Primary hepatocytes cultured in vitro are a powerful tool to study the functions of hepatocytes and to evaluate the metabolism and toxicity of new drugs. However, in vitro culture of hepatocytes has proven to be very difficult. Ordinary culture conditions lead to dedifferentiation of hepatocytes, resulting in rapid change in cell morphology and significant reduction in specific cell functions. In the current study, we show that hepatocyte dedifferentiation is a rapid process under 21% O2 conditions. Hepatocytes cultured in 21% O2 undergo epithelial-to-mesenchymal transition (EMT), obtain fibroblast-like morphology, and show decreased hepatic functions. In contrast, 5% O2 is very effective in maintaining the epithelial morphology and many functions of the primary hepatocytes cultured in vitro for up to five days. These functions include albumin production, glycogen storage, LDL-uptake and CYP450-mediated drug metabolism. Furthermore, we find that 5% O2 can relieve the production of reactive oxygen species (ROS) and decrease the level of DNA damage in primary cultured hepatocytes. In addition, we also show that blocking the ERK and GSK-3β pathways can inhibit the dedifferentiation of hepatocytes to a certain extent. Lowering the oxygen tension in cell culture is easily achievable, we believe it could be combined with other methods, such as the use of small molecule cocktails and 3D culture, to maintain proliferation and functions of primary hepatocytes in vitro.

SummaryLiver or hepatocytes transplantation is limited by the availability of donor organs. Functional hepatocytes independent of the donor sources may have wide applications in regenerative medicine and the drug industry. Recent studies have demonstrated that chemical cocktails may induce reprogramming of fibroblasts into a range of functional somatic cells. Here, we show that mouse fibroblasts can be transdifferentiated into the hepatocyte-like cells (iHeps) using only one transcription factor (TF) (Foxa1, Foxa2, or Foxa3) plus a chemical cocktail. 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.

Hepatocytes are very difficult to expand in vitro. A few studies have demonstrated that chemical cocktails with growth factors or Wnt ligands can support long-term expansion of hepatocytes via dedifferentiation. However, the culture conditions are complex, and clonal expansion of hepatic progenitors with full differentiation capacity are rarely reported. Here, we discover IL6, combined with EGF and HGF, promotes long-term expansion (>30 passages in ~150 days with theoretical expansion of ~1035 times) of primary mouse hepatocytes in vitro in simple 2D culture, by converting hepatocytes into induced hepatic progenitor cells (iHPCs), which maintain the capacity of differentiation into hepatocytes. IL6 also supports the establishment of single hepatocyte-derived iHPC clones. The summation of the downstream STAT3, ERK and AKT pathways induces a number of transcription factors which support rapid growth. This physiological and simple way may provide ideas for culturing previously difficult-to-culture cell types and support their future applications.

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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 hepatocytelike 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.