Project Summary The corneal limbal stem cells (LSC) play a vital function in homeostasis and wound healing. Damage to the LSC and other pathologies associated LSC deficiency (LSCD) cause cornea neovascularization, corneal opacification, and vision loss. Current treatment paradigm is based on the transplantation of LSC into patients to restore the LSC niche. Substantial effort over the last few years aims at identifying sources of LSC for transplantation with varying degree of success. However, our recent work indicates that transplantation of LSC might not be the the only useful treatment paradigm for LSCD. We showed that committed epithelial cells that have lost the stem cell marker K15 are able to dedifferentiate and repopulate the stem cell niche. If this process can be controlled and manipulated it could provide alternative treatment that can avoid the need for complex stem cell transplantation. We have shown that the niche is important for the process of dedifferentiation demonstrating that dedifferentiation and repopulation of the niche require communication between cells that remain in the niche and corneal epithelial cells. Here we will use cutting edge long term multi-day imaging of corneal organ culture to investigate both sides of the communication between the niche and epithelial cells. In Aim #1 we will take a pharmacological approach and screen 99 different compounds and acquire long timelapse movies of the restoration of the LSC niche in the presence of different drugs. The high content analysis will provide key information on the different signaling pathways used by the niche to recruit and induce the dedifferentiation of epithelial cells. In Aim #2 we will focus on the receiver side of the communication and analyze the source of cells used to repopulate the niche. Using advanced light sheet microscopy approaches we will track individual cells and discover the identity of the cells that are capable of dedifferentiation. Single cell tracking will show whether the ability to dedifferentiate is ubiquitous or whether only a small subset of cells maintain the ability to dedifferentiate. The successful completion of these aims will provide key insights into the physiological process of LSC niche recovery and will pave the way to the development of new treatments to LSCD.