Fast and Efficient Neural Conversion of Human Hematopoietic Cells

Cristina Bruzos‐Cidon1, Julio Castaño2, Maria Torrecilla1, Rosario Sanchez‐Pernaute3, Alessandra Giorgetti4

1 Department of Pharmacology, Faculty of Medicine, University of the Basque Country (UPV/EHU), 2 Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, 3 Laboratory of Stem Cells and Neural Repair, Fundación Inbiomed, San Sebastian, 4 Center for Regenerative Medicine in Barcelona, (CMRB), Barcelona
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.15
DOI:  10.1002/cpsc.16
Online Posting Date:  November, 2016
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Abstract

A major challenge in regenerative medicine is the generation of functionally effective target cells to replace or repair damaged tissues. The finding that most somatic cells can be directly converted into cells of another lineage by the expression of specific transcription factors has paved the way to novel applications. Induced neurons (iNs) represent an alternative source of neurons for disease modeling, drug screening, and potentially, for cell replacement therapy. This unit describes methods for the efficient conversion of blood cells into iNs, including protocols to isolate cord blood CD133+ cells, infect them with Sendai virus vectors that express SOX2 and c‐MYC, and differentiate the infected cells (PB‐MNCs) into mature neurons. A method to reprogram peripheral blood mononuclear cells into iNs is also described. Support protocols describe how to culture rat astrocytes and characterize the electrophysiology of iNs. © 2016 by John Wiley & Sons, Inc.

Keywords: cord blood; direct conversion; hematopoietic cells; induced neurons; reprogramming; Sendai virus

     
 
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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: Reprogramming CB CD133+ Cells to Neural Cells by SOX2 and C‐MYC SEV Vectors
  • Basic Protocol 2: Reprogramming PB‐MNCs Into Neural Cells Using SOX2 and C‐MYC Sendai Vectors
  • Support Protocol 1: Rat Primary Astrocyte Feeder Layers
  • Support Protocol 2: Whole‐Cell Patch‐Clamp Recording of Induced Neural Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Reprogramming CB CD133+ Cells to Neural Cells by SOX2 and C‐MYC SEV Vectors

  Materials
  • Fresh human cord blood (e.g., obtained from Hospital Donastia in San Sebastian, Spain, with signed informed consent from patients)
  • PBS‐EDTA buffer (see recipe)
  • Lympholyte‐H (Cederlane, cat. no. CL5016)Toxic by skin contact; wear gloves when handling.
  • Trypan blue stain (Invitrogen, cat. no. 15250‐061)
  • CD133 MicroBead Kit (Miltenyi Biotec, cat. no. 130‐050‐801), store at 4°C
  • PBS–0.2% (w/v) HSA (see recipe)
  • DMEM complete medium (see recipe)
  • Mouse IgG1 antihuman CD133‐PE (Miltenyi Biotec, cat. no.130‐080‐801)
  • Mouse IgG1 and k antihuman CD45‐APC (Becton Dickinson, cat. no. 555485)
  • Human recombinant SCF (see growth factors and cytokines recipe)
  • Human recombinant Flt3 (Flt3; see growth factors and cytokines recipe)
  • Human interleukin 6 (IL‐6; see growth factors and cytokines recipe)
  • Human thrombopoietin (TPO; see growth factors and cytokines recipe)
  • Human epidermal growth factor (EGF; see recipe)
  • CytoTune‐iPSC Sendai Reprogramming Kits (Life Technologies, cat. no. A13780‐01 or A13780‐02)
  • Anti‐SeV (MBL International Corporation, cat. no. IM‐0833)
  • DMEM‐F12 medium (Gibco, cat. no. 11320033)
  • Fetal bovine serum (FBS; Gibco, cat. no. 261440079)
  • 10 mg/ml polybrene (Chemicon, cat. no. TR‐1003‐6)
  • 100 ng/ml FGF2 (Miltenyi, cat. no. 130‐093‐842)
  • Neural induction medium, containing SMAD and GSK3β inhibitors (see recipe)
  • Accutase (Sigma, cat. no. A6964)
  • Neural expansion medium, containing BDNF and bFGF (see recipe)
  • 15 μg/ml polyornithine solution (Sigma, cat. no. P4957)
  • 1 μg/ml laminin (Sigma, cat. no. L2020)
  • Neural differentiation medium (see recipe), with GDNF, dibutyryl cAMP, and ascorbic acid
  • 500‐ml storage bottle (Corning, cat. no. 430282)
  • 15‐ and 50‐ml conical tubes
  • Mini‐MACS MS columns (Miltenyi Biotec, cat. no. 130‐041‐301)
  • Mini‐MACS separator (Miltenyi Biotec, cat. no. 130‐090‐312)
  • 2.5‐ml syringe (PentaFerte, cat. no. 08L01)
  • Preseparation filters (Miltenyi Biotec, cat. no. 130‐041‐407)
  • Class‐II tissue culture cabinet with aspirator (Bio‐II‐A, Telstar)
  • Inverted tissue culture microscope, with phase contrast and epifluorescence capabilities, and 5×, 10×, 20× and 40× objectives (Leica DMIL, Leica)
  • Tissue culture centrifuge with temperature control (Beckman Coulter)
  • 6‐well tissue culture plates
  • Slide flask (Nunc, cat. no. 170920)
  • 500‐ml bottle‐top filter system, 0.22‐μm (Millipore, cat. no. SCGPU05RE)

Basic Protocol 2: Reprogramming PB‐MNCs Into Neural Cells Using SOX2 and C‐MYC Sendai Vectors

  Materials (see also protocol 1)
  • PB‐MNCs (patient‐derived)
  • PBS–EDTA buffer (see recipe)
  • DMEM complete medium (see recipe)
  • Trypan blue stain (Life Technologies, cat. no. 15250‐061)
  • 10 mg/ml polybrene (Chemicon, cat. no. TR‐1003‐6)
  • Neural induction medium (see recipe)

Support Protocol 1: Rat Primary Astrocyte Feeder Layers

  Materials
  • Rat primary astrocytes (Lonza, cat. no. R‐CPAS‐522)
  • Astrocyte growth medium (see recipe)
  • Accutase
  • 0.1% gelatin (Millipore, cat. no. ES‐006‐B)
  • 500 μg/ml mitomycin C (see recipe)
  • PBS, Ca2+‐ and Mg2+‐free (Life Technologies, cat. no. 10010023)
  • Cryostor CS10 freezing medium (BioLife Solutions, cat. no. 210102)
  • 15‐ml conical tubes
  • T25 and T75 flasks
  • Cryovials

Support Protocol 2: Whole‐Cell Patch‐Clamp Recording of Induced Neural Cells

  Materials
  • ACSF (see recipe)
  • Glass coverslip with iNCs (from Basic Protocols protocol 11 or protocol 22)
  • Internal salt solution (see recipe)
  • L‐glutamic acid (Research Biochemicals International)
  • DNQX (Tocris Bioscience)
  • Specific equipment for patch‐clamp recordings
  • Fluorescence microscope (Nikon, Eklipse FN‐1), equipped with immersion objectives (Nikon 60×), micromanipulator (e.g., Siskiyou MC1000e), infrared camera and controller (Hamamatsu, C7500‐51 and C2741‐62), video monitor (HP, ZR30w), fluorescence light source (Nikon, C‐SHG1), and light lamp and controller (Nikon, Sumita LS‐DWL‐N and RC09‐ DWL/N)
  • Amplifier (Axo CNS, Multiclamp 700B)
  • Digidata (Axon CNS, 1440 A)
  • Needle puller (Narishige, model PC‐10)
  • Borosilicate glass capillaries (A‐M Systems, cat. no. 617000)
  • Head stage (Axon Instruments, CV‐7B)
  • Data acquisition software (Axon Instruments (Molecular devices), pClamp 10 Software)
  • Osmometer (Wescor, 5520)
  • Peristaltic pump (Gilson, Minipuls 3)
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Figures

Videos

Literature Cited

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