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AR Djalilian, CA Koch, RB Nussenblatt, Z Zhuang, EJ Holland, D Shen, CC Chan; The Use of Microsatellite Analysis to Determine the Survival of Donor Epithelial Cells After Limbal Stem Cell Transplantation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2232.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the survival and distribution of donor epithelial cells in patients who have undergone limbal allograft transplantation for stem cell deficiency. Methods:Corneal buttons were obtained at the time of penetrating keratoplasty from patients who had previously undergone limbal stem cell transplantation. Routine histology was performed to evaluate the pathology in 4 different quadrants of the corneal button. The central and peripheral epithelium from the corneal buttons were carefully microdissected and procured either manually or by laser capture microdissection (Arcturus). The DNA was isolated from the epithelial samples and from the patients' peripheral blood monocytes in proteinase K enriched buffer. For each DNA sample, selected microsatellite markers on different chromosomes including 4S1825, 5S820, 8S1128, and 14S306 were amplified and labeled with P32. The PCR products were subjected to 6% polyacrylamide gel electrophoresis. Results:The first corneal button analyzed was removed 4 months after limbal allograft transplantation. There were healed penetrating corneal scars with mild neovascularization in the periphery and focal corneal epithelial defect in every quadrant of the corneal button. Microsatellite analysis revealed the peripheral epithelial cells to be exclusively donor derived, while the central epithelium appeared to be mostly recipient cells. Conclusion:Corneal buttons removed at the time of keratoplasty from patients with previous limbal allograft transplantation can be used to identify molecular fingerprints of recipient vs. donor epithelial cells. This technique may be useful for determining the long-term survival of the donor epithelial cells and the need for continued systemic immunosuppression. It is also helpful for better understanding the mechanism of stem cell transplantation and corneal surface disorders.
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