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RETINA Publication-derived

IGF-1 Signaling in the Formation of Three-Dimensional Laminated Neural Retina and Ocular Structures From Human Embryonic Stem Cells

Source Mellough et al., 2015 · Institute of Genetic Medicine, Newcastle University · 10.1002/stem.2023

👤 Carla B. Mellough, Joseph Collin, Mahmoud Khazim, Kathryn White, Evelyne Sernagor, David H. W. Steel, Majlinda Lako ⏱ 90 days 📋 7 phases 🧫 Human ESC (H9, WiCell)

Abstract

A protocol for differentiating human embryonic stem cells (hESCs) into three-dimensional laminated neural retina with photoreceptor cells displaying advanced maturation features including inner and outer segment-like structures and phototransduction capacity by 6.5 weeks of differentiation. The protocol employs IGF-1 supplementation in minimal serum-free media and also generates accessory ocular structures including retinal pigmented epithelium, lens, and corneal-like epithelium.

Cell source
Human ESC (H9, WiCell)
Application
Disease modeling; Developmental study; Cell replacement therapy

Protocol overview

24 steps across 7 phases

Embryoid Body Formation and Initial Neural Induction Days 0–37
  1. 1 hESC Expansion and Embryoid Body Formation
  2. 2 Ventral Neural Induction Media Supplementation
  3. 3 Time-Lapse Morphological Documentation
Neural Retinal Differentiation and Maturation Days 37–90
  1. 4 Transition to Basal Knockout Serum-Free Media with IGF-1 Continuation
  2. 5 Extended Maturation Culture
  3. 6 Media Changes
Tissue Harvest and Immunocytochemistry Analysis Days 30–90
  1. 7 Embryoid Body Fixation
  2. 8 Cryostat Sectioning
  3. 9 Immunocytochemical Staining
  4. 10 Microscopy and Image Acquisition
Transmission Electron Microscopy (TEM) Analysis Days 30–90
  1. 11 Tissue Preparation for Electron Microscopy
  2. 12 TEM Imaging and Analysis
Functional Electrophysiology Studies Days 30, 45, 90
  1. 13 Embryoid Body Dark Adaptation
  2. 14 Fura-2-AM Loading
  3. 15 Perfusion with Oxygenated Artificial Cerebrospinal Fluid (aCSF)
  4. 16 cGMP Stimulation and Control Recordings
  5. 17 Calcium Imaging Acquisition
  6. 18 Fluorescence Data Analysis
  7. 19 Confirmation of Photoreceptor Identity by Immunocytochemistry
IGF-1 Receptor Signaling Interference Studies Days 0–90
  1. 20 IGF-IR Inhibition Experiments
  2. 21 IGF-IIR Neutralization Experiments
  3. 22 Optic Vesicle and Cup Counting
Statistical Analysis and Data Reporting Throughout differentiation (days 0–90)
  1. 23 Data Collection and Presentation
  2. 24 Statistical Significance Testing

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Mellough et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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