Schewe Matthias, Sacchetti Andrea, Schmitt Mark, Fodde Riccardo
Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center;
Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center.
J Vis Exp. 2017 Nov 20(129):56329. doi: 10.3791/56329.
The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberkühn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate. Throughout the small intestine, Lgr5 stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5 stem cells through several key ligands as Wnt3, EGF, and Dll1. When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5 and Paneth cells. Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5 cells to associate and form organoids when co-cultured. This approach, here referred to as "organoid reconstitution assay" (ORA), allows the genetic and biochemical modification of Paneth or Lgr5 stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.
肠上皮的特点是更新速度极快。在哺乳动物中，整个上皮衬里在4 - 5天内更新。成体肠干细胞位于利伯kühn隐窝底部，通过Lgr5基因的表达来标记，并通过其特征性的高增殖率维持体内平衡。在整个小肠中，Lgr5干细胞与一种称为潘氏细胞的特殊分泌细胞混合在一起。潘氏细胞分泌抗菌化合物（如溶菌酶和隐窝素/防御素），并对肠道菌群发挥控制作用。最近，人们发现潘氏细胞有一个新功能，即它们能够通过几种关键配体（如Wnt3、表皮生长因子和Dll1）为Lgr5干细胞提供生态位支持。当在体外分离并在特定生长因子和细胞外基质成分存在的情况下培养时，整个肠隐窝会产生称为类器官的长寿且自我更新的三维结构，其高度类似于成年小肠的隐窝 - 绒毛上皮结构。从整个隐窝建立的类器官培养物可以研究肠干细胞生态位的自我更新和分化，尽管没有探讨其各个组成部分（即Lgr5细胞和潘氏细胞）的作用。在这里，我们描述了一种新的类器官检测方法，该方法利用了潘氏细胞和Lgr5细胞在共培养时结合并形成类器官的能力。这种方法，这里称为“类器官重建检测”（ORA），允许对潘氏或Lgr5干细胞进行基因和生化修饰，然后重建为类器官。因此，它允许对肠干细胞生态位的两个主要组成部分进行功能分析。
