Abstract
Purpose :
In order to advanced understanding the pathogenesis of macular corneal dystrophy (MCD), we created a cell model in vitro by using induced pluripotent stem cells (iPSCs), which carrying compound heterozygous mutations in the CHST6 gene.
Methods :
In this sturdy, we recruited an MCD family with three siblings. The phenotype of each patient was demonstrated by detailed clinical examination. Genetic analysis by targeting next-generation sequencing (NGS), which verified compound heterozygous variation in the CHST6 gene (NM_021615): c.62_63delTinsGA (p. L21delinsRfs) and c.C892T (p. Q298X). Generation of MCD-iPSCs from specific urine cells and passed through neural crest stem cells (NCSCs) contribute to the corneal stromal-like cells (CSCs). CSCs characteristic was confirmed by immunofluorescence staining and real-time polymerase chain reaction(qPCR).
Results :
The pedigree of the MCD-family. Genotype information of compound heterozygous mutations performed sanger sequencing. Pre-operative photograph of the eyes of II.5 demonstrating ill-defined corneal stromal despaired. Confocal microscopy images (Fig. 1,2 A-B). The morphology of MCD-iPSC, which be able to differentiated into all three layers (Fig. 3A). We adopted adherent culture(2D) and three-dimensional (3D) spheroid culture of NCSC differentiation medium, qPCR displayed 3D state promoted the expression of the characteristic markers of NCSCs (Fig. 3B). The characterization of iPSC-Derived CSC (Fig. 3C).
Conclusions :
NCSCs undergo general and coordinated movements away from folds of the neural ectoderm to different regions of the embryo to give rise to ocular cells. We use patient-derived iPS cells via regulating NCSCs induction to simulating a MCD disease model in vitro. This information serves as platform for further analyses of the molecular networks involved in MCD, and provide insights into cell-based therapies, drug discovery.
This is a 2020 ARVO Annual Meeting abstract.