Project Grant R21EY035735

THE ROLE OF LIMBAL FIBROBLASTS FOR THE MAINTENANCE OF LIMBAL STEM CELLS - PROJECT SUMMARY / ABSTRACT CORNEAL EPITHELIUM IS MAINTAINED BY STEM CELLS RESIDING IN THE LIMBUS, THE AREA BETWEEN THE CORNEA AND CONJUNCTIVA. THESE STEM CELLS ARE KNOWN AS LIMBAL STEM CELLS (LSCS) AND GIVE RISE TO PROGENITOR CELLS, WHICH MIGRATE TOWARD CENTRAL AND APICAL CORNEA TO FORM THE STRATIFIED CORNEAL EPITHELIAL CELL LAYERS. LOSS OF LSCS RESULT IN LIMBAL STEM CELL DEFICIENCY (LSCD); THE PATIENTS SUFFER FROM SEVERE VISION LOSS DUE TO THE INGROWTH OF CLOUDY CONJUNCTIVA. WHILE UNILATERAL LSCD CAN BE TREATED BY TRANSPLANTATION OF AUTOLOGOUS LIMBAL TISSUE, PATIENTS WITH BILATERAL LSCD HAVE TO RELY ON ALLOGENEIC TRANSPLANTATION AND THE CLINICAL OUTCOME IS RELATIVELY POOR. IN ADDITION, THERE ARE MULTIPLE POST-OPERATIVE COMPLICATIONS REPORTED INCLUDING RECURRENT/PERSISTENT EPITHELIAL EROSION, INTRAOCULAR PRESSURE ELEVATION, AND REJECTION. MOREOVER, THE SHORTAGE OF CORNEAL DONORS REDUCES THE OPPORTUNITY FOR ALLOGENEIC TRANSPLANTATION. THERE IS, THEREFORE, A CRITICAL NEED TO DEVELOP A LESS INVASIVE THERAPY TO TREAT LSCD THAT ALSO ADDRESSES THE DONOR SHORTAGE. BESIDES THE GRAFT CONDITION, IT IS IMPORTANT TO CONTROL RECIPIENTS' LIMBAL NICHE MICROENVIRONMENT FOR A SUCCESSFUL TRANSPLANTATION, SINCE THERE HAS BEEN GROWING AWARENESS OF THE FACT THAT LIMBAL NICHE CELLS PLAY A CRITICAL ROLE FOR THE MAINTENANCE OF LSCS. OF THE NICHE CELLS, LIMBAL FIBROBLASTS ARE THOUGHT TO CONTRIBUTE TO THE MAINTENANCE OF CORNEAL EPITHELIAL PHENOTYPE BY SECRETING FIBROBLAST GROWTH FACTOR 7 (FGF7), WHOSE SECRETION IS PROMOTED BY PLATELET-DERIVED GROWTH FACTOR (PDGF)-BB AND INTERLEUKIN 1 BETA (IL1B). HOWEVER, IT REMAINS LARGELY UNKNOWN HOW LIMBAL FIBROBLASTS ARE AFFECTED IN THE LSCD AND HOW THE RECOVERY OF LIMBAL FIBROBLASTS CONTRIBUTES TO THE SUCCESS OF THE TREATMENT OF LSCD. OUR LONG-TERM GOAL IS TO DEVELOP A NOVEL TREATMENT FOR LSCD BY RECOVERING THE FUNCTION OF LIMBAL NICHE. OUR OVERALL OBJECTIVE IN THIS PROPOSAL IS TO REVEAL THE CONTRIBUTION OF LIMBAL FIBROBLASTS IN NORMAL AND LSCD CONDITIONS, AND TO ESTABLISH A LIMBAL FIBROBLAST INJECTION AS A NOVEL LSCD THERAPY. OUR CENTRAL HYPOTHESIS IS THAT LIMBAL FIBROBLASTS SECRETE FGF7 IN RESPONSE TO PDGF AND IL1B SECRETED BY LIMBAL DENDRITIC CELLS TO MAINTAIN THE LIMBAL STEM/PROGENITOR CELLS. MOREOVER, WE HYPOTHESIZE THAT LIMBAL NICHE CELLS INCLUDING LIMBAL FIBROBLASTS ARE DISRUPTED IN LSCD AND INJECTION OF LIMBAL FIBROBLASTS REGENERATE THE CORNEAL PHENOTYPE IN LSCD. IN ADDITION, WE WILL UTILIZE A NOVEL TECHNIQUE OF EYE-LIKE ORGANOID FORMATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS (IPSCS) TO CREATE LIMBAL FIBROBLASTS AS A CELL SOURCE OF INJECTABLE CELLS TO RESOLVE THE CORNEAL DONOR SHORTAGE. PROPOSED STUDY WILL REVEAL THE CHARACTERISTICS OF LIMBAL FIBROBLASTS AND LIMBAL DENDRITIC CELLS (AIM 1), EVALUATE THE INTERACTION OF LIMBAL NICHE CELLS AND LIMBAL EPITHELIAL CELLS (AIM 2), AND ESTABLISH LIMBAL FIBROBLAST INJECTION METHOD FOR THE TREATMENT OF MOUSE LSCD (AIM 3). SUCCESSFUL COMPLETION OF THIS PROJECT WILL IMPROVE OUR UNDERSTANDING OF LIMBAL NICHE CELLS IN NORMAL AND LSCD CONDITIONS, AND PROVIDE US WITH ANOTHER STRATEGIC OPTION TO TREAT LSCD, NAMELY LIMBAL FIBROBLAST INJECTION. THIS WILL ALLOW US TO CONDUCT LARGER ANIMAL EXPERIMENTS AND ULTIMATELY, APPLY THIS TREATMENT TO LSCD PATIENTS.