Sato Toshiro, Clevers Hans
Department of Gastroenterology, School of Medicine, Keio University, Tokyo, Japan.
Methods Mol Biol. 2013;945:319-28. doi: 10.1007/978-1-62703-125-7_19.
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have been made to establish primary small intestinal culture systems. However, no defined, reproducible and robust culture system had been developed. To establish such a system, we screened for optimal growth factor combinations based on genetic evidence of self-renewal regulation, differentiation, and carcinogenesis of intestinal stem cells. Here, we describe methods that we have established for the isolation and culture of primary small intestinal epithelial stem cells. In this culture system, isolated crypts form "organoid structures" with a histological hierarchy recapitulating in vivo small intestinal epithelium. Single isolated Lgr5+ intestinal stem cells also form these organoid structures, in which stem cells are maintained by self-renewal and give rise to all lineages of the intestinal epithelium. This culture system is particularly useful for studying the regulation of intestinal stem cell self-renewal and differentiation.
肠上皮是成年哺乳动物中自我更新最快的组织。我们最近通过体内遗传谱系追踪策略表明，Lgr5（富含亮氨酸重复序列的G蛋白偶联受体）在肠干细胞中表达。过去，人们为建立原代小肠培养系统付出了巨大努力。然而，尚未开发出明确、可重复且稳定的培养系统。为建立这样一个系统，我们基于肠干细胞自我更新调节、分化和致癌作用的遗传学证据筛选了最佳生长因子组合。在此，我们描述了我们建立的用于分离和培养原代小肠上皮干细胞的方法。在这个培养系统中，分离出的隐窝形成“类器官结构”，其组织学层次与体内小肠上皮相似。单个分离的Lgr5 +肠干细胞也能形成这些类器官结构，其中干细胞通过自我更新得以维持，并产生肠上皮的所有谱系。这个培养系统对于研究肠干细胞自我更新和分化的调节特别有用。
