OSTEO-REGENERATIVE ACTIVITY OF THE MESENCHYMATOUS MURINE BONE MARROW STEM CELL: PERSPECTIVES FOR REPAIRING BONE LOSS

Abstract

Purpose: The bone marrow (BM) mesenchymatous stem cell (MSC) is an osteochondrogenic stem cell which produces fibroblastic colony forming units (fCFU) in vitro. The regenerative activity of MSC could be useful for repairing extensive bone loss. A specific method for isolating human MSC (hMSC) is based on the expression of a membrane molecule, the very late antigen 1 (VLA1), was developed in the laboratory for possible cell therapy. Sorted VLA1-positive cells generate 100% of the fCFU and thus contain all of the MSC. The method used to isolate MSC was adapted to the mouse to elaborate an in vivo model.

Material and methods: VLA1-positive cells were isolated by direct sorting of BALB/cByJ@Ico and C57BL/6J@Ico male mouse BM. Total BM cells and VLA1-positive and negative cells were submitted to in vitro characterisation tests: i) expression of stem-cell-specific markers determined by flow cytometry and ii) cloning efficacy (CE=number of fCFU per 105 seeded cells) after ten days culture. VLA-positive cells not tested in vitro were cryopreserved for later use in vivo. When thawed, the VLA1-positive cells from BALB/c mice were characterised in vitro as described above and injected intravenously into female BALB/c mice. After three months, the recipient mice were sacrificed and fluorescent in situ hybridisation was used to search for the injected cells (with Y chromosome) or their progeny in different tissues.

Results: Sorted VLA1-positive cells expressed stem-cell-specific markers (CD34, Scal). Culture of the VLA1- positive and negative fractions revealed that all the fCFU were found in the VLA1-positive fraction. In the BALB/c stem line, sorting enabled a 10-fold enrichment of fCFU compared with total BM cells. As the C57 stem line exhibited only 2-fold enrichment, the BALC/c line was used for the in vivo study. Moreover, cryopreservation did not alter the CE of VLA1-positive cells nor their expression of specific markers. We report the in vivo results.

Conclusion: The method used for specific sorting of hMSC can be applied to murine BM. The mouse thus appears to be a good model for preclinical research on bone reconstruction.