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

Sep 30, 2026

Protocol modeling occult macular dystrophy uses induced pluripotent stem cells (iPSCs) generated from patient blood cells to produce photoreceptor-like cells. The method introduces a tetracycline-inducible PiggyBac system to stably deliver doxycycline-inducible CRX and NEUROD1, then differentiates iPSCs into iPRCs and validates photoreceptor marker expression. The workflow includes guidance for doxycycline-inducible induction, neomycin selection, single-cell cloning, colony expansion, and marker-based assessment to support mutation-focused studies of inherited retinal dystrophies.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages20

Reading Time50 min

AccessFree to download

Summary

Before you begin

iPSC generation and culture background

CRX/NEUROD1 induction rationale

Innovation

Institutional permissions

Preparation of iPSC culture

Timing

ll OPEN ACCESS Protocol Protocol for differentiating patient-derived iPSCs into photoreceptor-like cells 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. Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Yang Pan, Takeshi Iwata [takeshi.iwata@kankakuki.jp](takeshi.iwata@kankakuki.jp) Highlights Steps for inducing iPSCs with PiggyBac delivery of doxycyclineinducible CRXNEUROD1 Instructions for neomycin selection of stably transducediPSCs Guidance on singlecell cloning and colony expansion Steps for differentiating selected iPSC clones into photoreceptorlike cells (iPRCs) Pan & Iwata, STAR Protocols 7, 104283 March 20, 2026 © 2025 The Author(s) . Published by Elsevier Inc. [https://doi.org/10.1016/](https://doi.org/10.1016/)[ ](https://doi.org/10.1016/)[j.xpro.2025.104283](j.xpro.2025.104283) ll OPEN ACCESS Protocol Protocol for differentiating patient-derived iPSCs into photoreceptor-like cells Yang Pan 1,2 and Takeshi Iwata 1,3,* 1Division of Molecular and Cellular Biology, National Institute of Sensory Organs, NHO Tokyo Medical Center, Tokyo 152- 8902, Japan 2Technical contact 3Lead contact *Correspondence: [takeshi.iwata@kankakuki.jp](takeshi.iwata@kankakuki.jp) [https://doi.org/10.1016/j.xpro.2025.104283](https://doi.org/10.1016/j.xpro.2025.104283) SUMMARY 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 inves tigation of IRD pathogenesis. For complete details on the use and execution of this protocol, please refer to Pan et al.1 BEFORE YOU BEGIN Induced pluripotent stem cells (iPSCs) can be generated from patient-derived cells.2 We have established multiple iPSC lines from circulating T cells of patients with inherited retinal diseases and their unaffected family members using the Sendai virus reprogramming system.1 ,3–5 Initially, iPSCs are cultured on mitomycin C-inactivated mouse embryonic fibroblast feeder cells, then transferred to a feeder-free DEF-CS culture system. Although direct feeder-free human iPSC generation methods are now available,6 feeder cells still improve reprogramming efficiency, especially for older patients commonly seen in inherited retinal diseases. Overexpression of CRX and NEUROD1 in human iPSCs enables reproducible generation of induced photoreceptor-like cells (iPRCs) .7 This protocol describes an induction of iPRCs from patient-derived iPSCs using a PiggyBac delivery system to stably introduce a doxycycline-inducible CRXT2A-NEUROD1 construct (Figures 1A and 1B) . The protocol is optimized for iPSCs derived from an R45W-carrying occult macular dystrophy (OMD) patient and a related control, 1 but is also applicable to other iPSC lines. All cell culture steps should be performed under sterile conditions in a biosafety cabinet, and cells should be maintained in a humidified 37◦ C incubator with 5% CO2. Innovation

FAQs

What is the main purpose of this protocol?

To differentiate patient-derived iPSCs into photoreceptor-like cells using a doxycycline-inducible CRX and NEUROD1 PiggyBac system for inherited retinal dystrophy research.

How are CRX and NEUROD1 introduced into iPSCs?

They are delivered via a tetracycline-inducible PiggyBac vector using doxycycline induction, enabling stable expression of a doxycycline-inducible CRX-T2A-NEUROD1 construct.

What steps are included to obtain and verify differentiated photoreceptor-like cells?

The workflow includes neomycin selection of stably transduced iPSCs, single-cell cloning and colony expansion, differentiation of selected clones, and validation of photoreceptor markers to assess differentiation.

Sep 30, 2026

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