Kuske Beatrice, Savkovic Vuk, zur Nieden Nicole I
Department of Cell Biology & Neuroscience, University of California Riverside, Riverside, CA, USA.
Methods Mol Biol. 2011;690:195-215. doi: 10.1007/978-1-60761-962-8_14.
Differentiation procedures leading to osteogenic and chondrogenic differentiation of embryonic stem cells (ESCs) have been established and well upgraded over the past decade. Novel cell-culture conditions, signaling inducers, and chemical modifications of cellular environment have been found and optimized for use as steering or supporting modules in ESC differentiation. While most of the novel studies of osteoblasts or chondrocytes differentiated from ESCs deal with their regenerative potential, the "childhood diseases" of basic differentiation have not yet been quite solved. Purification procedures are still facing a lack of exclusive markers for osteogenic progenitors and a collateral development of other cell types at the end points of differentiation that possibly lead to teratomas. This chapter discusses the role of novel markers and inducers in osteogenic and chondrogenic differentiation, their effect on signaling pathways, particularly on that of Wnt/beta-catenin, and the time-specific manner of their action. We present an improved osteogenic differentiation protocol based on the hanging drop method and a time-optimized use of 1α,25-(OH)(2) vitamin D(3), all-trans retinoic acid, and bone morphogenetic protein 2 (BMP-2) with an end point efficiency increased up to 90% and a protocol for chondrogenic differentiation, which employs BMP-2 and transforming growth factor β1 as chondrogenic inducers, with 60% chondrogenic end point efficiency.
在过去十年中，已建立并大幅改进了促使胚胎干细胞（ESC）向成骨细胞和软骨细胞分化的分化程序。人们发现并优化了新型细胞培养条件、信号诱导剂以及细胞环境的化学修饰，将其用作ESC分化中的引导或支持模块。虽然大多数关于从ESC分化而来的成骨细胞或软骨细胞的新研究都涉及其再生潜力，但基本分化的“幼年疾病”尚未完全解决。纯化程序仍面临缺乏成骨祖细胞的特异性标志物以及在分化终点出现其他细胞类型的附带发育问题，这可能导致畸胎瘤。本章讨论了新型标志物和诱导剂在成骨和软骨分化中的作用、它们对信号通路的影响，特别是对Wnt/β-连环蛋白信号通路的影响，以及它们作用的时间特异性方式。我们提出了一种基于悬滴法的改进的成骨分化方案，以及对1α,25-(OH)₂维生素D₃、全反式维甲酸和骨形态发生蛋白2（BMP-2）的时间优化使用，终点效率提高到90%；还提出了一种软骨分化方案，该方案采用BMP-2和转化生长因子β1作为软骨诱导剂，软骨终点效率为60%。
