Generation Of Endodermal Progenitors By Lineage Reprogramming And Induction Of Them Into Functional Hepatocytes

The liver is the major organ responsible for protein synthesis, metabolic transformation, and detoxification of xenobiotics as well as for metabolically handling endogenous substrates. End-stage liver disease caused by liver metabolic disorders or acute liver failure is one of the most health-threatening diseases in the world, and currently, liver transplantation is the only effective treatment for end-stage liver diseases. However, the scarcity of donor livers and serious side effects of chronic immunosuppression are major obstacles to liver transplantation. As an alternative to organ transplantation, cell-based therapies that include hepatocyte transplantation, engineered hepatic tissue constructs, and the bioartificial liver, may potentially provide effective treatment for many liver diseases. However, primary human hepatocyte(PHH) is also a severely limited resource given the shortage of donor livers. The rapid development of stem cell and regenerative medicine brings hope for the cell therapybased treatment of end stage liver diseases.Pluripotent stem cells(PSCs), including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs), are capable of generating tissues from all three developmental germ layers with unlimited self-renewal capacity, providing great value in clinical application. Hepatocytes could be induced from either ESCs or iPSCs in vitro through sequential exposure to cytokines that mimics that in embryonic morphogenesis. However, the tumorigenic risk of ESCs and iPSCs caused safety concerns in their clinical applications. In addition, PSC-derived hepatocytes usually carried fetal hepatocyte-like phenotype and functions, which were not fully functional. Moreover, it is difficult to produce pure monolineage cultures of a desired cell type from PSCs.The discovery of iPSCs reprogramming demonstrated that a combination of defined transcription factors was sufficient to reprogram diverse somatic cell types to a pluripotent state in vitro. These studies have raised the possibility that a combination of cell-type-specific transcription factors could change cell fates between developmentally distant cell types, which ignited the field of lineage reprogramming. Ectopic expression of specific transcription factors in fibroblasts led to the generation of hepatocytes(iHeps) or hepatic stem cells(iHepSCs) of human or mouse origin. Other reports also indicated that fibroblasts could also be converted to human endodermal progenitors(iMPC-EPCs) by a reprogramming shortcut through transcription factor-forced induction assistent with small molecules. Reprogramming mediated by transcription factors completely or partially raised hopes for cell-based therapy. However, its application is still hampered by potential risks of genetic aberrations and tumorigenicity because of mistakes in genetic or epigenetic regulations.Small molecule-based reprogramming has recently attracted much interest due to its safety and efficiency in activations, which were suggested in controlling and changing cell fates. Successful reprogrammings solely induced by small molecules, such as, from mouse fibroblasts to iPSCs and from mouse or human fibroblasts into neurons, represented a reprogramming strategy involving with a mix of defined small molecules. However, there is still no report about successful generation of any lineagedetermined stem cells in any of the three embryonic germ layers solely by using small molecules thus far.In this study, we successfully converted human gastrointestinal epithelial cells to human induced endodermal progenitor cells(hiEndoPCs) using a mix of defined small molecules in presence of human gastric subepithelial myofibroblasts(GSEMFs) as feeders, and then differentiated hiEndoPCs into functional hepatocytes. The gastrointestinal epithelial cells were isolated from macroscopically normal mucosa of surgical specimens and endoscopic biopsies, serving as the starting cells for reprogramming. With the support of GSEMFs, a combination of four small molecules, Bay K 8644, Bix01294, RG108 and SB431542, was sufficient to reprogram the gastrointestinal epithelial cells to hiEndoPCs. The converted hiEndoPCs expressed endodermal stem/progenitor-associated transcription factors GATA4, FOXA2, SOX9, HNF1 B, PDX1, ONECUT2 and HOXA3. And hiEndoPCs were proved to be particular endodermal progenitor cells in terms of transcriptional and epigenetic signatures, and, maybe, are intermediates between primitive gut tube(PGT) and posterior foregut(PFG). Moreover, hiEndoPCs could be expanded clonogenically up to 4- 6 passages and demonstrated consistent morphologies. And hiEndoPCs could retained their proliferation and differentiation properties after thawing from cryopreservation.When using defined soluble signals for guiding to hepatocytes, hiEndoPCs were able to give rise to cells expressing typical hepatic markers. Global DNA methylation analysis demonstrated that the promotors of hepatocyte-specific genes, including TF, CYP2E1, APOBEC1, were demethylated in hiEndoPC-derived hepatocyte like cells(hiEndoPC-Heps). And RNA-seq data showed that genes involved in liver development and maturation, in fatty acid metabolism, in glucose metabolism, in lipoprotein metabolism and in drug metabolism were upregulated in hiEndoPC-Heps. Importantly, hiEndoPC-Heps carried functions of hepatocytes, including glycogen storage, accumulation of fatty droplets, uptake of Indocyanine green(ICG) and low density lipoprotein(LDL), albumin secretion, cytochrome P450 activity. Most significantly, they are able to rescue liver failure in Fah-/-Rag2-/- mice after transplantation.Additionally, hiEndoPCs could differentiated to pancreatic endocrine cells which expressed pancreatic-specific transcription factors(NKX6.1, PDX1 and NGN3) and hormones(GCG, INS and SST), and intestinal epithelial cells which expressing intestinal markers(CDX2, VIL1, MUC2, CHGA and LYSO). Moreover, epithelial cells of lung and thyroid could also be derived from hiEndoPCs in vitro. Whereas, hiEndoPCs were unable to differentiate to cells of mesodermal or ectodermal origin, suggesting that the differentiation potential of hiEndoPCs is restricted to endodermal fates. Finally, we evaluated the safety of hiEndoPCs, and confirmed that hiEndoPCs were non-tumorigenic in vivo and maintained a normal karyotype during 5 continuous passages in vitro.Overall, we successfully generated hiEndoPCs from human gastrointestinal epithelial cells via a small molecule-only lineage reprogramming process. The converted hiEndoPCs have the capacities of clonal expansion and of differentiating into multiple derivatives of endodermal cells including functional hepatocytes. Therefore, hiEndoPCs may become a new cell source for the production of therapeutically relevant endodermal cell types to meet the potential needs of various clinical applications and basic studies.