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Protocol for differentiating patient-derived iPSCs into photoreceptor-like cells

Year: 2025
Type: article
Abstract: Retinal biopsies are rarely feasible, limiting direct studies of inherited retinal dystrophies (IRDs). We present a protocol modeling occult macular dystrophy, an autosomal-dominant IRD, using induced pluripotent stem cells (iPSCs) generated from patient blood cells. We describe steps to differentiate iPSCs into photoreceptor-like cells by transducing CRX and NEUROD1 via a tetracycline-inducible PiggyBac vector. We also detail procedures for the validation of photoreceptor markers to assess differentiation. This protocol enables mutation-focused investigation of IRD pathogenesis. For complete details on the use and execution of this protocol, please refer to Pan et al. 1 • Steps for inducing iPSCs with PiggyBac delivery of doxycycline-inducible CRX-NEUROD1 • Instructions for neomycin selection of stably transduced iPSCs • Guidance on single-cell cloning and colony expansion • Steps for differentiating selected iPSC clones into photoreceptor-like cells (iPRCs) Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Retinal biopsies are rarely feasible, limiting direct studies of inherited retinal dystrophies (IRDs). We present a protocol modeling occult macular dystrophy, an autosomal-dominant IRD, using induced pluripotent stem cells (iPSCs) generated from patient blood cells. We describe steps to differentiate iPSCs into photoreceptor-like cells by transducing CRX and NEUROD1 via a tetracycline-inducible PiggyBac vector. We also detail procedures for the validation of photoreceptor markers to assess differentiation. This protocol enables mutation-focused investigation of IRD pathogenesis.
DOI: 10.1016/j.xpro.2025.104283
Authors: Yang Pan, Takeshi Iwata
Language: English
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W7116124205
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Cites: 7
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