Mouse Models for Human Hemato‐Lymphopoiesis

Patrick Ziegler1, Markus G. Manz1

1 Institute for Research in Biomedicine, Bellinzona
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.13
DOI:  10.1002/0471140856.tx0213s31
Online Posting Date:  February, 2007
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Abstract

By transplanting human hematopoietic stem and progenitor cells in newborn immunodeficient mice, researchers have recently achieved development of a human adaptive immune system, consisting of B cells, T cells, dendritic cells, and some cells of the myeloid lineage, which together form respective structured lymphoid organs as thymus, spleen, and lymph nodes, and produce some functional immune responses. These will provide valuable models to analyze the impact of chemicals, pharmaceuticals, or species‐specific infections on human hematolymphoid cells in vivo. This unit describes methods for isolation of the progenitor human cells and their injection into mouse to constitute an adaptive immune system.

Keywords: xenogeneic mouse models; immunology; hemato‐lymphopoiesis

     
 
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Table of Contents

  • Basic Protocol 1: Intrahepatic Injection Of Cord Blood CD34+ Cells into Newborn BALB/c‐Rag2nullIL2R‐Ynull Mice
  • Support Protocol 1: Purification of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood
  • Support Protocol 2: Tattooing Transplanted Newborns for Identification
  • Support Protocol 3: Lymphoid Organ Removal from Transplanted Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Intrahepatic Injection Of Cord Blood CD34+ Cells into Newborn BALB/c‐Rag2nullIL2R‐Ynull Mice

  Materials
  • BALB/c‐Rag2nullIL2rγnull mice, newborn
  • Cord blood HSCs, purified (see protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Ethanol
  • Cesium 137 source (e.g., Biobeam 8000, STS)
  • 37°C waterbath
  • 14‐ml polypropylene tube
  • 1.5‐ml polyethylene microcentrifuge tubes
  • Sterile tissue
  • 25‐µl syringe (e.g., 1702 RN, Hamilton)
  • 30‐G needle (e.g., Hamilton)

Support Protocol 1: Purification of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood

  Materials
  • Human umbilical cord blood (local cord blood banking facility), fresh blood (within 24 hr after birth, stored at 4°C or at room temperature)
  • Ficoll‐Hypaque isotonic solution, density 1.077 g/ml (e.g. Biochrom AG), stored in the dark
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS/2% (v/v) FCS/2 mM EDTA; degassed at room temperature, 4° to 8°C
  • Direct CD34 progenitor cell isolation kit, human (Miltenyi Biotec) containing:
    • CD34 MicroBeads (colloidal super‐paramagnetic MicroBeads conjugated to monoclonal mouse anti‐human CD34 antibody)
    • FcR blocking reagent (human IgG)
  • Cryopreservation solution: 10% (v/v) DMSO in FCS
  • 50‐ml polypropylene tubes
  • MACS magnetic columns (Miltenyi Biotec)
  • Magnetic column adaptor (Miltenyi Biotec)

Support Protocol 2: Tattooing Transplanted Newborns for Identification

  Materials
  • Tattooing paste (e.g., Hauptner‐Herberholz)
  • 95% ethanol
  • Newborn mice
  • Tissue oil (e.g., Ballistol‐Animal, Klever)
  • Permanent make‐up pen (ask local tattoo dealer)
  • 1‐ to 5‐prong needles, individually packed (e.g., B. Braun Medicals)
  • 1.5‐ml polyethylene microcentrifuge tubes
  • Paper towel

Support Protocol 3: Lymphoid Organ Removal from Transplanted Mice

  Materials
  • Cord‐blood transplanted mice
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A), cold
  • RPMI medium, supplemented with 10% (v/v) FCS
  • 0.15 U/mg collagenase D (e.g., Roche)
  • 2000 U/mg DNase I, bovine pancreas grade II (e.g., Roche)
  • Ammonium chloride solution (NH 4Cl; see recipe)
  • Freezing solution: 90% (v/v) FGS/10% (v/v) DMSO
  • 22‐ and 24‐G needles
  • Thick Styrofoam box covers
  • Scissors, tweezers, and scalpel suitable for dissection of small animals (e.g., Fine Science Tools)
  • 100‐µm cell strainer (e.g., Falcon)
  • Mortar and pestle
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Figures

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Literature Cited

Literature Cited
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