High-Purity Functional Corneal Endothelial Cells From Human Induced Pluripotent Stem Cells via a Novel Wash-Out Method

Abstract

Corneal endothelial failure can cause blindness, with transplantation as the only treatment. Due to donor shortages, establishing robust methods for generating corneal endothelial-like cells (CECs) from induced pluripotent stem cells (iPSCs) is critical. Differentiation protocols included iPSC-to-CEC induction with or without neural crest cell differentiation. CECs directly differentiated from iPSCs demonstrated robust expression of CEC-specific markers and a hexagonal morphology. The wash-out protocol is a novel, efficient, noncytotoxic method for removing undifferentiated iPSCs and obtaining CEC populations with high purity. Single-cell sequencing data showed that iPSC–CECs with wash-out were similar to human primary CECs. In vivo transplantation of iPSC–CECs into a corneal endothelial dysfunction (CED) rabbit model demonstrated their safety and therapeutic efficacy, with improved corneal transparency. 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. This work establishes an effective stem cell-based platform for producing corneal endothelium-like cells with clinical-grade quality, offering a scalable and regenerative alternative to conventional transplantation.

Keywords

animal model / cell therapy / clinical-grade human induced pluripotent stem cells / corneal endothelial-like cells / corneal endothelial dysfunction / single-cell RNA sequencing

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