University of Connecticut, Department of Molecular and Cell Biology, 91 North Eagleville Road, Unit 3125 Storrs, CT 06269-3125, USA.
Differentiation. 2011 Jan;81(1):1-10. doi: 10.1016/j.diff.2010.09.182. Epub 2010 Oct 8.
Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.
从胚胎干细胞 (ESC) 中生成谱系定向的肠干细胞，可以为理解肠道分化途径提供一个可行的实验系统，并且最终可能为再生受损肠道组织提供细胞。我们测试了两步分化程序，其中 ESC 首先用激活素 A 培养，以有利于形成确定的内胚层，然后用成纤维细胞条件培养基处理，有或没有 Wnt3A。确定的内胚层在胚胎发育的第 8.5 天（Sox17、Foxa2 和 Gata4 表达，Id2 沉默）表达了许多与肠道管发育相关的基因。肠干细胞标记基因 Lgr5 也在内胚层细胞中被激活，而 Msi1、Ephb2 和 Dcamkl1 肠干细胞标记物没有被激活。将内胚层暴露于含有 Wnt3A 的成纤维细胞条件培养基中，导致 Id2、其余肠干细胞标记物和后期肠道标记物 Cdx2、Fabp2 和 Muc2 的激活。有趣的是，Wnt3A 还模拟了与远端肠道相关中胚层相关的基因（Foxf2、Hlx 和 Hoxd8）。两步分化方案生成了具有隐窝样结构的肠道体，其中包括 Lgr5 表达的增殖细胞区域和细胞分化区域。这些肠道体还有平滑肌成分，有些会进行蠕动。这些细胞分化成肠道上皮的能力得到了这些细胞在小鼠结肠黏膜中的体内移植的支持。这些发现表明，ESC 衍生的确定内胚层可以进行肠道细胞分化途径，并可能为修复受损的肠道组织提供细胞。
