DESCRIPTION (provided by applicant): Many mammalian organs (skin, stomach, intestines, colon, and eye) possess a source of adult stem cells that continually replenishes their rapidly self-renewing epithelial surface. One important challenge in regenerative medicine is replacing these stem cells when they are eliminated following an injury or disease. The eye contains two highly specialized stratified squamous epithelia- the conjunctival epithelium and the corneal epithelium. A healthy corneal epithelium is essential for maintaining a clear cornea and normal vision. The limbus contains a small subpopulation of rare limbal stem cells (LSC) that continually repopulates the corneal epithelium. Patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in "conjunctivalization" of the corneal stroma that triggers neovascularization, chronic inflammation, and ultimately blindness due to an irreversibly opaque cornea. Several approaches have been used to replace LSC by transplanting limbal tissue or ex vivo expanded limbal cells. These procedures have obtained some success, mainly using autologous limbal tissue from patients with unilateral LSCD. Patients with bilateral LSCD have no source of autologous LSC and much less success was observed with allogeneic limbal tissue transplants. However, success of all these procedures was severely limited by the inability to prospectively identify and purify LSC. This problem was recently addressed by a new collaborative research group from the departments of: Ophthalmology, Genetics, and Transplantation Research (Mass Eye & Ear, Brigham & Women's Hospital and Boston Children's Hospital) that comprises the three PIs of this proposal, who discovered that the ABCB5 gene, a new member of the ATP-binding cassette (ABC) superfamily of active transporters, is expressed by stem cells of the limbus in both mouse and human tissues. Normal function of ABCB5+ LSC is required for corneal development and repair, through critical roles in stem cell maintenance and survival, and knockout mice that lack ABCB5 do not develop a fully differentiated mature corneal epithelium. Importantly, ABCB5 is a cell surface protein and specific monoclonal antibodies developed by the laboratories of Co-PIs Drs. M. Frank and N. Frank are capable of isolating pure ABCB5-positive cells from the limbus. Transplantation of purified human ABCB5+ (but not ABCB5-) LSC onto the corneal stroma of immunodeficient mice with induced LSCD restored the corneal epithelium, indicating that this purified LSC population has the potential to significantly improve therapy for corneal disease associated with LSCD. The current application builds upon these results to address the important challenges that prevent successful stem cell therapy for patients with unilateral or bilateral LSCD. Our overall hypothesis is that ABCB5+ stem cells from the limbus can be isolated and expanded ex vivo as a source of stem cells to...