Human Primary Mensenchymal Stromal Progenitor Cells Are Highly Enriched in Both, the CD271⁺/CD146⁺ and CD271⁺/CD146⁻ Bone Marrow Population with the Latter Acquiring CD146 Expression upon Culture in-Vitro

Culture-derived mesenchymal stromal cells (MSC), which are attractive candidates for clinical cell therapy approaches, arise from primary MSC progenitor/stem cells in the bone marrow. Recently, several groups have reported markers (CD271, CD146, GD2, SSEA4, etc.) that allowed for an enrichment of CFU-F, i.e. primary MSC progenitors. However, the exact phenotype of the bona fide mesenchymal stem/progenitor cells has not yet not been sufficiently defined. We therefore aimed to investigate primary MSC in bone marrow subpopulations defined by the expression of CD271 and CD146, as both markers have been reported to contain all assayable CFU-F and stromal stem cells, respectively (Quirici et al., Exp. Hematol. 2002; Sacchetti et al, Cell, 2007). Utilizing multi-color flow cytometry, unfractionated human bone marrow mononuclear cells (BM-MNC) were found to contain 0.05 ± 0.05% CD271⁺/CD146⁺ cells, whereas 0.82 ± 0.43% and 0.71 ± 0.23% were single-positive for CD271 and CD146, respectively. CD271/CD146 subpopulations were FACS sorted from lineage-depleted BM-MNC (RosetteSep) and assayed for CFU-F content (n=3). CFU-F could not be detected in the CD271⁻/CD146⁻ fraction. In contrast, CFU-F initiating cells were highly enriched in the CD271⁺/CD146⁺/CD45^−/low fraction (1.1 ± 0.2 CFU-F per 10 plated cells), which corresponds to a ca. 400-fold enrichment compared to the entire lineage-depleted fraction (2.7 ± 3.4 CFU-F per 1 × 10⁴ plated cells). Of note, CFU-F could also be assayed at high frequency from CD271⁺/CD146⁻ cells (20.4 ± 22.6 CFU-F per 1 × 10⁴ plated cells). Generally, CFU-F were not found in the CD271⁺/CD146⁺/CD45⁺ and the CD271⁻/CD146⁺ fractions, and were also not detectable within the whole CD271⁺/CD45⁺ population of unfractionated BM-MNC (n=4), which, however, gave rise to erythropoietic colonies. The two CFU-F enriched populations, i.e. CD271⁺/CD146⁺/CD45^−/low and CD271⁺/CD146⁻ cells, were then cultured under standard MSC growth conditions. MSC derived from both populations exhibited a typical MSC surface marker profile (CD105⁺, CD90⁺, CD73⁺, HLA-class I⁺, CD45⁻, CD34⁻, CD19⁻, CD14⁻, HLA-DR⁻) and typical MSC differentiation (adipocytes, osteoblasts, chondrocytes). Interestingly, MSC generated from CD271⁺/CD146⁻ cells became positive for CD146 in culture and stable CD146 expression over time was observed for MSC from both populations (up to the 5^th passage, average 82 ± 11%). In contrast, over the same culture period CD271 expression decreased with passage number and an average of only 10 ± 4% of the cultured cells remained positive for CD271. To further characterize the CFU-F enriched subpopulations, single cells from CD271⁺/CD146⁺/CD45^−/low and CD271⁺/CD146⁻/CD45^−/low cells were sorted into fibronectin-coated 96-well plates to investigate colony growth and differentiation potential. CFU-F frequencies in this assay were 4 per 96 seeded cells for both populations and all but one of the CD271⁺/CD146⁺/CD45^−/low clones could be further expanded in culture. Subpopulation-derived clones were capable of typical MSC differentiation and MSC derived from CD271⁺/CD146⁻/CD45^−/low clones–similar to the bulk cultures–became CD146 positive (89 ± 12%) after 2 passages, whereas here CD271 expression was not lost. Taken together, CD271⁺/CD146⁺/CD45^−/low and CD271⁺/CD146⁻/CD45^−/low bone marrow cells are highly enriched for primary MSC progenitor cells. The difference in CD146 expression, which disappears in culture, might relate different localizations of the primary cells in the marrow but might possibly also reflect functional differences, e.g. in stemness. Accordingly, experiments addressing in-situ location, in vivo differentiation potential, gene expression and surface-marker expression profiling of primary MSC are currently under way.