Transplantation of human induced pluripotent stem cell-derived neural crest cells for corneal endothelial regeneration

BackgroundThe corneal endothelium maintains corneal hydration through the barrier and pump function, while its dysfunction may cause corneal edema and vision reduction. Considering its development from neural crest cells (NCCs), here we investigated the efficacy of the human induced pluripotent stem cell (hiPSC)-derived NCCs for corneal endothelial regeneration in rabbits.MethodsDirected differentiation of hiPSC-derived NCCs was achieved using the chemically defined medium containing GSK-3 inhibitor and TGF-beta inhibitor. The differentiated cells were characterized by immunofluorescence staining, FACS analysis, and in vitro multi-lineage differentiation capacity. For in vivo functional evaluation, 1.0x10(6) hiPSC-derived NCCs or NIH-3T3 fibroblasts (as control) combined with 100 mu M Y-27632 were intracamerally injected into the anterior chamber of rabbits following removal of corneal endothelium. Rabbit corneal thickness and phenotype changes of the transplanted cells were examined at 7 and 14days with handy pachymeter, dual-immunofluorescence staining, and quantitative RT-PCR.ResultsThe hiPSC-derived NCCs were differentiated homogenously through 7days of induction and exhibited multi-lineage differentiation capacity into peripheral neurons, mesenchymal stem cells, and corneal keratocytes. After 7days of intracameral injection in rabbit, the hiPSC-derived NCCs led to a gradual recovery of normal corneal thickness and clarity, when comparing to control rabbit with fibroblasts injection. However, the recovery efficacy after 14days deteriorated and caused the reappearance of corneal edema. Mechanistically, the transplanted cells exhibited the impaired maturation, cellular senescence, and endothelial-mesenchymal transition (EnMT) after the early stage of the in vivo directional differentiation.ConclusionsTransplantation of the hiPSC-derived NCCs rapidly restored rabbit corneal thickness and clarity. However, the long-term recovery efficacy was impaired by the improper maturation, senescence, and EnMT of the transplanted cells.