Isolation and Assessment of Single Long‐Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow

David G. Kent1, Brad J. Dykstra2, Connie J. Eaves3

1 Department of Haematology, University of Cambridge, 2 Harvard Medical School, Boston, Massachusetts, 3 Department of Medical Genetics, University of British Columbia, Vancouver
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.4
DOI:  10.1002/cpsc.10
Online Posting Date:  August, 2016
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Abstract

Hematopoietic stem cells with long‐term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45+EPCR+CD150+CD48 (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision. © 2016 by John Wiley & Sons, Inc.

Keywords: stem cell isolation; flow cytometry; hematopoiesis; single‐cell transplants; multi‐lineage reconstitution

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of CD45+EPCR+CD150+CD48− (ESLAM) Cells from Adult Mouse Bone Marrow
  • Basic Protocol 2: Transplantation of Single Cells and Tracking Their Regenerated WBC Progeny
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of CD45+EPCR+CD150+CD48− (ESLAM) Cells from Adult Mouse Bone Marrow

  Materials
  • C57B1/6J (B6) mice, 8 to 12 weeks old
  • HBSS/2% FBS: Hanks’ balanced salt solution containing 2% (w/v) fetal bovine serum (FBS)
  • 0.8% (w/v) ammonium chloride (NH 4Cl) solution (STEMCELL Technologies, cat no. 07850), ice cold
  • 0.1 mg/ml DNase I (Sigma; optional)
  • HBSS/2% FBS plus 1.2 µl mouse FcR blocking antibody (2.4G2; STEMCELL Technologies, cat. no. 01504)CD45‐FITC (Clone 30‐F11; Biolegend, cat. no. 103108)
  • EPCR‐PE (Clone RMEPCR1560; STEMCELL Technologies, cat. no. 60038PE)
  • CD150‐PECy7 (Clone TC15‐12F12.2; Biolegend, cat. no. 115914)
  • CD48‐APC (Clone HM48‐1; Biolegend, cat. no. 103412)
  • HBSS/2% FBS plus 1 µM 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma, cat no. D9542; note that 7‐AAD, PI, or other viability markers can be used as well)
  • Serum‐free medium (SFM): see recipe or purchase StemSpan serum‐free expansion medium (STEMCELL Technologies, cat. no. 09600)
  • Dissecting instruments including scissors and forceps, sterile
  • Mortar and pestle (optional)
  • 70‐µm or 100‐µm filters for 50‐ml tubes (optional)
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Corning Falcon)
  • 21‐G, 1‐in. general‐use sterile hypodermic needles (BD, cat. no. 305165) and syringes
  • Tabletop centrifuge
  • Flow cytometer sample tubes (e.g., BD, cat. no. 352058 or 352063)
  • 5‐ml filter‐capped tubes (BD, cat. no. 358235)
  • Flow cytometer with sorting capability (e.g., Influx or Aria equipped with a 405, 488, and 640 nm lasers, BD)
  • 96‐well U‐bottom plates (e.g., Nunc, cat. no. 163320; optional, necessary if performing single‐cell isolation as in protocol 2)
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Basic Protocol 2: Transplantation of Single Cells and Tracking Their Regenerated WBC Progeny

  Materials
  • Single HSCs sorted into 100 to 200 µl medium in a 96‐well plate ( protocol 1)
  • Irradiated recipient mice (strain W41/W41 or B6, >7 weeks old; see annotation to step 6 of this protocol regarding irradiation)
  • 0.8% (w/v) ammonium chloride (NH 4Cl) solution (STEMCELL Technologies, cat no. 07850) ice cold
  • HBSS/2% FBS: Hanks’ balanced salt solution containing 2% (w/v) fetal bovine serum (FBS)
  • 2× blocking reagent (see recipe)
  • Antibody cocktails for peripheral blood analysis (see recipe)
  • HBSS/2% FBS plus 1 µM 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma, cat no. D9542)
  • Tabletop centrifuge with microtiter plate carrier
  • Inverted microscope (preferably with movable stage)
  • Insulin syringes with 28‐G, 0.5‐in needles (BD, cat no. 329465)
  • 137Cs γ‐ray irradiation machine (or X‐ray irradiator)
  • Infra‐red heat lamp
  • Heparinized capillary tubes (e.g., Fisher, cat no. 22‐362‐56)
  • 12 × 75–mm tubes with caps (BD cat. no. 352057)
  • 96‐well U‐bottom microtiter plates (e.g., Nunc, cat. no. 163320)
  • ScreenMates 1.4‐ml round‐bottom storage tubes in snap rack (Thermo Scientific, cat. no. 4246)
  • Flow cytometer (e.g., BD Fortessa equipped with 405, 488, and 640 nm lasers)
  • Additional reagents and equipment for blood collection (Donovan and Brown, ) and flow cytometry (Robinson et al., )
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Figures

Videos

Literature Cited

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