Generation of Highly Enriched Populations of Optic Vesicle−Like Retinal Cells from Human Pluripotent Stem Cells

Sarah K. Ohlemacher1, Clara L. Iglesias1, Akshayalakshmi Sridhar1, David M. Gamm2, Jason S. Meyer3

1 Department of Biology, Indiana University−Purdue University Indianapolis, 2 Department of Ophthalmology and Visual Sciences, University of Wisconsin−Madison, Wisconsin, 3 Department of Medical and Molecular Genetics, Indiana University, Indianapolis
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.8
DOI:  10.1002/9780470151808.sc01h08s32
Online Posting Date:  February, 2015
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The protocol outlined below is used to differentiate human pluripotent stem cells (hPSCs) into retinal cell types through a process that faithfully recapitulates the stepwise progression observed in vivo. From pluripotency, cells are differentiated to a primitive anterior neural fate, followed by progression into two distinct populations of retinal progenitors and forebrain progenitors, each of which can be manually separated and purified. The hPSC‐derived retinal progenitors are found to self‐organize into three‐dimensional optic vesicle−like structures, with each aggregate possessing the ability to differentiate into all major retinal cell types. The ability to faithfully recapitulate the stepwise in vivo development in a three‐dimensional cell culture system allows for the study of mechanisms underlying human retinogenesis. Furthermore, this methodology allows for the study of retinal dysfunction and disease modeling using patient‐derived cells, as well as high‐throughput pharmacological screening and eventually patient‐specific therapies. © 2015 by John Wiley & Sons, Inc.

Keywords: human pluripotent stem cells (hPSCs); retina; development; differentiation; optic vesicle

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Table of Contents

  • Introduction
  • Basic Protocol 1: Enzymatic Passaging of hPSCs
  • Basic Protocol 2: Induction of hPSCs to a Primitive Anterior Neuroepithelial Fate
  • Basic Protocol 3: Differentiation of Primitive Anterior Neuroepithelial Cells to a Retinal Pigment Epithelial Fate
  • Basic Protocol 4: Differentiation and Long‐Term Maintenance of Retinal Progenitor Cells
  • Basic Protocol 5: Induction of Retinal Progenitors to Specific Retinal Subtypes
  • Support Protocol 1: Coating Coverslips with Laminin and Poly‐D‐Ornithine
  • Reagents and Solutions
  • Commentary
  • References
  • Figures
  • Tables
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Basic Protocol 1: Enzymatic Passaging of hPSCs

  • hPSCs growing on Matrigel‐coated six‐well plate
  • 2 mg/ml dispase solution (see recipe)
  • DMEM/F12, 1:1 (Life Technologies)
  • mTeSR1 (Stemcell Technologies)
  • Inverted light microscope
  • 15‐ml conical tubes
  • Matrigel‐coated six‐well plate (see recipe)

Basic Protocol 2: Induction of hPSCs to a Primitive Anterior Neuroepithelial Fate

  • Harvested and washed undifferentiated hPSCs (see protocol 1, steps 1‐8)
  • mTeSR1 (Stemcell Technologies)
  • Neural induction medium (NIM; see recipe)
  • Fetal bovine serum (FBS)
  • T75 culture flask (Falcon)
  • 15‐ml conical tube
  • Six‐well culture plate

Basic Protocol 3: Differentiation of Primitive Anterior Neuroepithelial Cells to a Retinal Pigment Epithelial Fate

  • Primitive anterior neuroepithelial cells in six‐well plate (see protocol 2)
  • Retinal differentiation medium (RDM; see recipe)
  • Epidermal growth factor (EGF)
  • Fibroblast growth factor 2 (FGF2)
  • Heparin
  • Inverted light microscope
  • Tungsten needle, pipet tip, or other pointed object
  • Laminin/polyornithine−coated coverslips in a 4‐ or 24‐well plate (see protocol 6Support Protocol)

Basic Protocol 4: Differentiation and Long‐Term Maintenance of Retinal Progenitor Cells

  • Primitive anterior neuroepithelial cells in six‐well plate (see protocol 2)
  • Retinal differentiation medium (RDM; see recipe)
  • 60 × 15−mm polystyrene Petri dishes
  • Six‐well plates (Falcon)

Basic Protocol 5: Induction of Retinal Progenitors to Specific Retinal Subtypes

  • Retinal neurospheres at 40 days total differentiation (see protocol 4)
  • Accutase (BD Biosciences)
  • Retinal differentiation medium (RDM; see recipe)
  • Laminin/polyornithine−coated coverslips (see protocol 6Support Protocol)
  • Inverted light microscope
  • 1.5‐ml tube

Support Protocol 1: Coating Coverslips with Laminin and Poly‐D‐Ornithine

  • 12‐mm glass coverslips, washed with ethanol and subsequently autoclaved
  • 4‐ and/or 24‐well plates
  • 100 μg/ml poly‐D‐ornithine solution (see recipe)
  • 20 μg/ml laminin solution (see recipe)
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