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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE298619 | GEO | 2025/05/30

REPOSITORIES: GEO

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Project description:Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e. personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECM) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned co-culture (iMPCC) platform in a multi-well format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel™). We assessed iHep maturity via global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal CYP450 activities, phase-II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. Conclusion: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multi-organ responses to compounds. We used Affymetrix microarrays to profile the global gene expression of co-culture stabilized iHeps (iMPCCs) relative to freshly isolated and co-culture stabilized primary human hepatocytes (2 donors). To assess the transcriptomic stability of iHeps in iMPCCs, RNA was extracted following 9 and 21 days of culture for hybridization to Affymetrix microarrays. The hepatic maturation state of iHeps was assessed by comparing gene expression against microarrays containing data from two primary human hepatocyte donors, both following hepatocyte isolation (day 0) and after stabilization in the micropatterened co-culture platform (day 6 and day 42 MPCCs), as previously described.

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

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