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RETINA Publication-derived

Modeling early retinal development with human embryonic and induced pluripotent stem cells

Source Meyer et al., 2009 · Stem Cell Research Program, Waisman Center, University of Wisconsin-Madison

👤 Jason S. Meyer, Rebecca L. Shealer, Elizabeth E. Capowski, Lynda S. Wright, Kyle A. Wallace, Erin L. McMillan, Su-Chun Zhang, David M. Gamm ⏱ 80 days 📋 5 phases 🧫 Human ESC and Human iPSC

Abstract

This protocol describes a stepwise differentiation method for generating retinal cell populations from human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. The method produces cells representing all major stages of early eye and retinal development—from eye field specification through optic vesicle/cup formation to photoreceptor and retinal pigment epithelium differentiation—in a timeline that recapitulates normal human retinal development.

Cell source
Human ESC and Human iPSC
Application
Developmental study and disease modeling of retinogenesis

Protocol overview

38 steps across 5 phases

Eye Field Specification Days 0–16
  1. 1 hESC maintenance and expansion
  2. 2 Initiate neural differentiation
  3. 3 Promote neural rosette formation
  4. 4 Mechanical isolation of rosettes
  5. 5 Culture as neurospheres
  6. 6 Verify eye field specification by RT-PCR
  7. 7 Immunocytochemical verification of eye field markers
  8. 8 Monitor Pax6 and Rx coexpression by FACS
Optic Vesicle and Optic Cup Formation Days 16–50
  1. 1 Continue culture as neurospheres
  2. 2 Monitor Mitf expression by immunocytochemistry
  3. 3 Quantify Mitf gene expression by qPCR
  4. 4 Monitor Chx10 appearance and Mitf/Chx10 dynamics
  5. 5 Quantify Chx10 gene expression by qPCR
  6. 6 Assess Chx10 protein expression by FACS
  7. 7 Verify neural retinal progenitor markers in Chx10+ cells
  8. 8 Verify non-retinal neurospheres retain anterior neural fate
  9. 9 Test FGF signaling requirement (optional manipulation)
Retinal Pigment Epithelium (RPE) Differentiation Days 30–50
  1. 1 Maintain Pax6+/Rx+ eye field rosettes as adherent cultures
  2. 2 Observe RPE morphology and pigmentation
  3. 3 Verify Mitf and ZO-1 expression by immunocytochemistry
  4. 4 Quantify RPE population by FACS
  5. 5 Verify RPE gene expression by RT-PCR
Photoreceptor Differentiation Days 40–80
  1. 1 Maintain neural retinal progenitor neurospheres
  2. 2 Monitor Crx expression onset by immunocytochemistry
  3. 3 Quantify Crx expression within Crx+ neurospheres
  4. 4 Detect mature photoreceptor proteins (recoverin and opsin)
  5. 5 Verify sequential gene expression by RT-PCR
  6. 6 Verify timing against normal human retinogenesis
  7. 7 Analyze Pax6 isoform expression (optional)
Human iPS Cell Differentiation (Parallel Protocol) Days 0–80
  1. 1 Select high-competency iPS cell lines
  2. 2 Apply identical hESC differentiation protocol
  3. 3 Confirm eye field specification by day 10
  4. 4 Monitor Mitf and Chx10 progression (days 30–50)
  5. 5 Quantify Chx10+ population at day 40
  6. 6 Monitor photoreceptor marker appearance (day 80)
  7. 7 Detect mature photoreceptor proteins in iPS cells
  8. 8 Verify sequential gene expression in iPS cells by RT-PCR
  9. 9 Generate RPE from iPS cell cultures

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Meyer et al., 2009. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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