Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype

Human bone marrow mesenchymal stem cells (MSC) generate, via a fibroblast colony-forming unit (CFU-F), osteo-chondroblastic cells as well as adipocytes and stromacytes. To date, these stem cells are isolated indirectly using a cell culture method and phenotyped as CD45 negative while the in vivo counterparts are undetermined. Our aim was to develop a direct selection method and to determine the phenotype of the MSC isolated in this way. Mesenchymal cells were selected with anti-CD49a and/or anti-CD45 antibodies using either flow cytometry or a magnetic beads method. All CFU-F were always detected in the small population of CD49a-positive cells. These CFU retained their differentiation potential and gave rise to osteo-chondroblastic cells, adipocytes and stromacytes. Phenotypic studies on uncultured cells revealed a CD45(med,low) , CD34(low) , HLA-II- cell population. Flow cytometry cell sorting showed that MSC with CFU-F potential were obtained only from a CD49a(+) /CD45(med,low) population. In addition, when cultured, they clearly became CD45(-) , CD34(-) , HLA-II- , CD49a(+) . These results confirmed that MSC can be directly selected easily from human bone marrow using magnetic beads without altering their differentiation potential. These cells expressed mildly the haematopoietic marker CD45, which was dramatically downregulated by in vitro culture. The expression of CD45 coupled to CD49a thus enabled direct selection of the MSC.