title: Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector.
abstract: BACKGROUND
  The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment.
  
  
  METHODS
  In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry.
  
  
  RESULTS
  Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene.
  
  
  CONCLUSIONS
  This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nanba
authors.forename: Natsuki
authors.initials: N
authors.lastname: Matsushita
authors.forename: Fujio
authors.initials: F
authors.lastname: Toki
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
mesh_terms: D016475:3T3 Cells; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006494:Heparin Antagonists / Q000494:pharmacology; D006583:Hexadimethrine Bromide / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D011725:Pyridines / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D006494:Heparin Antagonists; D011725:Pyridines; C485184:enhanced green fluorescent protein; C108830:Y 27632; D049452:Green Fluorescent Proteins; D006583:Hexadimethrine Bromide; D054460:rho-Associated Kinases