Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy

Objective. Bone marrow stromal cells (BMSC) are an attractive target for no vel strategies in the gene/cell therapy of hematologic and skeletal patholo gies, involving BMSC in vitro expansion/transfection and reinfusion. We inv estigated the effects of in vitro expansion on BMSC pluripotentiality, prol iferative ability, and bone-forming efficiency in vivo. Material and Methods. BMSC from three marrow donors were cultured to determ ine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or coll agen II. First passage cells were compared to fresh marrow for their bone-f orming efficiency in vivo. Stromal cell clones were isolated from five dono rs and characterized for their multidifferentiation ability. The lifespan a nd differentiation kinetics of five of these clones were determined. Results. After the first passage, BMSC had a markedly diminish proliferatio n rate and gradually lost their multiple differentiation potential. Their b one-forming efficiency in vivo was reduced by about 36 times at first confl uence as compared to fresh bone marrow. Experiments on the clones yielded c omparable results. Conclusions. Culture expansion causes BMSC to gradually lose their early pr ogenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.