Massive and reproducible production of liver buds entirely from human pluripotent stem cells

Organoid technology provides a revolutionary paradigm towards therapy, yet to be applied in humans mainly because of the reproducibility and scalability challenges. Here, we overcome these limitations by evolving scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive ‘reverse’ screen experiments, we identified effective triple progenitor populations for generating liver buds in a highly reproducible manner: hepatic endoderm, endothelial and septum mesenchyme progenitors. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass-producing homogenous and miniaturized liver buds on a clinically relevant large scale (>108-cell scale). Vascularized and functional liver tissues generated entirely from iPSC significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacture platform for multi-cellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. Transcriptome profiling of iPSC derived liver buds (iPSC-LB), progenitor populations for generating liver buds (iPSC-tHE/iPSC-EC/iPSC-MC) and human liver tissue of various developmental stages.

类器官（organoid）技术为治疗领域带来了革命性范式，但目前仍因可重复性与可扩展性瓶颈难以应用于人体临床。本研究通过完全基于人类诱导多能干细胞（human induced pluripotent stem cells, iPSC）构建可规模化的器官芽生产平台，攻克了上述局限。通过开展大规模“反向”筛选实验，我们以极高可重复性筛选出了可用于构建肝芽的有效三群祖细胞：肝内胚层祖细胞、内皮祖细胞以及隔膜间充质祖细胞。此外，我们开发了全孔阵列培养平台，可在临床相关的大规模（>10⁸细胞量级）下批量生产均质化微型肝芽，实现了面向人类应用的可规模化生产。完全由iPSC构建的血管化功能性肝组织，可通过发育阶段匹配的祖细胞间互作显著增强后续肝组织功能成熟，最终可通过移植实现急性肝衰竭的功能挽救。综上，本研究为多细胞类器官的规模化供应提供了严苛的制造平台，通过跨产业合作可有力推动其临床与制药应用，尤其在肝脏疾病治疗领域。本研究同时对iPSC来源肝芽（iPSC-LB）、肝芽构建相关祖细胞群（iPSC-tHE/iPSC-EC/iPSC-MC）以及不同发育阶段的人类肝组织开展了转录组谱分析。