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RETINA Publication-derived

Retinal Organoids for Disease Modelling (2020, No. 2)

Full SOP with Academic or a plan

Source Chichagova et al., 2020 · Institute of Genetic Medicine, Newcastle University · 10.1002/stem.3116

Authors: Valeria Chichagova, Gerrit Hilgen, Ali Ghareeb, Maria Georgiou, Madeleine Carter, Evelyne Sernagor, Majlinda Lako, Lyle Armstrong Duration: 180 days 7 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of retinal organoids from human iPSC lines using two distinct differentiation methods that modulate either BMP4 (Method I) or IGF1 (Method II) signaling pathways. The protocol produces three-dimensional retinal structures containing photoreceptors, retinal ganglion cells, Müller glia, and other retinal cell types with variable efficiency depending on iPSC line and method chosen. The organoids demonstrate light-responsive functional properties and serve as platforms for disease modeling and developmental studies.

Cell source
Human iPSC
Application
Disease modeling and developmental study of retinal organogenesis

Protocol overview

13 steps across 7 phases

Phase 1 · iPSC Maintenance and Pluripotency Confirmation Prior to differentiation
  1. a Expand iPSC lines to required cell density
Phase 2 · Method I: BMP4 Pathway Activation Days 0-18 (initial phase)
  1. a Initiate BMP4-dependent retinal differentiation
Phase 3 · Method II: IGF1 Pathway Activation Days 0-18 (initial phase)
  1. a Initiate IGF1-dependent retinal differentiation
Phase 4 · Maturation Phase with Supplementary Factors (Day 18 onwards) Days 18-180+
  1. a Add supplementary maturation factors
  2. b Monitor morphological development through day 85 and day 169
  3. c Continue culture to day 180 for gene and protein expression analysis
Phase 5 · Gene Expression Analysis (Day 180) Day 180
  1. a Extract RNA and perform quantitative PCR
Phase 6 · Protein Expression and Localization Analysis (Day 180) Day 180
  1. a Perform immunofluorescence microscopy for photoreceptor markers
  2. b Stain for non-photoreceptor retinal cell types
Phase 7 · Functional Assessment: Light-Driven Responses (Days 150-180+) Days 150-180+
  1. a Prepare organoids for electrophysiological recording
  2. b Apply white light stimulus and record RGC responses
  3. c Apply sustained blue light stimulus to identify intrinsically photosensitive RGCs
  4. d Quantify change of firing (COF) and compare between methods

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Attribution

This SOP was written by Organthis based on the published method in Chichagova et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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