Abstract
Purpose :
With this study, our aim is to demonstrate that differentiation of limbal epithelial stem cells (LESCs) from human pluripotent stem cells (hPSC) provides new tools for modeling corneal epithelial renewal in addition to the great potential as novel treatment modality to restore functional LESC.
Methods :
Two hPSC lines were subjected to LESC differentiation with previously established method. Phenotypic characterization of undifferentiated and differentiated cells was conducted using immunofluorescent labeling (IF) with selected limbal/corneal epithelial and pluripotency markers. IF results in most relevant time points were verified with additional hPSC line. The number of cells expressing OCT3/4, ABCG2, p63α, ΔNp63 (p40), CK15 and CK14 were analyzed at days 10 and 24. Quantitative PCR and flow cytometry analysis were used to confirm the ABCG2 expression levels in the early (day 10-11) and more differentiated (day 24) cell populations. Importantly, the culture conditions supporting the differentiation towards the p63α-positive phenotype were modified to support the expansion of ABCG2-positive cell population. Furthermore, the functionality of this cell population was analyzed with population doubling and colony forming efficiency assays without mouse 3T3 cells and serum.
Results :
With established method, we are able to produce corneal progenitor populations highly expressing p63α/p40 markers. Interestingly in this study, ABCG2, a universally proposed marker of limbal stemness, is only transiently expressed in cells cultured in epithelial differentiation medium. Thus, this cell population represented a noticeably different LESC- like phenotype (being strongly ABCG2-positive) as compared to later arising cell population with high level of progenitor marker expression (ABCG2-negative/p63a-positive). In the novel stem cell preservation conditions, we were able to expand ABCG2-positive cells over several passages. Also, clear cell line dependent functional differences were observed.
Conclusions :
Phenotypical characterization of hPSC-LESC differentiation revealed a consecutive emergence of two distinct limbal cell -like populations. Based on our current research hypothesis and results, these two cell populations may represent functionally different stem/progenitor populations with implications in their regenerative efficacy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.