title: Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study.
abstract: BACKGROUND
  Cell therapy for large burns is dependent upon autologous epidermis reconstructed in vitro. However, the effectiveness of current procedures is limited by the delay needed to culture the patient's own keratinocytes. To assess whether the keratinocyte progeny of human embryonic stem cells (hESCs) could be used to form a temporary skin substitute for use in patients awaiting autologous grafts, we investigated the cells' capability of constructing a pluristratified epidermis.
  
  
  METHODS
  hESCs from lines H9 and SA01 were seeded at least in triplicate on fibroblast feeder cells for 40 days in a medium supplemented with bone morphogenetic protein 4 and ascorbic acid. Molecular characterisation of cell differentiation was done throughout the process by quantitative PCR, fluorescence-activated cell sorting, and immunocytochemical techniques. Keratinocyte molecular differentiation and functional capacity to construct a human epidermis were assessed in vitro and in vivo.
  
  
  RESULTS
  From hESCs, we generated a homogeneous population of cells that showed phenotypic characteristics of basal keratinocytes. Expression levels of genes encoding keratin 14, keratin 5, integrin alpha6, integrin beta4, collagen VII, and laminin 5 in these cells were similar to those in basal keratinocytes. After seeding on an artificial matrix, keratinocytes derived from hESCs (K-hESCs) formed a pluristratified epidermis. Keratin-14 immunostaining was seen in the basal compartment, with keratin 10 present in layers overlying the basal layer. Involucrin and filaggrin, late markers of epidermal differentiation, were detected in the uppermost layers only. 12 weeks after grafting onto five immunodeficient mice, epidermis derived from K-hESCs had a structure consistent with that of mature human skin. Human involucrin was appropriately located in spinous and granular layers and few Ki67-positive cells were detected in the basal layer.
  
  
  CONCLUSIONS
  hESCs can be differentiated into basal keratinocytes that are fully functional--ie, able to construct a pluristratified epidermis. This resource could be developed to provide temporary skin substitutes for patients awaiting autologous grafts.
  
  
  BACKGROUND
  Institut National de la Santé et de la Recherche Médicale, University Evry Val d'Essonne, Association Française contre les Myopathies, Fondation René Touraine, and Genopole.
authors.affiliation: INSERM/UEVE U-861, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic diseases, Evry Cedex, France.
authors.forename: Hind
authors.initials: H
authors.lastname: Guenou
authors.forename: Xavier
authors.initials: X
authors.lastname: Nissan
authors.forename: Fernando
authors.initials: F
authors.lastname: Larcher
authors.forename: Jessica
authors.initials: J
authors.lastname: Feteira
authors.forename: Gilles
authors.initials: G
authors.lastname: Lemaitre
authors.forename: Manoubia
authors.initials: M
authors.lastname: Saidani
authors.forename: Marcela
authors.initials: M
authors.lastname: Del Rio
authors.forename: Christine C
authors.initials: CC
authors.lastname: Barrault
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Bernard
authors.forename: Marc
authors.initials: M
authors.lastname: Peschanski
authors.forename: Christine
authors.initials: C
authors.lastname: Baldeschi
authors.forename: Gilles
authors.initials: G
authors.lastname: Waksman
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D000078404:Epidermal Cells; D000091344:Filaggrin Proteins; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology*; D017768:Skin, Artificial; D023822:Tissue Engineering
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007633:Keratins