Endocrine Abstracts

Background: The increasing global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) emphasizes its significant health implications, including liver inflammation, cirrhosis, and malignancy. Thyroid hormones (TH) play a vital role in hepatic energy metabolism, and their reduced local availability in the liver is associated with MASLD development. This study presents the comprehensive characterization of functional components of the TH system in human hepatocyte-like cells (HLCs), a refined hepatic model derived from human induced pluripotent stem cells (iPSCs).

Methods: A robust differentiation protocol was developed to efficiently generate hepatocyte-like cells from three iPSC donor lines. Consistent and comparable culture qualities were achieved through a 28-day differentiation period. A publicly available human iPS cell line from the Allen Cell Collection was also successfully differentiated with comparable results. RNA sequencing and network analysis from enriched gene set data examined maturation under continuous 5 nM T3 exposure compared to control differentiation media containing basal T3 concentrations ≤ 1 nM. Critical components of the TH system were identified by radioactive T3 uptake and DIO1 enzyme activity assays. Hepatocyte functionality was evaluated by assessing liver-specific enzyme activities (ALAT, ASAT, GPX1, CYP3A4) and secreted proteins (ALB, TTR, SELENOP). Additionally, exposure to 300 µM oleic and palmitic acid (1:1) for five days mimicked fatty acid accumulation.

Results: Transcriptomics revealed a refined hepatocyte model following T3 exposure, with increased THRB expression, reduced AFP levels, and an expression shift towards less DIO2/3 and more DIO1. Gene set enrichment analysis indicated T3-dependent downregulation of Hedgehog and NOTCH4 signaling, marking a more mature hepatocyte state. Decreases in extracellular matrix-receptor interactions and the PI3K-AKT pathway were associated with altered hepatic glucose supply. Increased ribosomal and proteasomal activities, along with enhanced oxidative phosphorylation, suggested cellular restructuring and heightened energy turnover associated with T3 exposure. Liver-specific enzyme activity and the secretion of binding proteins were in the comparative range of two hepatic cancer cell lines. The accumulation of lipid droplets after exposure to free fatty acids was also achieved.

Conclusions: This thoroughly characterized HLC model, expressing an intact and functional TH system, serves as an ideal platform for investigating T3-associated treatments and mechanisms involved within the context of MASLD.