Improve the reprogramming of human adult skin-derived fibroblasts into functional hepatocytes by directed evolution of hepatocyte nuclear factors (FWOSB54)

Large numbers of functional human hepatocytes are required for both biomedical research and pharmaceutical applications. Recent studies have shown that the ectopic expression of hepatic reprogramming factors leads to the direct conversion of human adult skin-derived fibroblasts into induced hepatocyte-like cells (iHeps). Although significant advancements have been made over the years, the reported conversion efficiencies and hepatic functionality of the obtained iHeps remain low. To overcome this problem, we propose to use directed protein evolution tools to drastically enhance the reprogramming performance of hepatocyte nuclear factors (HNF). Directed protein engineering is a technology platform that accelerates evolution by more than six orders of magnitude and tailors the functionality of proteins to specific applications. In a Darwinian
evolution spirit, we anticipate that applying selection pressure will allow to evolve HNFs (eHNFs) to unprecedented reprogramming performance, capable of generating iHeps with superior hepatic functionality. We will target specific domains involved in dimerization, DNA binding and
coactivator interactions and identify candidate eHNFs based on high-throughput phenotypic readouts of successful hepatic conversion, and genotyping by amplicon sequencing. We will challenge the performance of selected eHNF-iHeps in vitro and in an immune deficient murine liver disease model.

Keywords:FIBROBLASTS

Disciplines:Cell growth and development