ADSC which was slightly lower at 92 plusmn23) and low HSC marker expression (lt3 positive withthe exception of 8 plusmn 02 of early-passage ADSC whichexpressed monocyte-related CD14) (Figure 4) The mostavidly expressed markers (high signal intensity) were CD44(H-CAM) and CD105 (endoglin) for WJSC and ADSCrespectively But although expression levels of CD surfacemarkers remained constant over time in culture clonogenic-ity was reduced ForWJSC about 1 in 45 cells was clonogenicand this ability diminished by less than twofold over the first10 passages (+24 PD) but almost by a factor of five in thenext 10 passages (+28 PD) (Figure 3(c) and Table 3) ADSCformed robustly clones at all densities tested however theof CFU-F ratios were lower than those of WJSC that hadundergone many more PD Differentiation capacityinducedlineage specific differentiation (osteoblasts) also diminishedwith ageing in culture (Figures 3(c) and 3(d) Table 3) ForWJSC again this decline was more acute for the last 10passages although spontaneous differentiation to CFU-ALPincreased as a percentage relative to CFU-F probably asign of relative lineage commitmentrestriction In passage-2 cultures calcium concentration was marginally higher forADSC probably owing to the presence of bigger and bettermineralized nodules but was undetected in later passages

Last it is worth noting an increased resistance to trypsindigestion for ADSC The time period required for 50detachment at passage 4 was 10min (60) longer forADSC as compared to WJSC passaging did not significantlyincrease trypsinization time (see online Supplementary Fig-ure 4) Since the cells have similar sizes (at respectivepassages) it is suggested that the increased resistance totrypsinization may be due to differential adhesion moleculeexpression

4 Discussion

MSC populations share common characteristics howeverphenotypic diversification is bound to exist especially inan in vitro environment Indeed microarray studies show

6 Stem Cells International

Table 2 Synopsis of proliferation kinetics data of WJSC and ADSC during long-term in vitro expansion

Mean PDT(hrs)

Mean PDper passage

Mean cell yield perpassage (times106)

Theoretical total yield(times108) after five passages

Average time (days)required for obtaining

80 times 106 cells

WJSC 319 plusmn 68 384 plusmn 52lowast

29 plusmn 063 28 plusmn 059

30 plusmn 082 225 plusmn 08 1205 178

ADSC 4023 plusmn 2151 077 plusmn 024 103 plusmn 035 103 348WJSC data pairs in the first three columns correspond to cultures in the first 10 passages and in passages 11 to 20 respectively lowast119875 lt 0001 Values in the lasttwo columns have been calculated on the basis of the assumption that all isolated cells are serially subcultivated with no intermediate freezing of cell stocksbetween successive passages

Table 3 Overview of phenotypic characteristics of ADSC and WJSC at early (p2) and late (p10 and p1020 resp) passages

Passage (cumPD)WJSC ADSC

2 (45) 10 (289) 20 (567) 2 (24) 10 (81)Mean cell size (120583m2

plusmn SEM) 2340 plusmn 180 4882 plusmn 646 5003 plusmn 712 2407 plusmn 183 4907 plusmn 653 giant cellslowast 0 44 plusmn 4 30 plusmn 7 20 plusmn 5 31 plusmn 8Predominant morphology types () III (4638) III (3832) IIII (4436) IIII (5726) IIII (4233)Intracellular complexity (SSC) 130 85 77 132 115Senescence (120573-gal positive) 21 plusmn 09 113 plusmn 83 129 plusmn 110 17 plusmn 62 246 plusmn 97Self-renewal (CFU-F cells seeded) 1 46 1 73 1 366 1 68 1 140Osteogenic differentiation

CFU-ALP cells seeded( of CFU-F)

1 7860 plusmn 63

1 9876 plusmn 26

1 45083 plusmn 12

1 14946 plusmn 62

1 44831 plusmn 15

Bone nodule formation +++ ++ + +++ +(Ca2+) (mgdL) 108 plusmn 004 ND ND 156 plusmn 038 ND

lowastGiant cells gt3880120583m2 (WJSC) gt4600 120583m2 (ADSC)Bone nodule count scoring + (lt20 nodules) ++ (20ndash50) and +++ (gt50)ND not detected

that phenotypic diversification can actually in many cases beattributed more to differential adaptation to in vitro cultureconditions than to inherent donor sample heterogeneity dueto age sex pathology and so forth [29] In the present studywe have focused on the in vitro characterization of postnatalhumanMSC populations originating from sources that differboth developmentally as well as anatomically that is cellsisolated from the matrix (Whartonrsquos jelly) of the fetal UCtissue (WJSC) [14] and from the abdominal adipose tissue ofadults (ADSC) [15] We have chosen these two diverse popu-lations not only because they are representative of the adultand fetal human stem cell phenotype allowing comparisonsto be made but also for practical reasons sincemdashin contrastto other MSC populationsmdashhigh numbers can be relativelyeasily isolated from the respective tissues with no (as in thecase of WJSC) or minimal donor site morbidity We thenserially propagated these two MSC types over an extendedperiod under standardized culture conditions in an effortto investigate whether and to what extent they sustain theirproliferative capacity and phenotypic stability

With regard to ADSC their isolation is uncomplicatedand efficient especially for smaller tissue samples providingon average about two million cells per gram lipoaspirateEstablishment of primary cultures was however not 100efficient and propagation was slow Culture threshold waspinpointed at passage 5 up to which the cells divide on

average every eight days giving a total of 52 PD in a 40-dayperiod This equates following serial passaging to 103 times 108cells enough for a single administration to an 80 kg patientThis yield is comparable to that reported by other studiesfocusing on ADSC [27] as well as to the yield obtained bythe expansion of other adult MSC populations (eg BM-MSCs) [30] Naturally as in the case of other adult MSCssuch as peripheral blood- and BM-MSCs ADSC can beused autologously following ex vivo expansion Moreoverthey bear the additional advantage of probably being themost abundantly available and readily obtainable MSCscompared to the aforementioned cell types A comparativecharacterization of ADSC and various fetal and adult stemcell populations has been accounted by a number of studieshowever a comprehensive comparison to WJSC especiallywith respect to long-term culture is lacking

As highlighted earlier an accumulating number of pub-lications show that fetal (prenatal as well as perinatal) stemcells bear advantages in comparison to their adult counter-parts These concern increased proliferation rates prolongedcapacity for differentiation enhanced sensitivity to chemicalstimuli in the extracellular milieu and adaptation to cultureconditions [8 31ndash33] Moreover there is strong evidence thatfetal tissue MSCs are hypoimmunogenic and can be well tol-erated in allogeneic transplantation [1 34] Possible sourcesof fetal MSCs that can be easily harvested perinatally are

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

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200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

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481

321

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394

263

131

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276207138

690

209

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0

149

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49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

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282

141

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99

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159106

530

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92

46

0

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102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

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WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

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499374249124

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270203135

670

150

75

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110

73

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Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 3

was replaced Adherent cells were grown at 37∘C in 5 CO2

at saturating humidity with medium changes twice weeklyOnce 70ndash80 confluence was reached cells were detachedwith 005 trypsin-EDTA and replated (passaged) in 75 cm2flasks at a seeding density of 6000 cellscm2 Frozen stocks of05ndash2 million cellscryovial were maintained in liquid N

2 as

described previously

23 Determination of Proliferation Kinetics and Cell Size Thenumber of doublings in cell populations (PD) and the timeperiod (in hrs) in which they occur (PDT) were calculatedaccording to the following formulas

PD = ( 1log102) times log

10(NtNo)

PDT = 119905 times [log102

(log10Nt minus log

10No)]

(1)

where No is number of viable (as determined by trypan blueexclusion) cells at seeding Nt is number of viable cells atharvest and t is time (in hrs) between seeding and harvest Aproliferation index (PI) was used as a standardized measureof the proliferative capacity of the cells PI was defined as theratio of the number of population doublings (PD) in a specificpassage over the respective time period (PDT) in which theformer take place

To determine cell size (surface area in 120583m2) photos of sixrandom nonoverlapping fields were captured at 10x magnifi-cation using an Olympus E520 digital camera mounted on anAxiovert 40 CFL (Zeiss) inverted microscope The area of atleast five different cells per field was measured using AdobePhotoshop CS5 Extended Cells with sizes in the top 25 ofeach size range were considered as ldquogiantrdquo cells (gt4600 120583m2and gt3880120583m2 for ADSC and WJSC resp)

24 Characterization of Isolated Stem Cell Populations

241 Immunophenotyping Cultured WJSC and ADSC werecharacterized for the expression of the following mark-ers CD29 (120573

1-integrin) CD44 (H-CAM) CD73 (ECTO-

51015840 nucleaseSH3) CD90 (THY-1) CD105 (endoglinSH2)CD14 (LeuM3MY4) CD34 (HPCA1gp105ndash120) and CD45(LCA) Cells were detached using StemPro Accutase (Invit-rogen) washed in PBS and stained using specific FITC- PE-ECD- or PC5-conjugated monoclonal antibodies or isotype-matched (IgG1aIgG2a) controls (all by Beckman-Coulterexcept CD105-PE which was purchased from BD) 7-AAD(Beckman-Coulter) was used as a viability stain Analysiswas performed on a Beckman-Coulter EPICS XL-MCL flowcytometer At least 20000 live events were acquired

242 CFU-F Assay The capacity of ADSC and WJSC forself-renewal was evaluated by seeding cells at clonal densities(20ndash80 cellscm2) in 10 cm dishes and fixing and stainingthe resulting CFU-F two weeks later with 05 crystal vio-letmethanol solution (Digirolamo et al 1999) [20] Coloniesgt 2mm in diameter were counted manually

243 ALP Assay Alkaline-phosphatase-(ALP-)-positiveCFU which represent clonal populations of cells with os-teoprtogenitor properties were detected in duplicate culturesof ADSCWJSC (set up as described for CFU-F assaypreviously mentioned) using the Sigma leukocyte ALP histo-chemistry staining kit (Kaplow 1968) [21] following themanufacturerrsquos recommendations Colonies counterstainedwith hematoxylin and those gt 2mm in diameter werecounted manually

244 Osteogenic Differentiation The potency of WJSC andADSC for directed differentiation towards osteoblast lineagewas tested by alizarin red staining of mineralized bonenodules [22] Cells were grown in triplicate in 6-well plate for21 days in the presence of osteogenic supplements (50120583gmL120573-glycerophosphate 10mML-ascorbic acid 2-phosphate and5 times 10

minus7M dexamethasone all from Sigma) and then fixedin 10 formol saline and stained with 2 alizarin red Bonenodules gt 1mm were manually counted and scored in threedifferent categories + (lt 20) ++ (20ndash50) and +++ (gt 50)

245 Calcium Assay The Ca content of mineralized culturelysates was measured by a colorimetric assay (Cayman) [23]following the manufacturerrsquos recommendations Cells wereseeded at a density of 500cm2 in duplicate in 10 cm dishesand grown in the presence of osteogenic supplements (seeaforementioned) for 21 days Following a wash in CaMg-free PBS they were lysed in 100mM ice-cold Tris buffer spunat 10000 g and the supernatants stored at minus80∘C until usedSample absorbances were measured at 570 nm using a Tecanmicroplate reader and compared across a reference curveconstructed using calcium standards The assay detectionlimit was 05mgdL

246 Senescence Assay Cellular senescence was determinedby 120573-gal staining of subconfluent day 4-5 cultures set up in6-well plates using the Sigma cell senescence histochemicalkit [24] according to the manufacturerrsquos instructions Brieflycells were fixed and incubated with the staining mix contain-ing X-gal overnight at 37∘C in a low CO

2environment The

occurrence of 120573-gal positive blue-green cells was determinedby counting a minimum of 50 cells in at least five randomnonoverlapping fields under a microscope Each assay wasperformed in duplicate

247 Adherence Assay Cell detachment was measured intime series experiments set up in 6-well plates in triplicateCells were seeded at 4000ndash6000cm2 and left to grow to70ndash80 confluence upon which they were washed in PBSand incubated with 600 120583L warm 005 trypsin-EDTA (TE)per well for up to 30min without agitation Every 5min thedigested cell suspensions were drawn out into 15mL tubesand an aliquot was mixed with trypan blue and live cellscounted on a Neubauer plate while 600120583L of fresh warm TEwas added per well for the next measurement Control cellswere incubated with TE at 37∘C with frequent tapping until100 detached from culture plastic

4 Stem Cells International

25 Statistical Analysis Parametric and nonparametric tests(Spearman correlation analysis Mann-Whitney pairwisedata comparisons and ANOVA) were conducted usingGraphPad Prism 504 statistical analysis software Data aredepicted as means plusmn SD unless otherwise stated A P valuelt 005 was considered as statistically significant

3 Results

31 Primary Cell Isolation and Culture Initiation Efficiency

311 WJSC Mean nucleated cell yield following UC tissueprocessing was about 23 times 106 and was found to positivelycorrelate (Spearman 119903 = 0722119875 lt 0001) with tissue proper-ties (ie UC length) (Table 1 and see in SupplementaryMate-rial available online at httpdxdoiorg1011552013246134Figure 1) The isolation protocol involved removal of thevessels that due to the supercoiled anatomy of the UCincreases tissue processing time and hence results in some cellloss compared to protocols based on digestion of whole UCNevertheless our protocol produces a more homogeneouspopulation than the latter which results in the isolation ofa mixed population of at least four stem cell types [25 26]Five out of five (100) cultures of adherent cells derived fromplated UC nucleated cell samples led to the establishment ofcultures that grew successfully beyond passage 3 (Table 1)The three of these cultures with the shortest PDT (see thefollowing) were further subcultured and used for the exper-iments described in this study The percentage of adherentcells in the primary cultures was estimated at 12

312 ADSC ForADSCmeannucleated cell yield at isolationwas 312 times 106 cells (Table 1) Regression analysis of samplesshowed that this yield was not significantly associated withinitial tissue quantity (donor adipose weight) (see Supple-mentary Figure 1) or other donor characteristics such asage or sex Nevertheless there was a strong linear correlationof the number of isolated cells with adipose tissue weightfor samples up to 200 g (Spearman 119903 = 0952 119875 lt0001) highlighting the increased efficiency of the protocolfor samples in that range The eight adipose tissue sampleswith the highest mononuclear cell yields were plated out andused to establish cultures of ADSC Five of these samplestested proliferated very slowly (less than 20 increase incell yield between passages) and failed to grow beyondpassage 3 whereas 38 (375) proliferated further and wereused for the experiments ADSC yield was approximately18 times 10

6 cellsg lipoaspirate with the percentage of adherentcells in the primary cultures being much lower than thatof WJSC at 093 This yield is slightly higher than thatreported elsewhere (1 times 106 cellsg) [27] Viability of isolatedcells and resuscitated cells following initial cryopreservationwas consistently high (gt92) and similar to that of WJSC(Table 1)

32 Proliferation Kinetics of Long-Term Cultures Figures1(a)ndash1(c) depict the proliferation dynamics of the two MSCpopulations during prolonged in vitro subcultivation while

Table 2 summarises the main findings The mean cell yieldshown in Table 2 is the one that arises from the serial prop-agation of cells following the subculture protocol describedin Section 2 This was higher (by more than twofold) forWJSC and related to a constant number of about three PD perpassage (depicted by the linearity of the graph in Figure 1(a))accumulating to a total of 58 PD in the 100-day cultureperiod Mean PDT was 384 plusmn 86 hrs over that period Peakproliferative rate was observed at passage 6 (considering PIand PDT Figures 1(b) and 1(c) resp) which equated tonearly 19 PD over a month in culture Maximum PI of WJSC(observed at passage 6) was over 15-fold higher than that ofADSC and coincided with a maximum number of 372 plusmn032 PD WJSC showed no signs of slowdown even after 15passages (45 PD) (119875 gt 005 ANOVA on PDT of passages 2ndash16)There was nevertheless a significant increase in PDT afterpassage 17 which exceeded 50 hrs by passage 20

ADSC proliferation capacity was maximum at passage 2(see Figures 1(b) and 1(c)) and remained above average untilpassage 5 although related to a poor number of cumulativePD (just over five) Signs of senescence and essentially ces-sation of proliferation appeared after passage 6 Thencefortha plateau in PD accumulation a consistently high PDT (ofnearly 37 days) and low expansion rate (lt20 per passage)were observed Mean cell yield per passage in our cultureprotocol was two-three times lower for ADSC (Table 2)

The growth curves (Figure 1(d)) depict the kinetics ofproliferation within a single passage Both cell types followa typical sigmoid growth pattern but with different phasedurations WJSC exhibit a lag phase similar to that of ADSC(about 25 days) but a much shorter log phase (35 as opposedto 75 days) during which they expand more promptly(about fivefold expansion of cell population) WJSC becomeconfluent by day 7 while they continue to grow by 15-20 even when superconflluent forming dense cultures ofcompacted cells whereas ADSC undergo contact inhibitionafter day 11 not exceeding 90 confluency GM containingthe dipeptide form of glutamine increases cell yield duringlog phase by up to 200 (especially for ADSC 119875 lt 001 day9) while it enhances survival of postconfluentWJSC cultures(119875 lt 005 day 11 see online Supplementary Figure 2) Thisis in concordance with the effects of glutamine dipeptide onbone-marrow-derived MSC survival and growth [28]

33 Phenotypic Characteristics of Long-Term CulturesFigure 2(a) illustrates the change in cell size as a phenotypiccharacteristic during prolonged subculture A markedgreather than 300 change in cell size over culture timewas observed for both MSC populations ADSC mean sizewas 3876 120583m2 with minimum (1648) and maximum (6130)values observed at passages 1 and 8 respectively (Figure 2(a))WJSC had an average size of 2926120583m2 (min 1437 max 5183at passages 1 and 11 resp Figure 2(a)) WJSC exhibited aperiodicity in cell size that might be attributed to smallrapidly proliferating cells undergoing rounds of symmetricand asymmetric divisions which on one hand give rise todifferentiating daughter cells that gradually increase in sizeand probably become senescent (see also Figures 2(c) and 3)

Stem Cells International 5

Table 1 Cell isolation and culture establishment efficiency

WJSC ADSC 119875 value

Mean tissue length-weight 133 cm plusmn 2lowast 1738 g plusmn 1357 mdashMean nucleated cell yield (times106)

(per unit length-weight)228 plusmn 155(017cm)

312 plusmn 198(18g) mdash

Mean viability at isolation () 943 plusmn 22 966 plusmn 16 gt005Mean viability at resuscitation () 929 plusmn 43 927 plusmn 49 gt005 of adherent cells in starting culture 173 plusmn 46 093 plusmn 045 lt0001Adherent cultures beyond p3 100 (55) 375 (38) lt005Mean passage-to-passage expansion rate (p1ndash5) 974 plusmn 317 175 plusmn 56 lt001Mean subculture viability (p1ndash5) 976 plusmn 05 964 plusmn 19 gt005119875 value WJSC versus ADSC lowastA statistically significant association was found between UC tissue sample length and the yield of isolated nucleated cellsfollowing tissue processing (see text for details)

while at the same time replenishing the culture with moresubsets of small rapidly proliferating stem cells In support ofthis is the association of kinetics (PDT) with cell size (Figures2(b) and 2(c)) Spearman analysis showed that small cell sizecorrelates better with low PDT while cells that take longerto undergo rounds of division (senescent) tend to be largerSpecifically the cells with sizes in the top 25 (arbitrarilydenoted as giant cells) have also the highest PDT and thelatter correspond to mid to late passages (p7 or greater forADSC and p9 or greater for WJSC)

Besides cell size a marked change was observed in otherphenotypic properties of MSCs over in vitro expansionFigure 3 outlines these differences between early cultures andculture endpoints In terms of morphological characteristicsthree main morphologies were observed (a) spindle-shapedelongated (type I) (b) polygonal with few small membraneprotrusions (type II) (c) flattened with irregular shape andlarge (frequently multiple) nuclei (type III) Early-passage(p2) WJSC and ADSC have mainly type II and type Imorphologies respectively (see Table 3 and Figure 3(a))Serial passaging of both MSC populations results in gradualacquisition of type III morphology which especially inthe case of WJSC increases by 28-fold in occurrence andbecomes predominant by passage 20 In these culture pointscells with either type I or type III morphologies accountedfor over 70 of the cultures (see Table 3) with at least one-in-three cells being a giant cell (see Methods section for sizedefinition) bearing enlarged often multiple nuclei and beinglargely senescent staining positive for 120573-gal (Figure 3(b))The occurrence of senescent cells in ADSC cultures was fairlyfrequent with roughly one cell out of five staining positiveirrespective of the culture point (Table 3) On the contrarythe senescence rate of early WJSC culture which is very lowincreases 5-6 fold in later passages albeit with considerableinterculture variability An interesting observation was alsothat cytoplasmic granularity ofWJSC decreased by 35ndash40with passaging probably relating to rearrangements in thecytoskeleton and protein synthesis machinery (Table 3 andonline Supplementary Figure 3)

In terms of stemness properties both cell types have acapacity for clonogenicityself-renewal and lineage-specific

differentiation and express a typical immunophenotypic pro-file with high MSC marker (gt97 positive except CD44of late-passage ADSC which was slightly lower at 92 plusmn23) and low HSC marker expression (lt3 positive withthe exception of 8 plusmn 02 of early-passage ADSC whichexpressed monocyte-related CD14) (Figure 4) The mostavidly expressed markers (high signal intensity) were CD44(H-CAM) and CD105 (endoglin) for WJSC and ADSCrespectively But although expression levels of CD surfacemarkers remained constant over time in culture clonogenic-ity was reduced ForWJSC about 1 in 45 cells was clonogenicand this ability diminished by less than twofold over the first10 passages (+24 PD) but almost by a factor of five in thenext 10 passages (+28 PD) (Figure 3(c) and Table 3) ADSCformed robustly clones at all densities tested however theof CFU-F ratios were lower than those of WJSC that hadundergone many more PD Differentiation capacityinducedlineage specific differentiation (osteoblasts) also diminishedwith ageing in culture (Figures 3(c) and 3(d) Table 3) ForWJSC again this decline was more acute for the last 10passages although spontaneous differentiation to CFU-ALPincreased as a percentage relative to CFU-F probably asign of relative lineage commitmentrestriction In passage-2 cultures calcium concentration was marginally higher forADSC probably owing to the presence of bigger and bettermineralized nodules but was undetected in later passages

Last it is worth noting an increased resistance to trypsindigestion for ADSC The time period required for 50detachment at passage 4 was 10min (60) longer forADSC as compared to WJSC passaging did not significantlyincrease trypsinization time (see online Supplementary Fig-ure 4) Since the cells have similar sizes (at respectivepassages) it is suggested that the increased resistance totrypsinization may be due to differential adhesion moleculeexpression

4 Discussion

MSC populations share common characteristics howeverphenotypic diversification is bound to exist especially inan in vitro environment Indeed microarray studies show

6 Stem Cells International

Table 2 Synopsis of proliferation kinetics data of WJSC and ADSC during long-term in vitro expansion

Mean PDT(hrs)

Mean PDper passage

Mean cell yield perpassage (times106)

Theoretical total yield(times108) after five passages

Average time (days)required for obtaining

80 times 106 cells

WJSC 319 plusmn 68 384 plusmn 52lowast

29 plusmn 063 28 plusmn 059

30 plusmn 082 225 plusmn 08 1205 178

ADSC 4023 plusmn 2151 077 plusmn 024 103 plusmn 035 103 348WJSC data pairs in the first three columns correspond to cultures in the first 10 passages and in passages 11 to 20 respectively lowast119875 lt 0001 Values in the lasttwo columns have been calculated on the basis of the assumption that all isolated cells are serially subcultivated with no intermediate freezing of cell stocksbetween successive passages

Table 3 Overview of phenotypic characteristics of ADSC and WJSC at early (p2) and late (p10 and p1020 resp) passages

Passage (cumPD)WJSC ADSC

2 (45) 10 (289) 20 (567) 2 (24) 10 (81)Mean cell size (120583m2

plusmn SEM) 2340 plusmn 180 4882 plusmn 646 5003 plusmn 712 2407 plusmn 183 4907 plusmn 653 giant cellslowast 0 44 plusmn 4 30 plusmn 7 20 plusmn 5 31 plusmn 8Predominant morphology types () III (4638) III (3832) IIII (4436) IIII (5726) IIII (4233)Intracellular complexity (SSC) 130 85 77 132 115Senescence (120573-gal positive) 21 plusmn 09 113 plusmn 83 129 plusmn 110 17 plusmn 62 246 plusmn 97Self-renewal (CFU-F cells seeded) 1 46 1 73 1 366 1 68 1 140Osteogenic differentiation

CFU-ALP cells seeded( of CFU-F)

1 7860 plusmn 63

1 9876 plusmn 26

1 45083 plusmn 12

1 14946 plusmn 62

1 44831 plusmn 15

Bone nodule formation +++ ++ + +++ +(Ca2+) (mgdL) 108 plusmn 004 ND ND 156 plusmn 038 ND

lowastGiant cells gt3880120583m2 (WJSC) gt4600 120583m2 (ADSC)Bone nodule count scoring + (lt20 nodules) ++ (20ndash50) and +++ (gt50)ND not detected

that phenotypic diversification can actually in many cases beattributed more to differential adaptation to in vitro cultureconditions than to inherent donor sample heterogeneity dueto age sex pathology and so forth [29] In the present studywe have focused on the in vitro characterization of postnatalhumanMSC populations originating from sources that differboth developmentally as well as anatomically that is cellsisolated from the matrix (Whartonrsquos jelly) of the fetal UCtissue (WJSC) [14] and from the abdominal adipose tissue ofadults (ADSC) [15] We have chosen these two diverse popu-lations not only because they are representative of the adultand fetal human stem cell phenotype allowing comparisonsto be made but also for practical reasons sincemdashin contrastto other MSC populationsmdashhigh numbers can be relativelyeasily isolated from the respective tissues with no (as in thecase of WJSC) or minimal donor site morbidity We thenserially propagated these two MSC types over an extendedperiod under standardized culture conditions in an effortto investigate whether and to what extent they sustain theirproliferative capacity and phenotypic stability

With regard to ADSC their isolation is uncomplicatedand efficient especially for smaller tissue samples providingon average about two million cells per gram lipoaspirateEstablishment of primary cultures was however not 100efficient and propagation was slow Culture threshold waspinpointed at passage 5 up to which the cells divide on

average every eight days giving a total of 52 PD in a 40-dayperiod This equates following serial passaging to 103 times 108cells enough for a single administration to an 80 kg patientThis yield is comparable to that reported by other studiesfocusing on ADSC [27] as well as to the yield obtained bythe expansion of other adult MSC populations (eg BM-MSCs) [30] Naturally as in the case of other adult MSCssuch as peripheral blood- and BM-MSCs ADSC can beused autologously following ex vivo expansion Moreoverthey bear the additional advantage of probably being themost abundantly available and readily obtainable MSCscompared to the aforementioned cell types A comparativecharacterization of ADSC and various fetal and adult stemcell populations has been accounted by a number of studieshowever a comprehensive comparison to WJSC especiallywith respect to long-term culture is lacking

As highlighted earlier an accumulating number of pub-lications show that fetal (prenatal as well as perinatal) stemcells bear advantages in comparison to their adult counter-parts These concern increased proliferation rates prolongedcapacity for differentiation enhanced sensitivity to chemicalstimuli in the extracellular milieu and adaptation to cultureconditions [8 31ndash33] Moreover there is strong evidence thatfetal tissue MSCs are hypoimmunogenic and can be well tol-erated in allogeneic transplantation [1 34] Possible sourcesof fetal MSCs that can be easily harvested perinatally are

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

150

200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

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BioinformaticsAdvances in

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Signal TransductionJournal of

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International Journal of

Microbiology

4 Stem Cells International

25 Statistical Analysis Parametric and nonparametric tests(Spearman correlation analysis Mann-Whitney pairwisedata comparisons and ANOVA) were conducted usingGraphPad Prism 504 statistical analysis software Data aredepicted as means plusmn SD unless otherwise stated A P valuelt 005 was considered as statistically significant

3 Results

31 Primary Cell Isolation and Culture Initiation Efficiency

311 WJSC Mean nucleated cell yield following UC tissueprocessing was about 23 times 106 and was found to positivelycorrelate (Spearman 119903 = 0722119875 lt 0001) with tissue proper-ties (ie UC length) (Table 1 and see in SupplementaryMate-rial available online at httpdxdoiorg1011552013246134Figure 1) The isolation protocol involved removal of thevessels that due to the supercoiled anatomy of the UCincreases tissue processing time and hence results in some cellloss compared to protocols based on digestion of whole UCNevertheless our protocol produces a more homogeneouspopulation than the latter which results in the isolation ofa mixed population of at least four stem cell types [25 26]Five out of five (100) cultures of adherent cells derived fromplated UC nucleated cell samples led to the establishment ofcultures that grew successfully beyond passage 3 (Table 1)The three of these cultures with the shortest PDT (see thefollowing) were further subcultured and used for the exper-iments described in this study The percentage of adherentcells in the primary cultures was estimated at 12

312 ADSC ForADSCmeannucleated cell yield at isolationwas 312 times 106 cells (Table 1) Regression analysis of samplesshowed that this yield was not significantly associated withinitial tissue quantity (donor adipose weight) (see Supple-mentary Figure 1) or other donor characteristics such asage or sex Nevertheless there was a strong linear correlationof the number of isolated cells with adipose tissue weightfor samples up to 200 g (Spearman 119903 = 0952 119875 lt0001) highlighting the increased efficiency of the protocolfor samples in that range The eight adipose tissue sampleswith the highest mononuclear cell yields were plated out andused to establish cultures of ADSC Five of these samplestested proliferated very slowly (less than 20 increase incell yield between passages) and failed to grow beyondpassage 3 whereas 38 (375) proliferated further and wereused for the experiments ADSC yield was approximately18 times 10

6 cellsg lipoaspirate with the percentage of adherentcells in the primary cultures being much lower than thatof WJSC at 093 This yield is slightly higher than thatreported elsewhere (1 times 106 cellsg) [27] Viability of isolatedcells and resuscitated cells following initial cryopreservationwas consistently high (gt92) and similar to that of WJSC(Table 1)

32 Proliferation Kinetics of Long-Term Cultures Figures1(a)ndash1(c) depict the proliferation dynamics of the two MSCpopulations during prolonged in vitro subcultivation while

Table 2 summarises the main findings The mean cell yieldshown in Table 2 is the one that arises from the serial prop-agation of cells following the subculture protocol describedin Section 2 This was higher (by more than twofold) forWJSC and related to a constant number of about three PD perpassage (depicted by the linearity of the graph in Figure 1(a))accumulating to a total of 58 PD in the 100-day cultureperiod Mean PDT was 384 plusmn 86 hrs over that period Peakproliferative rate was observed at passage 6 (considering PIand PDT Figures 1(b) and 1(c) resp) which equated tonearly 19 PD over a month in culture Maximum PI of WJSC(observed at passage 6) was over 15-fold higher than that ofADSC and coincided with a maximum number of 372 plusmn032 PD WJSC showed no signs of slowdown even after 15passages (45 PD) (119875 gt 005 ANOVA on PDT of passages 2ndash16)There was nevertheless a significant increase in PDT afterpassage 17 which exceeded 50 hrs by passage 20

ADSC proliferation capacity was maximum at passage 2(see Figures 1(b) and 1(c)) and remained above average untilpassage 5 although related to a poor number of cumulativePD (just over five) Signs of senescence and essentially ces-sation of proliferation appeared after passage 6 Thencefortha plateau in PD accumulation a consistently high PDT (ofnearly 37 days) and low expansion rate (lt20 per passage)were observed Mean cell yield per passage in our cultureprotocol was two-three times lower for ADSC (Table 2)

The growth curves (Figure 1(d)) depict the kinetics ofproliferation within a single passage Both cell types followa typical sigmoid growth pattern but with different phasedurations WJSC exhibit a lag phase similar to that of ADSC(about 25 days) but a much shorter log phase (35 as opposedto 75 days) during which they expand more promptly(about fivefold expansion of cell population) WJSC becomeconfluent by day 7 while they continue to grow by 15-20 even when superconflluent forming dense cultures ofcompacted cells whereas ADSC undergo contact inhibitionafter day 11 not exceeding 90 confluency GM containingthe dipeptide form of glutamine increases cell yield duringlog phase by up to 200 (especially for ADSC 119875 lt 001 day9) while it enhances survival of postconfluentWJSC cultures(119875 lt 005 day 11 see online Supplementary Figure 2) Thisis in concordance with the effects of glutamine dipeptide onbone-marrow-derived MSC survival and growth [28]

33 Phenotypic Characteristics of Long-Term CulturesFigure 2(a) illustrates the change in cell size as a phenotypiccharacteristic during prolonged subculture A markedgreather than 300 change in cell size over culture timewas observed for both MSC populations ADSC mean sizewas 3876 120583m2 with minimum (1648) and maximum (6130)values observed at passages 1 and 8 respectively (Figure 2(a))WJSC had an average size of 2926120583m2 (min 1437 max 5183at passages 1 and 11 resp Figure 2(a)) WJSC exhibited aperiodicity in cell size that might be attributed to smallrapidly proliferating cells undergoing rounds of symmetricand asymmetric divisions which on one hand give rise todifferentiating daughter cells that gradually increase in sizeand probably become senescent (see also Figures 2(c) and 3)

Stem Cells International 5

Table 1 Cell isolation and culture establishment efficiency

WJSC ADSC 119875 value

Mean tissue length-weight 133 cm plusmn 2lowast 1738 g plusmn 1357 mdashMean nucleated cell yield (times106)

(per unit length-weight)228 plusmn 155(017cm)

312 plusmn 198(18g) mdash

Mean viability at isolation () 943 plusmn 22 966 plusmn 16 gt005Mean viability at resuscitation () 929 plusmn 43 927 plusmn 49 gt005 of adherent cells in starting culture 173 plusmn 46 093 plusmn 045 lt0001Adherent cultures beyond p3 100 (55) 375 (38) lt005Mean passage-to-passage expansion rate (p1ndash5) 974 plusmn 317 175 plusmn 56 lt001Mean subculture viability (p1ndash5) 976 plusmn 05 964 plusmn 19 gt005119875 value WJSC versus ADSC lowastA statistically significant association was found between UC tissue sample length and the yield of isolated nucleated cellsfollowing tissue processing (see text for details)

while at the same time replenishing the culture with moresubsets of small rapidly proliferating stem cells In support ofthis is the association of kinetics (PDT) with cell size (Figures2(b) and 2(c)) Spearman analysis showed that small cell sizecorrelates better with low PDT while cells that take longerto undergo rounds of division (senescent) tend to be largerSpecifically the cells with sizes in the top 25 (arbitrarilydenoted as giant cells) have also the highest PDT and thelatter correspond to mid to late passages (p7 or greater forADSC and p9 or greater for WJSC)

Besides cell size a marked change was observed in otherphenotypic properties of MSCs over in vitro expansionFigure 3 outlines these differences between early cultures andculture endpoints In terms of morphological characteristicsthree main morphologies were observed (a) spindle-shapedelongated (type I) (b) polygonal with few small membraneprotrusions (type II) (c) flattened with irregular shape andlarge (frequently multiple) nuclei (type III) Early-passage(p2) WJSC and ADSC have mainly type II and type Imorphologies respectively (see Table 3 and Figure 3(a))Serial passaging of both MSC populations results in gradualacquisition of type III morphology which especially inthe case of WJSC increases by 28-fold in occurrence andbecomes predominant by passage 20 In these culture pointscells with either type I or type III morphologies accountedfor over 70 of the cultures (see Table 3) with at least one-in-three cells being a giant cell (see Methods section for sizedefinition) bearing enlarged often multiple nuclei and beinglargely senescent staining positive for 120573-gal (Figure 3(b))The occurrence of senescent cells in ADSC cultures was fairlyfrequent with roughly one cell out of five staining positiveirrespective of the culture point (Table 3) On the contrarythe senescence rate of early WJSC culture which is very lowincreases 5-6 fold in later passages albeit with considerableinterculture variability An interesting observation was alsothat cytoplasmic granularity ofWJSC decreased by 35ndash40with passaging probably relating to rearrangements in thecytoskeleton and protein synthesis machinery (Table 3 andonline Supplementary Figure 3)

In terms of stemness properties both cell types have acapacity for clonogenicityself-renewal and lineage-specific

differentiation and express a typical immunophenotypic pro-file with high MSC marker (gt97 positive except CD44of late-passage ADSC which was slightly lower at 92 plusmn23) and low HSC marker expression (lt3 positive withthe exception of 8 plusmn 02 of early-passage ADSC whichexpressed monocyte-related CD14) (Figure 4) The mostavidly expressed markers (high signal intensity) were CD44(H-CAM) and CD105 (endoglin) for WJSC and ADSCrespectively But although expression levels of CD surfacemarkers remained constant over time in culture clonogenic-ity was reduced ForWJSC about 1 in 45 cells was clonogenicand this ability diminished by less than twofold over the first10 passages (+24 PD) but almost by a factor of five in thenext 10 passages (+28 PD) (Figure 3(c) and Table 3) ADSCformed robustly clones at all densities tested however theof CFU-F ratios were lower than those of WJSC that hadundergone many more PD Differentiation capacityinducedlineage specific differentiation (osteoblasts) also diminishedwith ageing in culture (Figures 3(c) and 3(d) Table 3) ForWJSC again this decline was more acute for the last 10passages although spontaneous differentiation to CFU-ALPincreased as a percentage relative to CFU-F probably asign of relative lineage commitmentrestriction In passage-2 cultures calcium concentration was marginally higher forADSC probably owing to the presence of bigger and bettermineralized nodules but was undetected in later passages

Last it is worth noting an increased resistance to trypsindigestion for ADSC The time period required for 50detachment at passage 4 was 10min (60) longer forADSC as compared to WJSC passaging did not significantlyincrease trypsinization time (see online Supplementary Fig-ure 4) Since the cells have similar sizes (at respectivepassages) it is suggested that the increased resistance totrypsinization may be due to differential adhesion moleculeexpression

4 Discussion

MSC populations share common characteristics howeverphenotypic diversification is bound to exist especially inan in vitro environment Indeed microarray studies show

6 Stem Cells International

Table 2 Synopsis of proliferation kinetics data of WJSC and ADSC during long-term in vitro expansion

Mean PDT(hrs)

Mean PDper passage

Mean cell yield perpassage (times106)

Theoretical total yield(times108) after five passages

Average time (days)required for obtaining

80 times 106 cells

WJSC 319 plusmn 68 384 plusmn 52lowast

29 plusmn 063 28 plusmn 059

30 plusmn 082 225 plusmn 08 1205 178

ADSC 4023 plusmn 2151 077 plusmn 024 103 plusmn 035 103 348WJSC data pairs in the first three columns correspond to cultures in the first 10 passages and in passages 11 to 20 respectively lowast119875 lt 0001 Values in the lasttwo columns have been calculated on the basis of the assumption that all isolated cells are serially subcultivated with no intermediate freezing of cell stocksbetween successive passages

Table 3 Overview of phenotypic characteristics of ADSC and WJSC at early (p2) and late (p10 and p1020 resp) passages

Passage (cumPD)WJSC ADSC

2 (45) 10 (289) 20 (567) 2 (24) 10 (81)Mean cell size (120583m2

plusmn SEM) 2340 plusmn 180 4882 plusmn 646 5003 plusmn 712 2407 plusmn 183 4907 plusmn 653 giant cellslowast 0 44 plusmn 4 30 plusmn 7 20 plusmn 5 31 plusmn 8Predominant morphology types () III (4638) III (3832) IIII (4436) IIII (5726) IIII (4233)Intracellular complexity (SSC) 130 85 77 132 115Senescence (120573-gal positive) 21 plusmn 09 113 plusmn 83 129 plusmn 110 17 plusmn 62 246 plusmn 97Self-renewal (CFU-F cells seeded) 1 46 1 73 1 366 1 68 1 140Osteogenic differentiation

CFU-ALP cells seeded( of CFU-F)

1 7860 plusmn 63

1 9876 plusmn 26

1 45083 plusmn 12

1 14946 plusmn 62

1 44831 plusmn 15

Bone nodule formation +++ ++ + +++ +(Ca2+) (mgdL) 108 plusmn 004 ND ND 156 plusmn 038 ND

lowastGiant cells gt3880120583m2 (WJSC) gt4600 120583m2 (ADSC)Bone nodule count scoring + (lt20 nodules) ++ (20ndash50) and +++ (gt50)ND not detected

that phenotypic diversification can actually in many cases beattributed more to differential adaptation to in vitro cultureconditions than to inherent donor sample heterogeneity dueto age sex pathology and so forth [29] In the present studywe have focused on the in vitro characterization of postnatalhumanMSC populations originating from sources that differboth developmentally as well as anatomically that is cellsisolated from the matrix (Whartonrsquos jelly) of the fetal UCtissue (WJSC) [14] and from the abdominal adipose tissue ofadults (ADSC) [15] We have chosen these two diverse popu-lations not only because they are representative of the adultand fetal human stem cell phenotype allowing comparisonsto be made but also for practical reasons sincemdashin contrastto other MSC populationsmdashhigh numbers can be relativelyeasily isolated from the respective tissues with no (as in thecase of WJSC) or minimal donor site morbidity We thenserially propagated these two MSC types over an extendedperiod under standardized culture conditions in an effortto investigate whether and to what extent they sustain theirproliferative capacity and phenotypic stability

With regard to ADSC their isolation is uncomplicatedand efficient especially for smaller tissue samples providingon average about two million cells per gram lipoaspirateEstablishment of primary cultures was however not 100efficient and propagation was slow Culture threshold waspinpointed at passage 5 up to which the cells divide on

average every eight days giving a total of 52 PD in a 40-dayperiod This equates following serial passaging to 103 times 108cells enough for a single administration to an 80 kg patientThis yield is comparable to that reported by other studiesfocusing on ADSC [27] as well as to the yield obtained bythe expansion of other adult MSC populations (eg BM-MSCs) [30] Naturally as in the case of other adult MSCssuch as peripheral blood- and BM-MSCs ADSC can beused autologously following ex vivo expansion Moreoverthey bear the additional advantage of probably being themost abundantly available and readily obtainable MSCscompared to the aforementioned cell types A comparativecharacterization of ADSC and various fetal and adult stemcell populations has been accounted by a number of studieshowever a comprehensive comparison to WJSC especiallywith respect to long-term culture is lacking

As highlighted earlier an accumulating number of pub-lications show that fetal (prenatal as well as perinatal) stemcells bear advantages in comparison to their adult counter-parts These concern increased proliferation rates prolongedcapacity for differentiation enhanced sensitivity to chemicalstimuli in the extracellular milieu and adaptation to cultureconditions [8 31ndash33] Moreover there is strong evidence thatfetal tissue MSCs are hypoimmunogenic and can be well tol-erated in allogeneic transplantation [1 34] Possible sourcesof fetal MSCs that can be easily harvested perinatally are

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

150

200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 5

Table 1 Cell isolation and culture establishment efficiency

WJSC ADSC 119875 value

Mean tissue length-weight 133 cm plusmn 2lowast 1738 g plusmn 1357 mdashMean nucleated cell yield (times106)

(per unit length-weight)228 plusmn 155(017cm)

312 plusmn 198(18g) mdash

Mean viability at isolation () 943 plusmn 22 966 plusmn 16 gt005Mean viability at resuscitation () 929 plusmn 43 927 plusmn 49 gt005 of adherent cells in starting culture 173 plusmn 46 093 plusmn 045 lt0001Adherent cultures beyond p3 100 (55) 375 (38) lt005Mean passage-to-passage expansion rate (p1ndash5) 974 plusmn 317 175 plusmn 56 lt001Mean subculture viability (p1ndash5) 976 plusmn 05 964 plusmn 19 gt005119875 value WJSC versus ADSC lowastA statistically significant association was found between UC tissue sample length and the yield of isolated nucleated cellsfollowing tissue processing (see text for details)

while at the same time replenishing the culture with moresubsets of small rapidly proliferating stem cells In support ofthis is the association of kinetics (PDT) with cell size (Figures2(b) and 2(c)) Spearman analysis showed that small cell sizecorrelates better with low PDT while cells that take longerto undergo rounds of division (senescent) tend to be largerSpecifically the cells with sizes in the top 25 (arbitrarilydenoted as giant cells) have also the highest PDT and thelatter correspond to mid to late passages (p7 or greater forADSC and p9 or greater for WJSC)

Besides cell size a marked change was observed in otherphenotypic properties of MSCs over in vitro expansionFigure 3 outlines these differences between early cultures andculture endpoints In terms of morphological characteristicsthree main morphologies were observed (a) spindle-shapedelongated (type I) (b) polygonal with few small membraneprotrusions (type II) (c) flattened with irregular shape andlarge (frequently multiple) nuclei (type III) Early-passage(p2) WJSC and ADSC have mainly type II and type Imorphologies respectively (see Table 3 and Figure 3(a))Serial passaging of both MSC populations results in gradualacquisition of type III morphology which especially inthe case of WJSC increases by 28-fold in occurrence andbecomes predominant by passage 20 In these culture pointscells with either type I or type III morphologies accountedfor over 70 of the cultures (see Table 3) with at least one-in-three cells being a giant cell (see Methods section for sizedefinition) bearing enlarged often multiple nuclei and beinglargely senescent staining positive for 120573-gal (Figure 3(b))The occurrence of senescent cells in ADSC cultures was fairlyfrequent with roughly one cell out of five staining positiveirrespective of the culture point (Table 3) On the contrarythe senescence rate of early WJSC culture which is very lowincreases 5-6 fold in later passages albeit with considerableinterculture variability An interesting observation was alsothat cytoplasmic granularity ofWJSC decreased by 35ndash40with passaging probably relating to rearrangements in thecytoskeleton and protein synthesis machinery (Table 3 andonline Supplementary Figure 3)

In terms of stemness properties both cell types have acapacity for clonogenicityself-renewal and lineage-specific

differentiation and express a typical immunophenotypic pro-file with high MSC marker (gt97 positive except CD44of late-passage ADSC which was slightly lower at 92 plusmn23) and low HSC marker expression (lt3 positive withthe exception of 8 plusmn 02 of early-passage ADSC whichexpressed monocyte-related CD14) (Figure 4) The mostavidly expressed markers (high signal intensity) were CD44(H-CAM) and CD105 (endoglin) for WJSC and ADSCrespectively But although expression levels of CD surfacemarkers remained constant over time in culture clonogenic-ity was reduced ForWJSC about 1 in 45 cells was clonogenicand this ability diminished by less than twofold over the first10 passages (+24 PD) but almost by a factor of five in thenext 10 passages (+28 PD) (Figure 3(c) and Table 3) ADSCformed robustly clones at all densities tested however theof CFU-F ratios were lower than those of WJSC that hadundergone many more PD Differentiation capacityinducedlineage specific differentiation (osteoblasts) also diminishedwith ageing in culture (Figures 3(c) and 3(d) Table 3) ForWJSC again this decline was more acute for the last 10passages although spontaneous differentiation to CFU-ALPincreased as a percentage relative to CFU-F probably asign of relative lineage commitmentrestriction In passage-2 cultures calcium concentration was marginally higher forADSC probably owing to the presence of bigger and bettermineralized nodules but was undetected in later passages

Last it is worth noting an increased resistance to trypsindigestion for ADSC The time period required for 50detachment at passage 4 was 10min (60) longer forADSC as compared to WJSC passaging did not significantlyincrease trypsinization time (see online Supplementary Fig-ure 4) Since the cells have similar sizes (at respectivepassages) it is suggested that the increased resistance totrypsinization may be due to differential adhesion moleculeexpression

4 Discussion

MSC populations share common characteristics howeverphenotypic diversification is bound to exist especially inan in vitro environment Indeed microarray studies show

6 Stem Cells International

Table 2 Synopsis of proliferation kinetics data of WJSC and ADSC during long-term in vitro expansion

Mean PDT(hrs)

Mean PDper passage

Mean cell yield perpassage (times106)

Theoretical total yield(times108) after five passages

Average time (days)required for obtaining

80 times 106 cells

WJSC 319 plusmn 68 384 plusmn 52lowast

29 plusmn 063 28 plusmn 059

30 plusmn 082 225 plusmn 08 1205 178

ADSC 4023 plusmn 2151 077 plusmn 024 103 plusmn 035 103 348WJSC data pairs in the first three columns correspond to cultures in the first 10 passages and in passages 11 to 20 respectively lowast119875 lt 0001 Values in the lasttwo columns have been calculated on the basis of the assumption that all isolated cells are serially subcultivated with no intermediate freezing of cell stocksbetween successive passages

Table 3 Overview of phenotypic characteristics of ADSC and WJSC at early (p2) and late (p10 and p1020 resp) passages

Passage (cumPD)WJSC ADSC

2 (45) 10 (289) 20 (567) 2 (24) 10 (81)Mean cell size (120583m2

plusmn SEM) 2340 plusmn 180 4882 plusmn 646 5003 plusmn 712 2407 plusmn 183 4907 plusmn 653 giant cellslowast 0 44 plusmn 4 30 plusmn 7 20 plusmn 5 31 plusmn 8Predominant morphology types () III (4638) III (3832) IIII (4436) IIII (5726) IIII (4233)Intracellular complexity (SSC) 130 85 77 132 115Senescence (120573-gal positive) 21 plusmn 09 113 plusmn 83 129 plusmn 110 17 plusmn 62 246 plusmn 97Self-renewal (CFU-F cells seeded) 1 46 1 73 1 366 1 68 1 140Osteogenic differentiation

CFU-ALP cells seeded( of CFU-F)

1 7860 plusmn 63

1 9876 plusmn 26

1 45083 plusmn 12

1 14946 plusmn 62

1 44831 plusmn 15

Bone nodule formation +++ ++ + +++ +(Ca2+) (mgdL) 108 plusmn 004 ND ND 156 plusmn 038 ND

lowastGiant cells gt3880120583m2 (WJSC) gt4600 120583m2 (ADSC)Bone nodule count scoring + (lt20 nodules) ++ (20ndash50) and +++ (gt50)ND not detected

that phenotypic diversification can actually in many cases beattributed more to differential adaptation to in vitro cultureconditions than to inherent donor sample heterogeneity dueto age sex pathology and so forth [29] In the present studywe have focused on the in vitro characterization of postnatalhumanMSC populations originating from sources that differboth developmentally as well as anatomically that is cellsisolated from the matrix (Whartonrsquos jelly) of the fetal UCtissue (WJSC) [14] and from the abdominal adipose tissue ofadults (ADSC) [15] We have chosen these two diverse popu-lations not only because they are representative of the adultand fetal human stem cell phenotype allowing comparisonsto be made but also for practical reasons sincemdashin contrastto other MSC populationsmdashhigh numbers can be relativelyeasily isolated from the respective tissues with no (as in thecase of WJSC) or minimal donor site morbidity We thenserially propagated these two MSC types over an extendedperiod under standardized culture conditions in an effortto investigate whether and to what extent they sustain theirproliferative capacity and phenotypic stability

With regard to ADSC their isolation is uncomplicatedand efficient especially for smaller tissue samples providingon average about two million cells per gram lipoaspirateEstablishment of primary cultures was however not 100efficient and propagation was slow Culture threshold waspinpointed at passage 5 up to which the cells divide on

average every eight days giving a total of 52 PD in a 40-dayperiod This equates following serial passaging to 103 times 108cells enough for a single administration to an 80 kg patientThis yield is comparable to that reported by other studiesfocusing on ADSC [27] as well as to the yield obtained bythe expansion of other adult MSC populations (eg BM-MSCs) [30] Naturally as in the case of other adult MSCssuch as peripheral blood- and BM-MSCs ADSC can beused autologously following ex vivo expansion Moreoverthey bear the additional advantage of probably being themost abundantly available and readily obtainable MSCscompared to the aforementioned cell types A comparativecharacterization of ADSC and various fetal and adult stemcell populations has been accounted by a number of studieshowever a comprehensive comparison to WJSC especiallywith respect to long-term culture is lacking

As highlighted earlier an accumulating number of pub-lications show that fetal (prenatal as well as perinatal) stemcells bear advantages in comparison to their adult counter-parts These concern increased proliferation rates prolongedcapacity for differentiation enhanced sensitivity to chemicalstimuli in the extracellular milieu and adaptation to cultureconditions [8 31ndash33] Moreover there is strong evidence thatfetal tissue MSCs are hypoimmunogenic and can be well tol-erated in allogeneic transplantation [1 34] Possible sourcesof fetal MSCs that can be easily harvested perinatally are

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

150

200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Stem Cells International

Table 2 Synopsis of proliferation kinetics data of WJSC and ADSC during long-term in vitro expansion

Mean PDT(hrs)

Mean PDper passage

Mean cell yield perpassage (times106)

Theoretical total yield(times108) after five passages

Average time (days)required for obtaining

80 times 106 cells

WJSC 319 plusmn 68 384 plusmn 52lowast

29 plusmn 063 28 plusmn 059

30 plusmn 082 225 plusmn 08 1205 178

ADSC 4023 plusmn 2151 077 plusmn 024 103 plusmn 035 103 348WJSC data pairs in the first three columns correspond to cultures in the first 10 passages and in passages 11 to 20 respectively lowast119875 lt 0001 Values in the lasttwo columns have been calculated on the basis of the assumption that all isolated cells are serially subcultivated with no intermediate freezing of cell stocksbetween successive passages

Table 3 Overview of phenotypic characteristics of ADSC and WJSC at early (p2) and late (p10 and p1020 resp) passages

Passage (cumPD)WJSC ADSC

2 (45) 10 (289) 20 (567) 2 (24) 10 (81)Mean cell size (120583m2

plusmn SEM) 2340 plusmn 180 4882 plusmn 646 5003 plusmn 712 2407 plusmn 183 4907 plusmn 653 giant cellslowast 0 44 plusmn 4 30 plusmn 7 20 plusmn 5 31 plusmn 8Predominant morphology types () III (4638) III (3832) IIII (4436) IIII (5726) IIII (4233)Intracellular complexity (SSC) 130 85 77 132 115Senescence (120573-gal positive) 21 plusmn 09 113 plusmn 83 129 plusmn 110 17 plusmn 62 246 plusmn 97Self-renewal (CFU-F cells seeded) 1 46 1 73 1 366 1 68 1 140Osteogenic differentiation

CFU-ALP cells seeded( of CFU-F)

1 7860 plusmn 63

1 9876 plusmn 26

1 45083 plusmn 12

1 14946 plusmn 62

1 44831 plusmn 15

Bone nodule formation +++ ++ + +++ +(Ca2+) (mgdL) 108 plusmn 004 ND ND 156 plusmn 038 ND

lowastGiant cells gt3880120583m2 (WJSC) gt4600 120583m2 (ADSC)Bone nodule count scoring + (lt20 nodules) ++ (20ndash50) and +++ (gt50)ND not detected

that phenotypic diversification can actually in many cases beattributed more to differential adaptation to in vitro cultureconditions than to inherent donor sample heterogeneity dueto age sex pathology and so forth [29] In the present studywe have focused on the in vitro characterization of postnatalhumanMSC populations originating from sources that differboth developmentally as well as anatomically that is cellsisolated from the matrix (Whartonrsquos jelly) of the fetal UCtissue (WJSC) [14] and from the abdominal adipose tissue ofadults (ADSC) [15] We have chosen these two diverse popu-lations not only because they are representative of the adultand fetal human stem cell phenotype allowing comparisonsto be made but also for practical reasons sincemdashin contrastto other MSC populationsmdashhigh numbers can be relativelyeasily isolated from the respective tissues with no (as in thecase of WJSC) or minimal donor site morbidity We thenserially propagated these two MSC types over an extendedperiod under standardized culture conditions in an effortto investigate whether and to what extent they sustain theirproliferative capacity and phenotypic stability

With regard to ADSC their isolation is uncomplicatedand efficient especially for smaller tissue samples providingon average about two million cells per gram lipoaspirateEstablishment of primary cultures was however not 100efficient and propagation was slow Culture threshold waspinpointed at passage 5 up to which the cells divide on

average every eight days giving a total of 52 PD in a 40-dayperiod This equates following serial passaging to 103 times 108cells enough for a single administration to an 80 kg patientThis yield is comparable to that reported by other studiesfocusing on ADSC [27] as well as to the yield obtained bythe expansion of other adult MSC populations (eg BM-MSCs) [30] Naturally as in the case of other adult MSCssuch as peripheral blood- and BM-MSCs ADSC can beused autologously following ex vivo expansion Moreoverthey bear the additional advantage of probably being themost abundantly available and readily obtainable MSCscompared to the aforementioned cell types A comparativecharacterization of ADSC and various fetal and adult stemcell populations has been accounted by a number of studieshowever a comprehensive comparison to WJSC especiallywith respect to long-term culture is lacking

As highlighted earlier an accumulating number of pub-lications show that fetal (prenatal as well as perinatal) stemcells bear advantages in comparison to their adult counter-parts These concern increased proliferation rates prolongedcapacity for differentiation enhanced sensitivity to chemicalstimuli in the extracellular milieu and adaptation to cultureconditions [8 31ndash33] Moreover there is strong evidence thatfetal tissue MSCs are hypoimmunogenic and can be well tol-erated in allogeneic transplantation [1 34] Possible sourcesof fetal MSCs that can be easily harvested perinatally are

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

150

200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 7

0123456789

1 11 21 31 41 51 61 71 81 91 101010203040506070

Cum

ulat

ive P

D (A

DSC

)

Time in culture (days)

Cum

ulat

ive P

D (W

JSC)

WJSCADSC

1198772 =1198772 = 0988409904

(a)

010203040506070

0100200300400500600700800900

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PDT

WJS

C (h

rs)

PDT

AD

SC (h

rs)

Passage

ADSCWJSC

(b)

0

50

100

150

200

02468

101214

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prol

ifera

tion

inde

x (W

JSC)

Prol

ifera

tion

inde

x (A

DSC

)

Passage

ADSCWJSC

(c)

0

300

600

900

1200

0306090

120150180

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days in culture

ADSCWJSC

103 )

WJS

C (times

103)

AD

SC ( times

lowast

(d)

Figure 1 Proliferation kinetics ofWJSC andADSCover extended invitro propagation (a) Number of cumulative population doublings(PD) as a function of time in culture (b) cell population doublingtimesmdashPDTmdash(hrs) at respective passages (c) proliferation index(PI ratio of PD over respective PDT) (d) growth curve of WJSCand ADSC within a single passage (3 to 4) The lowast and symbolshighlight the points when confluence was reached for WJSC andADSC respectively Dashed and long dashed horizontal linesdenote mean values for ADSC and WJSC respectively

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Passage

ADSCWJSC

Cel

lare

a(120583

m2)

(a)

0

200

400

600

800

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

ADSC

Cell size (120583m2)

1198772 = 08133

119910 = 01299119909 minus 1184

(b)

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

PDT

(hrs

)

WJSC

Cell size (120583m2)

119910 = 00044119909 + 2644

1198772 = 0338

(c)

Figure 2 Variation in cell size (surface area in 120583m2) over timein culture and its association with proliferation kinetics (a) Cellsize (mean area plusmn SEM) as a phenotypic property of WJSCand ADSC during serial propagation Dashed and long dashedhorizontal lines denote mean cell size for ADSC (3876 120583m2) andWJSC (2926 120583m2) respectively (b) regression analysis of ADSCsize versus PDT at respective passages (119875 lt 0001 Spearmanr correlation coefficient = 0915) Trendline equation and good-ness of fit (1198772) are shown Boxed data points correspond toADSC with sizes over 3880 120583m2 (top 25 of cells) (c) regressionanalysis of WJSC size versus PDT at respective passages (119875 lt001 119903 = 06) Open triangles p1ndash10 solid triangles p11ndash20Trendline equation and goodness of fit (1198772) are shown Boxeddata points correspond to WJSC with sizes over 4600120583m2 (top25)

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 Stem Cells International

WJSC ADSCPassage

(PD)2

(45)10

(289)20

(567)2

(24)10

(81)

(a) Morphology

(b) 120573-gal(senescence)

(c) CFU-F(self-renewal)

(d) CFU-ALP(osteoprogenitors)

(e) Bone nodules(osteogenesis)

Figure 3 Phenotypic properties of WJSC and ADSC at early and late passages (a) Light microscopy photos illustrating cell morphologyarrows show cells with type III morphology Top row phase contrast Mag = 25x (b) Senescence as depicted by 120573-gal staining Mag = 25xSubpanel shows a giant cell (gt650 120583m in length) with typical senescent phenotype (c) Crystal violet staining of CFU-F (self-renewal capacity)(d) Formation of CFU-ALP (osteoprogenitor colonies) (e) Calcified bone nodule formation in response to osteogenic stimulation Subpanelillustrates a typical alizarin red-stained bone nodule at high magnification (25x) Scale bars (a) and (b) 100 120583m (c) and (d) 10mm and (e)4mm

the embryologically derived placenta umbilical cord (UC)tissue and UC blood Historically MSCs from cord bloodhave been the most frequently studied and compared withfetal MSC types mainly due to their popularity in stem cellbanking [6 7] However their erratic isolation efficiency andcumbersome culture render them unattractive for clinicalapplications [7 35] In contrast in recent years Whartonrsquosjelly (WJ) the mesenchyme-like cushioning material foundbetween the vessels of the UC has been highlighted as one ofthe compartments of theUC tissue that harbors a particularlylarge population of MSCs (here termed WJSC) [14] Thesecells can be easily grown in culture without the need forspecific growth factors or feeder layers even in xeno-freeculture conditions [36 37] while at the same time they havebeen found to secrete soluble factors that are supportive ofhemopoiesis and ESC growth [38ndash41]

Results from our study clearly suggest that WJSC possesssuperior proliferation capacity and grow more predictablythan adult ADSC In particular they have a steeper log phase

and a short PDT (a mean PDT of nearly 32 hrs in the first10 passages is substantially shorter even when compared tocancer cells and hESC with average PDT of 46 and 72 hrsresp [42 43]) It is worth highlighting that most publicationsdescribe experiments with MSCs propagated up to passagefive Our data indicate that the theoretical total yield after fivepassages (provided that all cells are serially subcultured) is120 times higher for WJSC Furthermore with a PDT of justover 24 hrs in these early passages they divide seven timesfaster thanADSCWith respect toWJSC the seventh passagewas identified as a culture threshold By this stage the cellshave accumulated 22 PD in 33 days which equate to a totalyield of 165 times 1012 cells sufficient for 4000ndash20000 in vivoadministrations These kinetics are significantly faster thanan earlier study on WJSC which reported similar cumulativePD over a three-month period [44] With respect to theculture period required for getting a minimum number ofWJSC for transplantation this was half than in the case ofADSC All the above render WJSC preferable for laboratory

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 9

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

CD29

CD44

CD73

CD

90CD

105

CD14

CD34

CD45

441

294

147

0 0 0

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

423

282

141

28821514472

209

104

0

99

49

0

481

321

160

0

394

263

131

0

276207138

690

209

104

0

149

99

49

0

FL 1log FL 1log FL 1log FL 1log FL 1log

FL 2log FL 2logFL 2log FL 2logFL 2log

FL 3log FL 3log FL 3log FL 3logFL 3log

FL 4log FL 4log FL 4log FL 4log FL 4log

1218140

0

407

271

135

0

272

181

90

0

252

126

0

11073

36

0

423282141

0

423

282

141

0

299

199

99

0

159106

530

138

92

46

0

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 103 104

100 101 102 103 104 100 101 102 103 10 100 101 102

102

103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

0326597

130

WJSC ADSC

p 2 p 10 p 20 p 2 p 10

273

352

150

0

373

249

124

0

10

12

5

0

43

28

14

0

576

365

191

0

373

209

124

0

301

254121

0

60

50

42

0

175

113

90

0

222

153

76

0

243

145

72

0

166

111

55

0

499374249124

0

483362241120

0

225

150

75

0

384

256

128

0

270203135

670

150

75

0

110

73

36

0

Figure 4 Immunophenotypic analysis of WJSC and ADSC for the expression of typical mesenchymal and hemopoietic stem cell surfacemarkers (first five and last three markers resp) during subcultureThe vertical axes of the histograms depict event counts and the horizontalaxes represent fluorescence intensities of surface-bound conjugates that ismarker expression levels Staining ofWJSC andADSCwith specificmonoclonal antibody conjugates is illustrated by solid dark histograms (MSCmarkers CD-29 and -44 FITC CD-73 -90 and -105 PE) and byopen histograms (HSC markers CD-14 and -34 ECD CD-45 PC5) Stripped histograms correspond to isotype-matched (negative) controls

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Stem Cells International

experimentation as well as for clinical application Withrespect toMSCs-based gene therapy for exampleWJSC havebeen found to incorporate and express transgenes with highefficiency and increased yield allowing for a better clinicaloutcome than their adult counterparts [34 45]

In terms of phenotypic traits both populations exhibitedstable expression of typical MSC surface markers over timeNevertheless they also showed a gradual loss of self-renewaldifferentiation capacity and acquisition of signs of senescence(gradual change of morphology to type III increase in sizewith higher occurrence of giant and 120573 gal positive cells)Thissuggests that the expression pes se of typical CDmarkers usedforMSC characterization is not conclusive for defining ldquotruerdquostemness and should be used in tandem with other assaysThis has also been highlighted in the case of adult BM-MSCs[46] For ADSC the main phenotypic changes related toincrease in size partial change in morphology and a declinein clonogenicity For WJSC the changes were more dramaticin the late culture points which nevertheless correspond tomany more rounds of division in roughly the same cultureperiod compared to ADSCThe characteristics of phenotypicdrift (changes in morphology cytoplasmic complexity andresistance to trypsinization) are probably associated withunderlying changes in cellular metabolism (eg depletionof ATP levels) expression of cell adhesion molecules andrearrangement of the cytoskeleton [10 47] Clearly any in-depth discussion regarding mechanisms based on our datais largely speculative and beyond the scope of this paperMore work is required in that direction and for examplewhole-genome transcriptomics to detect global changes ingene expression followed by verification of selected markers(associated with cell cycle stemness and lineage-specificdifferentiation) by real-time PCR will aid in the dissectionof the molecular mechanisms mediating phenotypic driftAdditionally evaluation of telomere shortening expressionof hTERT and karyotypic analysis for mutationsmight revealcritical points in culture with respect to cellular ageingCombined these analyses may even provide important infor-mation on whether the drift is reversible for example bymodification of the culture conditions

In conclusion WJSC especially in the first six-sevenpassages not only exhibit obvious quantitative advantages butalso seem to possess superior qualitative traits (clonal abilityetc) compared to ADSC Owing to their high subcultivationefficiency and ease of manipulation in culture WJSC featureas ideal candidates for use in cytotherapy protocols Beforetheir ultimate use in the clinic though further optimizationof ex vivo expansion is required for example by maintainingcultures in chemically defined media free of animal productsand by employing the use of clinical-grade bioreactors orsuitable three-dimensional scaffolds [48 49] Furthermorebetter validation of their therapeutic potential is neededby carefully designed in vivo studies using suitable animalmodels With respect to this our group is currently workingtowards establishing protocols that minimize heterogeneityin MSC-based cancer cytotherapy studies using mouse mod-els On the contrary the clinical applicability of ADSC seemsquestionable especially in those cases where considerablescaleup of the ADSC-based tissue-engineered product would

be required this has also been highlighted in a recent review[50] Nevertheless it is possible that there is a potential forboosting their proliferation capacity and prolonging theirstem properties by modifying standard culture conditionsfor example by using a hypoxic environment or alternativemedia supplements (sera etc) [51ndash53] In any case estab-lishing a standardized culture environment and dissectingthe molecular mechanisms associated with retention of MSCphenotypic identity are pivotal not only for harnessing thefull potential of these cells in vitro but also for a betterunderstanding of key differences in their biology

Acknowledgments

The authors would like to thank Dr A Pintzas (NHRF)for critical review of the paper and Mrs N Konstantinidou(TAK-EIE) and Mr S Spyridakis (Medicon) for technicalassistance with cell isolation and flow cytometric analysisrespectivelyThis work was funded by a special research grantby TAK-EIE Stem Cell Bank an NHRF spinoff The authorsdeclare no conflict of interests

References

[1] P M Chen M L Yen K J Liu H K Sytwu and B LYen ldquoImmunomodulatory properties of human adult and fetalmultipotent mesenchymal stem cellsrdquo Journal of BiomedicalScience vol 18 no 1 article 49 2011

[2] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007

[3] S A Patel L Sherman J Munoz and P Rameshwar ldquoImmuno-logical properties of mesenchymal stem cells and clinical impli-cationsrdquo Archivum Immunologiae et Therapiae Experimentalis(Warsz) vol 56 no 1 pp 1ndash8 2008

[4] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[5] C Cavallo C Cuomo S Fantini et al ldquoComparison ofalternative mesenchymal stem cell sources for cell bankingand musculoskeletal advanced therapiesrdquo Journal of CellularBiochemistry vol 112 no 5 pp 1418ndash1430 2011

[6] S Kern H Eichler J Stoeve H Kluter and K Bieback ldquoCom-parative analysis of mesenchymal stem cells from bonemarrowumbilical cord blood or adipose tissuerdquo Stem Cells vol 24 no5 pp 1294ndash1301 2006

[7] J J Montesinos E Flores-Figueroa S Castillo-Medina etal ldquoHuman mesenchymal stromal cells from adult andneonatal sources comparative analysis of their morphologyimmunophenotype differentiation patterns and neural proteinexpressionrdquo Cytotherapy vol 11 no 2 pp 163ndash176 2009

[8] X Zhang M Hirai S Cantero et al ldquoIsolation and character-ization of mesenchymal stem cells from human umbilical cordblood reevaluation of critical factors for successful isolationand high ability to proliferate and differentiate to chondrocytesas compared tomesenchymal stem cells from bonemarrow andadipose tissuerdquo Journal of Cellular Biochemistry vol 112 no 4pp 1206ndash1218 2011

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 11

[9] A Bongso C Y Fong and K Gauthaman ldquoTaking stem cellsto the clinic major challengesrdquo Journal of Cellular Biochemistryvol 105 no 6 pp 1352ndash1360 2008

[10] V J Cristofalo A Lorenzini R G Allen C Torres and MTresini ldquoReplicative senescence a critical reviewrdquo Mechanismsof Ageing and Development vol 125 no 10-11 pp 827ndash8482004

[11] H Rubin ldquoCell aging in vivo and in vitrordquoMechanisms of Ageingand Development vol 98 no 1 pp 1ndash35 1997

[12] G Chamberlain J Fox B Ashton and J Middleton ldquoConcisereview mesenchymal stem cells their phenotype differen-tiation capacity immunological features and potential forhomingrdquo Stem Cells vol 25 no 11 pp 2739ndash2749 2007

[13] IMartinH Baldomero C Bocelli-Tyndall J PasswegD Sarisand A Tyndall ldquoThe survey on cellular and engineered tissuetherapies in europe in 2010rdquo Tissue Engineering A vol 18 no21-22 pp 2268ndash2279 2012

[14] D L Troyer and M L Weiss ldquoConcise review Whartonrsquos jelly-derived cells are a primitive stromal cell populationrdquo Stem Cellsvol 26 no 3 pp 591ndash599 2008

[15] J M Gimble A J Katz and B A Bunnell ldquoAdipose-derivedstem cells for regenerative medicinerdquo Circulation Research vol100 no 9 pp 1249ndash1260 2007

[16] B Puissant C Barreau P Bourin et al ldquoImmunomodulatoryeffect of human adipose tissue-derived adult stem cells compar-isonwith bonemarrowmesenchymal stem cellsrdquoBritish Journalof Haematology vol 129 no 1 pp 118ndash129 2005

[17] M L Weiss C Anderson S Medicetty et al ldquoImmuneproperties of human umbilical cord Whartonrsquos jelly-derivedcellsrdquo Stem Cells vol 26 no 11 pp 2865ndash2874 2008

[18] K Seshareddy D Troyer and M L Weiss ldquoMethod to isolatemesenchymal-like cells fromWhartonrsquos jelly of umbilical cordrdquoMethods in Cell Biology vol 86 pp 101ndash119 2008

[19] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[20] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[21] L S Kaplow ldquoLeukocyte alkaline phosphatase cytochemistryapplications and methodsrdquo Annals of the New York Academy ofSciences vol 155 pp 911ndash947 1968

[22] J N Beresford S E Graves and C A Smoothy ldquoFormation ofmineralized nodules by bone derived cells in vitro a model ofbone formationrdquoAmerican Journal of Medical Genetics vol 45no 2 pp 163ndash178 1993

[23] H V Connerty and A R Briggs ldquoDetermination of serumcalcium bymeans of orthocresolphthalein complexonerdquoAmer-ican Journal of Clinical Pathology vol 45 no 3 pp 290ndash2961966

[24] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[25] R C Schugar S M Chirieleison K E Wescoe et al ldquoHighharvest yield high expansion and phenotype stability of CD146mesenchymal stromal cells fromwhole primitive humanumbil-ical cord tissuerdquo Journal of Biomedicine and Biotechnology vol2009 Article ID 789526 11 pages 2009

[26] N Tsagias I Koliakos V Karagiannis M Eleftheriadou andG G Koliakos ldquoIsolation of mesenchymal stem cells using thetotal length of umbilical cord for transplantation purposesrdquoTransfusion Medicine vol 21 no 4 pp 253ndash261 2011

[27] Y Zhu T Liu K Song X Fan X Ma and Z Cui ldquoAdipose-derived stem cell a better stem cell than BMSCrdquo Cell Biochem-istry and Function vol 26 no 6 pp 664ndash675 2008

[28] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCell culture medium composition andtranslational adult bone marrow-derived stem cell researchrdquoStem Cells vol 24 no 5 pp 1409ndash1410 2006

[29] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005

[30] S P Bruder N Jaiswal and S E Haynesworth ldquoGrowth kinet-ics self-renewal and the osteogenic potential of purified humanmesenchymal stem cells during extensive subcultivation andfollowing cryopreservationrdquo Journal of Cellular Biochemistryvol 64 no 2 pp 278ndash294 1997

[31] I Christodoulou L D K Buttery G Tai L L Hench andJ M Polak ldquoCharacterization of human fetal osteoblasts bymicroarray analysis following stimulation with 58S bioactivegel-glass ionic dissolution productsrdquo Journal of BiomedicalMaterials Research B vol 77 no 2 pp 431ndash446 2006

[32] A A Ramkisoensing D A Pijnappels S F A Askar etal ldquoHuman embryonic and fetal mesenchymal stem cellsdifferentiate toward three different cardiac lineages in contrastto their adult counterpartsrdquo PLoS ONE vol 6 no 9 Article IDe24164 2011

[33] Z Y Zhang S H Teoh M S K Chong et al ldquoSuperiorosteogenic capacity for bone tissue engineering of fetal com-pared with perinatal and adult mesenchymal stem cellsrdquo StemCells vol 27 no 1 pp 126ndash137 2009

[34] K OrsquoDonoghue and N M Fisk ldquoFetal stem cellsrdquo Best Practiceand Research Clinical Obstetrics and Gynaecology vol 18 no 6pp 853ndash875 2004

[35] M F Manca I Zwart J Beo et al ldquoCharacterization ofmesenchymal stromal cells derived from full-term umbilicalcord bloodrdquo Cytotherapy vol 10 no 1 pp 54ndash68 2008

[36] I Hartmann T Hollweck S Haffner et al ldquoUmbilical cordtissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypicand functional propertiesrdquo Journal of Immunological Methodsvol 363 no 1 pp 80ndash89 2010

[37] T Hatlapatka P Moretti A Lavrentieva et al ldquoOptimization ofculture conditions for the expansion of umbilical cord-derivedmesenchymal stem or stromal cell-like cells using xeno-freeculture conditionsrdquo Tissue Engineering C vol 17 no 4 pp 485ndash493 2011

[38] DCDingWC Shyu S Z LinHW Liu SHChiou andT YChu ldquoHuman umbilical cord mesenchymal stem cells supportnon-tumorigenic expansion of human embryonic stem cellsrdquoCell Transplantation vol 21 no 7 pp 1515ndash1527 2012

[39] C Y Fong K Gauthaman S Cheyyatraivendran H D Lin ABiswas and A Bongso ldquoHuman umbilical cord Whartonrsquos jellystem cells and its conditioned medium support hematopoieticstem cell expansion ex vivordquo Journal of Cellular Biochemistryvol 113 no 2 pp 658ndash668 2012

[40] L L Lu Y J Liu S G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cells

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 Stem Cells International

with hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[41] A S Magin N R Korfer H Partenheimer C Lange AZander and T Noll ldquoPrimary cells as feeder cells for cocultureexpansion of human hematopoietic stem cells from umbilicalcord bloodmdasha comparative studyrdquo Stem Cells and Developmentvol 18 no 1 pp 173ndash186 2009

[42] B G Jeon B M Kumar E J Kang et al ldquoCharacterization andcomparison of telomere length telomerase and reverse tran-scriptase activity and gene expression in human mesenchymalstem cells and cancer cells of various originsrdquo Cell and TissueResearch vol 345 no 1 pp 149ndash161 2011

[43] Y B Park Y Y Kim S K Oh et al ldquoAlterations of prolifer-ative and differentiation potentials of human embryonic stemcells during long-term culturerdquo Experimental and MolecularMedicine vol 40 no 1 pp 98ndash108 2008

[44] C H Jo O S Kim E Y Park et al ldquoFetal mesenchymalstem cells derived from human umbilical cord sustain primitivecharacteristics during extensive expansionrdquo Cell and TissueResearch vol 334 no 3 pp 423ndash433 2008

[45] J Chan K OrsquoDonoghue J de la Fuente et al ldquoHuman fetalmesenchymal stem cells as vehicles for gene deliveryrdquo StemCells vol 23 no 1 pp 93ndash102 2005

[46] S Rallapalli D K Bishi R S Verma K M Cherian andS Guhathakurta ldquoA multiplex PCR technique to characterizehuman bonemarrow derivedmesenchymal stem cellsrdquoBiotech-nology Letters vol 31 no 12 pp 1843ndash1850 2009

[47] S Cho J Park and E S Hwang ldquoKinetics of the cell biologicalchanges occurring in the progression of DNA damage-inducedsenescencerdquoMolecules andCells vol 31 no 6 pp 539ndash546 2011

[48] T C Cardoso H F Ferrari A F Garcia et al ldquoIsolationand characterization of Whartonrsquos jelly-derived multipotentmesenchymal stromal cells obtained from bovine umbilicalcord andmaintained in a defined serum-free three-dimensionalsystemrdquo BMC Biotechnology vol 12 article 18 2012

[49] J E Dennis K Esterly A Awadallah C R Parrish G MPoynter and K L Goltry ldquoClinical-scale expansion of a mixedpopulation of bone marrow-derived stem and progenitor cellsfor potential use in bone tissue regenerationrdquo StemCells vol 25no 10 pp 2575ndash2582 2007

[50] M Locke V Feisst and P R Dunbar ldquoConcise review humanadipose-derived stem cells separating promise from clinicalneedrdquo Stem Cells vol 29 no 3 pp 404ndash411 2011

[51] V Tunaitis V Borutinskaite R Navakauskiene et al ldquoEffects ofdifferent sera on adipose tissue-derived mesenchymal stromalcellsrdquo Journal of Tissue Engineering and Regenerative Medicinevol 5 no 9 pp 733ndash746 2011

[52] S Yang L Pilgaard L G Chase et al ldquoDefined xenogeneic-free and hypoxic environment provides superior conditionsfor long-term expansion of human adipose-derived stem cellsrdquoTissue Engineering C vol 18 no 8 pp 593ndash602 2012

[53] M G Valorani EMontelatici A Germani et al ldquoPre-culturinghuman adipose tissue mesenchymal stem cells under hypoxiaincreases their adipogenic and osteogenic differentiation poten-tialsrdquo Cell Proliferation vol 45 no 3 pp 225ndash238 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology