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Zala Luznik, Petra Schollmayer, Marina Bertolin, Elvira Maličev, Sofija Andjelić, Andreja Nataša Kopitar, Alojz Ihan, Stefano Ferrari, Marko Hawlina; Effect of intact amniotic membrane orientation on the phenotypic potential of cultured limbal cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4346. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic membrane (AM) serves as a surrogate limbal niche for successful limbal epithelium (LE) expansion. The AM has an epithelial and a stromal side with different biological properties. The effect of intact AM orientation on the phenotypic potential of cultured limbal cells from limbal explants is still not well determined. Thus, we hypothesized that a difference in expression of epithelial, mesenchymal and putative stem cell markers would be observed between cultures cultivated on intact AM (on both sides) compared to cultures cultivated on plastic culture plates without AM (control group).
The preserved cadaveric human corneoscleral rim of 9 donors was cut into 2 mm2 sized explant samples. Each limbal explant was cultured on AM (N=11) or on culture plastic plates (N=13) using only human serum supplemented culturing medium. Intact AM was positioned with either AM intact epithelial side up (N=5) or the AM stromal side up (N=6). The expression of mesenchymal markers (CD73, CD90, CD105), proliferation and putative progenitor markers (CD184, CD117), and epithelial markers (MUC, CK7) was determined by flow cytometry. Immunohistochemistry on secondary cultures on AM was carried out with antibodies against pancytokeratin, p63, Ki67. Student’s t-test was used for statistical analysis.
On the 3rd culturing day cell outgrowth was observed in all the limbal explants. After one-week cell proliferation accelerated and a change in cell phenotype was observed. Cells cultivated on culture plates without AM became more elongated and spindle shaped. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Expression of mesenchymal markers was significantly higher (p < 0.05) in cultures cultivated without AM. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM. However, no statistically significant difference was observed between CD117 and CD184 positive cells in all tested conditions.
Limbal cells cultivated on AM exhibited a lower expression profile of mesenchymal cell markers than limbal cells cultivated on plastic plates, which is consistent with our hypothesis. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67 being observed between the two tested sides.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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