Biohistory Research Hall, Takatsuki, Osaka, Japan.
Institute for Comprehensive Research Kyoto Sangyo University, Kyoto, Japan.
Methods Mol Biol. 2022;2490:205-212. doi: 10.1007/978-1-0716-2281-0_15.
This chapter describes the protocol to derive definitive endoderm cells from epiblast stem cells (EpiSCs) via a process analogous to gastrulation in embryos. The basis of this protocol mimicking the in vivo gastrulation process makes a contrast with those using sequential administration of pharmacological molecules and recombinant signaling proteins even at nonphysiological levels. In the experimental setup, EpiSCs are first freed from the dish-adherent condition to form free-floating aggregates, where endoderm precursor pools are produced. Embedding the EpiSC aggregates in the Matrigel allows the endoderm precursors to interact with the Matrigel mimicking the laminin-rich basement membrane underlying the egg cylinder epiblast in embryos, and let the precursors migrate into the Matrigel-filled external zone and develop into endodermal epithelial tissues.
这一章描述了通过类似于胚胎原肠胚形成过程的方法从胚胎干细胞（EpiSCs）中获得确定的内胚层细胞的方案。该方案的基础是模仿体内原肠胚形成过程，与那些使用顺序给予药理学分子和重组信号蛋白的方案形成对比，即使在非生理水平下也是如此。在实验设置中，首先将 EpiSCs 从培养皿附着状态中释放出来，形成自由漂浮的聚集物，在那里产生内胚层前体池。将 EpiSC 聚集物嵌入 Matrigel 中，允许前体与 Matrigel 相互作用，模拟胚胎中卵圆柱外胚层下富含层粘连蛋白的基底膜，并使前体迁移到充满 Matrigel 的外部区，并发育成内胚层上皮组织。
