Functional improvement of stem cell derived hepatocyte-like cells after targeted FXR gene regulatory network manipulation

Abstract

Primary human hepatocytes (PHH) are important for clinical therapy as well as for studies in pharmacology and toxicology. Hepatocyte-like cells (HLC) derived from pluripotent stem cells offer the perspective of an unlimited supply of PHH, however, genome- and proteome-wide analyses demonstrated that HLC still show major differences compared to PHH. More recently it was shown that, HLC express hepatocyte- and non-hepatocyte-associated genes within the same cells, indicating that HLC reside in a hybrid state that can be targeted by bioinformatics-guided intervention [1]. In this context, it remains to be clarified whether cell line or differentiation protocol-specific differences lead to comparable hybrid states and if the reported hybrid state is a common feature among HLC. In this thesis, HLC obtained by two different protocols from three different induced pluripotent stem cell lines (iPSC) were compared using genome-wide transcriptomics. Furthermore, it was demonstrated that interventions to improve HLC differentiation by targeting the FXR gene regulatory network (GRN) increased the expression of hepatocyte-associated genes and suppressed undesired non-liver genes in HLC, thereby increasing their similarity to PHH. However, this has yet only been shown on the transcriptomic level. Here, functional assays of bile acid secretion and lipid droplet formation were performed to confirm that an FXR targeting intervention strategy does indeed increase the similarity of HLC to PHH.