Modeling Glaucoma: Retinal Ganglion Cells Generated from Induced Pluripotent Stem Cells of Patients with SIX6 Risk Allele Show Developmental Abnormalities

Abstract

Glaucoma represents a group of multifactorial diseases with a unifying pathology of progressive retinal ganglion cell (RGC) degeneration, causing irreversible vision loss. To test the hypothesis that RGCs are intrinsically vulnerable in glaucoma, we have developed an in vitro model using the SIX6 risk allele carrying glaucoma patient-specific induced pluripotent stem cells (iPSCs) for generating functional RGCs. Here, we demonstrate that the efficiency of RGC generation by SIX6 risk allele iPSCs is significantly lower than iPSCs-derived from healthy, age- and sex-matched controls. The decrease in the number of RGC generation is accompanied by repressed developmental expression of RGC regulatory genes. The SIX6 risk allele RGCs display short and simple neurites, reduced expression of guidance molecules, and immature electrophysiological signature. In addition, these cells have higher expression of glaucoma-associated genes, CDKN2A and CDKN2B, suggesting an early onset of the disease phenotype. Consistent with the developmental abnormalities, the SIX6 risk allele RGCs display global dysregulation of genes which map on developmentally relevant biological processes for RGC differentiation and signaling pathways such as mammalian target of rapamycin that integrate diverse functions for differentiation, metabolism, and survival. The results suggest that SIX6 influences different stages of RGC differentiation and their survival; therefore, alteration in SIX6 function due to the risk allele may lead to cellular and molecular abnormalities. These abnormalities, if carried into adulthood, may make RGCs vulnerable in glaucoma. Stem Cells 2017;35:2239-2252.

Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types

“…[31][32][33][34] Likewise, RGCs with specific mutations, differentiated from patientderived hiPSCs, have provided models for optic atrophy and glaucoma. 35,36 The relative ease of creating specific mutations with CRISPR/Cas9 genome editing should make it possible to take advantage of the cell typespecific reporters in PGP1 when modeling genetic retinal diseases. 32 PGP1-derived retinal organoids will provide a platform to screen drugs to treat retinal damage.…”

Retinal ganglion cells harboring the OPTN(E50K) mutation exhibit neurodegenerative phenotypes when derived from hPSC-derived three dimensional retinal organoids

“…Human pluripotent stem cells (hPSCs) provide an attractive option as a model for studies of cellular development and disease progression as they can be cultured indefinitely and induced to differentiate into all cell types of the body (Thomson et al, 1998), including RGCs (Deng et al, 2016; Gill et al, 2016; Langer et al, 2018; Ohlemacher et al, 2016; Riazifar et al, 2014; Tanaka et al, 2015; Teotia et al, 2017a). When harboring genetic mutations associated with disease states, the derivation of these cells from patient-specific sources allows for the ability to study mechanisms underlying diseases such as glaucoma (Inagaki et al, 2018; Minegishi et al, 2013; Ohlemacher et al, 2016; Teotia et al, 2017b). Additionally, gene editing approaches including CRISPR/Cas9 technology allow for the ability to create isogenic controls from these patient-derived cells and also introduce disease-causing mutations in unaffected cells, leading to the generation of new disease models (Cong et al, 2013).…”

Transcriptome analysis of the zebrafishatoh7−/−mutant,lakritz, highlights Atoh7-dependent genetic networks with potential implications for human eye diseases

“…These findings are in agreement with the present study reporting six6a being an Atoh7-upregulated gene. Remarkably variant forms of SIX6 (SIX9/OPTX2) have been linked to congenital microphthalmia as well as to the development of glaucoma20,29,32,[114][115][116][117][118][119] . Given that mutations in ATOH7 have been also associated with similar global eye disorders14,120 , these observations strongly point at the importance to further understand the interplay ATOH7, RX1 and SIX6 during eye development, and to assess how disruption of this evolutionarily conserved genetic network might be linked to such eye disorders 121 .Besides atoh7, rx1 and six6a, significant differentially expressed genes annotated with "neural retina development" were tubgcp4, gnl2, smarca5, mmp14a, znf503, and hsp70.1.…”