Bordelon Rachel C, Herath Madushani, Speer Allison L
Program in Children's Regenerative Medicine, Department of Pediatric Surgery, McGovern Medical School, The University of Texas Health Science Center at Houston.
Program in Children's Regenerative Medicine, Department of Pediatric Surgery, McGovern Medical School, The University of Texas Health Science Center at Houston;
J Vis Exp. 2025 Jan 17(215). doi: 10.3791/67702.
The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing. Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into >9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.
肠道细胞结构和功能的复杂性给生物工程小肠的创建带来了重大挑战。此前已有报道生成类似人类小肠的人肠道类器官（HIO）的技术。HIO包含上皮和间充质，但缺乏功能性肠道的其他关键组成部分，如肠神经系统（ENS）、免疫细胞、脉管系统和微生物群。两个独立的研究小组发表了不同的方法来用ENS使HIO获得神经支配。在此，我们讨论一种将ENS整合到源自HIO的生物工程小肠中的独特方法，该方法利用这些先前报道的组件来优化祖细胞身份以及发育时机。通过按照已发表的方案在几天时间内对分化标志物进行时间调控，使人多能干细胞（hPSC）分化以独立生成HIO和肠神经嵴细胞（ENCC）。一旦HIO达到中后肠球体阶段（大约第8天），将第15 - 21天的ENCC球体解离，与HIO共培养，并悬浮在透明的三维（3D）基底膜基质液滴中。HIO + ENCC共培养物在体外维持28 - 40天，然后移植到9周龄以上的免疫缺陷小鼠体内进行进一步发育和成熟。4 - 20周后可收获具有ENS的移植HIO（tHIO）。该方法整合了两种先前发表技术的要素，通过利用从hPSC生成的ENCC并在发育早期将它们与HIO共培养，以最大限度地暴露于可能有助于形成更成熟肠道形态的早期发育信号。
