title: Efficient differentiation of newly derived human embryonic stem cells from discarded blastocysts into hepatocyte-like cells.
abstract: OBJECTIVE
  To establish an optimal method to isolate and culture human embryonic stem (hES) cells from discarded blastocysts and to differentiate these cells into hepatocyte-like cells.
  
  
  METHODS
  Discarded human blastocysts of days 5-6 were cultured on mouse embryonic fibroblast cell feeder layers. Cells from the inner cell mass were isolated and subsequently cultured in vitro. To induce the formation of embryoid bodies (EB), dissociated ES cells were cultured in hanging drops for 5 days. The resulting EB were plated onto 100 mm plastic gelatin-coated dishes and allowed to attach for the outgrowth culture in the presence of hepatocyte growth factor for an additional 15 days.
  
  
  RESULTS
  The hES cells had typical morphological characteristics: round or elliptic nest-like colonies with distinct cell borders and a smooth surface, significant swelling growth, compact cell aggregation, and relatively big nucleus compared to a small cytoplasm. The hES cells were identified by positive alkaline phosphatase staining. All had normal karyotypes and expressed octamer-binding transcription factor and cell surface markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, and tumor rejection antigens TRA-1-60 and TRA-1-81. The cells could be differentiated to form teratomas in vivo. The hES cells could be induced to differentiate into hepatocyte-like cells and 70-80% of the cells expressed liver-associated proteins.
  
  
  CONCLUSIONS
  Three hES cell lines have been successfully isolated and cultured from discarded human blastocysts. The hES cells can be efficiently differentiated into hepatocyte-like cells using the EB system. These cells possess the potential for treatment of liver diseases.
authors.affiliation: Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Digestive Disease, Shanghai, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Lu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zheng
authors.forename: Jin Hui
authors.initials: JH
authors.lastname: Li
authors.forename: Xiao Qian
authors.initials: XQ
authors.lastname: Chong
authors.forename: Shu Dong
authors.initials: SD
authors.lastname: Xiao
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001757:Blastomeres / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D018920:Coculture Techniques / Q000379:methods; D058732:Embryoid Bodies / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D008107:Liver Diseases / Q000628:therapy; D051379:Mice; D016513:Mice, SCID; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000379:methods; D013724:Teratoma / Q000473:pathology
chemical_list: D015415:Biomarkers