Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

肝类器官可能为实现各种肝脏疾病的精准医疗提供千载难逢的机会。先前描述的多能干细胞的肝类器官方案依赖于复杂的多重分化步骤，包括 2D 和 3D 分化程序。因此，二维单层培养自发形成肝类器官与低通量生产相关，这可能会阻碍肝类器官生产的标准化，并阻碍该技术向临床或工业环境的转化。在这里，我们描述了从人类多能干细胞逐步、完全 3D 生产肝类器官。我们通过筛选每个分化步骤中分化信号的最佳浓度和时间来优化每个分化步骤。肝类器官可稳定扩展而不会丧失其肝功能。此外，在对已知肝毒性的药物进行治疗后，我们发现与PSC分化的2D肝细胞相比，肝类器官对药物引起的肝毒性更敏感，这使得它们非常适合候选药物的体外毒性筛选。当前研究中描述的用于生产功能性肝类器官的标准化全 3D 协议可能会成为肝类器官技术工业和临床转化的新平台。