Abstract
Purpose :
Differentiation of limbal epithelial stem cells (LESC) from human pluripotent stem cells (hPSC) provides new tools for studying cornea development in humans. Here, we have employed our established protocol for a minute examination of the hPSC-LESC differentiation process. With detailed phenotypical and functional analyses conducted in several time points, we aim to gain further knowledge of the underlying mechanisms that regulate the differentiation of hPSCs towards LESCs, and subsequently through transient amplifying cell (TAC) phase into stratified corneal epithelium.
Methods :
Two genetically distinct hPSC lines, one induced and one embryonic, were subjected to corneal differentiation in previously established, defined serum- and feeder cell-free conditions. Phenotypic characterization of undifferentiated hPSC and derivative cells at days 7, 9, 11, 14, 17, 21 and 24 was carried out using immunofluorescent labeling (IF) against a panel of selected limbal/corneal epithelial and pluripotency markers. Protein expression in each time point was first analyzed qualitatively. The expression development of p63α was quantified by counting the percentage of positive cells in each time point. The number of cells expressing OCT3/4, ABCG2, p63α, CK15 and CK14 were further quantified from cytospin samples acquired at days 10 and 24.
Results :
Qualitative analysis of IF demonstrated a decrease of pluripotency markers OCT3/4 and SSEA4, and ascending expression of putative LESC markers p63α, CK15 and CK14 during the 24 day examination period. Interestingly, ABCG2, a universally proposed marker of stemness, was only transiently expressed at days 9-11. The cell populations at day 10-11 as compared to day 24 represented a noticeably different phenotypes, the previous being ABCG2-positive, CK14/CK15-negative and the latter being ABCG2-negative, CK14/CK15-positive. Further molecular and functional characterization of these cell populations is currently in progress.
Conclusions :
Phenotypical characterization of hPSC-LESC differentiation revealed a consecutive emergence of two distinct LESC-like populations. We believe that these changes in hPSC-LESC phenotype in vitro represent the LESC differentiation process in vivo. Thus, hPSC-LESCs provide a potentially useful model for studying the mechanisms of corneal development and epithelial maintenance in vitro.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.