Center for Stem Cell and Organoid Medicine (CuSTOM), Perinatal Institute, Division of Developmental Biology, Cincinnati Children's Hospital, Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA.
Laboratory for Lung Development and Regeneration, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan.
Nat Protoc. 2022 Nov;17(11):2699-2719. doi: 10.1038/s41596-022-00733-3. Epub 2022 Aug 17.
Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a protocol to differentiate hPSCs into different types of organ-specific mesoderm, leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm, respectively, after 4-d cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7, the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60-90% pure liver septum transversum/mesothelium-like, 70-80% pure liver-like fibroblasts and populations of ~35% respiratory-like mesoderm, gastric-like mesoderm or esophageal-like mesoderm. This protocol can be performed by anyone with moderate experience differentiating hPSCs, provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.
内脏器官（如食管、肺、肝和胃）的发育是由胎儿前肠内胚层和相邻中胚层细胞之间的相互信号作用来协调的。尽管最近在体外重现发育信号方面取得了成功，使人类多能干细胞（hPSCs）能够分化为各种类型的器官特异性内胚层上皮细胞，但器官特异性中胚层的产生却受到了较少的关注。这是当前工程复杂人类组织的主要限制。在这里，我们描述了一种使用从小鼠前肠器官发生的单细胞转录组学中阐明的信号网络和分子标记来将 hPSCs 分化为不同类型的器官特异性中胚层的方案。基于已建立的方法，hPSC 衍生的侧板中胚层用视黄酸（RA）或 RA 与 Hedgehog（HH）激动剂处理，分别在 4 天培养后产生后或前前肠内脏中胚层。在培养的第 4 天至第 7 天，通过组合激活或抑制 WNT、BMP、RA 或 HH 通路，将这些细胞定向为器官特异性中胚层谱系。到第 7 天，培养物富含具有不同分子特征的不同类型的中胚层：60-90%纯肝隔 Transversum/间皮样，70-80%纯肝样成纤维细胞和~35%呼吸样中胚层、胃样中胚层或食管样中胚层的群体。该方案可由具有中等 hPSC 分化经验的任何人执行，提供了研究人类中胚层发育的新平台，并可用于工程更复杂的前肠组织，用于疾病建模和再生医学。
