Choo Andre, Lim Sai Kiang
Agency for Science Technology and Research, Bioprocessing Technology Institute, Singapore, Singapore.
Methods Mol Biol. 2011;690:175-82. doi: 10.1007/978-1-60761-962-8_12.
Mesenchymal stem cells (MSCs) have been isolated from many tissues including differentiating human embryonic stem cells (hESCs). Derivation of MSCs from hESCs consists of two major steps: differentiation and isolation. In our hands, differentiation of hESCs towards MSC-enriched culture can be induced by trypsinizing hESCs into single cells and plating them on gelatin-coated plates in a culture condition that enhances survival of hESC-derived MSCs and not hESCs. The trypsinized hESCs were grown with feeder support and the medium was supplemented with basic fibroblast growth factor (FGF2) and platelet-derived growth factor (PDGF)-AB. A highly enriched MSC culture could be obtained by repeated passaging by trypsinization. The enriched MSC cultures could be further purified by limiting dilution or FACS sorting for CD105(+) or CD73(+) and CD24(-).
间充质干细胞（MSCs）已从包括分化中的人类胚胎干细胞（hESCs）在内的多种组织中分离出来。从hESCs中获得MSCs主要包括两个步骤：分化和分离。在我们的实验中，将hESCs用胰蛋白酶消化成单细胞，然后接种到包被有明胶的培养皿上，在有利于hESC来源的MSCs而非hESCs存活的培养条件下，可诱导hESCs向富含MSCs的培养物分化。经胰蛋白酶消化的hESCs在饲养层支持下生长，培养基中添加碱性成纤维细胞生长因子（FGF2）和血小板衍生生长因子（PDGF）-AB。通过胰蛋白酶消化反复传代可获得高度富集的MSC培养物。富集的MSC培养物可通过有限稀释法或针对CD105(+)或CD73(+)以及CD24(-)的荧光激活细胞分选（FACS）进一步纯化。
