Differentiation of hepatocyte-like cells from human pluripotent stem cells

UoM administered thesis: Unknown

  • Authors:
  • Sarah Withey

Abstract

This thesis, titled 'Differentiation of hepatocyte-like cells from human pluripotent stem cells', is submitted to the University of Manchester for the degree of Doctor of Philosophy, by Sarah Withey, on 3rd March 2016. This thesis is focused on the directed differentiation of human embryonic stem cells (ESCs) towards hepatocyte-like cells (HLCs), for their use in predicting the hepatotoxicity of new chemical entities during drug development. To this end, this project focuses on the role of the liver in drug metabolism. ESCs were directed towards a hepatic phenotype by mimicking in vivo embryonic liver development. Liver-specific differentiation was confirmed by multiple functional and phenotypic analysis comparing HLCs to fetal and adult hepatocytes, as well as to non-liver tissue. HLCs were shown to be consistently more similar to hepatocytes than any other cell types, determined by increases in albumin secretion, alpha1-antitrypsin production, CYP3A activity and other transcript and protein parameters. Transcriptomic changes during differentiation mimicked human embryogenesis. By specifically including fresh human fetal and adult hepatocyte controls, HLC maturity was shown by transcript, protein and function to be fetal-like and short of the adult phenotype. Attempts were made to increase the efficiency of the differentiation protocol by passaging cells to increase cellular output part-way through the differentiation procedure. Expanding hepatoblast-like cells (hepatocyte precursor cells) increased cellular output by approximately 3x as much as non-passaged cells, but lost some hepatocyte-specific gene expression as determined by Q-PCR (including CYP450 related genes). To generate cells with a more adult phenotype, RNA-sequencing was undertaken on cells at various stages of the differentiation process, and compared to published data on fetal hepatocytes, various other non-liver fetal tissue and adult hepatocytes. This helped to identify differentially expressed genes between HLCs and adult hepatocytes. The list of genes was likely to contain the key factors that drive a mature hepatocyte phenotype from a fetal-like stage; those which the HLCs are failing to express. A variety of small molecules were identified which, based on the literature, are predicted to upregulate the 'adult-specific' transcription factors, which were less expressed in HLCs compared to adult liver. As determined by q-PCR, of the nine compounds taken through to dosing experiments on HLCs, an AKL5 inhibitor and thyroid hormone (ALK5i II and T3) increased the expression of xenobiotic metabolism related genes and various transcription factors, which have been shown to be important in liver development and maturation. These factors also increased serum albumin levels, another marker of a mature hepatocyte. These findings could be used to help to improve the differentiation of mature HLCs from human pluripotent stem cells.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2016