comparison of immunological properties of bone marrow stromal cells and adipose tissue–derived...

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Comparison of Immunological Properties of Bone Marrow

Stromal Cells and Adipose TissueDerived Stem Cells Beforeand After Osteogenic Differentiation In Vitro

PHILIPP NIEMEYER,1 MARTIN KORNACKER,2 ALEXANDER MEHLHORN,1

ANJA SECKINGER,2 JANA VOHRER,1 HAGEN SCHMAL,1 PHILIP KASTEN,3

VOLKER ECKSTEIN,2 NORBERT P. SU DKAMP,1 and ULF KRAUSE2

ABSTRACT

Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell

population for tissue-engineering purposes. MSCsfrom bonemarrow (bone marrow stromal cells[BMSCs])

are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells

in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immuno-

logical characteristics of BMSCs have been the subject of various investigations, those of stem cells

isolated from adipose tissue (ASCs) have not been adequately described. In addition, the influence of

osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject

of this article.

Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of

resting and activated allogenic peripheral blood mononuclear cells (PBMCs) was studied as a measure of

the immune response (mixed lymphocyte culture). At the same points, the expression of immunologically

relevant surface markers (e.g., major histocompatibility complex (MHC)-I, MHC-II, CD40, CD40L) was

measured, and correlations between the different sets of results were sought.

The pattern of surface antigen expression of BMSCs is the same as that of ASCs. Analogous to BMSCs,

undifferentiated cells isolated from adipose tissue lack expression of MHC-II; this is not lost in the course of

the osteogenic differentiation process. In co-culture with allogenic PBMCs, both cell types fail to lead to any

significant stimulation, and they both retain these characteristics during the differentiation process. BMSCs

and ASCs suppress proliferation on activated PBMCs before and after osteogenic differentiation.

Our results confirm that MSCs are immune modulating cells. These properties are retained even after

osteogenic induction in vitro and seem to be similar in BMSCs and ASCs. Our results suggest that allogenic

transplantation of BMSCs and ASCs would be possible, for example, in the context of tissue engineering.

INTRODUCTION

OWINGTO THEIRPLASTICITY and high proliferation capacityin vitro, human mesenchymal stem cells (MSCs)derived from bone marrow (bone marrow stromal cells

(BMSCs)) are promising candidate cells for tissue-engi-

neering approaches to such mesenchymal tissues as bone,

cartilage, and tendon.1 4 These cells can be isolated from

various tissues. Isolation from bone marrow aspirates,2,5

adipose tissue,6,7 and umbilical cord blood8,9 are now

1Department of Orthopaedic Surgery and Traumatology, University of Freiburg, Germany.

2Department of Internal Medicine V, University of Heidelberg, Germany.

3Department of Orthopaedic Surgery, University of Heidelberg, Germany.

TISSUE ENGINEERINGVolume 13, Number 1, 2007# Mary Ann Liebert, Inc.DOI: 10.1089/ten.2006.0114

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accepted as the most usual and most reliable methods, al-

though isolation from umbilical cord blood is a problem in

terms of clinical use insofar as such blood is not available for

regenerative therapy approaches, or only in exceptional

cases; it is therefore not considered further in this article.

Although the phenotypical and molecular characteristics

of MSCs from these source tissues were compared recently

and the properties of MSCs from bone marrow aspirates,

adipose tissue, and umbilical cord blood were found to be

similar,10,11 the particular immunological characteristics of

human BMSCs, which have been subjected to various in-

vestigations in past years, and of MSCs isolated from adipose

tissue (ASCs) have been compared only in a superficial

manner and only in cells in an undifferentiated condition.12

Against the backdrop of a possible application in terms of

tissue engineering, however, it seems that mesenchymal dif-

ferentiation of the cells in one direction or the other (e.g.,

osteogenic differentiation in the case of bone regeneration) in

vitro even before transplantation might be needed. BMSCs

retain their properties as immuno-privileged cells during this

differentiation process, but the immunological characteristics

of human ASCs exposed to osteogenic differentiation have

not been investigated. The purpose of the present work was

to compare the immune characteristics of human ASCs and

BMSCs, after accounting for the influence of osteogenic dif-

ferentiation in vitro, with the aim of deducing whether

the cells are suitable for human leukocyte antigen (HLA)-

incompatible tissue engineering in the case of bone tissue.

The nomenclature to be used for adult progenitor cells

with mesenchymal differentiation potential remains the ob-

ject of current discussion, and there is no definitive and

uniform terminology. In this article, the term BMSCs will be

used for MSCs isolated from bone marrow and the ASCs forthose isolated from adipose tissue, so as to keep the basic

sources from which they are isolated, which are the subject

of this article, to the fore.

In past years, various papers have described the phenotypic

and immunological characteristics of MSCs isolated from

bone marrow. Some of the expression patterns of cell surface

antigens that BMSCs are positive for are CD13, CD29, CD44,

CD73 (SH3 4), CD90, CD105 (SH2), CD166, and major

histocompatibility complex (MHC) class I. They are negative

for hematopoietic markers such as CD34, CD38, and CD45

and for antigens involved in immunological signal transduc-

tion, such as HLA-DR, DP, DQ (MHC class II), CD80,

CD86, CD40, and CD40L (CD154).5,1315

Although HLA-A,B, C/MHC-I and HLA-DR, DP, DQ/MHC-II are surface

antigens that are important for peptide presentation on the cell

surface,16 CD80 and CD86 facilitate recognition of the cells

byforeign T-cells.CD40,a memberof thetransforminggrowth

factor superfamily, completes the activation of the immune

system by facilitation of a B-cell response and macrophage

activation, whereas CD40L stimulates B-cells, leading to

secretion of important interleukins (IL-4, IL-5, IL-6) and

cytokines that precipitate an immune response.17 When co-

cultured with HLA-incompatible allogenic lymphocytes in

vitro, undifferentiated BMSCs do not lead to a significant

stimulation. BMSCs have even been reported to have a sup-

pressant effect on maximally stimulated allogenic lympho-

cytes.1820 In an animal model, intravenous administration of

HLA-incompatible BMSCs does not lead to an immunolog-

ical rejection reaction; in human subjects, co-transplantation

of mesenchymal with hematopoietic stem cells leads to a

lower incidence of graft-versus-host disease (GvHD) and thus

of transplant-associated complications.14 It is against this

backdrop that BMSCs are described as immunologically priv-

ileged or even immunomodulating.

These properties suggest that BMSCs could be used

to perform HLA-mismatched transplantation for tissue-

engineering purposes. This would be extremely beneficial,

and not only for economic reasons; it would also lead to

instant availability of these cells for tissue-engineering

purposes.

Although the immunological characteristics of BMSCs

thus seem to have been adequately and reliably determined,

there have been few attempts to investigate the immuno-

logical properties of ASCs even now. It has been suggested

that undifferentiated ASCs have properties similar to those of

BSMCs;12 the influence of osteogenic induction in vitro on

the immunological properties of ASCs has not so far

been examined; there has not yet been a direct comparison of

the immunomodulating or immunosuppressant potency of

mesenchymal progenitor cells from bone marrow and from

adipose tissue before and after osteogenic differentiation in

vitro, and this was our objective in conducting this study.

MATERIALS AND METHODS

Isolation and expansion of BMSCs

Bone marrow aspirates (1030 mL) were obtained from

five hematologically healthy donors during routine orthope-

dic surgery from the iliac crest; all had given informed con-

sent. The local ethics committee approved the donor program.

BMSCs were isolated as described elsewhere21 with minor

variations. Briefly, bone marrow mononuclear cells were

obtained using Biocoll density gradient centrifugation (d

1,077 g/cm3; Biochrom, Berlin, Germany) and plated in

fibronectin-coated tissue culture flasks (Nunc, Rochester,

NY). The expansion medium used was 58% low-glucose

Dulbeccos modified Eagle medium (DMEM; Cambrex,

East Rutherford, NJ), 40% MCDB201 (Sigma, Taufkirchen,Germany), 2% fetal calf serum (FCS; Stemcell Technolo-

gies, Inc., Vancouver, Canada), supplemented with 2 mM

L-glutamine, 100 U/mL penicillin/streptomycin, ITS media

supply, linoleic acid, 10 nM dexamethasone, 0.1mM L-

ascorbic-acid-2-phosphate (all from Sigma), platelet-derived

growth factorbb and epidermal growth factor (10 ng/mL

each; R&D Systems, Minneapolis, MN). To check their MSC

character, cells were successfully differentiated into bone,

cartilage, and fat following standard protocols;5 they were

found to express MSC-typical cell surface markers.2,11

112 NIEMEYER ET AL.


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Isolation of human ASC

ASCs were isolated from subcutaneous adipose tissue ta-

ken from five healthy donors undergoing abdominoplastic

surgery as previously described.22 The local ethical commit-

tee approved the donor program. Adipose tissue was finely

minced (12 mm3), washed with phosphate-buffered saline

(PBS), and digested with 2 mg/mL collagenase type I (Bio-chrom) for 90 min at 378C with continuous shaking. The

floating adipocytes were separated from the stromal cell frac-

tion using multiple centrifugation and washing steps. The

stromal cells were plated in a 175-cm2 tissue culture flask at

4,000 cells/cm2 filled with 25 mL DMEM/F12, 10% fetal calf

serum, 10 ng/mL basic fibroblast growth factor,23 100 U/mL

penicillin, and 100mg/mL streptomycin. The medium was

changed every third day, which washed out all nonadherent

cells. Once adherent cells had grown to confluence, they

were detached with trypsin-ethylenediaminetetraacetic acid

(EDTA) (Sigma), re-plated at a density of approximately

2,000 cells/cm2 and cultured for two further passages.

For all experiments, cells from passages 3 and 4 were usedfor the investigations.

Osteogenic differentiation in vitro

For osteogenic differentiation, similar conditions were used

for BMSCs and ASCs. A different medium was used: osteo-

genic medium consisting of low-glucose DMEM with 10%

FCS supplemented with 2 mM L-glutamine, and 100 U/mL

penicillin/1,000 U/mL streptomycin (all from Invitrogen) and

100 nM dexamethasone, 0.2mM L-ascorbic acid-2-phosphate,

and 10 mM b-glycerophosphate (all from Sigma) for a period

of up to 21 days; the medium was changed twice a week.

Mesenchymal differentiation potential has been demon-strated for BMSCs and ASCs. Cells from passage 3 to 6 were

stained for collagen type II (after chondrogenic differentia-

tion; antibody: ICN, Aurora, OH) as well as with von Kossa

(after osteogenic differentiation; Sigma, Taufkirchen, Ger-

many) and with oil red staining (after adipogenic differen-

tiation; Sigma, Taufkirchen). All stainings were performed

according to the manufacturers protocols.

Total ribonucleic acid extraction and

complementary deoxyribonucleic acid synthesis

Ribonucleic acid (RNA) from cultivated cells was ex-

tracted using the RNeasy Mini Kit (Qiagen, Hilden, Ger-many). Three hundredmL of lysis buffer (Buffer RLT,

RNeasy Mini Kit, Qiagen) was directly applied to the cells

after trypsination. Further RNA extraction was performed

according to the standard protocol. The efficiency of RNA

extraction was verified using mechanical homogenization of

the matrices and re-extraction of RNA. Residual RNA was

not detected in any of the samples analyzed. RNA was quan-

tified spectrometrically. One mg of total RNA was used for

first-strand complementary deoxyribonucleic acid (cDNA)

synthesis using 0.5 mM deoxyribonucleotide triphosphate,

1mM oligo-dT primer, 10 U RNase-inhibitor, and 4 U Om-

niscript Reverse Transcriptase (Qiagen) in a final volume

of 20mL.

Real-time quantitative PCR

cDNA (2mL) was used for PCR analysis. Real-time quan-

titative PCR (qPCR) was performed in a LightCycler in-strument (Roche Diagnostics, Grenzach-Wylen, Germany)

in a total volume of 20mL using the LightCycler FastStart

DNA Master SYBR Green I kit (Roche Diagnostics). Sam-

ples were heated to 958C for 10 min followed by 40 cycles of

denaturation at 958C for 0 s, annealing at 588C for 7 s, and

extension at 728C for 14 s. After the last cycle, a melting-

curve analysis was performed to verify the specificity of the

amplified PCR products. The following primers were used:

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH):

forward: 50-CCA CCC ATG GCA AAT TCC ATG

GCA-30

reverse: 50-ATG TTC GTC ATG GGT GTG AA-30

Alkaline phosphatase:

forward: 50-CAC GGG CAC CAT GAA GGA AAA-30

reverse: 50-TGG CGC AGG GGC ACA GGA GAC-30

Osteocalcin:

forward: 50-GGC AGC GAG GTA GTG AAG AGA C-3 0

reverse: 50-GGC AAG GGG AAG AGG AAA GAA G-30

The amount of PCR product was calculated using an external

GAPDH standard curve and LightCycler software. All val-

ues were normalized based on the GAPDH expression in the

corresponding samples.

Immunologic characterization of BMSCs

and ASCs

For BMSCs and ASCs, all experiments, including co-

culture with allogenic PBMCs and flow cytometry, were

performed before and after osteogenic differentiation for 14

days. To ensure that the duration of in vitro culture did not

influence theoutcomeparameters, undifferentiated cellswere

kept in expansion medium for the same period of time that

osteogenic medium was applied to the differentiated cells

(14 days). Therefore, time of in vitro culture was similar for

undifferentiated and differentiated cells. In the following, all

results are given for undifferentiated and differentiated

BMSCs and ASCs. Day 14 was chosen for immunological

experiments, because effective osteogenic differentiation

could already be observed at this stage of differentiation, but

extracellular matrix synthesis, which could influence flow

cytometry, had not occurred to full extent at this point of the

differentiation process.

Incubation of stem cells with allogenic PBMCs

After 14 days, osteogenic-induced cells and undifferen-

tiated controls were resuspended, 5104 BMSCs or ASCs

PROPERTIES OF BONE MARROW STROMAL CELLS AND ADIPOSE-DERIVED STEM CELLS 113


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were plated in wells of a 96-well plate (Nunc) in RPMI 1640

medium (50mL) supplemented with 2 mM L-glutamine,

100 U/mL penicillin/streptomycin, and 10% human AB se-

rum. PBMCs were used as responder cells and co-cultivated

at a 10:1 ratio with BMSCs/ASCs for 7 days. The ratio of

stem cells and PBMC was chosen in accordance with earlier

published studies.19 Pooled monocyte-derived allogeneic

dendritic cells (DC-14) from peripheral blood of five HLA-

A2 unrelated donors were obtained by culturing plastic

adherent PBMCs for 5 days with GM-CSF (800 U/mL,

Molgramostim, Essex Pharma, Munchen, Germany) and IL-

4 (500 U/mL, R&D Systems) and then induced into mature

DCs for 2 days with tumor necrosis factor alpha (10 ng/mL,

Sigma-Aldrich, Deisenhofen, Germany), IL-6 (1000 U/mL,

R&D Systems,Abingdon,Oxon, UnitedKingdom), and pros-

taglandin E2 (1 mg/mL, Sigma-Aldrich, Deisenhofen, Ger-

many).24,25 DCs were used as stimulator cells at a ratio of 1

DC:10 PBMCs when applicable. In a second set of experi-

ments, PBMCs and irradiated allogeneic DCs were incu-

bated as described above and irradiated ASCs or BMSCs at a

ratio of 1 stem cell:10 PBMCs were used as third-party cells

in the reaction.

Adherent MSCs and DC-14 were irradiated with 33Gy.

3H-Thymidine was added to the cultures 24 h before harvest

(2.5 LCi/well; Amersham, Buckinghamshire, UK). Thymi-

dine incorporation was measured using a b-scintillation

counter (Perkin Elmer, Wellesley, MA) and expressed as

counts per minute (cpm).

For quantitative analysis, proliferation rates (cpm) were

expressed as percentages of the maximum stimulation level

induced by OKT-3, IL-2, and allogeneic DCs (Fig. 1) as

percentages of the proliferation rate induced by allogenic

DCs alone in the case of the investigation of the influence onstimulated lymphocytes (ongoing mixed lymphocyte cul-

ture, Fig. 2). To determine the maximum level of activation,

OKT-3 (50 ng/mL; OrthoBioTech, Neuss, Germany), IL-2

(300 U/mL, OrthoBioTech), and DCs were added as de-

scribed previously.26 All experiments were done in triplicate.

In parallel, expression of cell surface markers was monitored

using flowcytometryat correspondingtime points (seebelow).

Flow cytometry

Differentiated and undifferentiated cells were screened

using an antibody panel against cell surface antigens (CD13,

CD14, CD29, CD31, CD34, CD38, CD44, CD45, CD56,CD73, CD90, CD105, CD106, and CD166) and co-stimula-

tory molecules (MHC-I [HLA-A, -B, -C], MHC-II [HLA-

DR, DP, DQ], CD80 [B71], CD86 [B72] CD40, CD40L

[CD154]) (all from Becton Dickinson, Franklin Lakes, NJ).

Then batches of 80,000 to 200,000 cells in 100 mL PBS with

1% FCS/2mM EDTA were each incubated for 30 min at 48C

in the dark with 1 to 5 mL of directly labeled antibody solu-

tion or matching isotype controls. After two washing steps

with PBS/1% FCS/2mM EDTA, cells were resuspended in

250mL buffer and analyzed with a fluorescence-activated cell

FIG. 1. Stimulation of allogenic lymphocytes. Lymphocytes

were stimulated maximally with allogenic dendritic cells (DCs),

OKT-3, and interleukin-2, and the resulting thymidine uptake was

set to 100%. Compared with this maximal stimulation, allogenic

DCs alone also stimulated responder cells on day 0 (black col-umn) and on day 14 (grey column) of culture, albeit at a lesser

degree. Alternatively, adipose tissuederived stem cells (ASCs)

and bone marrow stromal cells (BMSCs) showed only marginal

stimulation of responder cells in the undifferentiated state (day 0,

black column) and after in vitro osteogenic induction (day 14,

grey column). * indicates statistically significant changes between

different groups (p< 0.05).

FIG. 2. Inhibition of mixed lymphocyte reactions by undifferen-tiated and differentiated adipose tissuederived stem cells (ASCs)

and bone marrow stromal cells (BMSCs). Lymphocytes were stim-

ulated with allogenic dendritic cells, and the resulting thymidine

uptake was set to 100%. When undifferentiated (black columns) and

by differentiated (grey columns) ASCs were added as a third-party

cell population, the proliferation rate was significantly reduced.

Analogous results wereobtained for BMSCs (undifferentiated: black

columns; differentiated: grey columns). No significant differences

were found between ASCs and BMSCs. * indicates statistically

significant (p< 0.05) reduction of cell proliferation compared with

allogenous lymphocyte stimulation (100%).

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sorting(FACS)Vantage SE using CellQuest Software (Becton

Dickinson Immunocytometry Systems, Franklin Lakes, NJ).

Viable cells were determined using propidium iodine (PI)

staining. At least 10,000 PI-negative cells were acquired

(counted). PBMCs were used as positive controls for hema-

topoietic or MSC markers.

Immunofluorescence

To confirm flow-cytometry data, differentiated and undif-

ferentiated cells were plated in fibronectin-coated Lab-Tek II

chamber slides (Nunc). The next day, cells were fixed with

2% paraformaldehyde (Sigma) for 30 min, stained as above

and analyzed with an Olympus IX-70 microscope using

Analysis software AnalySIS (V3.2, Soft Imaging System

GmbH, Munster, Germany). Nuclei were counterstained with

Hoechst 33342.

Statistical analysis

Significant differences were identified using a 2-factorialunivariate analysis of variance (ANOVA) using SPSS soft-

ware (SPSS Inc., Chicago, IL). The Friedman test was per-

formed on all significant differences recognized, because all

changes over the study period of 24 days were followed and

analyzed for each series. Student t-tests were performed to

compare differences between the groups on days 0, 8, 16,

and 24. P< 0.05 (*) was considered significant, andp< 0.01

(**)was consideredstronglysignificant.Statisticswere based

on N 5 for all investigations.

RESULTS

Osteogenic, chondrogenic, and adipogenic differentiation

were successfully achieved in BMSCs and ASCs, confirming

their mesenchymal differentiation potential, and cells from

both sources had the surface antigen expression pattern typ-

ical of MSCs. After osteogenic induction, a definite miner-

alization (von Kossa reaction) was observed, whereas after

adipogenic induction, vacuoles were found to be positive us-

ing oil red staining and after chondrogenic differentiation

extracellular precipitation of collagen type II was detected in

the pellet culture. The ability to differentiate into osteogenic,

chondrogenic,andadipogeniclineageswasconfirmedinmono-

layer culture or in pellet culture at passages 3 and 6 after 2

weeks of differentiation; for osteogenic differentiation, oste-

ogenic marker genes were followed up to 4 weeks.

Similar results were obtained for ASCs and BMSCs.

Differentiation potential of ASCs is shown in Fig. 3.

Efficiency of osteogenic differentiation in vitro

To confirm the efficiency of osteogenic differentiation in

vitro, expression of alkaline phosphatase, and osteocalcin was

determined using real-time qPCR on day 0, 7, 14, and 21 of

FIG. 3. Fourteen days after osteogenic differentiation (B, von Kossa staining), chondrogenic differentiation (C, collagen type II

immunohistology), and adipogenic differentiation (D, oil red staining), adipose tissue derived stem cells (ASCs) (A, phenotype of

undifferentiated ASC) show tissue-specific differentiation in vitro. Color images available online at www.liebertpub.com /ten.



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the differentiation process (Light Cycler, Roche Diagnostics).

Normalized to GAPDH expression, expression of alkaline

phosphatasebecamesignificantlygreater(p< 0.01)inBMSCs

and ASCs over the period of osteogenic differentiation in vitro

than in controls kept in expansion medium over the same

period (results for control cells on day 14 are given in Fig. 4).

Analogous results for the expression of osteocalcin were ob-

tained in MSCs and ASCs (p< 0.01). Although a more pro-

nounced increase in the expression of alkaline phosphatase

and osteocalcin was found in ASCs than in BMSCs, this

difference was not statistically significant and can only be

regarded as a trend (p 0.30). Details are given in Fig. 4.

Parallel to molecular investigation on gene level, von Kossa

staining at 14 days after in vitro differentiation revealed sig-

nificant extracellular matrix synthesis (data not shown).

Expression of immunologically relevantsurface antigens

In addition to a MSC-associated cell surface marker

profile (including CD44, CD73, CD105), the occurrence of

immunologically relevant surface antigens was tested. FACS

analysis revealed a cluster of differentiation pattern typical

for MSCs: CD34; CD38; CD44;CD45; anti-HLA-

DR, DP, DQ; anti-HLA-ABC; CD80; CD66;

CD13; CD29; CD73; CD90; CD105; CD106;

and CD166.

Undifferentiated and differentiated ASCs and BMSCs are

HLA-ABC-positive but negative for the B- and T-cell co-

stimulatory molecules CD40, CD40L, CD80, CD86, andHLA-DR, DP, DQ. Osteogenic differentiation in vitro does

not have a significant influence on the expression of these

immunologically relevant surface antigens (Fig. 5). This

expression pattern was confirmed usingimmunofluorescence

microscopy (representative image is shown in Fig. 6).

Incubation of stem cells with allogenic PBMCs

Stem cells were incubated with allogenic PBMCs to de-

termine the immunogenicity of differentiated and undiffer-

entiated stem cells by measuring 3H thymidine uptake, similar

to a mixed lymphocyte reaction. The stimulation rates of al-

logenic DCs, undifferentiated BMSCs, osteogenically differ-entiated BMSCs, undifferentiated ASCs, and osteogenically

differentiated ASCs were compared with the maximal stim-

ulation rate that could be induced by the addition of OKT-3,

IL-2, and allogenic DCs. This value was set to 100% and

considered to be the maximal stimulation rate of the lym-

phocyte pool; all test values were compared with it. According

to this,there was 57.3% (18.3) stimulation of responder cells

by allogenic dendritic cells on day 0 and 50.9% (14.3) on

day 14. The stimulation rates achieved with BMSCs were

0.4% (0.2) in undifferentiated cells on day 0 and 6.8%

(4.2) after osteogenic induction on day 14. For ASCs, the

stimulation rates compared with the maximal stimulation rate

were 1.9% (0.8) on day 0 and 1.5% (0.5) on day 14.ANOVA with subsequent post hoc analysis for statistically

significant differences between individual groups showed sig-

nificantly lower stimulation rates in undifferentiated and dif-

ferentiated BMSCs (p(BMSC day 0 vs. allogenic DC) 0.002;

p(BMSC day 14 vs. allogenic DC) 0.02) and also in undifferentiated

and differentiated ASCs (p(ASC day 0 vs. allogenic DC) 0.002;

p(ASC day 14 vs. allogenic DC) 0.002) compared with allogenous

DCs. Neither on day 0 nor on day 14 were there significant

differences between the stimulation rates induced by ASCs

and those induced by BMSCs. The results are shown in Fig. 1.

FIG. 4. Expression of alkaline phosphatase (A) and osteocalcin

(B) normalized to the expression of glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) of adipose tissuederived stem cells

(ASCs, black columns) and mesenchymal stem cells (MSCs, grey

columns) on days 0, 7, 14, and 21 of osteogenic differentiationusing quantitative reverse transcriptase polymerase chain reaction.

As control, in parallel with osteogenic differentiation, some cells

were kept in expansion medium in vitro for 14 days and then an-

alyzed for expression of osteogenic markers. No significant in-

crease was detected in controls, whereas with alkaline phosphatase

and osteocalcin, a significant increase of ASCs and bone marrow

stromal cells (BMSCs) was detectable in specimens that underwent

osteogenic differentiation in vitro. * indicates significant differ-

ences in expression level compared with undifferentiated cells cul-

tured in expansion medium for 14 days (p< 0.05). No statistically

significant differences were found between ASCs and BMSCs.

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In a second step of the current experiment, BMSCs and

ASCs were added as third parties to responder cells (PBMCs)

co-incubated with allogenic DCs (ratio: 1 DC:10 PBMCs) toinvestigate possibleimmunosuppressive properties of BMSCs

and ASCs. In these tests, the stimulation rate without third-

party BMSCs/ASCs was set at 100%, and the proliferation

rates after addition of the cell population under investigation

were compared with these values. The addition of undiffer-

entiated ASCs and osteogenic differentiated ASCs (ratio

ASCs:PBMCs 1:10) caused the proliferation rate of the

responder cells to fall: to 60.6% (14.8) for undifferentiated

ASCs and to 46.7% (7.1) for osteogenically induced ASCs.

Similar results were found when BMSCs were added (undif-

ferentiated BMSCs: 55.6% (12.0); differentiated BMSCs:

61.9% (7.9)). Thus, a significant reduction in the prolifer-

ation rate, with a consequent reduction of allogenic stimula-tion (p< 0.05), was found with all cell types investigated. For

more detail, the reader is referred to Fig. 2.

DISCUSSION

In addition to the isolation of human BMSCs, which is

now regarded as an established procedure, isolation of ASCs

offers an attractive alternative for clinical application.6 Ad-

ipose tissue is easily accessible; it can be harvested using

liposuction, which is not highly invasive, and the in vitro and

in vivo mesenchymal differentiation potential of ASCs is

analogous to that of BMSCs. The phenotypical and molec-ular genetic properties of ASCs are similar to those of

BMSCs,10,11 although in recent years, the latter have been

described as immunologically privileged or even immuno-

suppressive in a number of studies and could possibly be

available for allogenic transplant applications.15,19 The use

of such in an allogenic setting is an attractive prospect, not

only from the economic point of view, but also medically;

cells could be stored in a universal donor bank, and a stem

cell therapy for regeneration of tissue (e.g. in bone) would

then be available when indicated in the context of acute care,

because individualized long-term cell expansion of autolo-

gous cells (which has already become an established part of

clinical routine for autologous chondrocyte transplantation)would be superfluous.

Although a number of the studies carried out so far to

examine the immunological properties of MSCs have been

performed on undifferentiated MSCs,12,19 the question of

whether the particular immunological features of these cells

persist after in vitro differentiation is of crucial importance

to tissue engineering, because in vitro differentiation could

be beneficial in the context of tissue regeneration. For

BMSCs, it has already been shown that effective differenti-

ation in vitro does not lead to any change in the expression

FIG. 5. Expression of immunologically relevant surface antigens. Undifferentiated and differentiated adipose tissue derived stem cells

(ASCs) and bone marrow stromal cells (BMSCs) were labeled with antibodies against cell surface antigens and analyzed using flow

cytometry. Representative histograms are shown (gray), and isotype controls are indicated (black). Mononuclear cells from peripheral

blood were used as control. Mesenchymal stem cells are to a greater or lesser extent positive for human leukocyte antigen ABC (major

histocompatibility complex I). No co-stimulatory molecules are expressed on any of the preparations.



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of immunologically relevant antigens on the cell surface

and that the proliferation-inhibiting effect on lymphocytes

in vitro is also maintained.15,27 So far, no analogous

experiments have been carried out with ASCs. The object of

the present study was therefore to compare the immuno-logical potential of BMSCs and ASCs, with special refer-

ence to their osteogenic differentiation in vitro.

Our investigation to characterize the immunological prop-

erties of BMSCs, ASCs, and the osteoprogenitors derived

from them in vitro was based on three sectors. First, ex-

pression of immunologically relevant surface markers was

investigated using FACS techniques during in vitro osteo-

genic differentiation over 14 days. Second, interaction be-

tween undifferentiated and differentiated BMSCs/ASCs and

resting HLA-incompatible allogenic PBMCs and their in-

fluence on such cells was examined. Third, the influence of

BMSCs/ASCs on activated allogenic PBMCs during in vitro

differentiation was determined with the aim of revealing anysuppressant influence of BMSCs/sASCs and BMSC-/ASC-

derived osteoprogenitors.

At this point, it has to be discussed critically whether

MSCs are fully differentiated after 14 days of osteogenic

induction in vitro. It is possible that, in the case of incom-

plete differentiation, some stem cell properties persist that

might be responsible for the immunological behavior of

MSC-derived precursors. Such effects cannot be definitively

excluded. As far as the efficiency ofin vitro differentiation is

concerned, an increase in characteristic osteogenic marker

genes (alkaline phosphatase and osteocalcin) was demon-

strated using RT-PCR, in addition to which matrix synthesis

in terms of mineralization was demonstrated using von

Kossa staining. These findings demonstrate efficient oste-

ogenic differentiation after 14 days. Furthermore, otherstudies that have investigated the influence of in vitro dif-

ferentiation on the immunological properties of BMSCs

performed experiments even earlier.15 This is unavoidable,

because after effective osteogenic and chondrogenic dif-

ferentiation for longer than 14 days, cells are embedded in

extracellular matrix, which might influence he immunolog-

ical behavior in terms of immunosuppression even more

than the cell phenotype.

For BMSCs, our investigations confirm earlier reports that

undifferentiated BMSCs were negative for the immuno-

logically relevant surface antigens HLA-DR, DP, DQ; CD40;

CD40L; CD80; and CD86.14 This surface antigen pattern

persists even in the face of effective osteogenic induction,thus confirming the data published by LeBlancs group15 in

their reports of similar experiments. In comparison with

HLA-incompatible immunocompetent cells, the stimulation

induced by HLA-incompatible BMSCs after incubation with

PBMCs is negligible. The addition of allogenic BMSCs

leads to only a slight enhancement of proliferation in the

responder cells, which the contamination of the culture

systems with cell types other than MSCs, which has been

described as typical for MSC culture systems, can explain. In

the case of lymphocytes that are already stimulated, the

FIG. 6. Flow cytometry results (fluorescence-activated cell sorting) have been confirmed using immunofluorescence microscopy.

Undifferentiated adipose tissuederived stem cells (ASCs) are negative for human leukocyte antigen DR, DP, and DQ (A, B) and positive for

CD44 (C, D; red). Nuclei are counterstained with DAPI (blue), scale bar 50mm. Color images available online at www.liebertpub.com /ten.

118 NIEMEYER ET AL.


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addition of allogenic BMSCs reduces proliferation approx-

imately 50%, emphasizing the immunomodulating property

of human BMSCs. As far as these properties are concerned,

our results are in keeping with those of other authors. 19,28

The mechanism of the immunosuppressant properties has

not been fully explained. The BMSC-induced inhibition

persisted in a setting in which BMSCs and responder lym-

phocytes were separated in different wells, from which we

can conclude that direct cell-to-cell contact is not necessary

for BMSC-induced immune suppression and that soluble

factors are responsible for immune suppression. Our results

and those of other studies thus suggest that MSCs induce a

suppressive local microenvironment through the production

of prostaglandins, IL-10, interferon gamma, and hepatocyte

growth factor and by the expression of indoleamine 2,3

dioxygenase.29,30 In this way, human BMSCs lead to direct

inhibition of T-cells31 and B-cells32 and can inhibit the

proliferation of NK cells33 and interfere with the maturation

and function of DCs.30,34 These complex mechanisms not

only suggest that applications in the context of allogenic

transplantation would be possible, but also indicate a po-

tential role for MSCs in the immunosuppressive treatment of

autoimmune diseases.

The immunological characteristics of human ASCs in the

undifferentiated state have been investigated only once.12 As

in Puissants studies, in our experiments, the immunological

characteristics of BMSCs andASCs were found to be similar.

Even ASCs that have been meticulously checked for mes-

enchymal differentiation potential are negative for the im-

portant immunologically relevant surface antigens MHC-II,

CD40, CD40L, CD80, and CD86. Nonetheless, human ASCs

remained negative for MHC-II, CD40, CD40L, CD80, and

CD86 and thus for important T- and B-cell-co-stimulatingsurface antigens, even after osteogenic induction in vitro. In

cultures with allogenic lymphocytes ASCs, like BMSCs, did

not lead to any significant enhancement of proliferation,

which supports the view that, also like BMSCs, they do not

elicit any rejection reaction. When allogenically activated

and proliferating lymphocytes are exposed to ASCs, the ef-

fect is similar to that of BMSCs in that proliferation is in-

hibited. In this point, our studies confirm Puissants results,

although it should be mentioned that the proportion of lym-

phocytes to stem cells was different in our experiments. In

contrast to the work done by Puissant (ratio 1:1), in our ex-

periments a ratio of 10 PBMCs to 1 stem cell led to quanti-

tatively similar suppression rates. The experiments wecarried outthe first after osteogenic differentiation of

ASCsshow that, as with BMSCs, there is no re-expression

of immunologically relevant antigens on the cell surface of

ASC, even after osteogenic induction there is no significant

activation to allogenic PBMCs after cultivation, and even the

immunosuppressive properties of human ASCs persist dur-

ing osteogenic differentiation. In contrast to BMSCs, the

mechanism of the immunomodulatory activity of ASCs has

not yet been explained, but a mechanism similar to that of the

immunosuppressant effect of BMSCs is likely.

In conclusion, our results support the hypothesis that

ASCs are immuno-privileged cells that may be available for

cell replacement therapy in HLA-incompatible hosts before

and after osteogenic differentiation in vitro; this last is of

great importance for tissue-engineering approaches involv-

ing regeneration of bone. As in the case of other in vitro

studies, however, the complexity of the human immune sys-

tem cannot be adequately represented in vitro, so that, ulti-

mately, engraftment of allogenic MSCs without a local or

systemic immune reaction has to be confirmed in vivo. In the

case of BMSCs, several studies have shown adequate cell

engraftment after allogenic3538 and even after xenogenic39

transplantation, but in one study, there was also a rejection

after xenogenic transplantation after earlier sensitization,40

so these experiments cannot be regarded as conclusive.

Furthermore, in the context of allogenic transplantation of

BMSCs, a thorough discussion of any possible side effects of

the immunosuppressive properties of BMSCs is necessary.

Recently, some groups reported tumor growth related to the

immunosuppressive effect of allogenic mesenchymal cells

in animals.4143 These investigations clarify that there are

still many questions to be answered before BMSCs can be

considered a safe and effective application for allogenic

transplantation. Also, it is not clear whether the capacity of

allogenic cells to form new tissue (e.g., bone) is inferior to

that of autologous cells. This also has to be determined in

further in vitro and in vivo experiments.

ACKNOWLEDGMENTS

We would like to thank Prof. Dr. A.D. Ho (Head, Med.

Klinik V, Heidelberg University) for provision of space andresources for the cell culture, Ms K. Horsch (Med. Klinik V,

Heidelberg University) for help and expertise with the

flow cytometry, and Mr. M. Herbst for technical assistance

with the MLR assays. The study was financed by grants

from the Albert-Ludwig-University Freiburg and the Uni-

versity of Freiburg. UK was supported by ADUMED-

foundation, Germany.

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Address reprint requests to:

Philipp Niemeyer, M.D.

Department of Orthopaedic Surgery and Traumatology

Albert Ludwig University Freiburg

University Hospital

Hugstetter Str. 55

D79095 Freiburg

Germany

E-mail: [email protected]



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