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RETINA Publication-derived

Retinal 2D Cell Cultures for Developmental Studies

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Source Meyer et al. · 10.1073/pnas.0905245106

Authors: Meyer et al. Duration: 40 days 4 phases Cell source: Human ESC (H9 line) and Human iPSC (IMR90 clone 4)

Abstract

This protocol describes the directed differentiation of human embryonic stem cells (H9) and induced pluripotent stem cells (IMR90 clone 4) toward retinal lineage fate, generating neuroepithelial rosettes that differentiate into either neural retinal progenitors or retinal pigment epithelium cells. The method employs feeder-free suspension culture with chemically defined media and specific growth factor supplementation to achieve eye field specification and subsequent retinal differentiation.

Cell source
Human ESC (H9 line) and Human iPSC (IMR90 clone 4)
Application
Retinal differentiation and developmental study

Protocol overview

14 steps across 4 phases

Phase 1 · Maintenance of hESCs and iPSCs Ongoing (passaged every 5-6 days)
  1. a Expand hESCs and iPSCs on feeder layer
  2. b Prepare embryonic stem cell medium (ESCM)
  3. c Passage cells every 5-6 days
Phase 2 · Initiation of Differentiation and Aggregate Formation Day 0-4
  1. a Enzymatically lift cells from MEF feeder layer
  2. b Culture cells as aggregates in suspension
Phase 3 · Neural Induction and Neuroepithelial Rosette Formation Day 4-16
  1. a Switch to chemically defined neural induction medium (NIM)
  2. b Allow aggregates to attach to culture dish with laminin
  3. c Allow formation of neuroepithelial rosettes
  4. d Optional: Add BMP4 and Wnt3A antagonists (days 0-10 variant)
  5. e Optional: Add FGF inhibitor SU5402 (variant experiment)
Phase 4 · Retinal Differentiation (Neural Retinal and RPE Pathways) Day 16-40
  1. a Switch to retinal differentiation medium (RDM)
  2. b Mechanically isolate rosettes for neural retinal progenitor pathway
  3. c Culture isolated rosettes in suspension for neurosphere formation
  4. d Maintain rosettes on substrate for RPE differentiation pathway

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Attribution

This SOP was written by Organthis based on the published method in Meyer et al.. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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