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Published on: November 10, 2023
Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low
Frederic Deschaseaux1, Florelle Gindraux, Rafika Saadi
1MEN EA 2284, Institut d'Etude et de Transfert de Gènes, Besançon, France. fdechaseaux@chu-besancon.fr
Abstract:
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 CD45med,low, CD34low, HLA-II- cell population. Flow cytometry cell sorting showed that MSC with CFU-F potential were obtained only from a CD49a+/CD45med,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.
Insights
Researchers developed a direct method to isolate human bone marrow mesenchymal stem cells (MSC) using CD49a and CD45 markers. This technique preserves their differentiation potential for various cell types.
Area of Science:
- Stem Cell Biology
- Hematology
- Immunophenotyping
Background:
- Human bone marrow mesenchymal stem cells (MSC) are crucial for generating various cell types.
- Current isolation methods are indirect and do not fully characterize in vivo MSC phenotypes.
Purpose of the Study:
- To develop a direct selection method for human bone marrow MSC.
- To determine the phenotype of MSC isolated via this direct method.
Main Methods:
- Mesenchymal cells were selected using anti-CD49a and anti-CD45 antibodies via flow cytometry and magnetic beads.
- Fibroblast colony-forming unit (CFU-F) potential and differentiation capacity were assessed.
- Phenotypic analysis of uncultured and cultured cells was performed using flow cytometry.
Main Results:
- Fibroblast colony-forming units (CFU-F) were exclusively found in the CD49a-positive cell population.
- Direct selection identified a CD49a+/CD45med,low population with MSC potential.
- In vitro culture downregulated CD45 expression while maintaining differentiation capacity.
Conclusions:
- A direct, efficient method using CD49a and CD45 markers for isolating human bone marrow MSC was established.
- This method preserves the multipotent differentiation capacity of MSC.
- The CD45 marker, downregulated upon culture, is key for direct MSC selection.
