title: Effects of isoproterenol and cholera toxin on human limbal epithelial cell cultures.
abstract: OBJECTIVE
  Cholera toxin and isoproterenol (β-adrenergic receptor agonist) are largely used to enhance cell proliferation. The aim of the study was to assess the effects of cholera toxin and isoproterenol on growth and differentiation of cells cultured from human superficial limbal explants.
  
  
  METHODS
  Limbal epithelial cells were cultured from superficial limbal explantsin basal medium either supplemented with cholera toxin or isoproterenol for 3 weeks. Growth kinetics and morphometry were studied by light and confocal microscopy. Progenitor and differentiated epithelial cell markers were studied by immunocytochemistry, flow cytometry, Colony Formation Assay, and reverse transcription and polymerase chain reaction.
  
  
  RESULTS
  Cell proliferation was significantly higher with 0.5 µg/ml (p = 0.049), 1 µg/ml (p = 0.005), and 2 µg/ml (p = 0.008) isoproterenol whereas, cholera toxin and 4 µg/ml isoproterenol did not significantly increase cell proliferation. Multilayered epithelial cell sheets were obtained in all culture conditions. Addition of isoproterenol resulted in smaller cell size (p < 0.05) 14 days after cells were cultured, whereas cholera toxin had no effects. Strong expression of cytokeratins 3 and 4/5/6/8/10/13/18 and lower expression of cytokeratin 19, vimentin, and Delta N p63α were observed after 3 weeks of culture with no significant differences in the percentage of positive cells according to culture medium. Colony-forming efficiencies were observed after 2 weeks in all culture condition but not after 3 weeks.
  
  
  CONCLUSIONS
  Isoproterenol was more efficient than cholera toxin for enhancing cell proliferation and resulted in smaller cell size. It appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency.
authors.affiliation: Institut de la Vision, UPMC Université Paris, Paris, France. djida.ghoubay@inserm.fr
authors.forename: Djida
authors.initials: D
authors.lastname: Ghoubay-Benallaoua
authors.forename: Florence
authors.initials: F
authors.lastname: Pécha
authors.forename: Pablo
authors.initials: P
authors.lastname: Goldschmidt
authors.forename: Anne
authors.initials: A
authors.lastname: Fialaire-Legendre
authors.forename: Christine
authors.initials: C
authors.lastname: Chaumeil
authors.forename: Laurent
authors.initials: L
authors.lastname: Laroche
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Borderie
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology*; D003114:Colony-Forming Units Assay; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007545:Isoproterenol / Q000494:pharmacology*; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D000318:Adrenergic beta-Agonists; D015415:Biomarkers; D012333:RNA, Messenger; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014746:Vimentin; D007633:Keratins; D002772:Cholera Toxin; D007545:Isoproterenol