Abstract
Purpose :
Ex vivo cultured limbal stem/progenitor cells is an effective alternative to other surgical treatments for limbal stem cell (LSC) deficiency, but a standard xenobiotic-free method for culturing the LSCs in vitro needs to be optimized. Because Wnt ligands are required for LSC expansion and preservation in vitro, it is theoretically possible to treat the LEC deficiency with recombinant Wnt proteins. However, Wnt ptoteins are highly hydrophobic and require detergents to purify, presenting challenges to effectively generate and study the therapeutic potential of recombinant Wnt proteins. Therefore, we decided to develop a small-molecule to mimic the functions of Wnt proteins.
Methods :
Activation of the Wnt signaling is initiated when secreted Wnt proteins bind to the transmembrane co-receptors Fzd and low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Therefore, to mimic Wnt proteins, we created a small molecule to stimulate Wnt protein-induced oligomerization of Lrp5/6 and Fzd.
Results :
In the study, we used a peptide derived from the N-terminal region of DKK1 (termed as ND) that binds to the first propeller domain of Lrp5/6 and a small molecule (termed as MFH) that binds to the CRD domain of Fzd. Both compounds were Wnt signaling inhibitors and separately they reduced progenitor cell properties in cultured LECs. However, a consolidated molecule linking MFH and ND together acts as a canonical Wnt mimic, the consolidated compound, MFH-ND, induced oligomerization of Lrp5/6 and Fzd and activated Wnt signaling. Moreover, the MFH-ND also enhanced LSC expansion in vitro.
Conclusions :
Our study provides evidence that generating small molecules that mimic growth factors required for LSC preservation and expansion is a feasible xenobiotic-free alternative to the NIH-3T3 feeder layer. As the maintenance of stem cell characteristics in the process of culture expansion is essential for the success of ocular surface reconstruction, the small molecules generated in this study may be helpful in the development of pharmaceutical reagents for treating corneal wounds.
This is a 2020 ARVO Annual Meeting abstract.