title: Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures.
abstract: OBJECTIVE
  To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC).
  
  
  METHODS
  Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67.
  
  
  RESULTS
  Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM.
  
  
  CONCLUSIONS
  Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.
authors.affiliation: Eye Hospital, University Medical Centre, Ljubljana, Slovenia.
authors.forename: Zala
authors.initials: Z
authors.lastname: Lužnik
authors.affiliation: Eye Hospital, University Medical Centre, Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Hawlina
authors.affiliation: Blood Transfusion Centre of Slovenia, Ljubljana. Slovenia.
authors.forename: Elvira
authors.initials: E
authors.lastname: Maličev
authors.affiliation: The Veneto Eye Bank Foundation (Fondazione Banca degli Occhi del Veneto), Zelarino-Venice, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Bertolin
authors.affiliation: Medical Faculty Ljubljana, Institute of Microbiology and Immunology, Ljubljana, Slovenia.
authors.forename: Andreja Nataša
authors.initials: AN
authors.lastname: Kopitar
authors.affiliation: Medical Faculty Ljubljana, Institute of Microbiology and Immunology, Ljubljana, Slovenia.
authors.forename: Alojz
authors.initials: A
authors.lastname: Ihan
authors.affiliation: The Veneto Eye Bank Foundation (Fondazione Banca degli Occhi del Veneto), Zelarino-Venice, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferrari
authors.affiliation: Eye Hospital, University Medical Centre, Ljubljana, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Schollmayer
mesh_terms: D000328:Adult; D000650:Amnion; D015703:Antigens, CD / Q000096:biosynthesis*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged
chemical_list: D015703:Antigens, CD