Fine Regulation of Early Lineage Bifurcations Improves Definitive Endoderm and Hepatocyte Differentiation

Human embryonic stem cell (hESC) can be differentiated into definitive endoderm (DE) through multiple branching lineage choices. 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-3α/β inhibitor and an mTOR inhibitor combined with TGF-β activation to generate an anterior primitive streak (progenitor to endoderm). Subsequently, a BMP inhibitor combined with TGF-β 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-virus interaction and hepatotoxicity testing.