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RETINA Publication-derived

Retinal Organoids from Rhesus Macaque iPSCs

Source Lopez et al., 2022 · Department of Ophthalmology & Vision Science, School of Medicine, U.C. Davis · 10.1111/cpr.13198

👤 Antonio Jacobo Lopez, Sangbae Kim, Xinye Qian, Jeffrey Rogers, J. Timothy Stout, Sara M. Thomasy, Anna La Torre, Rui Chen, Ala Moshiri ⏱ 100 days 📋 4 phases 🧫 Rhesus macaque iPSC (Macaca mulatta)

Abstract

This protocol generates three-dimensional retinal organoids from rhesus macaque induced pluripotent stem cells (rhiPSCs) through a stepwise neural induction and retinal differentiation process. The resulting organoids develop laminated retinal tissue containing photoreceptors, Müller glia, bipolar cells, and other retinal cell types, with differentiation occurring approximately 30–40% faster than human iPSC-derived retinal organoids due to the shorter gestational period of rhesus macaques.

Cell source
Rhesus macaque iPSC (Macaca mulatta)
Application
Disease modeling and developmental biology; preclinical retinal allograft transplantation studies

Protocol overview

22 steps across 4 phases

rhiPSC2431 Derivation and Stem Cell Culture Day 0–5+
  1. 1 Generate rhesus macaque fibroblasts from skin punches
  2. 2 Transfect fibroblasts with reprogramming plasmids using Neon system
  3. 3 Culture transfected cells for 4–5 days
  4. 4 Maintain rhiPSC2431 colonies in defined xeno-free medium
  5. 5 Maintain rhiPSC89 and rhiPSC90 on MEF feeders
  6. 6 Maintain human H9 ESCs for comparison
Embryoid Body Formation and Neural Induction Day 0–7
  1. 1 Prepare iPSC colonies for differentiation
  2. 2 Treat colonies with dispase
  3. 3 Prepare neural induction media
  4. 4 Plate fragmented colonies in non-adherent conditions
  5. 5 Sequential weaning of EBs into neural induction media
  6. 6 Treat EBs with BMP4 on day 6
  7. 7 Plate EBs on Matrigel on day 7
  8. 8 Media changes on days 9 and 12
2D Retinal Differentiation and Optic Vesicle Formation Day 7–30
  1. 1 Media change to retinal differentiation media on day 15
  2. 2 Continue RDM media changes through day 30
  3. 3 Observe optic vesicle-like structures
  4. 4 Pick retinal organoids on day 30
3D Retinal Organoid Culture and Maturation Day 30–100+
  1. 1 Resuspend organoids in 3D retinal differentiation media
  2. 2 Culture organoids with RA supplementation until day 100
  3. 3 Culture organoids beyond day 100 without RA
  4. 4 Monitor morphological stages

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lopez et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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