Project description:Recent studies have shown that defined hepatic factors could lead to the direct conversion of fibroblasts into induced hepatocytes (iHeps). However, reported conversion efficiencies are vey low and the underlying mechanism of the hepatic lineage conversion is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Throughout screening for additional factors that could potentially enhance the kinetics of the MET and hepatic programs, we have found that c-Myc and Klf4 (CK) dramatically accelerate the conversion kinetics, resulting in remarkably improved generation of iHeps (>87 fold). Furthermore, we identified small molecules that could replace the roles of CK and thus led to the highly efficient generation of iHeps without CK. Finally, we show that a single factor (Hnf1α) supported by small molecules is sufficient to robustly induce transprogramming of fibroblasts into functional hepatocyte-like cells with high yield. This novel approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into clinic.
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
Project description:Although transcription factor(TF)s regulate differentiation-related processes, including dedifferentiation and direct conversion, functional interactions between TFs regulating these processes are not well understood. Here we show that TFs preventing dedifferentiation are able to induce direct conversion. Using a neural lineage cell line and a large number of TFs expressed in it, we found a subset of TFs whose overexpression strongly interfered with dedifferentiation triggered by a procedure to induce induced pluripotent stem cells (iPSC), through a maintenance mechanism of the cell-type-specific transcriptional profile. Strikingly, the maintenance activity of the interfering TF set was strong enough to induce the cell line-specific transcriptional profile when overexpressed in a heterologous cell type. In addition, TFs that interfered with dedifferentiation in hepatic lineage cells involved known TFs with induction activity for hepatic lineage cells. Our results suggest that dedifferentiation suppresses a cell-type-specific transcriptional profile, which is primarily maintained by a small subset of TFs capable of inducing direct conversion. We anticipate that this functional correlation might be applicable in various cell types, which may include cancer cells, and might facilitate identification of TFs with induction activity to understand differentiation and tumorigenesis Examination of binding of 3 transcription factors and histone modifications in Neural progenitor like cells.
