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RETINA Publication-derived

Generation of Cone-Rich Human Retinal Organoids from hESCs

Source Kim et al., 2019 · Albert Einstein College of Medicine · 10.1073/pnas.1901572116

👤 Sangbae Kim, Albert Lowe, Rachayata Dharmat, Seunghoon Lee, Leah A. Owen, Jun Wang, Akbar Shakoor, Yumei Li, Denise J. Morgan, Andre A. Hejazi, Ales Cvekl, Margaret M. DeAngelis, Z. Jimmy Zhou, Rui Chen, Wei Liu ⏱ 270 days 📋 5 phases 🧫 Human ESC (H1) and Human iPSC

Abstract

This protocol describes the differentiation of human embryonic stem cells (H1) and induced pluripotent stem cells into cone-rich laminar retinal organoids with mature photoreceptor outer segments. The organoids resemble the human macula/fovea based on cone-to-rod ratio (1.4:1) and single-cell transcriptomes, and exhibit functional photoreceptor properties suitable for studying retinal development and disease.

Cell source
Human ESC (H1) and Human iPSC
Application
Retinal disease modeling and developmental study

Protocol overview

21 steps across 5 phases

hESC/hiPSC Maintenance Continuous
  1. 1 Culture hESCs on Matrigel-coated plates
Retinal Cyst Formation Day 0–5
  1. 1 Prepare small hESC aggregates
  2. 2 Gel the Matrigel suspension
  3. 3 Disperse in N2B27 medium for floating culture
  4. 4 Observe cyst formation
  5. 5 Passage cysts to new culture plates
Retinal Progenitor Cell Proliferation and Patterning Day 5–15
  1. 1 Culture cysts for attachment and growth
  2. 2 Monitor retinal progenitor zone formation
Retinal Organoid Formation and Expansion Day 15–30
  1. 1 Treat adherent cultures with dispase
  2. 2 Rinse and culture in B27 medium
  3. 3 Collect spontaneously detached floating organoids
  4. 4 Allow retinal organoid self-assembly
  5. 5 Transfer to orbital shaker culture
  6. 6 Add FCS to culture medium
  7. 7 Change medium every 3 days
Retinal Differentiation and Photoreceptor Maturation Day 30–270+
  1. 1 Verify retinal epithelial structure and early differentiation
  2. 2 Monitor stratified retinal tissue formation
  3. 3 Identify cone and rod photoreceptor differentiation
  4. 4 Confirm cone enrichment
  5. 5 Verify outer segment structure by electron microscopy
  6. 6 Continue long-term culture

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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