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RETINA Publication-derived

Transcriptome-based molecular staging of human stem cell-derived retinal organoids with accelerated photoreceptor differentiation by 9-cis retinal

Source Kaya et al., 2019 · National Eye Institute, National Institutes of Health

👤 Koray D. Kaya, Holly Y. Chen, Matthew J. Brooks, Ryan A. Kelley, Hiroko Shimada, Kunio Nagashima, Natalia de Val, Charles T. Drinnan, Linn Gieser, Kamil Kruczek, Slaven Erceg, Tiansen Li, Dunja Lukovic, Yogita K. Adlakha, Emily Welby, Anand Swaroop ⏱ 205 days 📋 8 phases 🧫 Human ESC (H9) and Human iPSC (PEN8E, NEI377, ESP1, ESP2)

Abstract

This protocol generates three-dimensional retinal organoids from human pluripotent stem cells (hESCs and hiPSCs) through a multi-stage differentiation process. The method enables molecular staging of organoid maturation by comparing transcriptome profiles to human fetal and adult retinal data, and demonstrates that 9-cis retinal supplementation accelerates rod photoreceptor differentiation more effectively than all-trans retinoic acid.

Cell source
Human ESC (H9) and Human iPSC (PEN8E, NEI377, ESP1, ESP2)
Application
Developmental study and disease modeling of retinal organoid differentiation

Protocol overview

58 steps across 8 phases

Pluripotent Stem Cell Maintenance Before D0
  1. 1 Culture hESCs in Essential 8 medium under hypoxia
  2. 2 Culture hiPSCs in appropriate media based on derivation method
  3. 3 Passage pluripotent stem cells
Embryoid Body Formation and Neural Induction D0–D7
  1. 1 Dissociate and form embryoid bodies
  2. 2 Add neural induction medium gradually (D1–D2)
  3. 3 Culture in 100% NIM (D3–D7)
  4. 4 Collect EBs at D7
Retinal Fate Specification and Optic Vesicle Formation D7–D42
  1. 1 Switch to Photoreceptor Induction Medium at D16
  2. 2 Excise optic vesicles (D21–D28)
  3. 3 Continue suspension culture in PIM (D28–D42)
Organoid Maturation with Retinoid Supplementation D42–D120+
  1. 1 Supplement PIM after D42
  2. 2 Add retinoid supplementation at D63 (Option A: 9-cis retinal)
  3. 3 Add retinoid supplementation at D63 (Option B: All-trans retinoic acid)
  4. 4 Add N2 supplement at D92
  5. 5 Continue culture until desired stage
Immunohistochemistry Analysis At harvest
  1. 1 Fix organoids
  2. 2 Cryoprotect in sucrose gradient
  3. 3 Embed in freezing medium
  4. 4 Section organoids
  5. 5 Perform immunostaining
  6. 6 Image immunostained sections
Immunoblot Analysis At harvest
  1. 1 Lyse individual organoids
  2. 2 Incubate lysate
  3. 3 Clarify lysate
  4. 4 Quantify protein
  5. 5 Prepare samples for electrophoresis
  6. 6 Perform SDS-PAGE
  7. 7 Transfer proteins to membrane
  8. 8 Block nonspecific binding
  9. 9 Incubate with primary antibody cocktail
  10. 10 Wash membrane
  11. 11 Incubate with secondary antibody
  12. 12 Wash membrane again
  13. 13 Detect chemiluminescence
  14. 14 Dry and re-probe membrane
  15. 15 Analyze and export images
Transmission Electron Microscopy At harvest (typically D120–D130)
  1. 1 Primary fixation
  2. 2 Wash after primary fixation
  3. 3 Osmium tetroxide fixation
  4. 4 Wash and buffer exchange
  5. 5 En bloc staining in uranyl acetate
  6. 6 Dehydration series
  7. 7 Transition with propylene oxide
  8. 8 Infiltration with resin mixture
  9. 9 Embedding in pure epoxy resin
  10. 10 Ultramicrotomy
  11. 11 Counter-stain sections
  12. 12 Stabilize sections
  13. 13 Electron microscopy imaging
RNA-seq and Transcriptome Analysis At harvest (various time points D25–D205)
  1. 1 Extract high-quality total RNA
  2. 2 Prepare directional mRNA library
  3. 3 Perform paired-end sequencing
  4. 4 Perform sequence alignment and quantitation
  5. 5 Perform differential expression analysis
  6. 6 Perform gene clustering
  7. 7 Perform Gene Ontology enrichment analysis
  8. 8 Perform molecular staging with dynamic time warping
  9. 9 Perform co-inertia analysis

Full SOP

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Attribution

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

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