Clonally selected hypoimmune limbal stem cells: an in vitro proof-of- concept towards an off-the-shelf therapy for limbal stem cell deficiency

Limbal stem cell deficiency (LSCD), a highly morbid and uncomfortable condition, causes progressive corneal opacification and vision loss through irreversible loss of the limbal epithelial stem cell pool. Current transplantation strategies are limited by significant challenges across both autologous and allogeneic approaches: allogeneic grafts require long-term systemic immunosuppression and carry poor long-term survival, while even autologous transplants, free from rejection, suffer approximately one-third graft failure, partially attributable to donor variability. Here, we report the generation of hypoimmune limbal stem cells (LSCs) engineered to evade allogeneic immune recognition while retaining full stem cell identity and differentiation competence. Using ROCK inhibitor-supported long-term culture, we demonstrate stable LSC expansion exceeding 202 population doublings, with preserved transcriptomic identity and differentiation capacity. Simultaneous CRISPR-Cas9-mediated knockout of B2M and CIITA, combined with lentiviral CD47 overexpression, conferred complete in vitro evasion of CD4⁺ and CD8⁺ T cell responses and abrogated macrophage-mediated phagocytosis. These findings establish an in vitro proof-of-concept framework towards a universal, off-the-shelf allogeneic LSC product that eliminates the need for systemic immunosuppression and reduces donor-dependent variability, with broad implications for the scalable treatment of both unilateral and bilateral LSCD.