MAPK1 mediates C/EBPß phosphorylation to promote maturation and inhibit EMT process in hESC-derived hepatocytes [CUT&Tag]

Human pluripotent stem cell (hPSC)-derived hepatocytes exhibit progressive differentiation toward a phenotype resembling primary human hepatocytes (PHHs). Nevertheless, they still display persistent functional deficiencies in key mature hepatocyte properties, particularly in albumin secretion and ammonia metabolism, remain challenge compared to PHHs. In this study, we elucidated a critical regulatory role of CCAAT/enhancer-binding protein beta (C/EBPß) in hepatocyte maturation. Forced expression of C/EBPß significantly enhanced hepatic functionality, as evidenced by increased albumin secretion, upregulation of hepatic-specific genes, and suppression of epithelial-mesenchymal transition (EMT) markers. Mechanistically, we found that C/EBPß directly binded to the promoter regions of E-cadherin (E-cad) and carbamoyl phosphate synthetase 1 (CPS1), enhancing their transcriptional activation. Furthermore, we identified a functional interaction between C/EBPß and MAPK signaling, where MAPK1-mediated phosphorylation at Thr-235 potentiated C/EBPß's transactivation capacity, thereby promoting hepatocyte differentiation and maturation. Our findings establish C/EBPß as a master regulator of hepatic maturation, orchestrating transcriptional networks and post-translational modification. These insights provide a theoretical framework for optimizing stem cell-derived hepatocyte differentiation, facilitating their applications in various fields. Overall design: Our previous work demonstrated that C/EBPß overexpression in hepatic progenitor cells (HPCs) promoted mitotic activation through cell cycle regulator induction. This study would uncover a stage-specific functional duality of C/EBPß in hESC-derived hepatocytes (hEHs). Quantitative assessments was used to determine how C/EBPß activation enhance hepatic functionality while suppressing EMT process, and whether MAPK1-C/EBPß regulatory axis also mechanistically play function in hPSC-derived hepatocytes by employing co-immunoprecipitation (Co-IP) assays to detect direct physical interaction between MAPK1 and C/EBPß. Then, MAPK1-mediated phosphorylation at C/EBPß Thr-235 to transcriptional activity of the downstream genes was further investigated, and finally CUT&Tag assays and luciferase activity was performed to confirm that C/EBPß could enhance promoter occupancy at target genes. The results of these studies would demonstrate that C/EBPß orchestrated hepatic maturation through dual mechanisms of transcriptional network regulation and dynamic post-translational modifications. These insights would provide a mechanistic basis for optimizing hPSC-to-hepatocyte differentiation strategies, offering new avenues for developing mature hepatocyte populations for applications in various fileds.