Project description:Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem/progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bi-directional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage-conversion into bi-potential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering. iHepSCs were converted form fibroblasts by transduction of Hnf1β and Foxa3. iHepSCs were induced to differentiate into hepatocyte-like cells and cholangiocytes in vitro. Totally, 9 samples including four clones of iHepSCS, one clone of LEPCs, two samples of MEFs and two samples of iHepSCs-derived cholangocytes were analyzed.
Project description:Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem/progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bi-directional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage-conversion into bi-potential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.
Project description:Recent studies have shown that defined sets of transcription factors could directly convert fibroblasts into induced hepatocytes (iHeps). However, the underlying mechanism of direct conversion process toward a hepatic lineage is largely unknown. Here, we report that the direct conversion kinetics from fibroblasts into iHeps throughout screening multiple additional factors that potentially rescue the delayed kinetics of MET and hepatic program. Mouse embryonic fibroblasts (MEFs) were efficiently converted into iHeps in the presence of c-Myc and Klf4 (CK), the activators for MET process, with the previously defined sets of hepatic transcription factors, resulting in remarkably improved generation of iHeps. Furthermore, in the presence of CK, Hnf4? alone could convert fibroblasts into iHeps within 5 days with a relatively higher efficiency. Cells transduced with different combinations of factors were cultured in standard Hep medium. Epithelial colonies were observed within 5 days with much higher numbers in the presence of additional factor, c-Myc and Klf4, compared to control group, indicating the number of epithelial colony was dramatically increased in the presence of additional stem cell factors