Human iPSC derived disease model of USH2A-associated retinitis pigmentosa and the retinal organoid in early stage

Abstract

Purpose : Retinitis pigmentosa (RP) is an irreversible inherited retinopathy. Here, we generated induced pluripotent stem cells (iPSCs) from a RP patient with mutations in the USH2A gene. The morphology changes and transcriptomic analysis of retinal organoid in early stage from such iPSC derived RP disease model is investigated.

Methods : IPS cell line was produced by integration-free CytoTune™-iPS 2.0 Sendai Reprogramming Kit from a RP patient with mutations in USH2A: c.8559-2A>G and c.9127_9129delTCC (Fig. 1A). 3D retinal organoids were generated from such iPSCs through induction-reversal culture as reported.

Results : IPSCs were successfully reprogramed from urine cells, which were positive for Oct4, Klf4, Sox2, and Nanog. They had the capacity to differentiate into cells representative of all three germ layers (Fig. 1B). The retinal organoids from iPSCs gradually grew larger. But the retinal organoids in the RP patient exhibited less diameter and thickness than those in the normal retinal organoids measured by image J software from 15d to 20d (Fig. 1C). Then, we examined transcriptome in retinal organoids on 34d by RNA-Seq analysis. The genes were selected from meaningful groups to obtain heatmaps. Compared with control of normal retinal organoids, there were 1808 down-regulated and 1853 up-regulated genes in the RP patient. The major upregulated genes in the retinal organoids of RP patient included retinal progenitor cells (RPC)-related (especially PAX6, RAX and VSX2), neuron apoptotic process-related (especially HIF1A, ADARB1 and CASP3), while the major downregulated genes involved USH2 complex-related (including WHRN, ADGRV1, PDZD7 and USH2A), basement membrane-related (especially COL4A6, LAMA3 and LAMb1) and tight junction interactions-related (especially CLDN2-7, CLDN19 and CLDN23) genes (Fig. 2).

Conclusions : The retinal organoids in early stage from an USH2A-associated retinitis pigmentosa patient display up-regulated mRNA of neuron apoptotic process-related as well as down-regulated mRNA of basement membrane-related and tight junction interactions-related, which are consistent with decreased diameter and thickness of organoids. Our study suggests some molecular biological characteristics of USH2A-associated retinitis pigmentosa.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Figure 1. Characterization of RP-UiPS cell line and retinal organoids.

 

Figure 2. The transcriptomic characteristics of retinal organoids in early stage.