title: Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.
abstract: OBJECTIVE
  Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites.
  
  
  METHODS
  Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays.
  
  
  RESULTS
  Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase.
  
  
  CONCLUSIONS
  We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation.
authors.affiliation: University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Terence N
authors.initials: TN
authors.lastname: Bukong
authors.forename: Tracie
authors.initials: T
authors.lastname: Lo
authors.forename: Gyongyi
authors.initials: G
authors.lastname: Szabo
authors.forename: Angela
authors.initials: A
authors.lastname: Dolganiuc
mesh_terms: D028341:Activins / Q000494:pharmacology*; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D005434:Flow Cytometry; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction; D012107:Research Design; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D004338:Drug Combinations; D014157:Transcription Factors; C422405:activin A; D028341:Activins; D005346:Fibroblast Growth Factors