Inhibiting FOXM1 Promotes Polyploidization and Metabolic Maturation in Human iPSC-Derived Hepatocytes by Modulating the Wnt/β-Catenin Pathway [Hi-C]

Differentiating induced pluripotent stem cells (iPSCs) into hepatocytes represents a key advancement in regenerative medicine and disease modeling. However, existing protocols mainly produce fetal cells, limiting accurate replication of liver functionality. Topoisomerase II (TOP2) and its transcription factor, forkhead box M1 (FOXM1), are silenced during late liver embryonic development; however, their roles in hepatocyte differentiation remain unexplored. We examined the effects of TOP2 and FOXM1 inhibition on hepatocyte differentiation. Subtoxic TOP2 inhibition reduces nuclear chromatin condensation without causing DNA damage. RNA-seq analysis showed that TOP2 inhibition induced cell cycle arrest in a TOP2A-selective manner with FOXM1 downregulation. ATAC-seq validation demonstrated that TOP2A inhibition decreases chromatin accessibility and modulates the Wnt/β-catenin pathway. Proteomic analysis revealed that FOXM1 inhibition modulated TOP2A expression, replicated TOP2A-mediated cell cycle arrest, and reduced the levels of fetal hepatocyte proteins (HBG1/2, UGT2B7, and AFP). Prolonged FOXM1 inhibition leads to hepatocyte polyploidization, increased CYP450 activity, and improved lipid metabolism. We demonstrated that FOXM1 inhibition is necessary for terminal differentiation of human iPSC-derived hepatocytes. This study suggests a role for FOXM1 and TOP2A in liver development, regeneration, and disease.