Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor

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Abstract

Background: Liver failure is a life-threatening illness and one of the most common causes of mortality. Liver or hepatocytes transplantation is limited by the availability of donor organs. To generate of functional and clinically applicable hepatocytes independent of donor organs is of great therapeutic interest. Here, we show that mouse fibroblasts can be transdifferentiated into the hepatocyte-like cells (iHeps) using only one TF (Foxa1, Foxa2 or Foxa3) plus a chemical cocktail.

Methods. MEFs (mouse embryonic fibroblasts) from E12.5 or TTFs (tail tip fibroblasts) from adult mice were isolated and transduced with Foxa1, Foxa2 or Foxa3 by viral infection and then treated with the chemical cocktail CRVPTD (C, CHIR99021; R, RepSox; F, Forskolin; V, VPA; P, Parnate; T, TTNPB; and D, Dznep). A two-stage optimization strategy was carried out to improve the induction efficiency and promote the proliferation and maturation of single TF induced iHeps. And the iHeps were characterized by various in vitro and in vivo studies.

Results. We found that transcription factor Foxa1, Foxa2 or Foxa3 could reprogram mouse fibroblasts into iHeps in the presence of the chemical cocktail CRVPTD. These iHeps show typical epithelial morphology, express multiple hepatocyte-specific genes and acquire hepatocyte functions. Genetic lineage tracing confirms the fibroblast origin of these iHeps. More interestingly, these iHeps are expandable in vitro and can reconstitute the damaged hepatic tissues of the fumarylacetoacetate hydrolase-deficient (Fah-/-) mice. Our study provides a strategy to generate functional hepatocyte-like cells by using a single TF plus a chemical cocktail, and is one step closer to generate the full-chemical iHeps.

Conclusion: In this study, we demonstrated that Foxa1, Foxa2, or Foxa3 alone is sufficient for generating in vitro expandable and in vivo functional iHeps in the presence of the chemical cocktail CRVPTD.