Derivation of Telencephalic Oligodendrocyte Progenitors from Human Pluripotent Stem Cells

Tamara Major1, Ann Powers2, Viviane Tabar3

1 Center for Basic and Translational Neuroscience, University of Copenhagen, Denmark, 2 Icahn Mount Sinai School of Medicine, New York, New York, 3 Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.10
DOI:  10.1002/cpsc.17
Online Posting Date:  November, 2016
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Abstract

Oligodendrocytes are the main myelinating cell of the adult CNS and are vulnerable to injury in diverse disorders, such as spinal cord injury, stroke, trauma, pharmacological and radiation toxicity, as well as neuroinflammation. Human pluripotent stem cells are attractive sources of oligodendrocyte lineage cells and provide a promising treatment strategy for exogenous myelin repair through transplantation. This unit describes a protocol for the step‐wise differentiation of forebrain late oligodendrocyte progenitor cells (OPCs) from human pluripotent stem cells in defined chemical in vitro culture conditions. It involves a stepwise progression of oligodendrocyte progenitors through their known developmental phases, starting with the expression of appropriate transcription factors (Olig2, Nkx2.2), the upregulation of PDGFRA, followed by the appearance of O4‐expressing cells, then O1 expression and finally mature myelin‐binding protein (MBP) expressing cells. Validation of cell fate is performed by extensive transcriptomal profiling, as well in vitro myelination essays with hESCs‐derived neuronal cells. Recapitulating forebrain oligodendrocyte development may generate cells more suitable for transplantation strategies for disorders primarily involving the telencephalon. © 2016 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells (hESCs); human‐induced pluripotent stem cells (hiPSCs); in vitro myelination; myelin; oligodendrocyte; oligodendrocyte differentiation

     
 
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Table of Contents

  • Introduction
  • Significance Statement
  • Basic Protocol 1: Generation of Oligodendrocytes from hESCs
  • Support Protocol 1: Characterization of the Cells at Different Stages of Differentiation by Immunostaining
  • Support Protocol 2: Selective Enrichment of Oligodendrocyte Population Using Fluorescence‐Activated Cell Sorting (FACS)
  • Support Protocol 3: Human Embryonic Stem Cell–Based in Vitro Myelination Assay
  • Support Protocol 4: Maintenance of hESCs and hiPSCs in Co‐Culture With Mouse Embryonic Fibroblasts (MEFs)
  • Support Protocol 5: Recipes for Coating of Tissue Culture Dishes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of Oligodendrocytes from hESCs

  Materials
  • hESCs/hiPSCs cultured on a feeder layer (see protocol 5) in 10‐cm culture dishes
  • Matrigel‐coated 6‐well cell culture plates (see protocol 6)
  • 0.05% Trypsin‐EDTA (Gibco‐Life Technologies, cat. no. 25300‐054)
  • hESC medium (see recipe)
  • 10 μg/ml FGF2 (R&D Systems, cat. no. 233‐FB)
  • Accutase (Innovative Cell Technologies, cat. no. AT104)
  • MEF‐conditioned hESC medium (CM; see recipe)
  • KSR medium (see recipe)
  • 500 μM LDN193189 (StemCell Technologies, cat. no. 72144)
  • 10 mM SB431542 (Tocris Bioscience, cat. no. 1614)
  • 10 mM XAV939 (Tocris Bioscience, cat. no. 3748)
  • N2 medium (see recipe)
  • 2 mM Purmorphamine (Stemgent, cat. no. 04‐0009)
  • 100 mM Sodium L‐ascorbate (AA; SIGMA cat. no. A4034)
  • Growth factors: 10 μg/ml BDNF (R&D Systems, cat. no. 248‐BD), 100 μg/ml FGF8 (R&D Systems, cat. no. 423‐F8), 10 μg/ml PDGF‐AA (R&D Systems, cat. no. 221‐AA), 10 μg/ml IGF‐1 (R&D Systems, cat. no. 291‐G1)
  • 10‐cm PO/Lam/FN‐coated cell culture dishes (see recipe)
  • DMEM/F12 medium (Gibco‐Life Technologies, cat. no. 11330‐032)
  • B27 Supplement (50×), minus vitamin A (Gibco‐Life Technologies, cat. no. 125870‐01)
  • 10 mM Y‐27632 dihydrochloride (Tocris Bioscience, cat. no. 1254)
  • 20 µg/ml 3, 3′, 5‐Triiodo‐L‐thyronine sodium salt (T3; Sigma, cat. no. T5516)
  • 100 mM Dibutyryl cAMP sodium salt (cAMP; Sigma, cat. no. D0260)
  • Ca and Mg free HBSS with 15 mM HEPES (see recipe)
  • Inverted microscope (Nikon EclipseTi‐U)
  • 15‐ml conical polypropylene centrifuge tubes
  • 5‐ and 10‐ml serological pipets
  • Centrifuge
  • 45‐μm cell strainers
  • Hemacytometer
  • 37°C incubator
  • Embedded dissecting microscope (Nikon SMZ1270)
  • 1‐ml syringe with a 27‐G needle
  • 200‐ and 1000‐μl pipets
  • Cell lifter

Support Protocol 1: Characterization of the Cells at Different Stages of Differentiation by Immunostaining

  Materials
  • PO/Lam/FN (see recipe)
  • NPCs/OPCs derived from hESCs/hiPSCs (see Basic Protocol)
  • Accutase (Innovative Cell Technologies)
  • N2 medium (see recipe)
  • Appropriate growth factors
  • Paraformaldehyde solution, 4% in PBS (4% PFA; Affymetrix, cat.no. 19943 1 LT)
  • Phosphate‐buffered saline (PBS; containing calcium and magnesium; Gibco‐Life Technologies, cat. no. 70011‐044), 1×, pH 7.4
  • 1% bovine serum solution 1% BSA (see recipe)
  • 0.1% (v/v) Triton‐X 100 in 1% BSA (see recipe)
  • 0.3% (v/v) Triton‐X 100 in 1% BSA (see recipe)
  • Appropriate primary antibody (see recipe for primary and secondary antibodies)
  • Fluorophore‐conjugated secondary antibody (see recipe for primary and secondary antibodies)
  • DAPI stain (see recipe)
  • 24‐well cell culture plates
  • 200‐ and 1000‐μl pipets
  • 1 ml syringe with a 27‐G needle
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • Centrifuge
  • 45‐μm cell strainers
  • Glass hemacytometer
  • Inverted microscope

Support Protocol 2: Selective Enrichment of Oligodendrocyte Population Using Fluorescence‐Activated Cell Sorting (FACS)

  Materials
  • Late OPCs derived from hESCs/hiPSCs (see Basic Protocol)
  • Accutase (Innovative Cell Technologies)
  • DMEM/F12 medium (see recipe)
  • 5% FBS‐HBSS (see recipe)
  • Ice
  • Anti‐O4 Antibody (see recipe for primary and secondary antibodies)
  • Alexa Fluor 488 secondary antibody (see recipe for primary and secondary antibodies)
  • DAPI stain (Invitrogen)
  • 100% fetal bovine serum (FBS; Gibco‐Life Technologies, cat. no. 16140‐071))
  • 24‐well cell culture plates coated with PO/Lam/FN (see recipe)
  • N2 medium (see recipe)
  • B27 Supplement (50×), minus vitamin A (Gibco‐Life Technologies)
  • 10 μg/ml BDNF
  • 100 mM AA
  • 20 μg/ml T3
  • 100 mM dibutyryl cAMP
  • Anti‐Myelin Basic Protein Antibody (MBP; see recipe for primary and secondary antibodies)
  • Anti‐Olig‐2 Antibody (see recipe for primary and secondary antibodies)
  • 37°C incubator
  • 15‐ml conical tubes
  • Centrifuge
  • 5‐ and 10‐ml serological pipets
  • 200‐ and 1000‐μl pipets
  • 45‐μm cell strainers
  • Hemacytometer
  • FACS tubes
  • Flow cytometer (e.g., MoFlo, Cytomation)
  • 1.5‐ml microcentrifuge tubes step 17

Support Protocol 3: Human Embryonic Stem Cell–Based in Vitro Myelination Assay

  Materials
  • hESCs/hiPSCs cultured on a feeder layer (see protocol 5) in 10‐cm culture dishes
  • KSR medium (see recipe)
  • 500 μM LDN193189
  • 10 mM SB431542
  • 10 mM XAV939
  • N2 medium (see recipe)
  • 10 mM CHIR99021
  • 24‐well cell culture plates coated with PO/Lam/FN (see recipe)
  • B27 Supplement (50×), minus vitamin A (Gibco‐Life Technologies)
  • 10 mM DAPT (see recipe)
  • FACS‐enriched O4+ OPCs derived in protocol 1Basic Protocol through the step 1‐step 35 and later FACS sorted according to protocol 3, step 1‐step 16. (see protocol 1Basic Protocol and protocol 3)
  • 10 μg/ml BDNF
  • 100 mM AA
  • 20 μg/ml T3
  • 100 mM dibutyryl cAMP
  • 4% paraformaldehyde (PFA)
  • Anti‐myelin basic protein antibody (MBP; see recipe for primary and secondary antibodies)
  • Anti‐neurofilament antibody (SMI312; see recipe for primary and secondary antibodies)
  • 37°C incubator
  • 15‐ml conical tubes
  • 5‐ml pipets
  • Centrifuge
  • 45‐μm cell strainers
  • Glass hemacytometer

Support Protocol 4: Maintenance of hESCs and hiPSCs in Co‐Culture With Mouse Embryonic Fibroblasts (MEFs)

  Materials
  • Mitotically inactivated mouse embryonic fibroblasts (MEFs; GlobalStem, cat. no. GSC‐6105 M)
  • hESCs/hiPSCs cultured on a feeder layer in 10‐cm culture dishes
  • hESC medium (see recipe)
  • 10‐cm cell culture dishes coated with gelatin (see recipe)
  • Dispase in Hank's Balanced Salt Solution (Stem Cell Technologies, cat. no. 7013)
  • FGF2 (see recipe)
  • 37°C incubator
  • 15‐ml conical tubes
  • Inverted microscope
  • 10‐ml serological pipets
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Figures

Videos

Literature Cited

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