Modeling retinal degeneration using induced pluripotent stem cells from patients with the common P347L mutation in the RHODOPSIN gene

Abstract

Purpose: Inherited retinal degenerations, associated with photoreceptor loss leading to blindness or visual impairment, affect more than one million people throughout the world. The recent discovery of direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells offers the opportunity to model in vitro the effects of mutations on human retinal development. We have focused on the most prevalent form of autosomal dominant retinitis pigmentosa (adRP) in European cohorts, resulting from a mutation (substitution P347L) on the gene coding for the visual pigment RHODOPSIN (RHO).

Methods: Reprogramming of fibroblasts is performed by a non integrative approach using Sendai Virus expressing the four reprogramming genes, OCT4, KLF4, SOX2 and C-MYC. The stemness status of the induced pluripotent stem (iPS) cells is assessed phenotypically and by the expression of specific markers both by qPCR and immunohistochemistry. Differentiation of iPS cell lines towards retinal and photoreceptor lineages is performed using a well-established protocol (Reichman et al. PNAS 2014; 111:8518).

Results: Fibroblasts of three patients from the same family (including one healthy patient) have been successfully reprogrammed into iPS cells. All generated iPS cell lines expressed the specific markers of pluripotency SSEA-4, TRA-1-81, OCT4 and NANOG. Q-PCR analysis confirmed the absence of the Sendai viral reprogramming genes and each iPS cell lines exhibited a normal karyotype. Normal and RHODOPSIN-mutated iPS cell lines were able to generate neural retina (NR)-like structures, containing retinal progenitors within 2 weeks. Culturing these NR-like structures in floating conditions, we have obtained photoreceptor (precursors and matures) from both normal and mutated cells. However after long-term cultures rod and cone cell numbers were significant lower in NR-like structures differentiated from RHODOPSIN-mutated iPS cell lines than in structures differentiated from normal iPS cells.

Conclusions: From these results we conclude that mature photoreceptors differentiated from RHODOPSIN-mutated iPS cells degenerate in a RP-specific manner, demonstrating the ability of patient-specific photoreceptors to recapitulate the disease phenotype in vitro.