IL22 supports Long-Term Expansion of Mouse and Human Hepatocytes in vitro.

Hepatocytes have wide applications in drug development, disease modeling, and cell therapy. However, to expand hepatocytes in large quantities in vitro without losing their functions remains a challenging task. Here we report that IL22, a cytokine highly upregulated after partial hepatectomy or hepatocyte transplantation, could support long-term expansion (>30 passages, with theoretical expansion of ~1025 times within ~150 days) of mice hepatocytes in vitro by dedifferentiate hepatocytes into hepatocyte progenitor cells (HPCs), which maintain the capacity of differentiation into mature hepatocytes. With transcriptomic analysis and lineage-specific deletion, we uncover the critical involvement of STAT3 pathway in IL22-mediated hepatocyte to HPC conversion. Two key transcription factors (TFs) down-stream of STAT3 pathway, Bhlha15 and Arntl2, govern IL22-induced hepatic dedifferentiation. Expression of these two TFs directly initiates the dedifferentiation of hepatocytes into HPCs, which could be expanded in long-term without the supplement of IL22. IL22 also supports human hepatocyte growth in simple culture condition. Within 30 days, human hepatocytes could be expanded by more than 10,000-fold by dedifferentiation into HPCs, which maintain the full capacity of maturation. Collectively, this study provides a simple culture system enables large-scale expansion of both mice and human hepatocytes in vitro, and elucidates the critical pathways and TFs involved in these processes. We use RNA sequencing to analyze the following four aspects:(1) To characterize the differences and similarities between IL22-induced hepatic progenitor cells (IL22-iHPCs) and IL22-induced mature hepatocytes (IL22-iMHs), we compared the gene expression profiles of td-Hepa (D0), IL22-td-iHPCs (D14), IL22-td-iHPCs (P30), and IL22-td-iMHs (P30).(2) To elucidate the role of STAT3 signaling, a key pathway activated by IL22, in the early stages of IL22-mediated hepatocyte proliferation, we conducted RNA sequencing on HepaSTAT3f/f and HepaSTAT3-/- cells cultured in IL22-conditioned hepatocyte culture medium (IL22-HCM) for a period of 0-7 days.(3) To understand the crucial roles of transcription factors (TFs) in hepatocyte dedifferentiation downstream of the IL22-STAT3 pathway, we analyzed the gene expression profiles of primary hepatocytes (D0), IL22-td-iHPCs (D14), and hepatocytes infected with GFP or Bhlha15+Arntl2 and cultured in hepatocyte culture medium (HCM) for 14 days. (4) To characterize the differences and similarities between IL22-induced human hepatic progenitor cells (IL22-hiHPCs) and IL22-induced human mature hepatocytes (IL22-hiMHs), we compared the gene expression profiles of hHepa (D0), IL22-hiHPCs(P5) and IL22-hiMHs (P5).