Pluripotent Stem Cell-Derived Hepatic Lineage

The clinical demand for end-stage liver failure, hereditary metabolic liver diseases, viral hepatitis, and drug-induced liver injury continues to increase, while donor liver resources remain extremely limited, restricting the application of liver transplantation and hepatocyte transplantation. Primary human hepatocytes are essential resources for hepatotoxicity screening, liver development studies, in vitro liver disease modeling, and regenerative medicine research. However, primary hepatocytes rely on donor liver availability, rapidly dedifferentiate and lose drug-metabolizing functions during in vitro culture, and cannot be stably expanded on a large scale, greatly limiting drug discovery and regenerative medicine development.

In 2010, a landmark serum-free differentiation protocol was reported in Hepatology, establishing a standardized system that faithfully recapitulates human liver embryonic development. This four-step, two-dimensional monolayer-directed differentiation system was developed for human embryonic stem cells (hESCs, H9) and induced pluripotent stem cells (hiPSCs). The entire process avoids embryoid body formation, animal serum, and feeder cells. Through sequential regulation of five key signaling molecules, including Activin A, BMP4, FGF2, HGF, and OSM, combined with precise hypoxic culture conditions, high-purity hepatocyte-like cells can be generated within approximately 20 days, with albumin-positive cells accounting for more than 80%. Multiple functional assays demonstrated that the induced cells exhibit typical hepatocyte physiological functions, including glycogen storage, lipid metabolism, low-density lipoprotein uptake, indocyanine green clearance, and urea synthesis. These cells also highly express liver-specific markers such as albumin, HNF4α, and cytochrome P450 drug-metabolizing enzymes. In vivo transplantation studies further confirmed that these hepatocyte-like cells can stably engraft into mouse liver parenchyma, continuously secrete human albumin, and possess liver regenerative potential. This differentiation platform can generate multiple liver cell types, including hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. This fully defined, highly reproducible, and scalable differentiation system provides a standardized human hepatic cell platform. It can be widely applied in high-throughput hepatotoxicity screening of innovative drugs, patient-specific liver disease modeling, studies of human liver embryonic development, hepatocyte transplantation cell source development, and artificial liver biomaterial research, providing stable, compliant, and functionally mature human hepatic cell resources for liver-related basic research and preclinical regenerative medicine development.

The differentiation process of pluripotent stem cells toward functional hepatocyte-like cells. hESCs/hiPSCs are first induced by Activin A to form definitive endoderm, followed by hepatic lineage specification under BMP4/FGF2 stimulation. Hepatoblast expansion is promoted by HGF, and the cells are finally matured into functional hepatocyte-like cells through OSM induction. The derived cells express hepatocyte markers including ALB, HNF4α, and CYP450, and exhibit hepatic functions such as albumin secretion, metabolism, and drug biotransformation, providing a valuable platform for liver disease modeling, drug screening, and regenerative medicine research.