Molecular and Cellular Characterization of Human Embryonic Stem Cell Derived Hepatocytes

Abstract

Primary human hepatocytes are commonly used to evaluate liver drug metabolism and toxicity. Pluripotent stem cell derived hepatocytes (SCDHs) have the potential to overcome access and function-related limitations associated with primary hepatocytes. However, in order for SCDHs to become routinely used in preclinical drug metabolism and toxicity screening, they must demonstrate reproducible activity of drug metabolism proteins, particularly the oxidative CYP enzymes, and at a level comparable to that of primary human hepatocytes. The work presented in this dissertation explores the potential of SCDHs to be used as an in vitro pre-clinical model for drug metabolism studies. Namely, we focus on further identifying how similar SCDHs are to primary human hepatocytes with regard to drug-metabolizing enzymes. Moreover, we performed genotype profiling on the commonly used human embryonic stem cell lines (hESCs) to further identify their utility in pharmacogenetic screening. In addition, we investigated the miRNA expression profile of SCDHs to identify miRNA candidates that could improve the maturity of SCDHs. Our data showed that SCDHs are immature compared to primary human hepatocytes in terms of the expression and activity of drug-metabolizing enzymes, notably the CYP enzymes. We found that SCDHs more closely resemble fetal hepatocytes regarding expression of hepatocyte markers, drug-metabolizing enzymes, transporters, and transcription factors. This translated into minimal CYP activity for only CYPs 1A and 3A when examining metabolite formation. In addition, we show that the miRNA expression profile of SCDHs compared to stem cells, cryopreserved hepatocytes and human liver tissue indicates global changes associated with immaturity in SCDHs. Taken together, our findings provide a more thorough characterization of SCDHs with regards to their use in drug metabolism studies, and provide insight into a possible mechanism to enhance the maturity and functionality of SCDHs. Additional studies are warranted to further evaluate the effect of the miRNAs identified here on the differentiation of hepatocytes.