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Praveen Joseph Susai Manickam, Divya Pidishetty, Gopal Kushawah, Puja Sarkar, Vinay Kumar Pulimamidi, Savitri Maddileti, Sreedhar Rao Boyenpally, Dilip Kumar Mishra, Rakesh Mishra, Indumathi Mariappan; Generation of in vitro and in vivo models of LCA12. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3788.
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© ARVO (1962-2015); The Authors (2016-present)
To establish developmental model systems to evaluate the effects of LCA12 mutations in RD3 gene
To establish an in vitro model system of LCA12, a patient-specific iPSC line (hiPSC-VS-3F) harboring c.296+1G>A mutation in human RD3 iPSC line (hiPSC-F2-3F) . Direct differentiation of 80% confluent cultures was done using the differentiation medium (DM: DMEM/F12, 4% KOSR, 4% FBS, 1X NEAA, 1X Glutamax, 1X Pen-Strep) for 3 days. Subsequently, the cultures were shifted to retinal differentiation medium (RDM: DM + 2% B27) for a month to induce eye field specification. The distinct eye field primordial (EFP) clusters that emerged at 4 weeks were excised and grown as suspension cultures in neuro-retinal medium (NRM: RDM + 1% N2 + 5 ng/mL bFGF) to obtain patient-specific retinal organoids (hiPSC-VS-OC) and healthy control retinal organoids (hiPSC-F2-OC). Simultaneously, an in vivo model system of LCA12 was created by CRISPR/Cas9 editing of zebrafish rd3 gene to generate homozygous mutant fishes (rd3-/-). The normal and mutant retinal tissues were further characterized by RT-PCR and IHC.
Both hiPSC-F2-3F and hiPSC-VS-3F maintained their stemness, pluripotency, genetic identity and genome stability. Our optimized eye field differentiation protocol yielded about 40.05±3.89 EFPs per million cells/well of a 6-well plate (n=3) in case of hiPSC-F2-3F and a similar efficiency was observed in hiPSC-VS-3F. The EFPs excised from both the lines readily formed 3D retinal organoids in suspension cultures and expressed CHX10, CRX, NEUROD1, RCVRN and RLBP1. However, RD3 expression could be detected only in hiPSC-F2-OC. Upon further culture and maturation, the neuro retinal precursors within patient-specific hiPSC-VS-OCs showed delayed differentiation and lamination defects. Similarly, the developing rd3-/- zebrafish displayed lamination defects and underdeveloped outer segments. Thinning of outer nuclear layer and possible loss of rod cells were also observed.
A patient-specific iPSC line and a homozygous mutant zebrafish line were generated as in vitro and in vivo models of LCA12. Lamination and maturation defects in both the model systems indicate possible roles of RD3 in early retinal development. Detailed molecular characterization of these disease models would help in further understanding of the role(s) of RD3 mutations in LCA12 pathogenesis.
This is a 2020 ARVO Annual Meeting abstract.
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