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Summary: Hepatocytes are the primary functional cells of the liver, performing its metabolic, detoxification, and endocrine functions. Functional hepatocytes are extremely valuable in drug discovery and evaluation, as well as in cell therapy for liver diseases. However, it has been a long-standing challenge to maintain the functions of hepatocytes in vitro. Even freshly isolated hepatocytes lose essential functions after short-term culture for reasons that are still not well understood. In the present study, we find that mechanical tension-induced yes-associated protein activation triggers hepatocyte dedifferentiation. Alleviation of mechanical tension by confining cell spreading is sufficient to inhibit hepatocyte dedifferentiation. Based on this finding, we identify a small molecular cocktail through reiterative chemical screening that can maintain hepatocyte functions over the long term and in vivo repopulation capacity by targeting actin polymerization and actomyosin contraction. Our work reveals the mechanisms underlying hepatocyte dedifferentiation and establishes feasible approaches to maintain hepatocyte functions. : It has been a long-standing challenge to maintain the functions of hepatocytes in vitro. Sun et al. find that mechanical tension-induced Yap activation triggers hepatocyte dedifferentiation. Alleviation of mechanical tension by confining cell spreading or treatment with a small molecule cocktail targeting actin/actomyosin dynamics could maintain hepatocyte functions. Keywords: hepatocytes, dedifferentiation, small molecules, mechanical tension

Somatic stem cells play crucial roles in organogenesis and tissue homeostasis and regeneration and may ultimately prove useful for cell therapy for a variety of degenerative diseases and injuries; however, isolation and expansion of most types of somatic stem cells from tissues are technically challenging. Human pluripotent stem cells are a renewable source for any adult cell types, including somatic stem cells. Generation of somatic stem cells from human pluripotent stem cells is a promising strategy to get these therapeutically valuable cells. Previously, we developed a chemically defined condition for mouse hepatoblast self-renewal through a reiterative screening strategy. In the present study, we efficiently generated hepatoblasts from human embryonic stem cells by a stepwise induction strategy. Importantly, these human embryonic stem cell-derived hepatoblasts can be captured and stably maintained using conditions previously established for mouse hepatoblast self-renewal, which includes basal media supplemented with insulin, transferrin, sodium selenite, epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor β receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. The cells can stably retain hepatoblast phenotypes during prolonged culture and can differentiate into mature hepatocytes through in vitro provision of hepatocyte lineage developmental cues. After being embedded into three-dimensional Matrigel, these cells efficiently formed bile duct-like structures resembling native bile duct tissues. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases. Significance Somatic stem cells have been proposed as promising candidates for cell-based therapy; however, isolation of somatic stem cells from adult tissues is usually invasive and technically challenging. In the present study, hepatoblasts from human embryonic stem cells were efficiently generated. These human hepatoblasts were then stably captured and maintained by a growth factor and small molecule cocktail, which included epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor β receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases.