Efficient Generation of Viral and Integration‐Free Human Induced Pluripotent Stem Cell‐Derived Oligodendrocytes

Araceli Espinosa‐Jeffrey1, Bruno Blanchi1, Juan Carlos Biancotti2, Shalini Kumar1, Megumi Hirose1, Berhan Mandefro3, Dodanim Talavera‐Adame3, Nissim Benvenisty4, Jean de Vellis1

1 Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, David Geffen School of Medicine at UCLA, Los Angeles, 2 Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, 3 Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, 4 Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.18
DOI:  10.1002/cpsc.19
Online Posting Date:  November, 2016
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Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation—our protocol generates viral‐ and integration‐free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain‐derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.

Keywords: chemically defined media; human induced pluripotent stem cells; neural stem cells; NSC; neurospheres; oligodendrocyte specification; oligodendrocyte maturation; lineage progression; oligospheres

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of EBs from hiPS Cells while Starting Neural Instruction
  • Support Protocol 1: Immunopanning (Alternative to Matrigel)
  • Support Protocol 2: Cell Viability Assay
  • Support Protocol 3: Freezing and Thawing NSCs
  • Basic Protocol 2: Propagation of Rodent and Human Neural Stem Cells (NSCs)
  • Basic Protocol 3: Propagation of Rodent and Human Neural Stem Cells (NSCs) and OL Specification in 2‐D Cultures
  • Support Protocol 4: Oligodendrocyte Lineage Progression and Maturation
  • Support Protocol 5: Preparation of Cortical Neurons
  • Support Protocol 6: Transplantation of OL Progenitors into Neonatal Rats
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of EBs from hiPS Cells while Starting Neural Instruction

  • Human induced pluripotent stem cells [hIPS cells; see unit 4.2 (Ohnuki et al., )]
  • 5 mM (1000×) Rho‐associated protein kinase (ROCK) inhibitor
  • hiPS medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca or Mg (Thermo Fisher Scientific, cat. no. 14200075)
  • 0.25% trypsin/EDTA (available from various vendors)
  • Mouse embryonic fibroblast (MEF) medium (see recipe)
  • EB medium (see recipe)
  • 10 mM dorsomorphin stock solution (Tocris, cat. no. 3093; store frozen in aliquots)
  • 10 mM SB431542 (TGF‐β/Smad inhibitor) stock solution (Tocris, cat. no. 1614; store frozen in aliquots)
  • N2 medium (see recipe)
  • 100 μg/ml basic fibroblast growth factor (bFGF) stock (Thermo Fisher Scientific, cat. no. 13256‐029; store frozen in aliquots)
  • 250 µg/ml sonic hedgehog (Shh; e.g., R&D Systems; store frozen in aliquots) stock solution
  • 100 mM retinoic acid (RA; Sigma, cat. no. R2625) stock solution in DMSO
  • Glial Defined Medium (GDM; see recipe)
  • OLBN medium (see recipe)
  • IGF‐1(88 to 100 µg/ml stock); use 1 µl/ml of medium
  • 6‐well tissue culture treated dishes (Corning Falcon, cat. no. 353046)
  • 15‐ml conical centrifuge tubes
  • Matrigel‐coated 6‐well plates or 75‐ or 115‐cm2 culture flasks (see recipe); alternatively use PSA‐NCAM IgM–coated plates (see protocol 2)
  • Poly‐D‐lysine‐coated coverslips, flasks, 24‐well plates, 4‐well plates, or 16‐well glass chambers (see, e.g., unit 2.14 for coating technique)
  • Additional reagents and equipment for culturing hiPS on MEF feeder layers (Biancotti et al., )

Support Protocol 1: Immunopanning (Alternative to Matrigel)

  • 50 mM Tris⋅Cl, pH 9.5
  • Bovine serum albumin (BSA)
  • Anti‐human PSA‐NCAM IgM (IOWA DSHB; http://www.uiowa.edu/˜dshbwww/)
  • Phosphate‐buffered saline (PBS; Thermo Fisher Scientific, cat. no. 28372)
  • Flasks (12.5 cm2, 75 cm2), or TPP tissue culture flask (115 cm2) with recloseable lid (TPP, cat. no. 90652)

Support Protocol 2: Cell Viability Assay

  • Cells to be tested
  • 1 × Tris‐buffered saline (TBS; Thermo Fisher Scientific, cat. no. 28358)
  • SYTOX blue nucleic acid stain (Invitrogen Molecular Probes, cat. no. S‐7020)
  • Phosphate‐buffered saline (PBS; Thermo Fisher Scientific, cat. no. 28372)
  • Hemacytometer
  • Fluorescence microsope

Support Protocol 3: Freezing and Thawing NSCs

  • NSCs
  • Hanks’ Balanced Salt Solution (HBSS; without Ca2+ or Mg2+; Thermo Fisher Scientific, cat. no. 14175103)
  • Basal stem cell medium (STM‐II; see recipe)
  • Liquid N 2
  • Serum‐free freezing medium (Synth‐a‐Freeze Medium, Thermo Fisher Scientific, cat. no. A1371301)
  • 90:10 STMc (see recipe): conditioned medium (CM; collected at step 23 of protocol 1; save in frozen aliquots)
  • 75‐cm2 cell culture flasks
  • Cell scrapers
  • 14‐G needle
  • 1.2‐ml Cryogenic freezing vials
  • Cryogenic slow‐freezing chamber
  • Cell culture containers coated with anti–human PSA‐NCAM IgM ( protocol 2), anti‐human whole IgM molecule, or poly‐D‐lysine (see, e.g., unit 2.14 for coating technique)
  • Additional reagents and equipment for determining cell viability ( protocol 2)

Basic Protocol 2: Propagation of Rodent and Human Neural Stem Cells (NSCs)

  • NSCs growing in culture (see Basic Protocols protocol 11 and protocol 52)
  • Hanks’ Balanced Salt Solution (HBSS) without Ca2+ or Mg2+ (Thermo Scientific, cat. no. 14175103)
  • OSM‐II OL specification medium (see recipe)
  • STM conditioned medium (STM‐CM)
  • Cell scraper
  • 15‐ml conical tubes
  • 14‐G needle and 12‐ml syringe
  • Anti–human PSA‐NCAM IgM‐coated plates ( protocol 2)
  • Anti‐IgM‐coated plates: use anti‐IgM (Sigma) to coat the plates (same procedure as in protocol 2)
  • 25‐ or 50‐ml Erlenmeyer (EM) flask

Basic Protocol 3: Propagation of Rodent and Human Neural Stem Cells (NSCs) and OL Specification in 2‐D Cultures

  • OSM‐II OL specification medium (see recipe)
  • Glial Defined Medium (GDM; see recipe)
  • OLDEM (OL maturation medium; see recipe for GDM, but omit the transferrin)
  • Basic fibroblast growth factor (bFGF; Invitrogen, cat. no. 13256‐029)
  • Neuronal culture ( protocol 8)
  • Poly‐D‐lysine‐coated wells or plates (see, e.g., unit 2.14 for coating technique)
  • Cell scraper
  • 25‐µm sieve (25‐μm Cellector, E‐C Apparatus Corp; if not available, use 40‐μm mesh; Corning Falcon, cat. no. 352340)
  • 24‐well plates
  • Additional reagents and equipment for plating cells in OSM‐II medium (see protocol 6) and counting viable cells ( protocol 3)

Support Protocol 4: Oligodendrocyte Lineage Progression and Maturation

  • Animals: timed‐pregnant (ED14 to 16) Sprague‐Dawley rats (Charles River)
  • Isoflurane
  • STM‐II basal stem cell medium (see recipe)
  • Bovine serum albumin (BSA)
  • Neurobasal‐B27 medium (NB‐B27; see recipe)
  • Neurobasal‐N medium for cortical neurons (see recipe)
  • Anesthesia apparatus
  • Scissors
  • Forceps
  • Needle
  • 15‐ml conical tubes
  • 230‐µm and 140‐µm sieves (Cellector, E‐C Apparatus Corp.)
  • Centrifuge
  • Poly‐D‐lysine‐coated 24‐ or 12‐well plates (see, e.g., unit 2.14 for coating technique)
  • Combustion Test Kit (Bacharach, cat. no. 10‐500)
  • Additional reagents and equipment for cell viability assay ( protocol 3)

Support Protocol 5: Preparation of Cortical Neurons

  • Neonatal rat pups (P0 to P5)
  • Isoflurane
  • Oxygen
  • 70% ethanol
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca or Mg (Thermo Fisher Scientific, cat. no. 14200075)
  • Microcentrifuge tube with cell sample ( protocol 8)
  • Borosilicate glass tube (Sutter Instruments, cat. no. B100–75‐15)
  • Micropipet puller (Sutter Instrument Co., Model P‐87)
  • Aspirator tube assembly (Sigma, cat. no. A5177–5EA)
  • Transillumination light source
  • Warming pad or warm water glove balloon
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