Abstract
Purpose :
Ex-vivo expanded limbal stem cells(LSCs) transplantation face challenges in replicating an artificial niche that excludes the use of xenobiotic free products. A new small molecule Wnt mimic, FZD7L-ND, which specifically targets Wnt receptor FZD7, was hypothesized to have both activating effect on the Wnt pathway and improvement of LSCs culture in-vivo using experimental models of luciferase reporter assay and cultured LSC's.
Methods :
The ability of FZD7-ND on inducing Wnt signaling was evaluated by TOP-Flash assay. HEK 293 cells were treated with FZD7L-ND(1-20 uM) and varying concentrations of Wnt3a(2-10.7 nM). Cell viability(fluorescence) and Wnt pathway activity(luciferase) was measured using Microplate Reader. The Wnt pathway activity was expressed as ratio of fluorescence to luciferase. Shapiro-Wilk was used to determine normal distribution. Ordinary one-way ANOVA was used to determine statistical significance. Standard deviation shown as error bars. P-values 0.05 ≤ were considered statistically significant. LSC’s were isolated and cultured from fresh tissue. At nine days, the wells were harvested, counted, and stained for K12, K14, and p63 bright cells. In separate wells, the colonies were stained with 0.5% rhodamine B to determine colony forming efficiency.
Results :
TOPFlash results show significant activation of the Wnt pathway(p value < 0.05), with a significant value around 10 uM of FZD7L-ND. At higher concentrations of Wnt3a(6.7 and 10.7 nM) there is a large exponential increase in the activation[ZJJ1] . In vivo, 10 uM FZD7L-ND has 1.74 fold change improvement in cell proliferation and 2.12 improvement in %p63 bright cells.
Conclusions :
The novel FZD7L-ND has very significant activation of the Wnt pathway in reporter assays. Compared to previous small molecule, FZD7L-ND displays greater selectivity, denoted by the lower concentration needed in the luciferase assay. Greater selectivity helps to create a targeted response while also decreasing concentration required for treatment. While more testing is needed to determine in vivo significance, preliminary tests show that FZD7L-ND influences limbal stem cell proliferation and a key stem cell marker, %p63. FZD7L-ND can potentially have a crucial role in advancing limbal stem cell deficiency therapeutics by helping to maintain and proliferate LSCs in vivo via Wnt pathway activation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.