Novo Nordisk Foundation Center for Stem Cell Biology, University of Copenhagen, Copenhagen, Denmark.
Methods Mol Biol. 2022;2416:105-116. doi: 10.1007/978-1-0716-1908-7_8.
In human, endoderm is induced in two waves, with the first being the extra-embryonic primitive endoderm (PrE), otherwise known as hypoblast, induced during blastocyst development, and the second being gastrulation-stage definitive endoderm (DE). The PrE gives rise to the primary and secondary yolk sac, and has supportive functions during pregnancy for nutrient provision, with descendants of this extra-embryonic lineage also playing a role in embryonic patterning. As in DE specification, we recently found that PrE could be induced in vitro by Wnt and Nodal-related signaling, but that the critical difference was in the pluripotent starting point for differentiation. Thus, blastocyst-like naïve human pluripotent stem cells retain the unique capacity to differentiate into PrE cultures, a cell type resembling the pre-implantation hypoblast. The PrE cells could then be expanded as stable naïve extra-embryonic endoderm (nEnd) cell lines, capable of indefinite self-renewal. Here, we describe detailed protocols to differentiate naïve pluripotent stem cells into PrE and then expand the cultures as nEnd, including descriptions of morphology, passaging technique, and troubleshooting.
在人类中，内胚层分两波诱导产生，第一波是胚胎外原始内胚层（PrE），也称为下胚层，在囊胚发育过程中诱导产生，第二波是原肠胚期的确定内胚层（DE）。PrE 产生初级和次级卵黄囊，并在妊娠期间为营养供应提供支持功能，该胚胎外谱系的后代也在胚胎模式形成中发挥作用。与 DE 特化一样，我们最近发现 PrE 可以通过 Wnt 和 Nodal 相关信号在体外诱导，但关键区别在于分化的多能起始点。因此，类囊胚样原始人多能干细胞保留了独特的分化为 PrE 培养物的能力，这种细胞类型类似于植入前的下胚层。然后可以将 PrE 细胞扩增为稳定的原始胚胎外内胚层（nEnd）细胞系，能够无限自我更新。在这里，我们描述了将原始多能干细胞分化为 PrE 并将培养物扩增为 nEnd 的详细方案，包括形态描述、传代技术和故障排除。
