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Although different DE differentiation methods have been established, there are still several limitations, such as the yield of heterogeneous cell populations containing undifferentiated or non-DE cells. Therefore, this study aimed to suppress the alternate fates at branch points and establish a robust and highly efficient differentiation protocol for hESC-derived hepatocytes (hESC-Heps). We developed a two-step DE induction protocol. First, hESCs were treated with a GSK-3\u03b1\/\u03b2 inhibitor and an mTOR inhibitor combined with TGF-\u03b2 activation to generate an anterior primitive streak (progenitor to endoderm). Subsequently, a BMP inhibitor combined with TGF-\u03b2 activation was used to abolish the mesoderm lineage. The resulting DE cells were further differentiated into hESC-Heps to evaluate their functionality. By regulating the branching lineage choices, we established an efficient two-step method that yielded up to 96% DE cells with minimal expression of pluripotency and mesodermal markers. Notably, this method reduced the dosage of Activin A, which makes it cost-effective for future applications. The derived hESC-Heps exhibited mature hepatocyte characteristics, including glycogen storage, indocyanine green uptake, and cytochrome P450 activity. Additionally, these cells demonstrated robust liver-specific functions such as sensitive innate immune responses and permissiveness to hepatitis B virus infection. In summary, we developed a novel and cost-effective method that achieves high-purity DE by precisely modulating cell fate decisions in the early stages. The derived hESC-Heps can serve as a model for further studies, such as host\u2013virus interaction and hepatotoxicity testing.<\/jats:p>","DOI":"10.1177\/15473287261436294","type":"journal-article","created":{"date-parts":[[2026,4,4]],"date-time":"2026-04-04T07:13:40Z","timestamp":1775286820000},"page":"125-134","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":0,"title":["Fine Regulation of Early Lineage Bifurcations Improves Definitive Endoderm and Hepatocyte Differentiation"],"prefix":"10.1177","volume":"35","author":[{"given":"Qi","family":"Zhou","sequence":"first","affiliation":[{"name":"Department of Gynecology of the First Affiliated Hospital, Shantou University Medical College, Shantou, China."},{"name":"Stem Cell Research Center, Shantou University Medical College, Shantou, China."},{"name":"The Center for Reproductive Medicine, Shantou University Medical College, Shantou, 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