Wong Raymond C B, Pera Martin F, Pébay Alice
Department of Biological Chemistry, University of California Irvine, Irvine, CA, USA.
Methods Mol Biol. 2012;874:167-75. doi: 10.1007/978-1-61779-800-9_13.
Embryonic stem cells are pluripotent and capable of indefinite self-renewal in vitro. Human embryonic stem cells (hESC) have generally been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in media supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of stem cells in FCS complicates experimental approaches to define the intracellular mechanisms required for hESC maintenance. This chapter describes the serum-free maintenance of hESC in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). This complete protocol provides a chemically defined serum-free system that is advantageous for studying signaling pathways involved in hESC pluripotency.
胚胎干细胞具有多能性，能够在体外无限自我更新。人胚胎干细胞（hESC）通常在添加胎牛血清（FCS）的培养基中，培养于原代小鼠胚胎成纤维细胞（MEF）饲养层上。然而，血清含有多种生物活性化合物，可能会对hESC的生长和分化产生不利影响。因此，在FCS中培养干细胞会使确定hESC维持所需细胞内机制的实验方法变得复杂。本章介绍了通过添加鞘氨醇-1-磷酸（S1P）和血小板衍生生长因子（PDGF），在无血清条件下培养hESC。这个完整的方案提供了一个化学成分明确的无血清系统，有利于研究参与hESC多能性的信号通路。
