Abstract
Purpose :
Descemet’s membrane (DM) is a promising substrate for cultivated limbal epithelial transplantation (CLET) due to its tranparency and resistance to degradation. The purpose of this study is to compare the stemness of donor limbal stem cells cultured (LSC) on DM versus human amniotic membrane (HAM).
Methods :
Limbal explants fragments were obtained from the corneoscleral rim of donor corneas after peeling off DM. The isolated explant fragments were then cultured in serum-free media on tissue culture plastic for one week to support LSC outgrowth from the fragments. The cultured LSCs were then trypsinized, resuspended, and then reseeded on either HAM or DM. Reseeded LSCs were then cultured on both HAM and DM for an additional week. After one week, culture specimens were fixed in formulin. Immunohistochemistry was then used to compare expression of putative stem cell markers (p63, ABCG2) between each culture substrate. In-cell western was performed to further quantify the relative expression of p63, ABCG2, and ABCB5. Celltag 700 (LI-COR Biosciences, Lincoln, NE) was used to normalize the infrared fluorescence to cell number in the in-cell western assay.
Results :
In total, five donor corneas were obtained for testing. LSCs cultured from each individual donor cornea were reseeded on separate sets of DM and HAM for comparison. Two sets of LSC cultures were assayed for putative stem cell marker expression (p63, ABCG2) using immunohistochemistry. Three sets of LSC cultures were assayed for marker expression (p63, ABCG2, ABCB5) using in-cell western. For cells cultured on DM, 82% of cells expressed p63, 70% expressed ABCG2, and 60% showed co-localized expression by immunohistochemistry. In contrast, for cells cultured on HAM, only 57% expressed p63, 16% expressed ABCG2, and 15% showed co-localized expression. Similarly, on in-cell western, there was greater expression of ABCG2 (0.094 vs. 0.028 relative infrared fluorescence) and p63 (0.011 vs 0.009 relative infrared fluorescence) among cells cultured on DM compared HAM. However, there was also less expression of ABCB5 among cells cultured on DM compared to HAM (0.090 vs 0.14 relative infrared fluorescence).
Conclusions :
DM is a viable culture substrate for ex vivo expansion of limbal stem cells. DM preserved ABCG2 and p63 expression in cultured LSCs better than HAM. However, HAM preserved expression of ABCB5 better than DM.
This is a 2020 ARVO Annual Meeting abstract.