Highly Expandable Human iPS Cell–Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High‐Throughput Screening

Chialin Cheng1, Daniel M. Fass1, Kat Folz‐Donahue2, Marcy E. MacDonald3, Stephen J. Haggarty1

1 Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 2 Currently at FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, 3 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.8
DOI:  10.1002/cphg.33
Online Posting Date:  January, 2017
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Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high‐throughput drug screening requires implementation of methods for the large‐scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell–derived CNS neural progenitor cells (NPC) using multi‐dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical‐like neurons from these NPC through inducible expression of the pro‐neural transcription factor Neurogenin 2 (iNgn2‐NPC). Finally, we describe methodology for the use of iNgn2‐NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high‐content, single‐cell‐level automated microscopy assays. © 2017 by John Wiley & Sons, Inc.

Keywords: drug discovery; high‐throughput screening; high‐content imaging; induced pluripotent stem cell; neuroplasticity; neurodegeneration; neuropsychiatric

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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintenance and Neural Induction of iPS Cells
  • Basic Protocol 2: Fluorescence‐Activated Cell Sorting (FACS) and Maintenance of Nascent NPC
  • Basic Protocol 3: Generation of iNgn2‐NPC Stable Line for Precisely Timed and Scalable Initiation of Differentiation into Excitatory Forebrain Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Maintenance and Neural Induction of iPS Cells

  • Human iPS cells (e.g., Silva et al., )
  • mTeSR1 medium (StemCell Technologies, cat. no. 05850)
  • DMEM/F12 medium (Gibco, cat. no. 11330)
  • Neural induction medium (NIM, StemCell Technologies cat. no. 05831)
  • Y‐27632 (EMD Millipore, cat. no. 688001)
  • Accutase (Sigma, cat. no. A6964‐100ML)
  • Phosphate‐buffered saline (PBS, MediaTech, cat. no. 21‐040‐CV)
  • Neural Rosette Selection Reagent (NRSR, StemCell Technologies, cat. no. 05832)
  • Neural proliferation medium (NPM; see recipe)
  • Matrigel‐coated 6‐well plates (see recipe)
  • Centrifuge
  • AggreWell 800 plate (StemCell Technologies, cat. no. 27865)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Countess automated cell counter [Thermo Fisher Scientific, cat. no. C1022; alternatively count cells using a hemacytometer and trypan blue exclusion as described in appendix 3G (Phelan, )]
  • Poly‐ornithine/laminin (POL)–coated 6‐well tissue culture plates (see recipe for POL‐coated plates)
  • Cell strainer, 40‐µm nylon mesh (BD Falcon, cat. no. 352340)
  • Angled, fire‐polished Pasteur pipet

Basic Protocol 2: Fluorescence‐Activated Cell Sorting (FACS) and Maintenance of Nascent NPC

  • FACS buffer (see recipe)
  • TrypLE Select (Gibco, cat. no. 12563‐029)
  • Day 20 to 21 rosette cluster cells ( protocol 1)
  • DNase I (Sigma, cat. no. D4527‐40KU)
  • Bovine serum albumin (BSA; Sigma, cat. no. A7906‐100 G)
  • PE‐CD133 (Miltenyi Biotec, cat. no. 130‐080‐801)
  • APC‐CD184 (BD Biosciences cat. no. 555976)
  • PerCP‐Cy5.5‐CD271 (BD Biosciences cat. no. 560834)
  • Sheath fluid: 0.9% NaCl (normal saline)
  • Reference NPC line (e.g., we use 8330‐8 NPC; Sheridan et al., )
  • 200 μg/ml DAPI (Molecular Probes, cat. no. D1306)
  • Neural proliferation medium (NPM, see recipe)
  • Freezing medium: 10% (v/v) DMSO in NPM
  • Liquid nitrogen
  • 35‐μm mesh strainer cap with FACS tube (BD Falcon Tube with Cell Strainer Cap, BD Falcon, cat. no. 352235)
  • Countess automated cell counter [Thermo Fisher Scientific, cat. no. C1022; alternatively count cells using a hemacytometer and trypan blue exclusion as described in appendix 3G (Phelan, )]
  • FACS machine (e.g., BD FACSAria IIu in biosafety cabinet)
  • FACS collection tubes (BD Falcon, cat. no. 352063)
  • Cryovials
  • Cell freezing container (e.g., Corning CoolCell)
  • Liquid nitrogen cryotank set up to accommodate cryovials

Basic Protocol 3: Generation of iNgn2‐NPC Stable Line for Precisely Timed and Scalable Initiation of Differentiation into Excitatory Forebrain Neurons

  • pTetO‐mNgn2‐TA‐puro (Zhang et al., ; Addgene plasmid ID# 52047)
  • pLX‐304 (Addgene plasmid ID# 25890)
  • Phusion Hot Start II DNA polymerase (ThermoFisher Scientific, cat. no. F549L)
  • PacI restriction endonuclease (NEB, cat. no. R0547S)
  • Calf intestinal alkaline phosphatase (NEB cat. no. M0290S)
  • PCR purification kit (e.g., Qiagen)
  • Quick Ligation kit (NEB cat. no. M2200S)
  • Stbl3 competent E. coli strain (Thermo Fisher Scientific cat. no. C737303)
  • Plasmid Maxi Kit (Qiagen, cat. no. 12162)
  • HEK293T cells (ATCC #293 T/17)
  • D10 medium (see recipe)
  • Transactivator plasmid pFUW‐M2rtTA (Hockemeyer et al., ; Addgene plasmid ID# 20342)
  • Lentiviral packaging plasmid pCMV‐dR8.2 dvpr (Stewart et al., ; Addgene plasmid ID# 8455)
  • VSV‐G envelope expressing plasmid pMD2.G (Addgene plasmid ID# 12259)
  • Opti‐MEM medium (Gibco, cat. no. 31985)
  • Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 11668‐019)
  • p24 ELISA kit: e.g., Lenti‐X P24 Rapid Titer Kit (Clontech, cat. no. 632200)
  • NPC growing in culture ( protocol 2)
  • Blasticidin (Gibco, cat. no. A11139‐03)
  • Neural proliferation medium (NPM, see recipe)
  • Phosphate‐buffered saline (PBS, MediaTech, cat. no. 21‐040‐CV)
  • iNgn2 neural medium (N3aM; see recipe)
  • Puromycin (Sigma, cat. no. P8833‐25MG)
  • Cytosine arabinoside (AraC; Sigma, cat. no. C6645)
  • Poly‐L‐lysine‐coated culture flasks (see recipe)
  • 0.45‐μm low‐protein‐binding filter (e.g., PES membrane)
  • POL‐coated plates (see recipe)
  • POLS‐coated plates (see recipe)
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), Sanger sequencing (Slatko et al., ), and other basic molecular biology techniques (Ausubel et al., )
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Literature Cited

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