Chronic liver and metabolic diseases inflict substantial morbidity and mortality in the U.S. Only a minority of patients with end-stage liver disease receive liver transplantation. Derivation of hepatocytes from human induced pluripotent stem cells (hiPSCs) is a promising transplant alternative, however this technology has not reached patients due to immature phenotype and inadequate engraftment of the cells. Interestingly, hiPSC derivation of hepatocytes offers a powerful tool for studying liver development, which in turn will provide insight as to how hepatocellular and metabolic diseases arise when developmental processes are subverted. Although specific mechanisms are unknown, the pioneer chromatin remodeling factor, FoxO1, is implicated in metabolic disorders, liver fibrosis, and nonalcoholic fatty liver disease. FoxO1 function is well characterized in matur hepatocytes where it regulates blood glucose levels. In addition to contributing to energy homeostasis FoxO1, and closely related FoxO3 (FoxO1/3), regulate cell cycle inhibition genes to promote maturation of adipocytes and cardiomyocytes. Despite these findings, FoxO1/3 have never been studied in the developing liver. This proposal is the first to demonstrate FoxO1/3 expression throughout all stages of human hepatocyte differentiation. We hypothesize that FoxO1 contributes to human hepatocyte differentiation through chromatin remodeling at metabolic genes and that FoxO1/3 regulate antiproliferative genes early in development. An innovative hiPSC-to-hepatocyte differentiation process and techniques unique to labs studying chromatin remodeling will be used to study roles of FoxO1/3 in human liver development.
Aim 1 will derive hepatocytes from hiPSCs and evaluate the impact of FoxO1 knockdown on metabolic gene expression at each stage by qRT-PCR. FoxO1 chromatin binding and opening will be assessed by chromatin immunoprecipitation and DNase I digestion, respectively. Rescue of FoxO1 knockdown cells by FoxO1 wild type or C-terminal deletion mutants will elucidate the mechanism by which FoxO1 opens chromatin.
Aim 2 will characterize phosphorylation states of FoxO1/3 during the early stages of hepatocyte differentiation. Cytoplasmic sequestration of FoxO1/3 due to phosphorylation will be confirmed by western blots of nuclear and cytoplasmic fractions. Two prominent liver development pathways will be assessed for contribution to phosphorylation. Small molecule inhibitors will inhibit each pathway and changes in FoxO1/3 phosphorylation status will be assessed. Alteration of FoxO1/3 target cell cycle inhibition gene expression will be measured. Phosphomutant FoxO1/3 transgenes expressed in differentiating cells will confirm the significance of regulation by phosphorylation during liver development by comparing gene expression profiles in microarray analysis. This proposal will establish specific molecular mechanisms by which FoxO proteins contribute to human liver development through regulation of metabolic and cell fate genes.
Nearly 15% of Americans are living with chronic liver disease that may result in liver failure. A lack of available livers for transplantation and the potential or infections and complications due to invasive surgery has prompted the scientific community to respond by developing cellular therapy alternatives. To improve alternatives to transplantation, scientists need to better understand the how a normal liver develops and harness those insights to improve new therapeutic technologies.
