Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BM MSCs) are defined as adherent culture-amplified cells giving rise, when cultured in appropriate conditions, to adipocytes, osteoblasts, and chondrocytes.¹  The phenotype of cultured MSCs remains limited in spite of several decades of study.^2,,–5  In addition, there has been only scattered data on the phenotype of the native BM MSCs because different markers have been used for MSC isolation in independent studies.^{6,,,,,,,,,,–17}  In this work we have studied the plasma membrane protein phenotype of cultured cells, screening for molecules not detected in hematopoietic cells. Hierarchical and principal components analyses of the transcripts coding for membrane proteins and distribution of the membrane antigens indicate lineage-homogeneity. Nine of the proteins detected specifically on cultured mesenchymal cells have proven useful to sort native MSCs from BM mononuclear cells (MNCs). This study should help select the optimal marker for BM MSCs and help discriminate MSCs from other stem/progenitor cells present in the bone marrow (hematopoietic stem cells, multipotent adult progenitor cells,¹⁸  SSEA-1⁺ cells,¹⁹  etc).

Approval for these studies was given by the Comité Consultatif de Protection des Personnes participant à la Recherche Biomédicale (CPPRB) for the University Hospital in Tours. Informed consent was obtained in accordance with the Declaration of Helsinki. Details of materials and methods used are in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article).

Human BM cells obtained from iliac crest aspirates were amplified as described.²⁰  After culture in adipogenic, chondrogenic, and osteogenic media, differentiation was evaluated by histochemical methods²⁰  and reverse transcription–polymerase chain reaction (RT-PCR) analysis.

Synovial tissues were collected postmortem from normal joints. Periosteal autografts were harvested from mastoids of patients undergoing mastoidectomy. Tissues were digested with collagenase, cells were cultured as described in Document S1.

Cells were incubated with conjugated monoclonal antibodies (mAbs) or with purified mAbs (Table S7). Acquisitions were performed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

CD235a⁺, CD45⁺, and CD11b⁺ hematopoietic fractions were isolated from BM samples using magnetic-assisted cell sorting (MACS; Miltenyi Biotec, Auburn, CA). The remaining cells were labeled with phycoerythrin (PE)-conjugated mAbs and sorted using a MoFlo cell sorter (Dako, Glostrup, Denmark). For fibroblast colony-forming unit (CFU-F) assays, mononuclear cells were seeded at 2000 to 40 000 cells/cm², and sorted cells at density 100 to 1000 cells/cm². Colonies were counted after 10 days.

Microarray data are available in the Gene Expression Omnibus (GEO)²¹  at http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE9894.

RNA was extracted from 6 P1 MSC samples and 3 samples each of CD235a⁺, CD45⁺, and CD11b⁺ cells. Hybridization on HG-U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA) was performed according to standards supplied by Affymetrix. Affymetrix GCOS 1.2 software was used to generate DAT, CEL, and EXP files and to process raw data for signal calculation and pairwise chip comparison. Group comparison and gene retrieval was performed using the online database SiPaGene.²²  Hierarchical clustering and principal components analyses were performed using Genesis software (http://www.genesis-softwareonline.com/).

Micro-macroporous biphasic calcium phosphate (MBCP) ceramic discs loaded with CD200⁺ culture-amplifed MSCs were implanted subcutaneously in the backs of nude mice.²³  Implants were harvested at 4 weeks and examined by histology (Goldner trichrome) and by scanning electron microscopy (back-scattered electrons mode).

We determined the transcripts encoding outer plasma membrane proteins (excluding Golgi, mitochondriae, etc.) using Affymetrix microarrays in 6 P1 samples. Of 1624 inventoried molecules, we detected 464 transcripts including 98 CDs (Tables S1,S3). Among these transcripts were 118 channel/transporter proteins, 102 cell-cell or cell-matrix adhesion receptors, 57 cytokine receptors and 20 junction molecules (tight, gap, etc.). Most receptors specific for immune cells (T cells, B cells, NK lymphocytes, dendritic cells, and monocytes/macrophages), and receptors for chemokines, hormones, neuromediators, and neuropeptides were not detected (Tables S2 and S4).

Of 114 membrane proteins studied by flow cytometry, we detected 51 proteins (Figure 1A,B, Tables S6,S7), among which were adhesion receptors (integrins, immunoglobulin superfamily members, tetraspanins, etc), diverse receptor tyrosine kinases, and HLA-ABC (but not HLA-DR). Remarkably, antigens expressed at high (relative mean fluorescence intensity [rMFI] ≥ 100), moderate (10 ≤ rMFI < 100) or low (2 ≤ rMFI < 10) levels remained at the same level from one sample to the other.

We then investigated whether levels of detected mRNAs were correlated with those of proteins by plotting the rMFI given by flow cytometry studies versus the signal intensity (S) determined by microarrays. The highly significant (r² = 0.453, P < .001) regression plot of log(rMFI) versus log(S) indicated a parabolic relationship.

A panel of 147 CDs was selected (genes identified as “absent” by GCOS 1.2 in all samples were excluded). Hierarchical clustering indicated that the population of cultured MSCs belonged to a cluster clearly distinct from populations of hematopoietic cells (Figure 1C). Moreover, the population of cultured MSCs did not contain discernible cell subsets and was distinguishable from other skeletal, but non–BM-derived, cultured mesenchymal cells, that is, periosteal cells (POCs) and synovial fibroblasts (SFbs). The first 3 components of the principal components analysis (PCA) confirmed the distinctions (Figure 1D): hematopoietic cells were discriminated from mesenchymal cells according to the x-axis, while y- and z-axes allowed us to distinguish the different hematopoietic (CD45⁺, CD11b⁺, and CD235a⁺) and mesenchymal (MSC, POC, and SFb) populations.

Of the 464 transcripts (including 98 CDs) expressed in cultured MSCs, the 113 (including 20 CDs) that were not detected in hematopoietic cells (except for 4 transcripts detected at low level) were defined as specific (Table S5). Of the 20 specific CDs (Figure 1E), 17 could be studied at the protein level and were expressed at the plasma membrane: CD49b, CD90, CD73, CD105, CD130, CD140a, CD140b, CD146, CD151, CD200, CD202b, CD266, CD295, CD325, CD332, and integrin alphaV(CD51)/beta5(ITGB5). Five of these (CD146, CD200, CD295, CD325, CD332) discriminated also at the transcript level MSCs from POCs and SFbs.

Our data demonstrate that cultured cells constitute a lineage-homogenous cell population. Lineage specificity is defined by a set of markers estimated on the MSC population as a whole. The mesenchymal lineage was clearly distinct from the hematopoietic lineage because (1) 24.5% (113/464) of the transcripts for membrane protein antigens were detected specifically on cultured MSCs, and (2) hierarchical clustering and PCA showed that the different hematopoietic cell populations segregated apart from cultured MSCs and showed no major gene expression variability from one MSC sample to the other. Moreover, clustering also discriminated MSCs from other non–BM-derived, cultured skeletal mesenchymal cells. Lineage-homogeneity is a population characteristic that does not preclude clonal heterogeneity within the population. Clonal heterogeneity has been evidenced by the study of CFU-Fs whose differentiation potential, albeit variable, remains restricted to the mesenchymal lineages.^1,24,25 

For cell therapy it is essential to transplant a well-defined and lineage-homogenous cell population, as obtained in our study. Our standardized protocol has been upscaled to provide sufficient cells with known phenotype and differentiation potential for therapeutic administration. Using this protocol, a randomized trial for prevention of acute graft-versus-host disease is in progress, including 12 patients thus far.

We hypothesized that some of the specific membrane antigens present on cultured MSCs would define the BM mesenchymal cell population of origin containing the CFU-Fs. Of the 17 CD markers specific for cultured MSCs, 9 (53%) reproducibly allowed us to determine a minute population of BM mononuclear cells containing the CFU-Fs: CD49b, CD73, CD90, CD105, CD130, CD146, CD200, and integrin alphaV/beta5 (Figure 2A). The percentage of BM cells recovered varied from 1.9% (CD105) to 0.014% (alphaV/beta5) of the total MNCs, indicating that some of the antibodies, such as CD49b and CD105, selected a population encompassing, but not being restricted to, native MSCs (Figure 2B). The enrichment in CFU-Fs was low (23- to 60-fold) for CD49b, CD105, and CD90; high (100- to 333-fold) for CD73, CD130, CD146, and CD200; and very high (1750) but also highly variable for alphaV/beta 5 (Figure 2B).

Finally, after adipogenic and osteogenic induction, the protein expression of CD49b, CD73, CD105, CD146, and CD200 was decreased, if not collapsed (Figure 2C). After chondrogenic induction, the mRNA expression (evaluated by quantitative RT-PCR) of these markers was also clearly decreased and was completely abolished for CD200 (not shown).

According to these results, CD200, a new marker for MSCs not expressed on bone marrow hematopoietic cells in healthy individuals (this report and Moreaux et al²⁶ ), appeared to be one of the most efficient markers to reproducibly purify native MSCs. The in vitro adipogenic, osteogenic, and chondrogenic potential of CD200⁺ cells was similar to that observed for cells separated by adherence or according to CD146 expression (Figure 2D,E). CD200⁺ MSCs also generated ectopic bone in vivo in nude mice (Figure 2F). Whether MSCs exert their immunosuppressive activity through CD200, a known immunomodulatory molecule, has to be studied.

In conclusion, we have established the extensive and specific membrane phenotype of culture-amplified BM MSCs and started to identify several specific surface markers to sort native MSCs. Many other specific candidates remain to be tested (Table S5). Nonprotein markers such as SSEA-4 or GD2^14,17  have also to be included. For transplantation, the advantage of native MSCs might reside not so much in better differentiation and proliferation potential as in improved homing capacity to different tissues.²⁷ 

The authors are grateful to Jorge Domenenech and members of the laboratory for their helpful discussions, Julien Gaillard and Anja Wachtel for technical assistance, and Christelle Gauthier for secretarial help. We thank BioRetis (Berlin, Germany) for technical support in data analysis and sharing.

This work was supported by the European Community (Key action 1.2.4-3 Integrated Project Genostem, contract No. 503161), by a grant from Inserm (Contract ProA No. A04069FS), and by the national genome research network in Germany (German Federal Ministry of Education and Research, grant 01GS0413).

Contribution: B.D. devised the experiments and carried out the cell cultures and flow cytometric analyses with the help of N.G. and J.R. T.H. devised and carried out the microarray experiments. Y.L.V. and D.K. performed the flow cytometry sorting experiments. P.L. performed the experiments for ectopic bone formation in vivo. C.J. contributed to the overall methodology. P.R. and L.S. provided the bone marrow samples and contributed to the overall methodology. P.C. analyzed the microarray data and directed the work.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Bruno Delorme, Laboratoire d'Hématopoièse, Faculté de médecine, Batiment Dutrochet, 10 Bvd Tonnellé, Tours 37032, France, e-mail: delorme@med.univ-tours.fr.