Abstract
Adult Bone Marrow (BM) mesenchymal stem /progenitor cells (MS/PC) are a potentially useful tool for cell therapy and tissue repair. Most of the studies related to their characterization have been carried out on cells selected by adherence and expanded in vitro. We have developed a simple means of directly identifying mesenchymal subsets from fresh BM.
First, we adapted our tools on MS/PC enriched by short time adherence (D1-3) before any cell division. Acquisition and analysis of the CD45-CD14- cells was performed on a EPICS Elite flow cytometer (Coulter, Miami, FL, USA). We have already shown that all CFU-F were included in adherent CD45-CD14- subset. When there was a sufficient number of collected adherent cells, the sub populations, defined by the expression of CD73, CD105, CDw90, or CD49a on CD45-CD14- cells, were sorted and plated in CFU-F assay. Then, we adapted this procedure to un-manipulated fresh BM mononuclear cell (MNC).
Adherent CD45-CD14- cells expressing the CD73 or the CD49a antigen contained all the CFU-F even though the CD105+ and CDw90+ subsets comprised less than half the total. In fresh un-manipulated BM MNC, CD73 and CD49a were also highly discriminative and allowed up to a 3 log enrichment of CFU-F when compared to BM MNC. The expression of CD105 and CDw90 appeared non-specific on fresh mesenchymal progenitors. Usual culture conditions up-regulated most of the tested antigens, in particular CD73 and CD49a with a significant increase in mean of fluorescence.
Antigen expression on CD45-CD14- subset
Conclusion: The CD45-CD14-/CD73+ and CD45-CD14-/CD49a+ phenotypes identified subsets containing all mesenchymal progenitors and sufficiently enriched to detect them in fresh BM, enabling evaluation of mesenchymal content of BM collections for cell therapy. Futhermore, this study argues for the existence of differences in antigen expression between un-manipulated mesenchymal cells (MC) and in vitro expanded MC and for the feasibility of distinguishing cell subsets in MC populations.
| . | . | CD73+ . | CD105+ . | CDw90+ . | CD49a+ . |
|---|---|---|---|---|---|
| Phenotypic analysis and plating afficiency of adherent (D1-3) and un-manipulated CD45-CD14- cells. The proportion of each subset was expressed as a percentage of CD45-CD14- cells (a). The proportion of CFU-F obtained from the positive subset was reported in percentage of the total CFU-F obtained from the two subsets (positive and negative for the same antigen) (b). | |||||
| % (a) | adherent | 18.3 | 21.1 | 17.4 | 21.1 |
| un-manipulated | 5.2 | 17.4 | 4.5 | 4.4 | |
| CFU-F % (b) | adherent | 100.0 | 45.4 | 49.0 | 95.2 |
| un-manipulated | 100.0 | 50.5 | 65.6 | 99.7 | |
| . | . | CD73+ . | CD105+ . | CDw90+ . | CD49a+ . |
|---|---|---|---|---|---|
| Phenotypic analysis and plating afficiency of adherent (D1-3) and un-manipulated CD45-CD14- cells. The proportion of each subset was expressed as a percentage of CD45-CD14- cells (a). The proportion of CFU-F obtained from the positive subset was reported in percentage of the total CFU-F obtained from the two subsets (positive and negative for the same antigen) (b). | |||||
| % (a) | adherent | 18.3 | 21.1 | 17.4 | 21.1 |
| un-manipulated | 5.2 | 17.4 | 4.5 | 4.4 | |
| CFU-F % (b) | adherent | 100.0 | 45.4 | 49.0 | 95.2 |
| un-manipulated | 100.0 | 50.5 | 65.6 | 99.7 | |
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