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RETINA Publication-derived

Extracellular Matrix Component Expression in Human Pluripotent Stem Cell-Derived Retinal Organoids

Source Felemban et al., 2018 · Institute of Genetic Medicine, Newcastle University, UK · 10.1016/j.actbio.2018.05.023

👤 Majed Felemban, Birthe Dorgau, Nicola Claire Hunt, Dean Hallam, Darin Zerti, Roman Bauer, Yuchun Ding, Joseph Collin, David Steel, Natalio Krasnogor, Jumana Al-Aama, Susan Lindsay, Carla Mellough, Majlinda Lako ⏱ 164 days 📋 7 phases 🧫 Human ESC (H9), Human iPSC (SB-Ad3)

Abstract

This protocol generates laminated retinal organoids from human pluripotent stem cells (hESCs and hiPSCs) with detailed characterization of extracellular matrix component expression, including Collagen IV, Fibronectin, Versican, Brevican, IMPG1, and CD44. The method recapitulates in vivo human retinogenesis and allows functional studies of ECM components critical for photoreceptor and interphotoreceptor matrix development.

Cell source
Human ESC (H9), Human iPSC (SB-Ad3)
Application
Developmental study; Disease modeling

Protocol overview

39 steps across 7 phases

Preparation of tissue samples and sections Day 0 (preparation phase)
  1. 1 Isolation and fixation of mouse and macaque retinae
  2. 2 Dehydration and embedding of animal tissues
  3. 3 Collection and storage of paraffin sections
  4. 4 Preparation of adult human retinal tissue
  5. 5 Preparation of organ culture medium
  6. 6 Fixation and cryoprotection of adult human eyes
  7. 7 Preparation of human developmental tissue sections
Immunohistochemistry for ECM protein detection Variable (typically 2 days per batch of slides)
  1. 1 Dewax and rehydrate tissue sections
  2. 2 Antigen retrieval by heat treatment
  3. 3 Block non-specific epitope binding
  4. 4 Primary antibody incubation
  5. 5 Secondary antibody incubation and autofluorescence quenching
  6. 6 Mounting with nuclear counterstain
  7. 7 Alternative immunostaining protocol (for organoid sections)
  8. 8 Specificity controls for primary antibodies
  9. 9 Specificity controls for secondary antibodies
Human pluripotent stem cell differentiation to retinal organoids Day 0 to day 150+ (differentiation period)
  1. 1 Culture and expansion of stem cells
  2. 2 Initiation of retinal differentiation (Days 0–2)
  3. 3 Medium supplementation from Day 18 onwards
  4. 4 Collection of retinal organoids at Day 35, 90, and 150
  5. 5 Post-fixation processing and cryoprotection
  6. 6 Cryostat sectioning of retinal organoids
  7. 7 Storage of cryostat sections
Functional blocking studies with IMPG1 and CD44 Day 90–164 (14-day blocking period)
  1. 1 Addition of blocking antibodies at Day 90 or Day 150
  2. 2 Culture with blocking antibodies for 14 days
  3. 3 Assessment of neuroepithelium thickness at Days 7, 10, and 14
  4. 4 Collection of organoids for IHC and qRT-PCR analysis
Image acquisition and analysis Variable (post-immunostaining)
  1. 1 Image capture using fluorescence microscopy
  2. 2 Tissue sample analysis
  3. 3 Image processing and cell segmentation
  4. 4 Quantification of immunostaining
qRT-PCR for gene expression analysis Variable (1–2 days)
  1. 1 Organoid homogenization and RNA extraction
  2. 2 Reverse transcription
  3. 3 qRT-PCR setup
  4. 4 qRT-PCR reaction parameters
  5. 5 Data analysis and relative expression calculation
Statistical analysis Variable (post-data collection)
  1. 1 Data compilation and statistical testing
  2. 2 Non-parametric statistical testing for RNA-seq data
  3. 3 Significance thresholds

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Felemban et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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