Generation of retinal models from two iPSC lines derived from patients harboring mutations in PDE6A and PDE6C genes.

Abstract

Purpose : Inherited retinal dystrophies are a large group of heterogeneous diseases that often lead to permanent vision loss due to dysfunction or death of photoreceptor cells. Retinitis pigmentosa (RP) and Achromatopsia (ACHM) have been related with pathogenic variants in rod and cone-specific cyclic guanosine monophosphate (cGMP) phosphodiesterase α (PDE6A) and α’ (PDE6C) subunit genes, respectively.
Our aim is to generate photoreceptor progenitors (PhRPs) and retinal organoids (ROs) derived from two patient-iPSC lines: FRIMOi001-A, harboring two heterozygous mutations (c.305G>A and c.1268delT) in PDE6A, and FRIMOi007-A, carrying a homozygous mutation (c.1670G>A) in PDE6C.

Methods : We followed a PhRPs differentiation protocol (2D), described by Barnea-Cramer et al., and ROs differentiation protocol, described by Gonzalez-Cordero et al.

Results : Through 2D protocol, there is no possibility to develop the outer segment of photoreceptor cells, where PDE6 proteins are located. However, PDE6A mRNA expression was detected in PhRPs, but not PDE6C, as the amount of rod PhRPs obtained was superior to cones. After 225 day of differentiation, ROs derived from our patient’s iPSC lines contained a big amount of cone and rod photoreceptor cells which enable us to study PDE6A and PDE6C genes (Figure 1). Furthermore, ROs generated photoreceptor outer segments, permitting us to study PDE6 family proteins location. We observed that in PhRPs and ROs derived from FRIMOi001-A iPSC line PDE6A mRNA expression was significantly lower than in control lines. In ROs-derived FRIMOi007-A iPSC line, PDE6C protein was observed in cone photoreceptor outer segments and PDE6C expression showed differences in patient ROs compared to control ROs.

Conclusions : PhRPs and ROs derived from a RP patient carrying PDE6A mutations allowed quantifying PDE6A mRNA expression, and it seemed lower than controls, however, more replicates are needed for conclusive results.
We also proved that ROs are useful to study cone photoreceptor cells-specific genes, as we demonstrated in our Achromatopsia patient ROs with PDE6C mutation. The preliminary results indicated a decrease in PDE6C expression in ROs derived from the patient.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Immune fluorescence staining of RO-derived FRIMOi007-A iPSC line shows expression of PDE6H (cone-specific cGMP phosphodiesterase γ subunit protein) and RHO (rhodopsin) at 33 weeks of retinal differentiation.