Skip to content
← Back to browse
RETINA Publication-derived

Production of Retinal Cells from Confluent Human iPS Cells

Source Reichman et al., 2014 · INSERM, Institut de la Vision, Université Paris Descartes · 10.1007/7651_2014_143

👤 Sacha Reichman, Olivier Goureau ⏱ 21 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of confluent human iPSCs into retinal cell types without embryoid body formation or exogenous molecules. In 2 weeks, iPSCs generate retinal pigmented epithelial cells and self-forming neural retina-like structures containing retinal progenitor cells that can be further differentiated into all retinal cell types, including retinal ganglion cells and photoreceptors, for regenerative medicine applications.

Cell source
Human iPSC
Application
Regenerative medicine and disease modeling of retinal degenerative diseases

Protocol overview

58 steps across 6 phases

Maintenance and Expansion of hiPS Cells Ongoing prior to differentiation
  1. 1 Coat culture dishes with gelatin
  2. 2 Plate feeders
  3. 3 Thaw hiPS cells
  4. 4 Transfer cells to culture medium
  5. 5 Centrifuge cells
  6. 6 Remove supernatant
  7. 7 Resuspend and plate cells
  8. 8 Culture cells in incubator
  9. 9 Change medium and passage
Generation of NR-Like Structures from hiPS Cells Day 0 to Day 14
  1. 1 Expand hiPS cells to confluence
  2. 2 Induce differentiation by FGF-2 withdrawal
  3. 3 Switch to ProN2 medium
  4. 4 Change medium every 2 days
  5. 5 Identify emergent NR-like structures
Maturation of NR-Like Structures by Floating Cultures Day 14 onwards
  1. 1 Isolate NR-like structures
  2. 2 Transfer to 24-well plates
  3. 3 Apply 3D shaker culture (D14-D16)
  4. 4 Transition to static floating culture (D16 onwards)
  5. 5 Change medium at D21
  6. 6 Continue medium changes
  7. 7 Maintain floating cultures
Generation and Amplification of hiPS-Derived RPE Cells Day 4 to Day 21 and beyond
  1. 1 Coat 24-well plates with gelatin
  2. 2 Remove gelatin and add ProN2 medium
  3. 3 Isolate hiRPE patches
  4. 4 Transfer RPE patches to gelatin-coated wells
  5. 5 Change ProN2 medium
  6. 6 Wash at confluence
  7. 7 Dissociate with TrypLE
  8. 8 Mechanically dissociate cells
  9. 9 Stop TrypLE activity
  10. 10 Centrifuge cell suspension
  11. 11 Resuspend cells
  12. 12 Distribute cells to new plates
  13. 13 Continue culture and passages
RNA Extraction and TaqMan Gene Expression Analysis As needed for molecular characterization
  1. 1 Collect samples for RNA extraction
  2. 2 Extract RNA
  3. 3 Check RNA quality and yields
  4. 4 Prepare RNA sample
  5. 5 Synthesize cDNA
  6. 6 Dilute cDNA
  7. 7 Prepare qPCR reaction mix
  8. 8 Run qPCR
  9. 9 Analyze gene expression
Immunohistochemistry Analysis As needed for morphological characterization
  1. 1 Fix NR-like structures
  2. 2 Rinse structures
  3. 3 Incubate in sucrose solution
  4. 4 Embed structures
  5. 5 Freeze embedded structures
  6. 6 Collect cryosections
  7. 7 Remove gelatin from slides
  8. 8 Block non-specific binding
  9. 9 Incubate with primary antibody
  10. 10 Wash slides after primary antibody
  11. 11 Incubate with secondary antibody
  12. 12 Wash slides after secondary antibody
  13. 13 Incubate with DAPI
  14. 14 Final wash
  15. 15 Mount coverslips

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Reichman et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence