Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is the most common primary corneal endothelial dystrophy and the leading indication for corneal transplantation globally. Generation of corneal endothelial cells (CEnCs) from induced pluripotent stem cells (iPSCs) can reduce the burden on the availability of corneal transplants. An iPSC-derived CEnC (iCEnC) model of FECD will allow investigation of its unclear etiology, thereby providing novel venues to treat the disease.
Methods :
Peripheral mononuclear blood cells (PBMCs) isolated from FECD patients and healthy volunteers were reprogrammed to iPSCs with the necessary institutional approvals. Characterization of the iPSCs lines was determined by their expression of pluripotent genes and ability to form embryoid bodies. The iPSC were differentiated to CEnC lineage in xeno-free and chemically defined culture conditions. The iCEnCs were characterized based on the expression of CEC markers by immunofluorescence and qPCR. The iCEnCs were evaluated ex-vivo for their integrative capacity on endothelium-denuded donor cornea. In-vitro FECD modelling was done by menadione (MN) treatment of the iCEnCs. Validation of the FECD model was done by fluorescence microscopy, qPCR and western blots.
Results :
Generated iPSCs lines expressed the pluripotency markers NANOG, OCT4 and SOX2. Embryoid bodies could be generated from all the iPSC lines and possessed tri-lineage differentiation potential. All the iPSC lines possessed normal karyotype and are being sequenced to identify the putative gene mutations. More than 90% of iCEnCs expressed CEnC markers. Post-transplantation of iCEnCs into the decellularized donor cornea, cultured for 7 days shows the presence of attached cells when visualized under the specular microscope. Immunofluorescence staining showed expression of Na+K+ATPase and Non-muscle myosin IIA in iCEnCs transplanted donor corneal flat mounts after 25 days in culture. iCEnC-based FECD model with MN recapitulated the disease phenotype with loss of iCENCs due to apoptosis along with the formation of typical excrescence positive for extracellular matrix (ECM) proteins
Conclusions :
iCENCs-based model of FECD will aid as a screening platform to identify newer therapeutic strategies. Allogenic iCENCs can serve as a potential source of transplant to address the loss of CEnCs in the near future.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.