Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring organs is a critical unmet challenge in an evolving organoid field. Here, we describe a protocol for the continuous patterning of hepatic, biliary and pancreatic (HBP) structures from a 3D culture of human pluripotent stem cells (PSCs). After differentiating PSCs into anterior and posterior gut spheroids, the two spheroids are fused together in one well. Subsequently, self-patterning of multi-organ (i.e., HBP) domains occurs within the boundary region of the two spheroids, even in the absence of any extrinsic factors. Long-term culture of HBP structures induces differentiation of the domains into segregated organs complete with developmentally relevant invagination and epithelial branching. This in-a-dish model of human hepato-biliary-pancreatic organogenesis provides a unique platform for studying human development, congenital disorders, drug development and therapeutic transplantation. More broadly, our approach could potentially be used to establish inter-organ connectivity models for other organ systems derived from stem cell cultures.

人体类器官正在成为研究人体器官发育和疾病的宝贵资源。人类类器官的适用性受到限制，部分原因是当前技术的架构过于简单，该技术会产生缺乏器官间结构连接的单组织类器官。因此，在不断发展的类器官领域中，包含相邻器官之间连接性的工程类器官系统是一个尚未解决的关键挑战。在这里，我们描述了一种用于从人类多能干细胞 (PSC) 的 3D 培养中连续模式化肝脏、胆道和胰腺 (HBP) 结构的协议。在将 PSC 区分为前肠道球体和后肠道球体后，两个球体在一个井中融合在一起。随后，多器官的自模式（即，HBP) 域出现在两个球体的边界区域内，即使在没有任何外在因素的情况下也是如此。HBP 结构的长期培养诱导结构域分化成分离的器官，并具有发育相关的内陷和上皮分支。这种人类肝胆胰器官发生的盘内模型为研究人类发育、先天性疾病、药物开发和治疗移植提供了一个独特的平台。更广泛地说，我们的方法可能用于为源自干细胞培养的其他器官系统建立器官间连接模型。HBP 结构的长期培养诱导结构域分化成分离的器官，并具有发育相关的内陷和上皮分支。这种人类肝胆胰器官发生的盘内模型为研究人类发育、先天性疾病、药物开发和治疗移植提供了一个独特的平台。更广泛地说，我们的方法可能用于为源自干细胞培养的其他器官系统建立器官间连接模型。HBP 结构的长期培养诱导结构域分化成分离的器官，并具有发育相关的内陷和上皮分支。这种人类肝胆胰器官发生的盘内模型为研究人类发育、先天性疾病、药物开发和治疗移植提供了一个独特的平台。更广泛地说，我们的方法可能用于为源自干细胞培养的其他器官系统建立器官间连接模型。