Regulation of Transplanted Fetal Hepatocyte Proliferation by Histone Post‐Translational Modifications

Introduction Hepatic cell transplantation (HCT) offers a potential therapeutic alternative to orthotopic liver transplantation for end-stage liver disease and could help relax the current reliance on scarce donor livers. While transplanted adult hepatocytes have shown a limited window of post-transplant efficacy, fetal hepatocytes have been demonstrated to durably persist and proliferate in animal models and human clinical trials. Ethical procurement concerns prevent human fetal cells from being used in the clinic, making it important to characterize the mechanisms responsible for their growth advantage in order to activate this phenotype in adult hepatocytes or induced hepatocyte-like cells. Our study examines histone post-translational modification (hPTM) and gene expression profiles of fetal-derived colonies and paired adult host tissue, compared to primary fetal and adult hepatocytes. Methods Post-transplant livers from our rat model were frozen, cryosectioned, and stained for fetal-derived colonies. Sections of colony and host tissue from the same liver were collected using laser capture microdissection (LCM), and pre-transplant adult and fetal rat hepatocytes were isolated or immunopurified using hepatic and ductal lineage markers. Histones were extracted from these tissue and cell samples for hPTM abundance quantification by mass spectrometry, and RNA was extracted for next-gen sequencing. Data was analyzed using R, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA). Results 13 PTMs of Histone H3 were found to have significantly different relative abundance between fetal-derived colonies and host tissue as well as between fetal and adult hepatocytes, with the majority of marks having consistent abundance trends. RNA-Seq expression profiles segregated the LCM samples by colony or host origin. Differential expression analysis found ≅1,000 significantly differentially expressed genes (DEGs) between colony and host tissue, and hundreds of these were found to also be similarly differentially expressed between fetal and adult hepatocytes. When analyzed by GO and IPA, the DEGs found to be expressed higher in fetal-derived colonies revealed enrichment of ion transmembrane transport and calcium signaling pathways. Conclusions For at least 10 months following transplantation into an adult liver microenvironment, fetal-hepatocyte-derived colonies retain a distinct subset of both hPTM abundance and mRNA expression patterns in common with pre-transplant fetal cells. These retained differences may drive the competitive growth advantage seen in fetal cells post-transplant, and could potentially be harnessed to improve HCT outcomes. The differentially-abundant hPTMs in colony tissue were all on Histone H3 and have known associations with gene transcription activity, and the colony DEGs reflect enrichment of biological processes associated with nutrient uptake, proliferation, and signaling. Our future work will explore the link between specific differentially expressed genes and the differentially abundant hPTMs that may be regulating them via ChIP-Seq analysis of these histone marks.