Telomere-Associated Proliferative Capacity in Expandable Porcine Hepatocyte-like Progenitor Cells

Primary hepatocytes are limited by poor proliferative capacity and a finite replicative lifespan, restricting their utility in long-term in vitro studies. Here, we report the generation of expandable hepatocyte-like progenitor cells from GGTA1 knockout pigs, a large-animal model with reduced immunogenicity. Porcine fibroblasts were directly reprogrammed using a non-integrative episomal system encoding hepatic transcription factors, enabling stable lineage conversion without genomic integration. A simplified two-vector configuration combined with codon optimization enabled evaluation of vector-dependent effects while maintaining genomic safety without viral integration. The resulting cells exhibited hepatocyte-like morphology and gene expression, and transcriptomic analysis revealed a progressive shift toward liver-associated profiles during extended culture. Chromosomal analysis revealed vector-dependent differences in genomic stability, with codon-optimized cells showing increased aneuploidy, indicating a trade-off between proliferative capacity and genomic integrity. The cells also demonstrated sustained proliferative capacity, supported by maintenance of telomere length, increased expression of TERT and MYC, and reduced CDKN1A levels. Importantly, sustained proliferation was supported by complementary evidence from chromosomal and telomeric analyses. Although chromosomal alterations were observed during long-term culture, their biological significance remains to be fully determined. These cells partially recapitulate hepatocyte functions and provide a renewable in vitro system for studies of hepatic biology, proliferation, drug metabolism, toxicity, and repeated in vitro applications.