Temporal Differences in the Expression of p63α and Proliferation of Limbal Epithelial Stem Cells From Donor Corneas.

PURPOSE: Limbal stem cell deficiency is a blinding corneal disease that requires transplantation to replenish the limbal epithelial stem cells (LESCs) and restore vision. Keratolimbal allografts from cadaveric donors are commonly used as a source for LESCs. The goal of this study was to verify the efficacy of Food and Drug Administration-approved hypothermic corneal storage solutions in preserving LESCs within human donor corneas over time.

METHODS

Limbal segments from deceased donor corneas were stored in Food and Drug Administration-approved corneal storage solutions for 0, 4, or 7 days. Tissues were then processed for immunohistochemistry, and number of p63α+ cells within the limbus were calculated. LESC density was calculated as the number of p63α+ cells per total number of cells. LESCs were isolated from donor corneas after 7 days of hypothermic storage and subjected to a colony formation assay, and the numbers of holoclones were calculated.

RESULTS

The average density of p63α+ cells was 37.4% on day 0, 27.3% on day 4, and 10.9% on day 7 ( P < 0.05) in limbal segments. Holoclones (15.33 ± 3.5) were formed by cells isolated from the limbal region of donor corneas after 7 days in the hypothermic storage conditions, indicating that viable LESCs are still present in human donor corneas after 7 days in storage.

CONCLUSIONS

Although the density of p63α+ LESCs significantly decreases over time, viable LESCs are present in the donor corneas after 7 days in hypothermic storage conditions. Thus, failure of detection or absence of expression of standard marker(s) does not necessarily indicate the loss of functional LESCs.