Human Hematopoietic Cell Culture, Transduction, and Analyses

Jesper Bonde1, Louisa Wirthlin2, Donald B. Kohn3, Jan A. Nolta2

1 Statens Serum Institut, Copenhagen, Denmark, 2 University of California at Davis, Sacramento, California, 3 University of Southern California School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.7
DOI:  10.1002/0471142905.hg1307s56
Online Posting Date:  January, 2008
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Abstract

This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34+ cells, transduction of these cells with a retroviral vector on fibronectin‐coated plates, assaying the efficiency of transduction, and establishing long‐term cultures. Support protocols describe methods for maintenance of vector‐producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long‐term cultures. Other protocols provide PCR‐based methods to analyze individual colonies for transduction, methods to analyze cells harvested from long‐term cultures, and procedures for freezing and thawing of hematopoietic cells. Curr. Protoc. Hum. Genet. 56:13.7.1‐13.7.31. © 2008 by John Wiley & Sons, Inc.

Keywords: hematopoietic progenitors; transduction; retroviral vector

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Retroviral‐Mediated Transduction of CD34+ Cells on Retronectin in Serum‐Free Culture with Cytokine Support
  • Support Protocol 1: Maintenance of Vector‐Producing Fibroblasts and Collection of Cell‐Free Supernatant for Transduction
  • Support Protocol 2: Preparation of Retronectin‐Coated Plates
  • Support Protocol 3: Enzymatic Removal of Magnetic Beads from Immunomagnetically Selected Cells
  • Support Protocol 4: Plating Colony‐Forming Cells
  • Support Protocol 5: Preparing Whole‐Cell Lysates of Individual CFU Colonies for PCR
  • Support Protocol 6: Analysis of Clonal Integration in Individual Colonies
  • Support Protocol 7: Establishing Primary Human Marrow Stromal Monolayers from Harvested Bone Marrow
  • Support Protocol 8: Harvesting Cells for Analysis of Long‐Term Bone Marrow Cultures
  • Support Protocol 9: Freezing and Thawing Hematopoietic Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Retroviral‐Mediated Transduction of CD34+ Cells on Retronectin in Serum‐Free Culture with Cytokine Support

  Materials
  • Human bone marrow, mobilized peripheral blood, or umbilical cord blood, treated with anticoagulant
  • Hanks' buffered saline solution (HBSS, Sigma; also see appendix 2D)
  • Phosphate‐buffered saline (PBS, Sigma; also see appendix 2D)
  • Ficoll‐Hypaque (GE Healthcare)
  • CD34 purification kit including magnetic column (MACS) system to isolate target cell population (e.g., Miltenyi Biotec, Dynal, or Stem Cell Technologies)
  • Serum‐free transduction medium: X‐Vivo 15 (Lonza Group, Basel, Switzerland) with recombinant human SCF, Flt3‐L, and TPO (each at 10 ng/ml)
  • Retroviral vector supernatant (see protocol 2)
  • 1 mg/ml protamine sulfate (Lyphomed; optional)
  • Cell dissociation buffer (Invitrogen)
  • Basal bone marrow medium (BBMM; see recipe)
  • Methylcellulose medium (see recipe)
  • 50 mg/ml geneticin (see recipe)
  • 100 mM HEPES buffer solution (Invitrogen)
  • Monolayer cultures of irradiated stromal cells in 25‐cm2 vent‐cap flasks (see protocol 8)
  • Long‐term bone marrow culture medium (LBMM; see recipe)
  • 50‐ml conical polypropylene centrifuge tubes
  • Beckman GS‐6R centrifuge and GH 3.7 rotor (or equivalent) with adaptors for 50‐ml tubes
  • Automated cell counter (optional)
  • 1‐ml syringes
  • Rotating platform
  • 35 × 10–mm gridded tissue culture dishes (Nunc)
  • 10‐ and 35‐cm tissue culture dishes
  • Additional reagents and equipment for retronectin coating of plates ( protocol 3) and counting cells by trypan blue exclusion ( appendix 3G)

Support Protocol 1: Maintenance of Vector‐Producing Fibroblasts and Collection of Cell‐Free Supernatant for Transduction

  Materials
  • Culture of vector‐producing fibroblasts (VPF; ATCC #CLR‐10686)
  • Trypsin (BioWhittaker)D10HG medium (see recipe), 32°C
  • 75‐cm2 tissue culture flasks with 0.2‐µm‐filter vented caps (Costar)
  • 32°C, humidified, 4.5% to 5.0% CO 2 incubator
  • 0.45‐µm Uniflo‐25 syringe filter with calcium acetate membrane (Schleicher & Schuell)
  • 10‐ml polypropylene tubes
  • Additional reagents and equipment for trypsinizing and subculturing monolayer cells ( appendix 3G)

Support Protocol 2: Preparation of Retronectin‐Coated Plates

  Materials
  • Recombinant fibronectin C‐terminal fragment (retronectin) containing CS‐1, RGD, and heparin‐binding domains, lyophilized (BioWhittaker)
  • PBS with Mg2+/Ca2+ (Sigma)
  • 2% (w/v) BSA: dilute deionized 10% BSA (see recipe) 1:5 in PBS (with Mg2+/Ca2+; Sigma)
  • 6‐well tissue culture plates, 25‐cm2 vent‐cap tissue culture flasks, or 35‐mm tissue culture dishes

Support Protocol 3: Enzymatic Removal of Magnetic Beads from Immunomagnetically Selected Cells

  Materials
  • Immunomagnetically selected CD34+ cells (see protocol 1)
  • RPMI 1640 medium containing 1% (w/v) human serum albumin (RPMI/1% HSA)
  • 2500 U/ml chymopapain (Chymodactin, Baxter)
  • Beckman GS‐6R centrifuge and GH 3.7 rotor (or equivalent)

Support Protocol 4: Plating Colony‐Forming Cells

  Materials
  • Long‐term bone marrow cultures, 5 weeks after plating or post‐transduction cultures
  • Long‐term bone marrow culture medium (LBMM; see recipe)
  • Methylcellulose medium (see recipe) with appropriate supplements (see protocol 1) or MethoCult (StemCell Technologies, cat. no. 4434).
  • 25‐cm2 tissue culture flasks with 0.2‐µm filter‐vented caps (Costar)
  • 15‐ml polypropylene centrifuge tubes
  • Beckman GS‐6R centrifuge and GH 3.7 rotor (or equivalent)
  • 35 ×10–mm gridded tissue culture dishes (Nunc)
  • Additional reagents and equipment for trypsinizing monolayer cultures and counting cells ( appendix 3G)

Support Protocol 5: Preparing Whole‐Cell Lysates of Individual CFU Colonies for PCR

  Materials
  • Cultures of cfu in methylcellulose medium (see protocol 1)
  • PBS (Sigma)
  • RBC lysis buffer (optional; see recipe)
  • Whole‐cell lysis buffer (see recipe)
  • 10 mg/ml proteinase K (Sigma)
  • Inverted phase‐contrast microscope
  • 20‐ to 200‐µl plugged pipet tips, sterile
  • 56° and 100°C water bath or heat block

Support Protocol 6: Analysis of Clonal Integration in Individual Colonies

  Materials
  • Individual well‐isolated colonies (see protocol 6)
  • Proteinase K digestion buffer (see recipe)
  • 10 mg/ml proteinase K (Sigma) in H 2O
  • 25:24:1 (v/v/v) buffered phenol/chloroform/isoamyl alcohol ( appendix 3B)
  • 20 mg/ml glycogen (Boehringer Mannheim)
  • 10 M ammonium acetate ( appendix 2D)
  • 100% and 70% ethanol
  • TE buffer, pH 7.5 ( appendix 2D)
  • 0.1 M spermidine (Sigma)
  • React 2 buffer (Invitrogen)
  • 5 U/µl TaqI restriction enzyme (Invitrogen)
  • 5 U/µl T4 DNA ligase and 5× buffer (Life Technologies)
  • 50 pmol/µl PCR primers (Operon):
    • INVa: 5′‐AGGAACTGCTTACCAACA‐3′
    • INVb: 5′‐CTGTTCCTTGGGAGGGT‐3′
    • INVc: 5′‐TCCTGACCTTGATCTGA‐3′
    • INVd: 5′‐CTGAGTGATTGACTACC‐3′
  • 10× PCR amplification buffer ( appendix 2D, or purchase from Perkin‐Elmer)
  • 25 mM MgCl 2
  • 2 mM 4dNTP mix ( appendix 2D)
  • 5 U/µl AmpliTaq DNA polymerase (Perkin‐Elmer)
  • Mineral oil (PCR grade)
  • SeaKem LE agarose (FMC Bioproducts)
  • NuSieve agarose (FMC Bioproducts)
  • 1 × 107 cpm [32P]ATP end‐labeled oligonucleotide probe ( appendix 3E) specific for LTR sequences: 5′‐GGCAAGCTAGCTTAAGT‐3′
  • Circumvent Thermal Cycle Sequencing Kit (New England Biolabs)
  • 56° and 65°C water baths or heat blocks
  • PCR tubes
  • Thermal cycler
  • Nylon membrane (e.g., Biotrace, Pall Biodyne)
  • Scalpel, sterile
  • Spin‐X columns (Costar)
  • Additional reagents and equipment for isolating colonies of cfu and lysing red blood cells (see protocol 6), extracting and precipitating DNA ( appendix 3C), and agarose gel electrophoresis, Southern blotting, and hybridization (unit 2.7)

Support Protocol 7: Establishing Primary Human Marrow Stromal Monolayers from Harvested Bone Marrow

  Materials
  • Screens used to filter harvested bone marrow or bone marrow aspirate
  • Dexter's original medium (DOM; see recipe)
  • PBS ( appendix 2D)
  • Trypsin/versene solution (BioWhittaker)
  • Hanks' buffered saline solution (HBSS; appendix 2D)
  • D10HG medium (see recipe)
  • Basal bone marrow medium (BBMM; see recipe)
  • 25‐cm2 and 75‐cm2 tissue culture flasks with 0.2‐µm filter‐vented caps (Costar)
  • Cesium radiation source (e.g., Gammacell 1000, Nordion International)
  • Additional reagents and equipment for trypsinizing and subculturing monolayer cells ( appendix 3G)

Support Protocol 8: Harvesting Cells for Analysis of Long‐Term Bone Marrow Cultures

  Materials
  • Bone marrow, cord blood, or peripheral blood cells
  • Autologous serum or heat‐inactivated human AB serum
  • Freezing medium (see recipe), 4°C
  • Liquid nitrogen
  • 1 mg/ml DNase (Sigma), filter sterilized
  • Thawing medium (see recipe)
  • PBS (Sigma)
  • 2‐ml cryovials (Nalge)
  • Rate‐controlled cell freezer
  • 15‐ml centrifuge tubes, sterile
  • Centrifuge
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Figures

Videos

Literature Cited

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Key References
   Hanenberg et al., 1997. See above.
  Describes the usefulness of and mechanism for using recombinant fibronectin to augment retroviral‐mediated gene transfer into hematopoietic cells.
   Nolta et al., 1995. See above.
  Describes methods directly precedent to the use of fibronectin in the current methods, as well as method for making support layers for long‐term culture. Also describes the benefits of using stromal cell layer to augment transduction and maintain the engraftment ability of human hematopoietic progenitor cells in an immune‐deficient murine xenograft system.
   Nolta et al., 1996. See above.
  First demonstration of retroviral‐mediated transduction of pluripotent human stem cells.
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