Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.
Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.
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DOI：
10.1038/nature09691
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发表时间：
2011-02-03
期刊：
影响因子：
64.8
通讯作者：
Wells, James M.
中科院分区：
文献类型：
--
作者：
Spence, Jason R.;Mayhew, Christopher N.;Rankin, Scott A.;Kuhar, Matthew F.;Vallance, Jefferson E.;Tolle, Kathryn;Hoskins, Elizabeth E.;Kalinichenko, Vladimir V.;Wells, Susanne I.;Zorn, Aaron M.;Shroyer, Noah F.;Wells, James M.
胚胎发育的研究已经成功地指导了人类胚胎和诱导多能干细胞（PSCs）在体外向特定器官细胞类型的分化。例如，人类PSCs已分化为肝脏肝细胞和胰腺内分泌细胞的单层培养物，分别在肝病和糖尿病动物模型中具有治疗效果。然而，在体外生成复杂的三维器官组织仍然是翻译研究的主要挑战。我们已经建立了一个强大而有效的过程，通过时间序列的生长因子操作来模拟胚胎肠道发育，在体外指导人类PSCs向肠道组织的分化（总结在补充图1中）。这包括激活素诱导的最终内胚层（DE）形成，FGF/Wnt诱导的后内胚层模式，后肠规格和形态发生；促进肠道生长、形态发生和细胞分化的促肠培养系统。由此产生的三维肠道“类器官”由极化柱状上皮组成，呈绒毛样结构和隐窝样增殖区，表达肠道干细胞标记物。上皮细胞包括功能性肠细胞、杯状细胞、Paneth细胞和肠内分泌细胞。利用该培养系统作为研究人类肠道发育的模型，我们发现Wnt3a和FGF4的联合活性是后肠规范所必需的，而FGF4单独就足以促进后肠形态发生。我们的数据表明，人类肠道干细胞在发育过程中是从头形成的。最后，我们确定在肠内分泌失调中发生突变的促内分泌转录因子NEUROG3是体外人类肠内分泌细胞发育的必要和充分条件。总之，psc衍生的人类肠道组织应该允许对人类肠道发育和疾病进行前所未有的研究。
Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro . For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes respectively. However the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. We have established a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development (Summarized in supplementary Fig. 1). This involved activin-induced definitive endoderm (DE) formation, FGF/Wnt induced posterior endoderm pattering, hindgut specification and morphogenesis; and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal “organoids” consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth, and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of Wnt3a and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data suggests that human intestinal stem cells form de novo during development. Lastly we determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. In conclusion, PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
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