Relevant Cells Derived Human iPSC: A Model To Study Leber Congenital Amaurosis

Abstract

Purpose: : The global objective of our project is based on the scientific concept that human pluripotent stem cells, which express a disease-related mutant gene, could be used to model human diseases. However, a key challenge in the field is the demonstration of disease-related phenotypes that could represent a relevant cellular model allowing analytic and therapeutic research for the disease.

Methods: : We have used this approach to obtain a pathological model of a rare monogenic disease, the Leber Congenital Amaurosis (LCA). LCA is the most severe retinal dystrophy causing blindness or severe visual impairment before the age of one year. Linkage analysis, homozygosis mapping and candidate gene analysis facilitated the identification of 14 genes mutated in patients with LCA and juvenile retinal degeneration, which together explain approximately 70% of the cases. By comparing native and mutant induced Pluripotent Stem Cells (iPSC) lines at the exon level, the aim of our study is to identify biomarkers which present alteration in gene expression, protein cell content or interaction, cellular or sub-cellular morphological abnormalities associated with the genotype leading to LCA pathology.

Results: : The first task of our study was to derive two iPSC lines, from fibroblasts of two LCA patients. The second step was to differentiate mutant and native iPSC into more specialized cells, Neural Stem Cells (NSC) that mimic the neural tube stage and Retinal Pigment Epithelial (RPE) cells that could be targeted by the disease. Indeed, biomarkers related to LCA are most likely identifiable in cells that have reached a certain stage of differentiation. These cells have also the advantage of being homogeneous and easily amplifiable. To find relevant biomarkers on these cells we performed transcriptome analysis using Affymetrix Exon Array GeneChip®.

Conclusions: : The third part of the project has begun and is dedicated to the validation of these biomarkers by qPCR and Western Blot. This will be the starting point of physiopathological analysis and to a large scale drug-screening in hope to find therapeutic for this form of LCA.

Keywords: pathology: human • differentiation • gene screening