Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells

Objective. It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Materials and Methods. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi -CRIP or ecotropic psi -CRE producer cells for 48 hours and grown in the presence of G418. Results. Cells incubated in psi -CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi -CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta -glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells, Conclusions. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.