Large Animal Models for Stem and Progenitor Cell Analysis

Robert E. Donahue1, Ken Kuramoto1, Cynthia E. Dunbar1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22A.1
DOI:  10.1002/0471142735.im22a01s69
Online Posting Date:  November, 2005
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Abstract

Extrapolation of an understanding regarding hematopoiesis and, in particular, hematopoietic stem cells (HSCs) from rodent models or in vitro human cell models to applications in humans has proven very difficult. This is not surprising, given the differences between rodent and human hematopoietic physiology and the lack of true in vitro assays for HSCs. Therefore, translational preclinical development of genetic and cellular therapies is dependent on the utilization of practical and well‐defined large animal models. This chapter will introduce the most commonly used model species, including macaques, baboons, dogs, cats, and sheep, and explain the particular advantages and limitations of each. Specific protocols for the support of macaques through ablative cell and gene therapy procedures will be included to introduce investigators to the types of resources and support required to maintain a large animal facility dedicated to high‐intensity experimentation, and also to introduce investigators to the types of procedures that are possible.

Keywords: stem cells; canine; feline; nonhuman primate; hematopoiesis; gene transfer; xenograft

     
 
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Table of Contents

  • Basic Protocol 1: Leukapheresis for the Collection of Hematopoietic Stem and Progenitor Cells for Transplantation
  • Alternate Protocol 1: Bone Marrow Harvesting for the Collection of Hematopoietic Stem and Progenitor Cells for Transplantation
  • Basic Protocol 2: Ficoll‐Hypaque Enrichment of Mononuclear Cells
  • Basic Protocol 3: Purification of Rhesus Macaque CD34+ Progenitor and Stem Cells
  • Basic Protocol 4: Transduction of Rhesus Macaque CD34+ Cells with Retroviral Vectors
  • Basic Protocol 5: Rhesus Macaque Autologous Stem Cell Transplantation
  • Basic Protocol 6: Supportive Care Post‐Transplantation
  • Support Protocol 1: Reverse Typing of Blood Groups
  • Support Protocol 2: Placement of an Indwelling Central Line Catheter
  • Support Protocol 3: Collection of Autologous Blood for Use at the Time of Bone Marrow or Mobilized Peripheral Blood Collection
  • Support Protocol 4: Rhesus Macaque Colony‐Forming Unit Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Leukapheresis for the Collection of Hematopoietic Stem and Progenitor Cells for Transplantation

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon) with indwelling central line catheter (see protocol 9) or without
  • Neupogen (granulocyte colony‐stimulating factor; Amgen)
  • Stem cell factor (R & D Systems)
  • Anticoagulant citrate dextrose formula A solution (ACDA; 500‐mL bag; Baxter Healthcare)
  • Normal saline (500‐ml bag; Baxter Healthcare)
  • 1000 U/ml preservative‐free heparin (American Pharmaceutical Partners)
  • Collected autologous blood (see protocol 10)
  • 1.88‐in., 18‐G angiocatheter
  • 12‐in., 19‐G intracatheter (if no central line catheter is in place)
  • CS‐3000 Plus blood cell separator (Baxter Healthcare) fitted with the following:
    • 42‐ml modified small‐volume collection chamber (American Fluoroseal)
    • Shunt chamber (Baxter Healthcare)
    • Apheresis tubing kit, open (Baxter Healthcare)
  • SCD 312 sterile connecting device (Haemonetics)
  • 150‐ and 600‐ml transfer packs (Baxter Healthcare)
  • Male luer from a 20‐in. extension set (Abbott Laboratories)
  • Kelly clamps
  • Blood component recipient set with 170‐µm filter and drip chamber (Baxter Healthcare)
  • 18‐G Needle*Lock device (Baxter Healthcare)
  • 19‐G needle
  • Sampling site coupler (Baxter Healthcare)
  • 60‐ml syringe
  • Additional reagents and equipment for preparing the animal (see protocol 9)
NOTE: The animal of interest is to receive no food for 12 hr prior to apheresis.

Alternate Protocol 1: Bone Marrow Harvesting for the Collection of Hematopoietic Stem and Progenitor Cells for Transplantation

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon)
  • Betadine surgical scrub
  • 1000 U/ml preservative‐free heparin (American Pharmaceutical Partners)
  • Normal saline (Baxter Healthcare) or Normosol (Abbott Laboratories)
  • Buprenex (Reckitt and Colman Pharmaceuticals)
  • 80‐mg acetaminophen tablets
  • Electric hair clippers
  • Sterile scrub brush or sponge
  • Sterile gloves
  • Sterile surgical drapes
  • 12‐ml slip‐tip syringes
  • Surgical air drill (Surgairtome Two; Zimmer/Hall)
  • 3frac14‐in., 16‐G angiocatheters
  • Bone wax (Ethicon)
  • 3‐0 or 4‐0 PDS sutures on cutting and taper needles
  • 16‐G Rosenthal BM aspiration needles (Popper and Sons)
  • Additional reagents and equipment for preparing the animal (see protocol 9)

Basic Protocol 2: Ficoll‐Hypaque Enrichment of Mononuclear Cells

  Materials
  • Collected peripheral blood (PB; see protocol 1) or bone marrow (BM; see protocol 2)
  • Hanks balanced salt solution ( appendix 22)
  • Ficoll‐Hypaque Plus (Amersham)
  • PBS ( appendix 22)
  • Freezing medium (see recipe)
  • 50‐ml conical tubes
  • Clinical centrifuge

Basic Protocol 3: Purification of Rhesus Macaque CD34+ Progenitor and Stem Cells

  Materials
  • Mononuclear cells isolated from PB or BM by Ficoll‐Hypaque gradient centrifugation (see protocol 3)
  • PBS containing 1% (w/v) BSA (Sigma)
  • Biotinylated anti‐CD34 antibody 12.8 ( )
  • Streptavidin‐coated microbeads (Miltenyi Biotec)
  • Freezing medium (optional; see recipe)
  • Clinical centrifuge
  • Tube rocker
  • 40‐µm cell strainers (BD Biosciences)
  • 50‐ml conical tubes
  • MidiMACS separation units (Miltenyi Biotec)
  • MACS MultiStand (Miltenyi Biotec)
  • MACS LS separation columns (Miltenyi Biotec)
  • 10‐ml round‐bottom Falcon tubes (BD Biosciences)
  • Additional reagents and equipment for counting viable cells ( appendix 3B)

Basic Protocol 4: Transduction of Rhesus Macaque CD34+ Cells with Retroviral Vectors

  Materials
  • RetroNectin (Takara)
  • Enriched CD34+ progenitor and stem cells (see protocol 4)
  • Vector‐containing supernatant (see recipe)
  • Recombinant human flt‐3/flk‐2 ligand (R & D Systems)
  • Recombinant human stem cell factor (R & D Systems)
  • Recombinant human thrombopoietin (R & D Systems)
  • PBS ( appendix 2A)
  • 0.25% (w/v) trypsin solution (Invitrogen)
  • 1× Dulbecco's modified Eagle medium (DMEM; Life Technologies) containing 10% (v/v) FBS (D10 medium)
  • Freezing medium (optional; see recipe)
  • T‐175 tissue culture flasks (BD Biosciences)
  • 50‐ml conical tubes
  • Clinical centrifuge
  • Additional reagents and equipment for counting viable cells ( appendix 3B)
NOTE: The authors use undiluted, freshly prepared vector‐containing supernatant on each day of culture and transduction. However, some investigators have reported diluting vector‐containing supernatant with an equal volume of D10 or serum‐free medium before use, and others have reported adding vector supernatant only for the final 48 hr of transduction.

Basic Protocol 5: Rhesus Macaque Autologous Stem Cell Transplantation

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon)
  • Diphenylhydramine (Benadryl; Pfizer)
  • Metoclopramide (Reglan; Wyeth‐Ayerst)
  • Ketamine, Telazol, or Telazol reconstituted with ketamine (Fort Dodge Animal Health)
  • Acepromazine (Vedco)
  • Granisetron hydrochloride (Kytril; Roche)
  • Normal saline (Baxter Healthcare)
  • Busulfan (Orphan Medical)
  • Autologous stem cells to be transplanted (see protocol 3, protocol 4, or protocol 5)
  • Hanks balanced salt solution (HBSS; appendix 22) containing 2% (v/v) FBS or 2% (v/v) autologous serum and containing 10 U/ml preservative‐free heparin (American Pharmaceutical Partners)
  • Eldorado 8 60Co teletherapy unit (Atomic Energy of Canada)
  • 18‐G (or larger) angiocatheter

Basic Protocol 6: Supportive Care Post‐Transplantation

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon) that has undergone autologous stem cell transplantation (see protocol 6), housed in a laminar flow cage unit (unit 1.2)
  • Magnesium sulfate
  • Potassium chloride
  • 5% (w/v) dextrose in half‐normal saline (D5/0.45% NaCl)
  • Neupogen (granulocyte colony‐stimulating factor; Amgen)
  • Vitamin K (2 mg/ml solution; Abbott Laboratories)
  • Folic acid (5 mg/ml solution; Boehringer Ingelheim)
  • Injectable iron dextran (100 mg/ml; Phoenix Pharmaceutical)
  • Ceftazidime (Ceptaz; GlaxoSmithKline)
  • Fluconazole (Diflucan; Pfizer)
  • Gentamicin (Abbott Laboratories)
  • Vancomycin (Abbott Laboratories)
  • Ciprofloxacin (Bayer)
  • Diphenylhydramine (Benadryl; Pfizer)
  • Amphotericin B (Boehringer Ingelheim)
  • Voriconazole (Pfizer)
  • Hydrocortisone sodium succinate (Solu‐Cortef; Pharmacia)
  • Normal saline (Baxter Healthcare)
  • 50% (w/v) magnesium sulfate solution in H 2O
  • Metronidazole (B. Braun Medical)
  • Blood group–matched donor animal
  • Citrate‐phosphate‐dextrose‐adenine preservative solution (CPDA; Baxter Healthcare)
  • Liposyn II 10% (Abbott Laboratories)
  • Aminosyn II 4.25% in 25% (w/v) dextrose (Abbott Laboratories)
  • 60‐ml syringe
  • Clinical centrifuge
  • Blood irradiator
  • 300‐ or 600‐ml transfer pack (Baxter Healthcare)
  • Blood component recipient set with 170‐µm filter and drip chamber (Baxter Healthcare)

Support Protocol 1: Reverse Typing of Blood Groups

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon) to be tested
  • Washed, packed human group O red blood cells
  • 2% (v/v) suspensions of human group O, group A1, group A2, and group B red blood cells (Gamma Biologicals)
  • 12 × 75–mm tubes
  • Clinical centrifuge

Support Protocol 2: Placement of an Indwelling Central Line Catheter

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon) to receive indwelling line catheter
  • Atropine sulfate (American Pharmaceutical Partners)
  • Ketamine, Telazol, or Telazol reconstituted with ketamine (Fort Dodge Animal Health)
  • Isoflurane (Abbott Laboratories)
  • Oxygen
  • Ketamine (Fort Dodge Animal Health, Fort Dodge, Iowa)
  • Telazol (Fort Dodge Animal Health, Fort Dodge, IO)
  • Saline or lactated normal Ringer's solution (Baxter Healthcare)
  • Betadine surgical scrub
  • Alcohol scrub solution
  • Betadine solution
  • Hypaque (diatrizoate sodium injection 50%; Nycomed)
  • Sterile water or half‐normal saline (0.45% w/v NaCl)
  • 100 U/ml sodium heparin in 0.45% (w/v) NaCl
  • Cefazolin sodium injection (Hanford)
  • Endotracheal tube (inner diameter, 4.0 to 6.0 mm; Hudson Respiratory Care)
  • Heated water blanket
  • Electric hair clippers
  • 1.88‐in. 8‐ to 19‐G angiocatheter
  • Surgical stapler or surgical pen or sponge
  • Surgical scissors or Kelly clamps
  • Surgical pick‐ups, various sizes
  • 6.6‐French single‐lumen Broviac catheter (Bard Access Systems)
  • 12‐in., 8‐G knitting needle, sterile
  • 3‐0 Prolene sutures and 3‐0 and 4‐0 PDS and Vicryl sutures on cutting and taper needles
  • Spinal catheter introducer (BD Medical Systems)
  • Fluoroscopy apparatus
  • Nonhuman primate jacket (Lomir or Alice King Chatham Medical Art)
  • 24‐in. stainless steel tether (Alice King Chatham Medical Art)
  • Intravenous infusion pump/monitor system (Omni‐Flow 4000 or Omni‐Flow 4000 Plus; Abbott Laboratories) with sterile intravenous tubing
NOTE: The animal of interest is to receive no food for 12 hr prior to catheter placement.NOTE: Aseptic technique must be used for line placement, as well as for all other survival surgical procedures.

Support Protocol 3: Collection of Autologous Blood for Use at the Time of Bone Marrow or Mobilized Peripheral Blood Collection

  Materials
  • Animal (e.g., rhesus macaque, cynomolgus macaque, baboon) from which blood is to be drawn
  • Sodium heparin (1000 U/ml; American Pharmaceutical Partners)
  • Citrate‐phosphate‐dextrose‐adenine preservative solution (CPDA; Baxter Healthcare)
  • Injectable iron dextran (100 mg/ml; Phoenix Pharmaceutical)
  • Adsol (Baxter Healthcare)
  • Normosol (Abbott Laboratories)
  • 300‐ml transfer pack (Baxter Healthcare)

Support Protocol 4: Rhesus Macaque Colony‐Forming Unit Assays

  Materials
  • Methylcellulose
  • Human recombinant granulocyte/macrophage colony‐stimulating factor (GM‐CSF; R & D Systems)
  • Human recombinant interleukin 3 (R & D Systems)
  • Human recombinant stem cell factor (R & D Systems)
  • Human recombinant erythropoietin (R & D Systems)
  • Whole or CD34‐enriched BM or mPB cells (see protocol 3 or protocol 4)
  • 1× Dulbecco's modified Eagle medium (DMEM; Life Technologies) containing 10% (v/v) FBS (D10 medium) or PBS ( appendix 22) containing 1% (w/v) BSA (Sigma)
  • PBS ( appendix 2A)
  • 15‐ml snap‐top tubes
  • Phosphate buffered saline
  • 3‐ml syringes fitted with 16‐G needles
  • 35‐ and 100‐mm‐diameter tissue culture dishes
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Literature Cited

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