TISSUE ENGINEERING Volume 13, Number 1, 2007 # Mary Ann Liebert, Inc. DOI: 10.1089/ten.2006.

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Comparison of Immunological Properties of Bone Marrow Stromal Cells and Adipose TissueDerived Stem Cells Before and 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 DKAMP,1 and ULF KRAUSE2 VOLKER ECKSTEIN,2 NORBERT P. SU

ABSTRACT Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (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 immunological 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 inuence 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 inuence 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 signicant 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 conrm 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

O
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WING TO THEIR PLASTICITY and high proliferation capacity

in vitro, human mesenchymal stem cells (MSCs) derived from bone marrow (bone marrow stromal cells

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

Department of Orthopaedic Surgery and Traumatology, University of Freiburg, Germany. Department of Internal Medicine V, University of Heidelberg, Germany. 3 Department of Orthopaedic Surgery, University of Heidelberg, Germany. 111

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NIEMEYER ET AL. vitro, undifferentiated BMSCs do not lead to a signicant stimulation. BMSCs have even been reported to have a suppressant effect on maximally stimulated allogenic lymphocytes.1820 In an animal model, intravenous administration of HLA-incompatible BMSCs does not lead to an immunological 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 privileged or even immunomodulating. These properties suggest that BMSCs could be used to perform HLA-mismatched transplantation for tissueengineering purposes. This would be extremely benecial, 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 immunological properties of ASCs even now. It has been suggested that undifferentiated ASCs have properties similar to those of BSMCs;12 the inuence 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.

accepted as the most usual and most reliable methods, although 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 investigations in past years, and of MSCs isolated from adipose tissue (ASCs) have been compared only in a supercial 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 differentiation 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 inuence of osteogenic differentiation 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 object of current discussion, and there is no denitive and uniform terminology. In this article, the term BMSCs will be used for MSCs isolated from bone marrow and the ASCs for those 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 transduction, such as HLA-DR, DP, DQ (MHC class II), CD80, CD86, CD40, and CD40L (CD154).5,13 15 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 by foreign T-cells.CD40, a member of the transforming growth 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 cocultured with HLA-incompatible allogenic lymphocytes in

MATERIALS AND METHODS Isolation and expansion of BMSCs

Bone marrow aspirates (10 30 mL) were obtained from ve hematologically healthy donors during routine orthopedic surgery from the iliac crest; all had given informed consent. The local ethics committee approved the donor program. BMSCs were isolated as described elsewhere21 with minor variations. Briey, bone marrow mononuclear cells were obtained using Biocoll density gradient centrifugation (d 1,077 g/cm3; Biochrom, Berlin, Germany) and plated in bronectin-coated tissue culture asks (Nunc, Rochester, NY). The expansion medium used was 58% low-glucose Dulbeccos modied Eagle medium (DMEM; Cambrex, East Rutherford, NJ), 40% MCDB201 (Sigma, Taufkirchen, Germany), 2% fetal calf serum (FCS; Stemcell Technologies, Inc., Vancouver, Canada), supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, ITS media supply, linoleic acid, 10 nM dexamethasone, 0.1 mM Lascorbic-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

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Isolation of human ASC

ASCs were isolated from subcutaneous adipose tissue taken from ve healthy donors undergoing abdominoplastic surgery as previously described.22 The local ethical committee approved the donor program. Adipose tissue was nely minced (12 mm3), washed with phosphate-buffered saline (PBS), and digested with 2 mg/mL collagenase type I (Biochrom) for 90 min at 378C with continuous shaking. The oating adipocytes were separated from the stromal cell fraction using multiple centrifugation and washing steps. The stromal cells were plated in a 175-cm2 tissue culture ask at 4,000 cells/cm2 lled with 25 mL DMEM/F12, 10% fetal calf serum, 10 ng/mL basic broblast growth factor,23 100 U/mL penicillin, and 100 mg/mL streptomycin. The medium was changed every third day, which washed out all nonadherent cells. Once adherent cells had grown to conuence, 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 used for the investigations.

1 mM oligo-dT primer, 10 U RNase-inhibitor, and 4 U Omniscript Reverse Transcriptase (Qiagen) in a nal volume of 20 mL.

Real-time quantitative PCR

cDNA (2 mL) was used for PCR analysis. Real-time quantitative PCR (qPCR) was performed in a LightCycler instrument (Roche Diagnostics, Grenzach-Wylen, Germany) in a total volume of 20 mL using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Samples 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 meltingcurve analysis was performed to verify the specicity of the amplied 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-30 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 values were normalized based on the GAPDH expression in the corresponding samples.

Osteogenic differentiation in vitro

For osteogenic differentiation, similar conditions were used for BMSCs and ASCs. A different medium was used: osteogenic 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, 0.2 mM 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 demonstrated for BMSCs and ASCs. Cells from passage 3 to 6 were stained for collagen type II (after chondrogenic differentiation; antibody: ICN, Aurora, OH) as well as with von Kossa (after osteogenic differentiation; Sigma, Taufkirchen, Germany) and with oil red staining (after adipogenic differentiation; Sigma, Taufkirchen). All stainings were performed according to the manufacturers protocols.

Immunologic characterization of BMSCs and ASCs

For BMSCs and ASCs, all experiments, including coculture with allogenic PBMCs and ow cytometry, were performed before and after osteogenic differentiation for 14 days. To ensure that the duration of in vitro culture did not inuence the outcome parameters, undifferentiated cells were 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 inuence ow cytometry, had not occurred to full extent at this point of the differentiation process.

Total ribonucleic acid extraction and complementary deoxyribonucleic acid synthesis

Ribonucleic acid (RNA) from cultivated cells was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Three hundred mL 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 efciency of RNA extraction was veried using mechanical homogenization of the matrices and re-extraction of RNA. Residual RNA was not detected in any of the samples analyzed. RNA was quantied spectrometrically. One mg of total RNA was used for rst-strand complementary deoxyribonucleic acid (cDNA) synthesis using 0.5 mM deoxyribonucleotide triphosphate,

Incubation of stem cells with allogenic PBMCs

After 14 days, osteogenic-induced cells and undifferentiated controls were resuspended, 5104 BMSCs or ASCs

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were plated in wells of a 96-well plate (Nunc) in RPMI 1640 medium (50 mL) supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, and 10% human AB serum. 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 ve HLAA2 unrelated donors were obtained by culturing plastic adherent PBMCs for 5 days with GM-CSF (800 U/mL, nchen, Germany) and ILMolgramostim, Essex Pharma, Mu 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, United Kingdom), and prostaglandin E2 (1 mg/mL, Sigma-Aldrich, Deisenhofen, Germany).24,25 DCs were used as stimulator cells at a ratio of 1 DC:10 PBMCs when applicable. In a second set of experiments, PBMCs and irradiated allogeneic DCs were incubated 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). Thymidine 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 inuence on stimulated lymphocytes (ongoing mixed lymphocyte culture, 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 described previously.26 All experiments were done in triplicate. In parallel, expression of cell surface markers was monitored using ow cytometry at corresponding time points (see below).

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 column) 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 signicant changes between different groups (p < 0.05).

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-stimulatory molecules (MHC-I [HLA-A, -B, -C], MHC-II [HLADR, 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 solution or matching isotype controls. After two washing steps with PBS/1% FCS/2mM EDTA, cells were resuspended in 250 mL buffer and analyzed with a uorescence-activated cell

FIG. 2. Inhibition of mixed lymphocyte reactions by undifferentiated and differentiated adipose tissuederived stem cells (ASCs) and bone marrow stromal cells (BMSCs). Lymphocytes were stimulated 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 signicantly reduced. Analogous results were obtained for BMSCs (undifferentiated: black columns; differentiated: grey columns). No signicant differences were found between ASCs and BMSCs. * indicates statistically signicant ( p < 0.05) reduction of cell proliferation compared with allogenous lymphocyte stimulation (100%).

PROPERTIES OF BONE MARROW STROMAL CELLS AND ADIPOSE-DERIVED STEM CELLS 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 hematopoietic or MSC markers.

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(**) was considered strongly signicant. Statistics were based on N 5 for all investigations.

RESULTS
Osteogenic, chondrogenic, and adipogenic differentiation were successfully achieved in BMSCs and ASCs, conrming their mesenchymal differentiation potential, and cells from both sources had the surface antigen expression pattern typical of MSCs. After osteogenic induction, a denite mineralization (von Kossa reaction) was observed, whereas after adipogenic induction, vacuoles were found to be positive using 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,andadipogeniclineageswasconrmedinmonolayer culture or in pellet culture at passages 3 and 6 after 2 weeks of differentiation; for osteogenic differentiation, osteogenic 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.

Immunouorescence
To conrm ow-cytometry data, differentiated and undifferentiated cells were plated in bronectin-coated Lab-Tek II chamber slides (Nunc). The next day, cells were xed 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 nster, Germany). Nuclei were counterstained with GmbH, Mu Hoechst 33342.

Statistical analysis
Signicant differences were identied using a 2-factorial univariate analysis of variance (ANOVA) using SPSS software (SPSS Inc., Chicago, IL). The Friedman test was performed on all signicant 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 signicant, and p < 0.01

Efciency of osteogenic differentiation in vitro

To conrm the efciency 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-specic differentiation in vitro. Color images available online at www.liebertpub.com /ten.

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NIEMEYER ET AL. difference was not statistically signicant 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 signicant extracellular matrix synthesis (data not shown).

the differentiation process (Light Cycler, Roche Diagnostics). Normalized to GAPDH expression, expression of alkaline phosphatasebecame signicantly greater( p < 0.01) in BMSCs 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 obtained in MSCs and ASCs ( p < 0.01). Although a more pronounced increase in the expression of alkaline phosphatase and osteocalcin was found in ASCs than in BMSCs, this

Expression of immunologically relevant surface antigens

In addition to a MSC-associated cell surface marker prole (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-HLADR, 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 costimulatory molecules CD40, CD40L, CD80, CD86, and HLA-DR, DP, DQ. Osteogenic differentiation in vitro does not have a signicant inuence on the expression of these immunologically relevant surface antigens (Fig. 5). This expression pattern was conrmed using immunouorescence microscopy (representative image is shown in Fig. 6).

Incubation of stem cells with allogenic PBMCs

Stem cells were incubated with allogenic PBMCs to determine the immunogenicity of differentiated and undifferentiated stem cells by measuring 3H thymidine uptake, similar to a mixed lymphocyte reaction. The stimulation rates of allogenic DCs, undifferentiated BMSCs, osteogenically differentiated BMSCs, undifferentiated ASCs, and osteogenically differentiated ASCs were compared with the maximal stimulation 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 lymphocyte 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 signicant differences between individual groups showed signicantly lower stimulation rates in undifferentiated and differentiated 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 signicant 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 differentiation using 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 analyzed for expression of osteogenic markers. No signicant increase was detected in controls, whereas with alkaline phosphatase and osteocalcin, a signicant increase of ASCs and bone marrow stromal cells (BMSCs) was detectable in specimens that underwent osteogenic differentiation in vitro. * indicates signicant differences in expression level compared with undifferentiated cells cultured in expansion medium for 14 days (p < 0.05). No statistically signicant differences were found between ASCs and BMSCs.

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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 ow 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.

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) to investigate possible immunosuppressive properties of BMSCs and ASCs. In these tests, the stimulation rate without thirdparty 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 undifferentiated 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 (undifferentiated BMSCs: 55.6% (12.0); differentiated BMSCs: 61.9% (7.9)). Thus, a signicant reduction in the proliferation rate, with a consequent reduction of allogenic stimulation ( 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 Adipose 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 molecular 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 immunosuppressive 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 autologous cells (which has already become an established part of clinical routine for autologous chondrocyte transplantation) would be superuous. 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 benecial in the context of tissue regeneration. For BMSCs, it has already been shown that effective differentiation in vitro does not lead to any change in the expression

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FIG. 6. Flow cytometry results (uorescence-activated cell sorting) have been conrmed using immunouorescence 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 50 mm. Color images available online at www.liebertpub.com /ten.

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 immunological potential of BMSCs and ASCs, with special reference to their osteogenic differentiation in vitro. Our investigation to characterize the immunological properties of BMSCs, ASCs, and the osteoprogenitors derived from them in vitro was based on three sectors. First, expression of immunologically relevant surface markers was investigated using FACS techniques during in vitro osteogenic differentiation over 14 days. Second, interaction between undifferentiated and differentiated BMSCs/ASCs and resting HLA-incompatible allogenic PBMCs and their inuence on such cells was examined. Third, the inuence of BMSCs/ASCs on activated allogenic PBMCs during in vitro differentiation was determined with the aim of revealing any suppressant inuence of BMSCs/sASCs and BMSC-/ASCderived 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 incomplete differentiation, some stem cell properties persist that might be responsible for the immunological behavior of MSC-derived precursors. Such effects cannot be denitively excluded. As far as the efciency of in vitro differentiation is concerned, an increase in characteristic osteogenic marker

genes (alkaline phosphatase and osteocalcin) was demonstrated using RT-PCR, in addition to which matrix synthesis in terms of mineralization was demonstrated using von Kossa staining. These ndings demonstrate efcient osteogenic differentiation after 14 days. Furthermore, other studies that have investigated the inuence of in vitro differentiation on the immunological properties of BMSCs performed experiments even earlier.15 This is unavoidable, because after effective osteogenic and chondrogenic differentiation for longer than 14 days, cells are embedded in extracellular matrix, which might inuence he immunological behavior in terms of immunosuppression even more than the cell phenotype. For BMSCs, our investigations conrm earlier reports that undifferentiated BMSCs were negative for the immunologically 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 conrming 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

PROPERTIES OF BONE MARROW STROMAL CELLS AND ADIPOSE-DERIVED STEM CELLS addition of allogenic BMSCs reduces proliferation approximately 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 lymphocytes 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 potential 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 and ASCs were found to be similar. Even ASCs that have been meticulously checked for mesenchymal differentiation potential are negative for the important 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-stimulating surface antigens, even after osteogenic induction in vitro. In cultures with allogenic lymphocytes ASCs, like BMSCs, did not lead to any signicant 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 effect is similar to that of BMSCs in that proliferation is inhibited. In this point, our studies conrm Puissants results, although it should be mentioned that the proportion of lymphocytes to stem cells was different in our experiments. In contrast to the work done by Puissant (ratio 1:1), in our experiments a ratio of 10 PBMCs to 1 stem cell led to quantitatively similar suppression rates. The experiments we carried outthe rst 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 signicant activation to allogenic PBMCs after cultivation, and even the immunosuppressive properties of human ASCs persist during 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.

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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 involving regeneration of bone. As in the case of other in vitro studies, however, the complexity of the human immune system cannot be adequately represented in vitro, so that, ultimately, engraftment of allogenic MSCs without a local or systemic immune reaction has to be conrmed 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.41 43 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 and resources for the cell culture, Ms K. Horsch (Med. Klinik V, Heidelberg University) for help and expertise with the ow cytometry, and Mr. M. Herbst for technical assistance with the MLR assays. The study was nanced by grants from the Albert-Ludwig-University Freiburg and the University of Freiburg. UK was supported by ADUMEDfoundation, 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: PhNiemeyer@aol.com