Low nanomolar Triiodothyronine Concentrations Drive Further Maturation of hiPSC-Derived Hepatocyte-Like Cells

Background The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) highlights the need for improved in vitro models to study disease mechanisms and therapeutic interventions. Thyroid hormones (TH) are key regulators of hepatic carbohydrate and lipid metabolism, and their reduced availability is implicated in MASLD pathogenesis. While human induced pluripotent stem cell-derived hepatocyte-like cells (hiPSC-HLCs) serve as liver models, TH interactions with the hepatic system remain largely unaddressed. Given the emerging therapeutic potential of TH and thyromimetics in steatotic liver disease (SLD), we aimed to develop an improved HLC model that incorporates physiological TH signaling. Methods HLCs were differentiated from two hiPSC lines using a protocol optimized by single-cell adaptation and hypoxic culture conditions. Differentiation was extended by seven days in specialized hepatocyte media with or without low nanomolar (nM) T3 from d21 to d28. Pathway analysis was conducted to assess molecular maturation signatures. Cells were analyzed for functional TH action, including TH uptake, metabolism (DIO1 activity), and T3-dependent gene regulation. Liver-specific enzyme activity relevant for redox signaling and drug metabolism was assessed, alongside the secretion of TH-distributor proteins. The model’s suitability for SLD research was evaluated through fatty acid loading experiments. Results Pathway analysis revealed downregulation of Hedgehog and NOTCH4 signaling, consistent with advanced hepatocyte maturation under low nM T3 conditions. Matured HLCs exhibited all key components of local TH action, including TH uptake, active metabolism via DIO1, and T3-dependent regulation of hepatic genes. The model demonstrated functional liver enzyme activity involved in redox signaling and drug metabolism, alongside the secretion of TH-distributor proteins relevant for systemic TH function. Notably, T3-matured HLCs retained the hepatocyte-typical capacity to accumulate fatty acids, supporting their use in SLD research. Conclusions This study establishes an improved HLC model that functionally integrates TH signaling, making it a valuable tool for investigating MASLD pathogenesis and potential therapeutic strategies targeting hepatic TH availability. The model’s ability to simulate TH-mediated metabolic regulation and fatty acid handling underscores its relevance as a translational platform for drug testing in SLD.