Small-Molecule Conversion of Neural Crest Cells to Corneal Endothelial Cells for Endothelial Regeneration

Purpose: Corneal endothelial dysfunction leads to irreversible blindness, yet treatment remains constrained by a global shortage of donor tissue. This study aimed to develop a donor-independent strategy for generating functional corneal endothelial cells (CECs) by reprogramming murine neural crest cells (NCCs) through pharmacological inhibition of TGF-β signaling. Methods: NCCs were genetically labeled and isolated from E13.5 Wnt1-Cre;Rosa26-LSL-tdTomato mouse embryos by fluorescence-activated cell sorting. Sorted NCCs were treated with the selective TGF-β inhibitor SB431542 to induce conversion into induced corneal endothelial–like cells (iCECs). Cell identity was validated by immunofluorescence, flow cytometry, wound-healing and endocytic uptake assays, and bulk RNA sequencing with comparison to primary CECs and NCCs. Therapeutic efficacy was assessed by transplanting iCECs into a rabbit model of corneal endothelial dysfunction induced by descemetorhexis. Results: SB431542 treatment efficiently converted NCCs into polygonal, cobblestone-like iCECs that expressed canonical CEC markers (ZO-1, Na⁺/K⁺-ATPase, AQP1, Laminin) while silencing neural crest genes. Approximately 85% of iCECs were Ki67-positive, and cells achieved complete wound closure within 24 hours. Transcriptomic profiling confirmed that iCECs closely resembled primary CECs and were clearly distinct from NCCs. Following transplantation, iCECs reconstituted a confluent endothelial monolayer, normalized central corneal thickness, and restored corneal transparency by Day 35, whereas untreated controls remained opaque. Conclusions: Targeted TGF-β inhibition enables efficient lineage conversion of NCCs into functional corneal endothelial–like cells with robust proliferative capacity and in vivo therapeutic efficacy. This pharmacologically defined strategy provides a scalable, donor-independent cell source and offers a promising therapeutic approach for corneal endothelial diseases.