Hepatic programming of human skin-derived precursor cells into functional hepatocyte-like cells for in vitro liver research

Primary human hepatocytes (PHHs) are the current gold standard for in vitro liver
research, but their use is hindered by scarce availability and dedifferentiation in culture. Alternatively, human skin-derived precursors (hSKPs) differentiated towards hepatic progenitor-like cells (hSKP-HPCs) serve as a valuable in vitro model for studying drug-induced liver steatosis and metabolic dysfunction-associated steatohepatitis. However, the hepatic phenotype of hSKP-HPCs is immature compared to PHH. Therefore, this study aims to improve the hepatic maturity of these cells by overexpressing hepatogenic programming factors.
Hepatogenic programming of undifferentiated hSKPs into hepatocyte-like cells (hSKP-HEPs) was achieved by co-overexpression of three liver-enriched transcription factors (LETFs), namely FOXA3, HNF1α and HNF4α using an all-in-one hepatic programming cassette. The induction of these LETFs was achieved using a CRE/LoxP-based flip excision system. After switching on the hepatic programming cassette in hSKPs, the previously established 24-day hepatic differentiation protocol was used. At day 24, the transcriptomic profile of the obtained hSKP-HEPs was evaluated using microarray and compared to the unswitched hSKP-HPCs (control) and paediatric PHHs. Results were confirmed using RT-qPCR.
Microarray analysis showed significant higher levels of multiple hepatic markers in switched hSKP-HEPs when compared to unswitched hSKP-HPCs, including serum markers such as ALB (655-fold), SERPINA1 (88-fold),… and biotransformation markers such as CYP2C9 (20-fold), UGT1A family (107-fold), ABCC2 (11-fold),… The gene expression changes of several of these markers were also verified and confirmed by RT-qPCR i.e. ALB (2246-fold), SERPINA1 (561-fold), CYP2C9 (682-fold), UGT1A9 (4679-fold) and ABCC2 (306-fold).
An improved hepatic phenotype was successfully achieved after overexpression of the hepatic programming cassette in hSKPs. Although further characterization at protein and functional levels is still required, our findings provide promising prospects for generating hSKP-HEPs with a superior hepatic phenotype compared to hSKP-HPCs, which would present as a valuable new tool for in vitro liver toxicity studies and disease modelling.