Abstract
Purpose :
Corneal endothelial cells (CEC) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSC). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model.
Methods :
iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected by Wesetern blot and immunocytochemistry, and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). CCK-8 assay was done to assess cell viability and proliferation.
Results :
After culturing iPSC-derived NCCs to induce CECs, we found the expression of zona occludens-1 (ZO-1), Na+/K+ ATPase, SLC4A11, N-Cadherin, NCAM, and a morphology of hexagonal shape. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation’s in vitro and in vivo efficacy.
Conclusions :
Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. The transplantation did not induce host immune responses, indicating the maintenance of immune privilege and no allograft rejection. Our results showed in vitro and in vivo capacity of iPSC-CEC to demonstrate the efficacy and safety of transplant that may be a powerful source of clinical therapy for patients with CED.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.