Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R
Centro di Biotecnologie Avanzate, Genova, Italy.
Exp Hematol. 2000 Jun;28(6):707-15. doi: 10.1016/s0301-472x(00)00160-0.
Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine 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 collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor 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.
骨髓基质细胞（BMSC）是血液学和骨骼疾病基因/细胞治疗新策略的一个有吸引力的靶点，涉及BMSC的体外扩增/转染和再输注。我们研究了体外扩增对BMSC多能性、增殖能力和体内成骨效率的影响。培养来自三名骨髓供体的BMSC以确定其生长动力学。在每次传代时，通过在诱导培养基中培养并用茜素红、阿尔辛蓝或苏丹黑染色，以及通过对骨钙素或胶原蛋白II进行免疫染色来验证其分化潜能。将第一代细胞与新鲜骨髓在体内的成骨效率进行比较。从五名供体中分离出基质细胞克隆，并对其多分化能力进行表征。确定了其中五个克隆的寿命和分化动力学。第一代传代后，BMSC的增殖率明显降低，并逐渐失去其多分化潜能。与新鲜骨髓相比，它们在首次汇合时的体内成骨效率降低了约36倍。对克隆的实验产生了类似的结果。培养扩增导致BMSC逐渐失去其早期祖细胞特性。培养的持续时间和条件对于这些细胞的成功临床应用可能至关重要，并且在设计涉及基质间充质祖细胞操作和再输注的新治疗策略时必须予以考虑。
