Abstract
Purpose :
Understanding effective limbal epithelial stem cell maintenance is necessary to improve limbal stem cell deficiency (LSCD) treatment using ex vivo cultured limbal stem/progenitor cells (LSCs). We have previously demonstrated that canonical Wnt signaling is crucial for limbal stem cell maintenance and that Wnt2, Wnt6, Wnt11, and Wnt16b are upregulated in the limbus relative to the cornea. The purpose of this study is to investigate the effect and mechanism of supplementing Wnt16b in the LSC cultures on the expansion of the stem/progenitor cell population in vitro.
Methods :
Wnt16b was overexpressed in 3T3 cells, which do not express Wnt16b, using a Wnt16b-IRES-GFP lentivirus vector. An IRES-GFP vector served as a control. Transduced 3T3 cells were sorted into low, medium, and high GFP expression groups. LSCs were isolated from five donors and cultured on low, medium, and high Wnt16b-expressing 3T3s compared to their respective GFP controls and an uninfected 3T3 control. After 12-14 days in culture, the cultivated LSCs were evaluated by quantifying cells expressing K12, K14, and high levels of p63α. Canonical and non-canonical Wnt pathway markers expressed in the cultivated LSCs were evaluated by quantitative real-time PCR (qRT-PCR).
Results :
LSCs cultured on low Wnt16b-3T3 cells, which most closely corresponded to levels of Wnt16b in the human limbus in vivo, contained significantly higher percentages of p63α-bright cells and the non-canonical Wnt marker phosphorylated cJun than LSCs cultured on low GFP-3T3 cells. LSCs cultured on Wnt16b-3T3s also differentially expressed Wnt6, Wnt7a, and Wnt7b. Proliferation, colony-forming efficiency, and cell size were not significantly affected.
Conclusions :
Wnt16b contributes to maintaining a fine balance of canonical and non-canonical pathways to regulate limbal stem cell proliferation, differentiation, and self-renewal in vitro.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.