Human heart-forming organoids recapitulate early heart and foregut development - single-cell RNA sequencing data

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice. Single-cell RNA sequencing of two HFOs derived from the HES3 NKX2.5-eGFP reporter line on day 13 of differentiation.

针对肠道、大脑、肾脏及其他器官的早期组织发育类器官（organoid）模型已被成功构建，但针对心脏的同类模型却长期缺失。本研究通过将人类多能干细胞（human pluripotent stem cell）聚集体包埋于基质胶（Matrigel）中，随后借助小分子化合物进行双相WNT通路（WNT pathway）调控以实现定向心脏分化，成功构建出结构复杂、高度规整的三维心脏形成类器官（heart-forming organoids, HFOs）。该类器官由以内皮样细胞（endocardial-like cells）衬里的心肌层（myocardial layer）构成，外围环绕横隔样原基（septum-transversum-like anlagen）；其内部还包含空间分布与分子特征均存在显著差异的前肠前部与后部内胚层组织，以及一套完整的血管网络（vascular network）。该类器官的组织结构与心脏管（heart tube）形成前的早期天然心脏原基（heart anlagen）高度相似，而已知心脏管形成过程需要与前肠内胚层发育存在相互调控作用。我们通过实验证实，NKX2.5基因敲除（NKX2.5-knockout）的心脏形成类器官会呈现出此前在转基因小鼠（transgenic mice）中观察到的心脏畸形表型，借此将HFOs应用于体外遗传缺陷（genetic defects）研究。本研究对分化第13天、源自HES3 NKX2.5-eGFP报告细胞系（reporter line）的2个心脏形成类器官开展了单细胞RNA测序（single-cell RNA sequencing）。