Department of Biomedical Engineering, University of Michigan College of Engineering, Ann Arbor, MI, United States.
Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Michigan Medical School, Ann Arbor, MI, United States.
Methods Cell Biol. 2020;159:143-174. doi: 10.1016/bs.mcb.2020.03.007. Epub 2020 May 4.
Human intestinal organoids (HIOs) derived from pluripotent stem cells were first described almost a decade ago as a method to differentiate intestinal tissue containing both epithelium and supporting mesenchymal cells. The original protocol documents a directed differentiation approach to first induce definitive endoderm from pluripotent stem cells, followed by hindgut specification, resulting in the self-organization of 3D hindgut spheroids. These hindgut spheroids are then embedded in a basement membrane extracellular matrix (ECM) such as Matrigel and mature into HIOs over about 4 weeks in culture. Since the initial HIO protocol was published, the methods to generate HIOs have been updated over time including revisions to the directed differentiation protocol and implementation of new culture methods for spheroids such as embedding in alginate or polyethylene glycol hydrogels as defined alternatives to Matrigel. Additionally, HIOs have been utilized for new applications such as co-culture with bacteria. This protocol compiles the most up to date information on HIO generation and presents alternative experimental applications.
人肠类器官（HIOs）最初是由多能干细胞分化而来，作为一种包含上皮和支持性间质细胞的肠组织分化方法，在近十年前被首次描述。原始方案文档描述了一种定向分化方法，首先从多能干细胞诱导出确定的内胚层，然后进行后肠特化，导致 3D 后肠球体的自组织。然后，这些后肠球体被嵌入基底膜细胞外基质（ECM）中，如 Matrigel，并在培养中约 4 周成熟为 HIOs。自最初的 HIO 方案发表以来，生成 HIOs 的方法随着时间的推移而不断更新，包括定向分化方案的修订以及为球体（如嵌入藻酸盐或聚乙二醇水凝胶）实施新的培养方法，作为 Matrigel 的替代方案。此外，HIOs 已被用于新的应用，如与细菌共培养。本方案汇集了最新的 HIO 生成信息，并提出了替代的实验应用。
