Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.
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. 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.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. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.
Kitano
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Y
Department of Surgery, Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA.
Radu
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A
Shaaban
A
A
Flake
A W
AW
D017667:Adipocytes / Q000166:cytology; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism; D002448:Cell Adhesion; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology; D005994:Glycerophosphates / Q000494:pharmacology; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009355:Neomycin; D010012:Osteogenesis; D012190:Retroviridae / Q000235:genetics*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis; D001616:beta-Galactosidase / Q000235:genetics
D005938:Glucocorticoids; D005994:Glycerophosphates; D019010:Vascular Cell Adhesion Molecule-1; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D009355:Neomycin; D001374:Azacitidine; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid