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RETINA Publication-derived

A highly reproducible and efficient method for retinal organoid differentiation from human pluripotent stem cells

Source Harkin et al., 2024 · Indiana University School of Medicine · 10.1073/pnas

👤 Jade Harkin, Kiersten H. Peña, Cátia Gomes, Melody Hernandez, Sailee S. Lavekar, Kaman So, Kelly Lentsch, Elyse M. Feder, Sarah Morrow, Kang-Chieh Huang, Kaylee D. Tutrow, Ann Morris, Chi Zhang, Jason S. Meyer ⏱ 150 days 📋 10 phases 🧫 Human hESC (H7, H9) and Human iPSC (IMR90-4, WTC11, PGP1, JM2019)

Abstract

This protocol describes a highly standardized and efficient method for generating 3D retinal organoids from human pluripotent stem cells (hPSCs) at 100% efficiency with enhanced reproducibility. By utilizing quick reaggregation methods to control early aggregate size and timed BMP4 signaling activation, the protocol enables pure populations of retinal organoids from multiple hPSC lines while also facilitating the study of earliest stages of human retinal fate specification.

Cell source
Human hESC (H7, H9) and Human iPSC (IMR90-4, WTC11, PGP1, JM2019)
Application
Disease modeling and developmental study of human retinogenesis

Protocol overview

61 steps across 10 phases

Biological Reagent Preparation Pre-protocol
  1. 1 Prepare BMP4 Stock Solution
  2. 2 Prepare DHA Stock Solution
  3. 3 Prepare Retinoic Acid Stock Solution
  4. 4 Prepare BDNF Stock Solution
iPSC Reprogramming and Cell Line Establishment Weeks 1–6
  1. 1 Obtain and Thaw PBMCs
  2. 2 Transduce PBMCs with Reprogramming Factors
  3. 3 Transition Transduced Cells to Pluripotent Medium
  4. 4 Identify and Manually Passage Primitive Colonies
  5. 5 Expand Colonies on Matrigel-Coated Plates
  6. 6 Transition to Automated Passaging at Passage 6
  7. 7 Karyotype Analysis and Cryopreservation
CRISPR/Cas9 Gene Editing (BRN3b-EGFP-Thy1.2 Reporter) Weeks 1–3 (post-reprogramming)
  1. 1 Design and Synthesize Targeting Plasmid
  2. 2 Prepare Plasmids for Electroporation
  3. 3 Electroporat Plasmids into JM2019 iPSCs
  4. 4 Culture Electroporated Cells
  5. 5 Dissociate and Sort GFP-Positive Cells
  6. 6 Clonal Expansion and PCR Validation
Early-Stage Cell Aggregation (Days 0–8) Days 0–8
  1. 1 Dissociate hPSC Colonies and Seed into 96-Well Plates
  2. 2 Initiate Neural Induction (Day 0)
  3. 3 Transfer Aggregates to 10 cm Dishes (Day 1)
  4. 4 Replace Neural Induction Medium (Day 2)
  5. 5 Scale Up to Complete Neural Induction Medium (Day 3)
  6. 6 Apply BMP Signaling Modulation (Day 6)
  7. 7 Collect and Plate Aggregates in 6-Well Plates (Day 8)
Early Retinal Differentiation (Days 9–16) Days 9–16
  1. 1 First Media Exchange with Reduced BMP4/LDN (Day 9)
  2. 2 Additional Media Exchanges (Days 12 and 15)
  3. 3 Transition to Retinal Differentiation Medium (Day 16)
Mid-Stage Retinal Development (Days 18–22) Days 18–22
  1. 1 Increase FBS to 3% (Day 18)
  2. 2 Increase FBS to 5% (Day 20)
  3. 3 Transition to Advanced-RDM (Day 22)
Late-Stage Maturation and Photoreceptor Development (Days 22–150+) Days 22–150+
  1. 1 Routine Medium Changes (Days 22–50)
  2. 2 Begin Late-Stage Supplementation (Day 50)
  3. 3 Remove Retinoic Acid (Day 90)
  4. 4 Extended Culture Beyond Day 90
Immunocytochemistry and Microscopy Analysis At designated timepoints (typically Days 8, 16, 30, 50, 70, 150)
  1. 1 Brightfield Imaging
  2. 2 Fix Organoids in Paraformaldehyde
  3. 3 Wash in PBS
  4. 4 Cryoprotection in Sucrose Solutions
  5. 5 Embed in OCT and Freeze
  6. 6 Cryosection at 12 µm
  7. 7 Permeabilize Tissue Sections
  8. 8 Block Non-Specific Binding
  9. 9 Apply Primary Antibodies Overnight
  10. 10 Wash After Primary Antibody
  11. 11 Apply Secondary Antibodies
  12. 12 Final Wash and Mounting
  13. 13 Confocal Microscopy Imaging
  14. 14 Time-Lapse Imaging of Aggregate Formation
Quantification and Statistical Analysis Post-imaging
  1. 1 Quantify Aggregate and Organoid Size
  2. 2 Calculate Circularity
  3. 3 Perform Statistical Testing
RNA-seq and Transcriptional Profiling (Optional) Days 6–8
  1. 1 Sample Preparation and Grouping
  2. 2 RNA Extraction
  3. 3 RNA Quality Assessment
  4. 4 cDNA Library Preparation
  5. 5 RNA Sequencing
  6. 6 Quality Control of Sequencing Data
  7. 7 Map Reads to Human Genome
  8. 8 Quantify Gene Expression
  9. 9 Differential Expression Analysis
  10. 10 Pathway Enrichment Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Harkin et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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