Toh Wei Seong, Lee Eng Hin, Richards Mark, Cao Tong
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
Methods Mol Biol. 2010;584:317-31. doi: 10.1007/978-1-60761-369-5_17.
Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative medicine applications. However, directing lineage-restricted differentiation of hESCs and obtaining a homogenous differentiated cell population is still a challenge. We previously described a micromass culture system as a model system to study chondrogenic commitment of the hESCs. Using this system, various growth factors including BMP2 and TGFbeta1 direct chondrogenic differentiation and modulate cartilage-specific matrix gene expression in a distinctive manner. Furthermore, a high percentage of differentiated cells exhibit typical morphological characteristics of chondrocytes and express cartilage matrix proteins such as type II collagen and proteoglycans. Chondrogenic cells can be further isolated and cultured to form functional cartilage tissue in vitro. Here, we describe in detail our established protocols to analyze chondrogenic differentiation of hESCs, and possible isolation of chondrogenic cells to form functional cartilaginous tissue.
人类胚胎干细胞（hESCs）具有自我更新能力，并能分化为三个胚层的任何细胞谱系，因此在再生医学应用方面具有巨大潜力。然而，引导hESCs进行谱系限制分化并获得同质的分化细胞群体仍然是一项挑战。我们之前描述了一种微团培养系统，作为研究hESCs软骨形成定向分化的模型系统。利用该系统，包括骨形态发生蛋白2（BMP2）和转化生长因子β1（TGFβ1）在内的各种生长因子以独特方式引导软骨形成分化并调节软骨特异性基质基因表达。此外，高比例的分化细胞表现出软骨细胞的典型形态特征，并表达软骨基质蛋白，如II型胶原蛋白和蛋白聚糖。软骨形成细胞可进一步分离和培养，以在体外形成功能性软骨组织。在此，我们详细描述了我们建立的分析hESCs软骨形成分化的方案，以及可能分离软骨形成细胞以形成功能性软骨组织的方法。
