A two-step de-differentiation and maturation of primary human hepatocytes in vitro with efficient metabolic and regenerative capacities

Hepatocytes hold significant promise for applications in drug screening, disease modeling, and clinical cell transplantation. Generating large amouts of hepatocyte in vitro for those application is still challenging. We have previously established a 5C medium for long-term culture and functional maintanace human primary hepatocytes (PHHs) in vitro. However, 5C condtion could not support hepatocyte expansion, which limited its application. In this study, we established a new hepatocyte expansion medium (NHEM) for efficient hepatocyte expansion, which turned the PHHs into hepatic progenitor-like cells (HPLCs). We also generated an optimized 6C condtion for HPLCs maturation. Overall, this study provide a new strategy to generate large amounts of hepatocytes in vitro. Overall design: Total of 24 samples were analyzed, which included fresh primary human hepatocytes (PHHs) and PHHs cultured in different condition in vitro. To figure out whether NHEM induce PHHs into proliferative hepatic progenitor-like cells (HPLCs), we conducted RNA-seq of PHHs cultured in NHEM from different donors. We also analyzed the 5C_PHHs and 6C_HHs to characterize their transcriptome as matured hepatocytes.