Author/Editor     Lužnik, Zala; Hawlina, Marko; Maličev, Elvira; Bertolin, Marina; Kopitar, Andreja Nataša; Ihan, Alojz; Ferrari, Stefano; Schollmayer, Petra
Title     Effect of cryopreserved amniotic membrane orientation on the expression of limbal mesenchymal and epithelial stem cell markers in prolonged limbal explant cultures
Type     članek
Vol. and No.     Letnik 11, št. 10
Publication year     2016
Volume     str. 1-17
ISSN     1932-6203 - PloS one
Language     eng
Abstract     Purpose 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 puta- tive 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 immunophenoty ped for the co-express ion of mesenchymal stem cell markers (CD73/CD90/ CD105 positive and CD45 negative), prolif- eration and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistoch emistry 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. Conclusion 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.
Keywords     stem cell
markers
limbal
matične celice
označevalci
rob med beločnico