Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.

The generation of large numbers of functional human hepatocytes for cell-based approaches to liver disease is an important and unmet goal. Direct reprogramming of fibroblasts to hepatic lineages could offer a solution to this problem but so far has only been achieved with mouse cells. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs, can be expanded in vitro, and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Upon transplantation into mice with concanavalin-A-induced acute liver failure and fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHeps restore the liver function and prolong survival. Collectively, our results demonstrate successful lineage conversion of nonhepatic human cells into mature hepatocytes with potential for biomedical and pharmaceutical applications.

[1]  George Q. Daley,et al.  Reprogramming Cellular Identity for Regenerative Medicine , 2012, Cell.

[2]  T. Yagi,et al.  Enhanced proliferative potential in culture of cells from p53-deficient mice. , 1993, Oncogene.

[3]  Philippe Morel,et al.  Improved Survival of Fulminant Liver Failure by Transplantation of Microencapsulated Cryopreserved Porcine Hepatocytes in Mice , 2009, Cell transplantation.

[4]  L. Donehower,et al.  In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. , 1993, Oncogene.

[5]  X Liu,et al.  Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[6]  L. Hui,et al.  Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors , 2011, Nature.

[7]  Jeroen S. van Zon,et al.  Direct cell reprogramming is a stochastic process amenable to acceleration , 2009, Nature.

[8]  H. Wong,et al.  Bioartificial Liver: Implanted Artificial Cells Microencapsulated Living Hepatocytes Increases Survival of Liver Failure Rats , 1986, The International journal of artificial organs.

[9]  L. Corey,et al.  Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes. , 2007, The American journal of pathology.

[10]  M. T. Donato,et al.  Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. , 2004, Current drug metabolism.

[11]  M. Grompe,et al.  Generation and Regeneration of Cells of the Liver and Pancreas , 2008, Science.

[12]  J. Shay,et al.  A role for both RB and p53 in the regulation of human cellular senescence. , 1991, Experimental cell research.

[13]  A. Wendel,et al.  A T cell-dependent experimental liver injury in mice inducible by concanavalin A. , 1992, The Journal of clinical investigation.

[14]  Sayaka Sekiya,et al.  Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors , 2011, Nature.

[15]  F. Oesch,et al.  Generation of human hepatocytes by stem cell technology: definition of the hepatocyte , 2005, Expert opinion on drug metabolism & toxicology.

[16]  Guoyu Pan,et al.  Assessment of biliary clearance in early drug discovery using sandwich-cultured hepatocyte model. , 2012, Journal of pharmaceutical sciences.

[17]  Thomas Vierbuchen,et al.  Direct Lineage Conversions: Unnatural but useful? , 2011, Nature Biotechnology.

[18]  F. Guengerich,et al.  Cytochrome p450 enzymes in the generation of commercial products , 2002, Nature Reviews Drug Discovery.

[19]  A. Dhawan,et al.  Human hepatocyte transplantation: current experience and future challenges , 2010, Nature Reviews Gastroenterology &Hepatology.

[20]  B. Nordlinger,et al.  Internal bioartificial liver with xenogeneic hepatocytes prevents death from acute liver failure: an experimental study. , 1998, Annals of surgery.

[21]  L. Hui,et al.  Liver xeno-repopulation with human hepatocytes in Fah-/-Rag2-/- mice after pharmacological immunosuppression. , 2010, The American journal of pathology.

[22]  Aarati R. Ranade,et al.  Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice , 2007, Nature Biotechnology.

[23]  D. Hay,et al.  Liver tissue engineering and cell sources: issues and challenges , 2013, Liver international : official journal of the International Association for the Study of the Liver.

[24]  Qiuyan Wang,et al.  Cytoskeletal reorganization and repolarization of hepatocarcinoma cells in APA microcapsule to mimic native tumor characteristics. , 2006, Hepatology research : the official journal of the Japan Society of Hepatology.

[25]  S. Nyberg,et al.  Cell therapies for liver diseases , 2012, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society.

[26]  C. Legallais,et al.  Artificial and bioartificial liver devices: present and future , 2009, Gut.

[27]  K. Hochedlinger,et al.  Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. , 2008, Cell stem cell.

[28]  M. Grompe,et al.  Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I , 1996, Nature Genetics.

[29]  Robert A. Weinberg,et al.  Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes , 1983, Nature.

[30]  H. Blau,et al.  Nuclear reprogramming to a pluripotent state by three approaches , 2010, Nature.

[31]  Gorka Orive,et al.  History, challenges and perspectives of cell microencapsulation. , 2004, Trends in biotechnology.

[32]  T. Kiyono,et al.  Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells , 1998, Nature.

[33]  T. Enver,et al.  Forcing cells to change lineages , 2009, Nature.

[34]  L. Hui,et al.  Cell fate conversion: Direct induction of hepatocyte‐like cells from fibroblasts , 2013, Journal of cellular biochemistry.

[35]  K. Zaret.  Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation , 2008, Nature Reviews Genetics.

[36]  J. Castell,et al.  Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies , 2006, Expert opinion on drug metabolism & toxicology.

[37]  Mike J. Mason,et al.  Role of the Murine Reprogramming Factors in the Induction of Pluripotency , 2009, Cell.

[38]  J Michael DiMaio,et al.  Making steady progress on direct cardiac reprogramming toward clinical application. , 2013, Circulation research.

[39]  B. Goodwin,et al.  Regulation of hepatic drug metabolism: role of the nuclear receptors PXR and CAR. , 2002, Seminars in liver disease.

[40]  Thomas Vierbuchen,et al.  Induction of human neuronal cells by defined transcription factors , 2011, Nature.

[41]  D. Gifford,et al.  Tissue-specific transcriptional regulation has diverged significantly between human and mouse , 2007, Nature Genetics.

[42]  G. Crabtree,et al.  A transcriptional hierarchy involved in mammalian cell-type specification , 1992, Nature.

[43]  J. C. Belmonte,et al.  Diseases in a dish: modeling human genetic disorders using induced pluripotent cells , 2011, Nature Medicine.

[44]  Jialiang Liang,et al.  A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. , 2010, Cell stem cell.

[45]  J. Carroll,et al.  Pioneer transcription factors: establishing competence for gene expression. , 2011, Genes & development.