Elsevier

Cytotherapy

Volume 11, Issue 2, 2009, Pages 114-128
Cytotherapy

Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets

https://doi.org/10.1080/14653240802716590Get rights and content

Background aims

Human mesenchymal stromal cells (MSC) are promising candidates for cell therapy because of their intriguing properties (high proliferation and differentiation capacity, microenvironmental function and immune modulation). However, MSC are heterogeneous and a better understanding of the heterogeneity of the cells that form the MSC cultures is critical.

Methods

Human MSC were generated in standard cultures and stained with carboxyfluorescein succinimidyl ester (CFSE) for cell division tracking. Gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics) was utilized to characterize potential MSC subpopulations (progenitor content and differentiation capacity) and identify potential MSC subpopulation markers.

Results

The majority of MSC had undergone more than two cell divisions (79.7 ± 2.0%) after 10 days of culture, whereas 3.5 ± 0.9% of MSC had not divided. MSC were then sorted into rapidly dividing cells (RDC) and slowly/non-dividing cells (SDC/NDC). Colony-forming unit–fibroblast (CFU-F) frequencies were lowest in NDC and highest in RDC with low forward-/side-scatter properties (RDClolo). Comparative microarray analysis of NDC versus RDC identified 102 differentially expressed genes. Two of these genes (FMOD and VCAM1) corresponded to cell-surface molecules that enabled the prospective identification of a VCAM1+/FMOD+ MSC subpopulation, which increased with passage and showed very low progenitor activity and limited differentiation potential.

Conclusions

These data clearly demonstrate functional differences within MSC cultures. Furthermore, this study shows that cell sorting based on proliferation characteristics and gene expression profiling can be utilized to identify surface markers for the characterization of MSC subpopulations.

Introduction

Culture-derived multipotent mesenchymal stromal cells (MSC; also commonly referred to as mesenchymal stem cells) are characterized by a number of intriguing properties (reviewed in references [1,2]). MSC have a high proliferation capacity, they can be expanded effectively in culture, and they have a high differentiation capacity (bone, cartilage, fat and possibly other cell types such as tendon and muscle). Furthermore, MSC possess hematopoiesis-supporting stroma capacity and they are capable of reconstituting the hematopoietic microenvironment upon transplantation in situ [3, 4]. Moreover, MSC are potent immune modulators in vitro and in vivo [5].

These exciting features have raised considerable interest in MSC as candidates for cell therapy approaches and, accordingly, clinical MSC cell transplantation and cell replacement studies have already been initiated (reviewed in references 5., 6., 7.). However, MSC derived from standard cultures, although defined by the homogeneous expression of surface marker molecules such as CD105, CD73, CD90 and HLA class I and lack of expression of CD34, CD45, CD14, CD19 and HLA-DR [8], are heterogeneous in terms of cell size, morphology, proliferation and differentiation potential [9, 10].

Considering the growing interest in MSC, especially for clinical application, it is critical to understand better the heterogeneity of the cells that form the MSC cultures. This information, together with a detailed and thorough characterization of their primary stem and progenitor cells in the bone marrow (BM), will also lead to a better understanding of MSC growth and differentiation characteristics.

With regard to primary MSC, considerable progress has been made in the enrichment and characterization of early stromal stem and progenitor cells from the BM. Simmons & Torok-Storb [11] were the first to develop an antibody (Ab), STRO-1, that allowed the isolation of stromal precursors in aspirated BM cell suspensions, but it was not until recently that additional surface markers for the prospective enrichment of early MSC progenitor cells have been reported. These markers/marker combinations include molecules such as nerve growth factor receptor (NGFR, CD271) 12., 13., 14., GD2 [15], CD49a [16], SSEA-4 [17], frizzled-9 (FZD-9) [18] and CD146 [4] for human cells, and SSEA-1 for primitive murine MSC [19].

The identification of human MSC subpopulations in culture, however, has been hampered by the lack of appropriate markers. Thus there is a clear need to develop strategies leading to the identification of potential MSC (sub)population markers that will allow study of the relationship of the different cell types as well as describing distinct developmental stages and their possible hierarchical structure.

We have applied gene expression profiling of functionally distinct subpopulations of cultured MSC as a tool to identify potential MSC subpopulation markers. Cultured MSC were sorted according to their divisional history into rapidly dividing cells (RDC) and slowly/non-dividing cells (SDC/NDC). Vascular cell adhesion molecule 1 (VCAM1) and fibromodulin (FMOD) were identified as potential candidates by microarray analysis, and these markers in fact defined a subpopulation of cells that expanded with passage number and exhibited reduced colony-forming cell content and reduced differentiation potential.

Section snippets

MSC cultures

BM mononuclear cells (MNC) were isolated from human vertebral body spongiosa as described elsewhere [20]. MNC were isolated by density-gradient centrifugation (Biocoll, Biochrom AG, Berlin, Germany), frozen in aliquots and thawed when needed for MSC generation. The colony-forming unit–fibroblast (CFU-F) frequency of vertebral body MNC has been reported previously (median CFU-F per 1 × 105 MNC, 11.6; range 6.0–20.0) [20]. For MSC cultures, MNC were seeded at 2.5–4 × 105 cells/cm2 in complete MSC

Surface marker profile, differentiation capacity and progenitor cell content of BM-derived MSC

Adherent cells generated from vertebral BM cells in serum-containing growth medium exhibited the typical characteristics of multipotent MSC, i.e. characteristic morphology, typical MSC surface marker profile (CD73+ CD90+ CD105+ CD45 CD14 CD34 HLA-II; Figure 2A) and could be induced to differentiate into adipogenic, osteogenic and chondrogenic lineages, as illustrated in Figure 2B and reported previously [20]. None of the standard MSC markers allowed identification of subpopulations within

Discussion

MSC are isolated from BM MNC by their adherence to cell culture-treated plastic surfaces and using selected batches of FCS. Cultured MSC are clonally derived from CFU-F and are characterized by combinations of typical homogeneously expressed surface markers, such as CD105, CD73 and CD90. [8]. However, it has long been noticed that CFU-F and MSC cultures contain cells that differ morphologically [9] and consist of a mixture of functionally and genetically different cells 23., 24., 25., 26..

Acknowledgements

This study was supported by funds from the Swedish Research Council, the Swedish Childhood Cancer Foundation, the Crafoord Foundation, Gunnar Nilsson's Cancer Foundation, the Lundgren Foundation, John Persson's Foundation, and ALF (Government Public Health Grant). The Lund Stem Cell Center is supported by a Center of Excellence grant from the Swedish Foundation for Strategic Research. The authors would like to thank Anna Fossum and Zhi Ma, Lund Strategic Research Center for Stem Cell Biology

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