Summary Worldwide, only 1 out of 70 patients with corneal endothelial dystrophies and injuries receive a corneal transplant due to the limited corneal donor pool, the need for advanced surgical facilities and talent, and expense. RashmiVu is developing a stem cell based regenerative technology that addresses this large unmet global need through a paradigm shift, wherein treatment could be as simple as an intracameral injection of stem cells as an outpatient procedure. RashmiVu has repeatedly demonstrated successful isolation and expansion of human corneal endothelial stem cells (hCESCs) into millions, confirmed that these cells are hCESCs by analysis of phenotypic markers, and demonstrated their regenerative potential in preliminary animal studies in both mice and rabbits. This Fast-Track STTR seeks to complete development and demonstrate potential products including (a) a single cell suspension for intracameral injection, (b) a single cell formulation that can be “painted” onto the corneal endothelium, and (c & d) sheets of hCESCs and/or their differentiated mature corneal endothelial phenotypes for use in DSEK and DMEK surgeries. Phase I Specific Aims: 1. Cell Yield: Determine tissue procurement inclusion/exclusion criteria that reliably yield 5M+ hCESCs/donor eye, and optimize differentiation to maximize yield of mature CEC phenotypes; 2. Definitive healing: Rapid innate healing in rabbits obfuscates effects of hCESCs (see Prelim Data). The team will complete development of a slow-healing cryoinjury rabbit model and demonstrate definitive hCESC efficacy; 3. Mechanisms: Determine mechanisms potentiated by or convertible to a pharmaceutical pathway. Phase II Specific Aims: 4. Cell suspension formulations: Optimize cell suspensions for injection and targeted delivery including ROCKi pre-treatment, and optimize media and methods for high cell survival after freezing, storage, thawing, and revival; 5. Cell Sheets: Develop monolayer sheets of hCESCs on transplantable substrates, develop a method for consistent differentiation of the hCESC monolayer into a mature CEC monolayer on these sheets, and demonstrate successful partial thickness transplantation (DSEK) in rabbits; 6. Dosage, Safety & Efficacy: Initiate FDA-recommended pre-clinical trials to demonstrate safety, efficacy, effectiveness of targeted delivery approaches including dose response, cell survival, engraftment, distribution, migration, proliferation, tumorigenicity, host immune response, and cellular toxicities. A successful regenerative treatment for corneal endothelial dystrophies and damages could dramatically reduce health care costs, eliminate blindness, and return people to fully functioning members of society, all with an attractive market opportunity.