Associate Research Professor University of Ulsan College of Medicine, United States
Abstract: Failure of corneal endothelial function can cause corneal transparency and blindness issues, and currently, corneal transplantation is the only available treatment. Given the shortage of donor corneas, establishing robust methods for generating corneal endothelial-like cells (CECs) from induced pluripotent stem cells (iPSCs) is critical. We developed and utilized a novel method to generate homogenous CEC populations by washing undifferentiated stem cells out of the differentiated populations and demonstrated their therapeutic efficacy and safety in an animal model of corneal endothelial dysfunction (CED). Using clinical GMP-grade human iPSCs derived from cord blood, CEC differentiation was induced with or without the neural crest cell (NCC) stage for 14 to 28 days. The wash-out protocol allows more differentiated CECs to adhere to a VTN-coated surface in the initial stage, whereas the remaining population, with relatively weaker attachment ability, is washed out and removed. We identified robust CEC-specific markers on iPSC-derived CECs with hexagonal morphology. Using a wash-out method, we significantly improved the purity and differentiation efficiency of the CEC population. Single-cell sequencing data showed that iPSC-CECs with wash-out were similar to human primary CECs based on the differentially expressed genes (DEGs) associated with focal adhesion, cell-substrate junctions, mitochondrial protein complex, metabolic activity, and extracellular vesicles. In vivo, transplantation of iPSC-CECs into a CED rabbit model demonstrated their safety and therapeutic efficacy, with improved corneal transparency. Surviving transplanted cells were observed on the endothelium for up to 16 weeks post-transplantation. We successfully differentiated clinical GMP-grade human iPSC into CEC in vitro. In vitro, results using a panel of iPSC- and CEC-markers proved the efficient and successful induction of CECs through direct differentiation from iPSCs using the novel wash-out method. Notable recovery of corneal clarity in the CED model, without graft rejection, highlights the in vitro and in vivo potential of iPSC-CECs as a powerful source for clinical therapy in patients with CED.
