title: High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium.
abstract: BACKGROUND
  We have previously differentiated hepatocyte like cells from deciduous tooth pulp stem and extracted third molar pulp stem cells with a protocol that used fetal bovine serum, but it showed high contaminations of nondifferentiated cells. Both the lower purity of hepatically differentiated cells and usage of serum are obstacles for application of cell therapy or regenerative medicine. Objective of this study was to investigate the capacity for and purity of hepatocyte-like differentiation of CD117-positive dental pulp stem cells without serum.
  
  
  METHODS
  Mesenchymal cells from human deciduous and extracted third molar pulp were isolated and expanded in vitro. We separated CD117-positive cells by using a magnetic-activated cell sorter. The cells were characterized immunofluorescently by using known stem cell markers. For hepatic differentiation, the media were supplemented with hepatic growth factor, insulin-transferrin-selenium-x, dexamethasone, and oncostatin M. Expression of hepatic markers alpha fetoprotein, albumin, hepatic nuclear factor-4 alpha, insulin-like growth factor-1, and carbamoyl phosphate synthetase was examined immunofluorescently after differentiation. The amount of differentiated cells was assessed by using flow cytometry. Glycogen storage and urea concentration in the medium were defined.
  
  
  RESULTS
  Both cell cultures demonstrated a number of cells positive for all tested hepatic markers after differentiation, ie, albumin-positive cells were almost 90% of differentiated deciduous pulp cells. The concentration of urea in the media increased significantly after differentiation. Significant amount of cytoplasmic glycogen storage was found in the cells.
  
  
  CONCLUSIONS
  Without serum both cell types differentiated into high-purity hepatocyte-like cells. These cells offer a source for hepatocyte lineage differentiation for transplantation in the future.
authors.affiliation: Department of Oral Health, Nippon Dental University, Tokyo, Japan.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Ishkitiev
authors.forename: Ken
authors.initials: K
authors.lastname: Yaegaki
authors.forename: Toshio
authors.initials: T
authors.lastname: Imai
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tanaka
authors.forename: Taka
authors.initials: T
authors.lastname: Nakahara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishikawa
authors.forename: Vanyo
authors.initials: V
authors.lastname: Mitev
authors.forename: Markus
authors.initials: M
authors.lastname: Haapasalo
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D019070:Cell Lineage; D016895:Culture Media, Serum-Free; D003782:Dental Pulp / Q000166:cytology*; D003907:Dexamethasone / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005938:Glucocorticoids / Q000494:pharmacology; D006003:Glycogen / Q000032:analysis; D006131:Growth Inhibitors / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D051557:Hepatocyte Nuclear Factor 4 / Q000032:analysis; D022781:Hepatocytes / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000032:analysis; D053683:Oncostatin M / Q000494:pharmacology; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D012643:Selenium / Q000494:pharmacology; D012709:Serum Albumin / Q000032:analysis; D013234:Stem Cells / Q000502:physiology*; D014168:Transferrin / Q000494:pharmacology; D014508:Urea / Q000032:analysis; D000509:alpha-Fetoproteins / Q000032:analysis
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free; D005938:Glucocorticoids; D006131:Growth Inhibitors; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D007328:Insulin; D012709:Serum Albumin; D014168:Transferrin; D000509:alpha-Fetoproteins; D053683:Oncostatin M; D017228:Hepatocyte Growth Factor; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone; D014508:Urea; D006003:Glycogen; D019009:Proto-Oncogene Proteins c-kit; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D012643:Selenium