title: MicroRNA-199a-5p inhibition enhances the liver repopulation ability of human embryonic stem cell-derived hepatic cells.
abstract: OBJECTIVE
  Current hepatic differentiation protocols for human embryonic stem cells (ESCs) require substantial improvements. MicroRNAs (miRNAs) have been reported to regulate hepatocyte cell fate during liver development, but their utility to improve hepatocyte differentiation from ESCs remains to be investigated. Therefore, our aim was to identify and to analyse hepatogenic miRNAs for their potential to improve hepatocyte differentiation from ESCs.
  
  
  METHODS
  By miRNA profiling and in vitro screening, we identified miR-199a-5p among several potential hepatogenic miRNAs. Transplantation studies of miR-199a-5p-inhibited hepatocyte-like cells (HLCs) in the liver of immunodeficient fumarylacetoacetate hydrolase knockout mice (Fah(-/-)/Rag2(-/-)/Il2rg(-/-)) were performed to assess their in vivo liver repopulation potential. For target determination, western blot and luciferase reporter assay were carried out.
  
  
  RESULTS
  miRNA profiling revealed 20 conserved candidate hepatogenic miRNAs. By miRNA screening, only miR-199a-5p inhibition in HLCs was found to be able to enhance the in vitro hepatic differentiation of mouse as well as human ESCs. miR-199a-5p inhibition in human ESCs-derived HLCs enhanced their engraftment and repopulation capacity in the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Furthermore, we identified SMARCA4 and MST1 as novel targets of miR-199a-5p that may contribute to the improved hepatocyte generation and in vivo liver repopulation.
  
  
  CONCLUSIONS
  Our findings demonstrate that miR-199a-5p inhibition in ES-derived HLCs leads to improved hepatocyte differentiation. Upon transplantation, HLCs were able to engraft and repopulate the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Thus, our findings suggest that miRNA modulation may serve as a promising approach to generate more mature HLCs from stem cell sources for the treatment of liver diseases.
authors.affiliation: Junior Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: Selina
authors.initials: S
authors.lastname: Möbus
authors.affiliation: Junior Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: Dakai
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany.
authors.forename: Qinggong
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: Institute for Molecular Biology, Hannover Medical School, Germany.
authors.forename: Timo H-W
authors.initials: TH
authors.lastname: Lüdtke
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany.
authors.forename: Asha
authors.initials: A
authors.lastname: Balakrishnan
authors.affiliation: Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Sgodda
authors.affiliation: Junior Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
authors.forename: Bhavna
authors.initials: B
authors.lastname: Rani
authors.affiliation: Institute for Molecular Biology, Hannover Medical School, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kispert
authors.affiliation: Computational Biology and Bioinformatics Group, Max Planck Institute for Molecular Biomedicine, Münster, Germany; Group of Computational Biology and Systems Biomedicine, Biodonostia Health Research Institute, 20014 San Sebastián, Spain; IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain.
authors.forename: Marcos J
authors.initials: MJ
authors.lastname: Araúzo-Bravo
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Vogel
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Manns
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Ott
authors.affiliation: Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Computational Biology and Bioinformatics Group, Max Planck Institute for Molecular Biomedicine, Münster, Germany. Electronic address: cantz.tobias@mh-hannover.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.affiliation: Junior Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address: sharma.amar@mh-hannover.de.
authors.forename: Amar Deep
authors.initials: AD
authors.lastname: Sharma
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016031:Liver Transplantation; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; C534780:Mirn199 microRNA, mouse; D012313:RNA
keywords: Embryonic stem cells; Hepatocyte differentiation; Liver repopulation; MicroRNA