Therapeutic Potency of Induced Pluripotent Stem-Cell-Derived Corneal Endothelial-like Cells for Corneal Endothelial Dysfunction

Abstract

Corneal endothelial cells (CECs) 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 (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the 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 using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. 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. 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. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

A p-Tyr42 RhoA Inhibitor Promotes the Regeneration of Human Corneal Endothelial Cells by Ameliorating Cellular Senescence

“…Corneal endothelial cells (CECs), which are located at the innermost layer of the cornea and dehydrate the corneal stroma, are different from vascular endothelial cells [ 55 ]. CECs originate from neural crest cells [ 55 ].…”

“…Corneal endothelial cells (CECs), which are located at the innermost layer of the cornea and dehydrate the corneal stroma, are different from vascular endothelial cells [ 55 ]. CECs originate from neural crest cells [ 55 ]. TGF-β is a multifunctional cytokine that plays a crucial role in regulating cell growth, differentiation, migration, and survival [ 20 , 56 ].…”

Current state of endothelial cell therapy

“…This significantly reduces the number and quality of HCECs available for culture [24]. Additionally, compared to cells from young donors, cells from older donors grow more slowly, and exhibit high heterogeneity as well as characteristics of senescence [26]. Also, the proliferative capacity of HCECSs is limited to an average of three-to-four subcultures, beyond which the cells become fibroblastic or senescent [27].…”

Early Visibility of Cellular Aggregates and Changes in Central Corneal Thickness as Predictors of Successful Corneal Endothelial Cell Injection Therapy

“…They are typically maintained in a non-proliferative state in vivo but have been shown to proliferate in vitro [ 4 , 14 ]. Numerous groups have reported a variety of cell sources and methods to achieve functional CECs, including deriving CECs from the expansion of primary donor cadaveric CECs [ 15 , 16 , 17 , 18 ], human embryonic stem cells (hESC) [ 19 , 20 , 21 , 22 ] and human induced pluripotent stem cells (hiPSC) [ 23 , 24 , 25 ]. Ong et al also recently reported functional outcomes with an alternative method that did not require complex cellular propagation techniques.…”