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RETINA Publication-derived

Retinal 2D Cell Cultures for Disease Modelling

Full SOP with Academic or a plan

Source Meyer et al., 2011 · Waisman Center, University of Wisconsin, Madison · 10.1002/stem.674

Authors: Jason S. Meyer, Sara E. Howden, Kyle A. Wallace, Amelia D. Verhoeven, Lynda S. Wright, Elizabeth E. Capowski, Isabel Pinilla, Jessica M. Martin, Shulan Tian, Ron Stewart, Bikash Pattnaik, James Thomson, David M. Gamm Duration: 120 days 8 phases Cell source: Human ESC (WA09, WA01) and Human iPSC (IMR90-4, 6-9-12T, iPS-12, iPS-12.4, patient-derived lentivirus-derived lines)

Abstract

This protocol describes the derivation of optic vesicle-like (OV) structures from human pluripotent stem cells (hESCs and hiPSCs) through directed neural differentiation. OV-like structures are enriched populations of retinal progenitor cells that can be isolated by morphological selection and differentiated into major neuroretinal cell types (photoreceptors, retinal ganglion cells, bipolar cells, amacrine cells) and retinal pigment epithelium (RPE). The method enables study of human retinal development and disease modeling, including functional assessment of disease-specific defects in patient-derived hiPSC lines.

Cell source
Human ESC (WA09, WA01) and Human iPSC (IMR90-4, 6-9-12T, iPS-12, iPS-12.4, patient-derived lentivirus-derived lines)
Application
Developmental study; Disease modeling; Retinal cell differentiation and characterization

Protocol overview

25 steps across 8 phases

Phase 1 · Pluripotent Stem Cell Maintenance Days 0-0
  1. a Culture hESCs and hiPSCs in ESC medium
Phase 2 · Initial Neural Induction and Aggregate Formation Days 1-7
  1. a Culture colonies as suspended aggregates
  2. b Switch to Neural Induction Medium
  3. c Optional: Treat with Noggin/Dorsomorphin and DKK1/XAV939
Phase 3 · Neural Cluster Formation and Rosette Isolation Days 7-16
  1. a Plate aggregates onto laminin-coated substrate
  2. b Mechanically isolate neural rosette-containing clusters
  3. c Culture isolated rosettes as cellular aggregates
Phase 4 · Optic Vesicle-like Structure Isolation Days 20-25
  1. a Visually identify and separate vesicle-like structures
  2. b Culture vesicle-like structures independently in RDM
  3. c Culture nonvesicular spheres independently in RDM
Phase 5 · Retinal Differentiation of OV-like Structures Days 25-120
  1. a Continue culture of OV-like structures in RDM
  2. b Optional: Sample cultures for gene expression analysis
Phase 6 · RPE Differentiation from OV-like Structures (Optional) Days 20-40
  1. a Add Activin A to OV-like structures
  2. b Identify and isolate pigmented structures
  3. c Plate pigmented structures on laminin-coated substrate
  4. d Isolate and dissociate RPE cell patches
  5. e Remove mitogens and assess RPE phenotype
Phase 7 · Photoreceptor-like Cell Characterization (Optional) Days 100-120
  1. a Differentiate OV-like structures for electrophysiology
  2. b Prepare patch-clamp recording setup
  3. c Perform whole-cell patch-clamp recordings
  4. d Analyze and plot current-voltage relationships
Phase 8 · Calcium Imaging of RPE Cells (Optional) Days 60-120
  1. a Load RPE cells with Fura-2 AM
  2. b Wash cells and prepare for imaging
  3. c Perform calcium imaging
  4. d Analyze calcium imaging data

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Attribution

This SOP was written by Organthis based on the published method in Meyer et al., 2011. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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