Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University;
Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University.
J Vis Exp. 2022 Oct 11(188). doi: 10.3791/64534.
The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.
肠道上皮细胞每 5-7 天更新一次，由位于隐窝区域底部的肠道上皮干细胞（IESC）群控制。IESC 包括活跃的干细胞，它们自我更新并分化为各种上皮细胞类型，以及静止的干细胞，它们在受到损伤时充当储备干细胞。肠道上皮的再生由这些活跃的 IESC 的自我更新和分化能力控制。此外，隐窝干细胞群体的平衡和干细胞龛的维持对于肠道再生至关重要。类器官培养是研究调节干细胞存活和功能的蛋白质、信号分子和环境线索的重要且有吸引力的方法。这种模型比动物模型更便宜、更耗时、更易于操作。类器官还模拟组织微环境，提供体内相关性。本方案描述了结肠隐窝的分离，将这些分离的隐窝细胞嵌入三维凝胶基质系统中，并培养隐窝细胞形成能够自我组织、增殖、自我更新和分化的结肠类器官。该模型允许人们操纵环境-敲除特定的蛋白质，如 Claudin-7，激活/失活信号通路等-以研究这些效应如何影响结肠干细胞的功能。具体来说，研究了紧密连接蛋白 Claudin-7 在结肠干细胞功能中的作用。Claudin-7 对于维持肠道内稳态和屏障功能及完整性至关重要。在小鼠中敲除 Claudin-7 会诱导类似于炎症性肠病的表型，表现为肠道炎症、上皮增生、体重减轻、粘膜溃疡、上皮细胞脱落和腺瘤。先前有报道称 Claudin-7 是小肠上皮干细胞功能所必需的。在本方案中，建立了结肠类器官培养系统来研究 Claudin-7 在大肠中的作用。
