Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells

Mohamad Khazaei1, Christopher S. Ahuja2, Michael G. Fehlings3

1 Division of Genetics and Development, Krembil Research Institute, Toronto, Ontario, 2 Division of Neurosurgery, University of Toronto, Toronto, Ontario, 3 Faculty of Medicine, University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.20
DOI:  10.1002/cpsc.31
Online Posting Date:  August, 2017
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Abstract

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o‐NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell‐derived neural progenitor cells (hiPS‐NPCs), and human induced pluripotent stem cell‐oligodendrogenic neural progenitor cells (hiPSC‐o‐NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic‐NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc.

Keywords: differentiation; human induced pluripotent stem cell; oligodendrocyte; neural progenitor; spine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Passaging and Maintenance of Human Induced Pluripotent Stem Cells in Culture
  • Basic Protocol 2: Differentiation of Human Induced Pluripotent Stem Cells to Neural Progenitor Cells
  • Basic Protocol 3: Differentiation of Human Induced Pluripotent Stem cell‐Derived Neural Progenitor Cells to an Oligodendrogenic Fate
  • Support Protocol 1: Preparing Coated Plates
  • Support Protocol 2: Freezing/Thawing Human Induced Pluripotent Stem Cell‐Derived Neural Progenitor Cells and Oligodendrogenic Neural Progenitor Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Passaging and Maintenance of Human Induced Pluripotent Stem Cells in Culture

  Materials
  • Matrigel
  • Human induced pluripotent stem cells (hiPSCs)
  • mTeSR medium
  • Accutase
  • DMEM/F12 medium
  • ROCK inhibitor Y‐27632
  • Growth medium without ROCK inhibitor
  • Cell scraper or rubber policeman
  • 15‐ml Falcon tubes
  • P1000 pipet
  • Sterile needle

Basic Protocol 2: Differentiation of Human Induced Pluripotent Stem Cells to Neural Progenitor Cells

  Materials
  • Human induced pluripotent stem cells (hiPSCs)
  • Leukemia inhibitory factor (LIF)
  • Accutase
  • DMEM/F12
  • Non‐essential amino acids
  • B‐27 supplement without vitamin A
  • N2 supplement
  • Y27632 (ROCK inhibitor)
  • Noggin (BMP inhibitor)
  • CHIR 9902 (GSK3 β inhibitor)
  • Compound C (AMP Kinase inhibitor)
  • SB431542 (TGFβ‐receptor inhibitor)
  • Neural induction medium (NIM; see recipe)
  • Neural expansion medium (NEM; see recipe)
  • Trypan Blue
  • Matrigel‐coated plates ( protocol 4)
  • Coverslips (optional)
  • 50‐ml Falcon tube
  • Hemocytometer or automated counting platform

Basic Protocol 3: Differentiation of Human Induced Pluripotent Stem cell‐Derived Neural Progenitor Cells to an Oligodendrogenic Fate

  Materials
  • Accutase or TrypLE
  • Retinoic acid (RA)
  • B‐27 supplement with vitamin A
  • B‐27 supplement without vitamin A
  • Sonic hedgehog (Shh)
  • N2 supplement
  • N1 supplement
  • PDGF‐AA
  • FGF
  • FGF2
  • EGF
  • Heparin
  • Thyroxine
  • Matrigel‐coated plates

Support Protocol 1: Preparing Coated Plates

  Materials
  • Matrigel coating
  • DMEM/F12 medium
  • Neurobasal medium (LifeTechnologies)
  • Poly L‐lysine (PLL)
  • Laminin
  • 0.15 M borate buffer (pH 8.3)
  • PBS
  • Incubator, 37 °C
  • 0.2‐µm filters

Support Protocol 2: Freezing/Thawing Human Induced Pluripotent Stem Cell‐Derived Neural Progenitor Cells and Oligodendrogenic Neural Progenitor Cells

  Materials
  • Human induced pluripotent stem cell (hiPSC)‐derived neural progenitor cells (NPCs) and/or oligodendrogenic neural progenitor cells (o‐NPCs; see protocol 3)
  • DMSO
  • DMEM/F‐12 plus Glutamax
  • FBS
  • TrypLE Express enzyme (1×; Thermo Fisher Scientific, cat. no. 12604021)
  • Neural expansion medium (NEM; see recipe)
  • Liquid nitrogen
  • Centrifuge tube, sterile
  • 2‐ml cryogenic storage vials
  • Cell freezing container
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Figures

Videos

Literature Cited

Literature Cited
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