Abstract
Purpose :
Deceased donor limbal stem cells (LSCs) are an under-utilized source of transplant tissue for limbal stem cell deficiency (LSCD). This study sought to determine if specific donor characteristics could be utilized to increase the number of viable LSCs provided for transplant. This will serve as a basis for improving the practicability of using eye-bank procured tissue as a suitable source of LSCs for allogenic limbal stem cell transplants.
Methods :
All work was completed in compliance with the Declaration of Helsinki and Eye Bank Association of America regulations. Research-consented donor (N=12, ages 30-67) eyes were collected and dissected to retain the limbus and a section of sclera in clock-hour sized pieces. Segments were stored in an FDA-approved corneal storage solution for 4 and 7 days. After storage, tissue was processed for immunohistochemistry. Cryosections of the limbus were incubated with p63-α primary antibody (4892S, Cell Signaling) to mark LSCs. The LSCs were quantified by counting all p63-α positive (+) cells in four 20X fields under a Leica DMi8 epifluorescence microscope. This was compared to all bisbenzimide+ nuclei in the same field to calculate the % of cells that are p63-α+. Two cohorts were developed separating Caucasian (n=6) and Non-Caucasian donors (n=6).
Results :
The average density of p63-α+ LSCs in the Caucasian cohort fell from 18% on day 4 to nearly 0% on day 7. In the non-Caucasian cohort, the average density of p63-α+ LSCs was 35% on day 4, and fell to 22% on day 7. A t-test indicated a significant difference between Caucasian vs non-Caucasian cohorts for both day 4 (P=0.023) and day 7 (P<0.001) time points.
Conclusions :
This data builds upon previous knowledge about LSC viability in eye-banked corneas, suggesting that LSCs stored according to U.S. eye bank practices lose their stemness within 4-7 days after procurement. Nonetheless, our data demonstrate that p63-α positive LSCs are viable at higher levels and for a longer duration in the non-Caucasian compared to the Caucasian cohort. Setting donor criteria, similar to how criteria is set for other specialized grafts, could improve LSC viability for surgical transplant to treat LSCD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.