In Vivo Evaluation of Leukemic Stem Cells through the Xenotransplantation Model

Dominique Bonnet1

1 Cancer Research UK, London Research Institute, Haematopoietic Stem Cell Laboratory, London, United Kingdom
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.2
DOI:  10.1002/9780470151808.sc0302s7
Online Posting Date:  December, 2008
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The xenotransplantation model has been instrumental for the identification and characterization of human leukemic stem cells. This unit describes our current method for the engraftment of human leukemic patients' samples in the xenotransplanted mouse model. We concentrate uniquely on the model of acute myeloid leukemia, as it was the first type of leukemia for which the xenotransplantation model was developed. Nevertheless, the Basic Protocol could be applied to other sorts of blood disorders. Curr. Protoc. Stem Cell Biol. 7:3.2.1‐3.2.11. © 2008 by John Wiley & Sons, Inc.

Keywords: hematopoietic stem cell (HSC); xenotransplantation; immunodeficient mice; leukemic stem cell (LSC)

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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of Leukemia Stem Cells Through Xenotransplantation
  • Support Protocol 1: Intra‐Bone Marrow Injection
  • Support Protocol 2: Thawing AML Cells
  • Support Protocol 3: Purification Strategy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Identification of Leukemia Stem Cells Through Xenotransplantation

  • Immunodeficient mice: NOD/SCID, NOD/SCID‐β 2 microglobulin null (β2m−/−), or NOD/SCID IL2R gammanull (Jackson Laboratory)
  • Acidified water: a solution of HCl at a final pH 2.8 to 3.2
  • AML sample: peripheral blood or bone marrow
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; see recipe)
  • Ammonium chloride solution (Stem Cell Technologies)
  • Fetal bovine serum (FBS; Stem Cell Technologies, cat. no. 06471)
  • Antibodies against human CD45, CD34, CD38, CD33, and CD19 (BD Biosciences Pharmingen)
  • 100 ng/ml 4′,6‐diamidino‐2‐phenylindole (DAPI; UV excited, Sigma‐Aldrich) or TOPRO‐3 [HeNe (633‐nm) excitable, Molecular Probes]
  • Irradiator: Cesium source is recommended, but an X‐ray system or Cobalt source can also be used
  • 29‐G, ½‐in. needle and insulin syringe (Tyco Healthcare)
  • Dissection tools: scissors and forceps
  • 5‐ml snap‐top polystyrene tubes
  • Benchtop centrifuge equipped with swing‐out bucket rotor for 15‐ and 50‐ml conical tubes
  • Hemacytometer
  • Fluorescent‐activated cell sorter, e.g., FACSAria (BD Biosciences) and/or a Moflow (Dako) equipped with 488‐nm, 633‐nm, and 404‐nm lasers
  • 440/40 bandpass (bp) filter for analysis of DAPI, a 530/30 bp filter for FITC, a 575/26 bp for PE, a 695/40 bp for PerCP, and a 660/20 bp for TOPRO‐3
  • Mouse depletion kit (e.g., StemCell Technologies, cat. no. 13066)
  • Additional reagents and equipment for assessing for AML engraftment ( protocol 4), parenteral injections (Donovan and Brown, ), euthanasia of mice (Donovan and Brown, ), and performing a cell count using a hemacytometer (unit 1.3)

Support Protocol 1: Intra‐Bone Marrow Injection

  • Anesthetic solution (see recipe)
  • Post‐operative analgesic (Vetergesic; Alstoe Animal health), diluted 1/10 in PBS and injected at 100 µl subcutaneously per mouse
  • 29‐G, ½‐in. needle (or 25‐G needle) and insulin syringe (Tyco Healthcare)

Support Protocol 2: Thawing AML Cells

  • AML cells, frozen in 1.8‐ to 2‐ml cryovials
  • DNase (Sigma, cat. no. D4513), thawed
  • Fetal bovine serum (FBS; Stem Cell Technologies, cat. no. 06471) or any other suppliers
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; see recipe)
  • 37°C water bath
  • 50‐ml centrifuge tubes, sterile
  • Table‐top centrifuge equipped with swing‐out bucket rotor for 15‐ and 50‐ml conical tubes
  • Cell strainer
  • Additional reagents and equipment for counting cells (unit 1.3)
NOTE: Before thawing the samples, ensure the water bath is at 37°C.NOTE: Before starting, ensure that the DNase is completely thawed.
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Literature Cited

Literature Cited
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