Genomics Analysis of Freshly-Purified Human Marrow Stromal Cells (MSCs): Comparison with Hematopoietic Lineage Cells and Culture-Expanded MSCs.

Gene and antigen expression profile of bone marrow MSCs in culture has been investigated, but these characteristics of MSCs in vivo are poorly understood. In this study we used microarrays to compare MSCs freshly-purified from human bone marrow aspirates (f-MSCs) with donor-matched hematopoietic lineage cells (HLCs) and with standard culture-expanded MSCs (e-MSCs). Following enrichment with Anti-Fibroblast MicroBeads, f-MSCs were purified by cell sorting as CD45lowCD235a-LNGFR+ [1] and HLCs as CD45+CD235a+LNGFR-. RNA was extracted, amplified and hybridized to an Affimetrix U133 Plus 2.0 chip. Raw fluorescence data were normalised to GAPDH for direct comparison with qPCR. Duplicate hybridizations were used to define 3 relative zones of sensitivity and select appropriate thresholds for significant changes in expression. The microarray data were validated by qPCR using several mesenchymal lineage genes (including Sox9 and Runx2) and by flow cytometry for several known MSC surface markers (including CD73, CD90, CD146, CD10, CD63 and CD130). Expression data were further searched for gene ontology using a custom-made, Perl-based program. Compared to HLCs, f-MSCs over-expressed genes encoding extracellular matrix proteins (including versican, fibronectin and collagen I), stromal cytokines/chemokines (including SDF-1, IL-7 and CXCL10), and molecules associated with cell junctions and communication with endothelium (including connexin 43, VCAM-1 and angiopoietin-1). Osteogenesis-related genes (BMPs and their receptors, alkaline phosphatase, osteonectin, osteocalcin, osteomodulin and others) were more abundant in f-MSCs than in HLCs. Some mature fat tissue products were also quite abundant in f-MSCs (particularly lipoprotein lipase and adipsin), suggesting the presence of cells committed to adipogenesis. Of note, the expression of stroma- and fat-related transcripts was significantly reduced in e-MSCs compared to f-MSCs. Novel surface antigens of f-MSCs identified by microarray, Junctional Adhesion Molecule 2 (normally expressed on vascular endothelial cells), Leptin receptor (implicated in fat and bone metabolism), and CD81 (tetraspanin family adhesion molecule) were confirmed at the protein level by 6-colour flow cytometry. Altogether, this work confirmed the CD45lowCD235a-LNGFR+ phenotype f-MSCs in human bone marrow, revealed a unique expression profile of f-MSCs in vivo and described new surface markers of f-MSCs. These data have implications for our understanding of the in vivo biology of non-hematopoietic bone marrow stromal cells and their role in hematopoiesis and skeletal remodelling in health and disease.