Telles-Silva, K. A. | bioRxiv Science

bioRxiv2025

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

Human induced pluripotent stem cell (iPSCs)-derived hepatocytes are widely used in regenerative medicine and disease modeling. However, existing protocols mainly produce fetal-like cells, limiting accurate modeling 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 unclear. Here, we examined the effects of TOP2 and FOXM1 inhibition on the terminal differentiation of hepatocytes. We found that subtoxic TOP2 inhibition reduced 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/{beta}-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 is correlated with increased hepatocyte polyploidization, enhanced CYP450 activity, and improved lipid metabolism, suggesting a potential role in these processes. Overall, our findings suggest that FOXM1 inhibition significantly promotes the terminal differentiation of human iPSC-derived hepatocytes, indicating a potential role for FOXM1 and TOP2A in liver development, regeneration, and disease.

cell biology↗

bioRxiv2025

iPSC-derived skeletal muscle spheroids for Duchenne Muscular Dystrophy modeling

BackgroundThe progressive skeletal muscle degeneration observed in Duchenne Muscular Dystrophy (DMD) patients requires multiple cycles of satellite cells (SCs) activation to promote tissue regeneration. Dystrophic SCs present intrinsic defects, and the disrupting fibrotic niche hinders appropriate muscle recovery. Traditional 2D culture systems face challenges in modeling the DMD muscle niche and SCs behavior. Our aim was to validate a 3D culture of skeletal muscle spheroids (iSMS) for DMD modeling, as compared to the traditional 2D culture, while investigating the pathophysiological mechanisms of dystrophin deficiency in vitro. MethodsTo compare iSMS with traditional 2D myogenic differentiation, we differentiated PAX7 reporter wild-type (WT), dystrophic (DMD) isogenic induced pluripotent stem cells (iPSCs), and patients iPSCs, characterized myogenic markers levels and assessed differences in proliferation and differentiation using RT-qPCR, immunofluorescence, and flow cytometry. ResultsOur data showed that although both 2D and iSMS culture systems generated myogenic progenitors positive for MYOD, MYOG, MYF5, and MYH3, iSMS improved PAX7 expression in vitro. Moreover, we identified a differential regulation of canonical Notch signaling genes between iSMS and 2D, which may influence the comparison between WT and DMD. We also characterized the differentiation of myogenic progenitors derived from 2D and iSMS towards elongated myofibers, providing a valuable comparison with muscle fibers differentiated from human primary myoblasts. Additionally, DMD iSMS derived progenitors proliferated at reduced levels compared with WT iSMS, a characteristic not observed in progenitors derived from 2D cultures. Finally, we performed iSMS and 2D myogenic differentiation of iPSC lines from three patients with DMD, thus validating the iSMS protocol for DMD modeling. ConclusionOur results highlight the important advantages of using the iSMS differentiation platform over 2D for disease modeling. Exploring these 3D systems may help to gain a deeper understanding of SCs behavior to advance in novel treatments for DMD, which might be applicable to other forms of muscular disorders.

cell biology↗