Characterization of Mouse Hematopoietic Stem and Progenitor Cells

Cindy L. Miller1, Brad Dykstra2, Connie J. Eaves3

1 StemCell Technologies, Vancouver, British, Columbia, 2 University Medical Centre Groningen, Groningen, The Netherlands, 3 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22B.2
DOI:  10.1002/0471142735.im22b02s80
Online Posting Date:  February, 2008
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Abstract

The unit describes functional assays for the quantification of mouse hematopoietic stem cells and progenitor cells. The competitive repopulating unit (CRU) assay detects transplantable mouse hematopoietic stem cells with the capacity to regenerate all of the blood cell lineages for extended time periods in vivo. The long‐term culture‐initiating cell (LTC‐IC) assay, founded on the bone marrow long‐term culture system, measures primitive hematopoietic progenitors based on their capacity to produce myeloid progeny for at least four weeks. Colony‐forming cell (CFC) assays, performed in semisolid medium cultures to assess mouse pre‐B, megakaryocyte, erythroid, granulocyte‐monocyte, and multipotential hematopoietic progenitors are also described. These assays are powerful tools for evaluating human stem cell (HSC) and progenitor content in various hematopoietic tissues, during development as well as in the adult animal, and in cell populations manipulated ex vivo. Curr. Protoc. Immunol. 80:22B.2.1‐22B.2.31. © 2008 by John Wiley & Sons, Inc.

Keywords: CRU assay; competitive repopulating unit assay; LTC‐IC assay; long‐term culture‐initiating cell assay; CFC assay; colony‐forming cell assay; methylcellulose‐based medium; collagen gels; acetylcholinesterase staining; hematopoietic stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Mouse Competitive Repopulating Unit (CRU) Assay
  • Alternate Protocol 1: CRU Assay Using Sublethally Irradiated W41/W41 Recipients
  • Alternate Protocol 2: CRU Assays Using Immunostaining in 96‐Well Plates
  • Support Protocol 1: Collection and Analysis of Flow Cytometry Data to Quantify Levels of Donor Reconstitution
  • Basic Protocol 2: Mouse Long‐Term Culture‐Initiating Cell (LTC‐IC) Assay
  • Basic Protocol 3: Mouse Colony‐Forming Cell (CFC) Assay
  • Alternate Protocol 3: Mouse Colony‐Forming Cell (CFC) Assay for Colony Forming Unit‐Megakaryocyte (CFU‐Mk) Using a Cytochemical Stain
  • Support Protocol 2: Isolation of Mouse Bone Marrow (BM) Cells
  • Support Protocol 3: Isolation of Day 14.5 Post‐Coitus Fetal Liver Cells
  • Support Protocol 4: Harvesting Colonies from Methylcellulose‐Based Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mouse Competitive Repopulating Unit (CRU) Assay

  Materials
  • Recipient mice: C57B1/6 (CD45.2) mice, 7 to 12 weeks old; healthy animals maintained under standardized pathogen‐free conditions; typically, between 12 and 48 mice per CRU experiment
  • Iscove's modified Dulbecco's medium ( appendix 2A) containing 2% (v/v) fetal bovine serum (IMDM/2% FBS), 2°C to 8°C
  • Helper cells: bone marrow (BM) cells from a minimum of two B6.SJL (CD45.1) mice ( protocol 8)
  • Test cells: adult BM cells ( protocol 8) or day 14.5 post‐coitus fetal livers (FL; three or more; protocol 9) from B6.SJL (CD45.1) mice
  • Acidified water, pH 3.0: prepared by adding 1 ml sterile 0.1 N HCl per 100 ml sterile water
  • Hanks' balanced salt solution ( appendix 2A) containing 2% (v/v) fetal bovine serum (HBSS/2% FBS)
  • 0.8% (w/v) ammonium chloride lysis solution (see recipe)
  • Unlabeled monoclonal antibody: anti‐mouse Fcγ receptor (clone 2.4G2)
  • Fluorochrome‐conjugated anti‐mouse monoclonal antibodies (available from, e.g., BD Biosciences, eBiosciences); titrated before use to determine appropriate concentrations (see Stewart and Stewart, ) and diluted in HBSS/2% FBS before use:
    • CD45.1‐fluorescein isothiocyanate (FITC; clone A20)
    • CD45.2‐allophycocyanin (APC; clone 104)
    • CD11b/Mac1‐PE
    • Ly6g‐PE (clone 1A8)
    • CD45R/B220‐phycoerythrin (PE; clone RA3–6B2)
    • CD5/Ly‐1‐PE (clone 53–7.3)
  • 1 µg propidium iodide (PI)/ml HBSS ( appendix 2A)/2% (v/v) FBS
  • Calibrated γ‐ or X‐ray irradiation source
  • Heat lamp
  • Restraining apparatus for mouse tail vein injections
  • 1‐cc syringes with 26‐G needles
  • #11 scalpel with pointed blade
  • Heparinized 100‐µl capillary tubes (e.g., Fisher)
  • 17 × 100–mm, 14‐ml polystyrene round‐bottom test tubes (e.g., Falcon, BD)
  • Refrigerated centrifuge, 2°C to 8°C
  • 12 × 75–mm, 5‐ml polystyrene round‐bottom test tubes (FACS tubes; e.g., Falcon, BD)
  • Dual laser flow cytometer (e.g., FACSCalibur, BD) and analysis software (e.g., Cell Quest Pro or FlowJo)
  • Limiting dilution analysis software (L‐Calc; free download from StemCell Technologies, http://www.stemcell.com)
  • Additional reagents and equipment for counting cells (e.g., see appendix 3A)

Alternate Protocol 1: CRU Assay Using Sublethally Irradiated W41/W41 Recipients

  • C57BL/6J‐KitW‐41J mice, 7 to 12 weeks of age

Alternate Protocol 2: CRU Assays Using Immunostaining in 96‐Well Plates

  • 96‐well U‐bottom plates (e.g., BD Biosciences)
  • Multidispensing pipettor
  • 100‐ to 200‐µl multichannel pipettor and troughs
  • Refrigerated centrifuge with rotor adapted for 96‐well plates, 2°C to 8°C
  • Automated 96‐well plate sampler for flow cytometer or 96‐well compatible 1.4‐ml round‐bottom FACS tubes and rack (e.g., Matrix technologies)

Support Protocol 1: Collection and Analysis of Flow Cytometry Data to Quantify Levels of Donor Reconstitution

  Materials
  • Immunostained cells from protocol 1, protocol 2, or protocol 3
  • Flow cytometer with CellQuest, FlowJo, or similar analysis software
  • L‐Calc or similar program (for calculating CRU frequency)

Basic Protocol 2: Mouse Long‐Term Culture‐Initiating Cell (LTC‐IC) Assay

  Materials
  • mLTCM/10−6 M HC (see recipe)
  • Mouse bone marrow (BM) cells (for feeder layers; protocol 8)
  • Iscove's modified Dulbecco's medium ( appendix 2A) containing 2% (v/v) fetal bovine serum (IMDM/2% FBS)
  • Test cells: Adult BM cells ( protocol 8)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • 0.25% (w/v) trypsin‐citrate or 0.25% (w/v) trypsin‐EDTA solution (StemCell Technologies)
  • FBS
  • MethoCult M3434 MC‐based medium (StemCell Technologies)
  • 96‐well flat‐bottom culture plates (e.g., Nunc, Corning)
  • Multichannel pipettors for accurate dispensing of 10 to 100 µl and >100 µl volumes
  • Plastic containers with loose cover (e.g., 20‐cm2 bacterial plates)
  • 35‐mm culture dishes, sterile
  • γ‐irradiation or X‐ray machine
  • 12 × 75–mm test tubes and racks
  • 1‐ and 3‐ml syringes and 16‐gauge blunt‐end needles for CFC assays (StemCell)
  • Inverted microscope
  • L‐Calc software program (free download from StemCell Technologies at http://www.stemcell.com)
  • Flow cytometer (e.g., FACSCalibur, BD Biosciences)
  • Additional reagents and equipment for counting cells ( appendix 3A) and enumerating CFC colonies ( protocol 6)

Basic Protocol 3: Mouse Colony‐Forming Cell (CFC) Assay

  Materials
  • Methylcellulose‐based culture (MC) media with cytokines (MethoCult, StemCell Technologies or prepared in‐house):
    • CFU‐E and mature BFU‐E assay: MethoCult M3334 (3 U/ml erythropoietin only)
    • BFU‐E, CFU‐GM, CFU‐GEMM assay: MethoCult M3434 (3 U/ml rh‐Epo, 10 ng/ml rmIL‐3, 50 ng/ml rmSCF, 10 ng/ml rhIL‐6)
    • CFU‐pre‐B assay: MethoCult M3630 (10 ng/ml rhIL‐7)
  • Test cells: adult BM cells ( protocol 8) or day 14.5 fetal livers (FL; three or more; protocol 9)
  • Iscove's modified Dulbecco's medium ( appendix 2A) containing 2% (v/v) fetal bovine serum (IMDM/2% FBS)
  • Exclusion dye (e.g., trypan blue)
  • 14‐ml test tubes
  • 3‐ml or 6‐ml syringes and 16‐G blunt‐end needles
  • 3‐ml Luer‐Lok syringe and 16‐G blunt‐end needle
  • 35‐mm low‐adherence petri dishes
  • 100‐mm (or larger) petri dishes or square bacterial dishes with loose‐fitting lids
  • Inverted microscope equipped with 2×, 4×, and 10× objectives
  • Additional reagents and equipment for counting cells ( appendix 3A)

Alternate Protocol 3: Mouse Colony‐Forming Cell (CFC) Assay for Colony Forming Unit‐Megakaryocyte (CFU‐Mk) Using a Cytochemical Stain

  • SF medium (containing cytokines; see recipe)
  • 3 mg/ml bovine collagen (StemCell Technologies), 2°C to 8°C (hold on ice during assay setup)
  • Acetone
  • Chamber slides with two wells, nylon separators, and adsorbent filters (StemCell Technologies)
  • Acetone‐resistant container
  • Additional reagents and equipment for performing acetylcholinesterase staining protocol (see http://www.stemcell.com)

Support Protocol 2: Isolation of Mouse Bone Marrow (BM) Cells

  Materials
  • B6.SJL (CD45.1) mice, at least two
  • 70% (v/v) isopropanol or ethanol
  • Medium: Hanks' balanced salt solution (HBSS; appendix 2A) containing 2% (v/v) fetal bovine serum (HBSS/2% FBS) or Iscove's modified Dulbecco's medium (IMDM; appendix 2A) containing 2% (v/v) fetal bovine serum (IMDM/2% FBS)
  • Fine‐point forceps and scissors (for bulk dissection), nonsterile
  • Fine‐point sharp scissors and forceps (for tissue isolation), sterile
  • 3‐cc syringes with 21‐ and 22‐gauge needles, sterile
  • 35‐mm petri dishes
  • 14‐ml tubes
  • 70‐µl cell filter
  • Refrigerated centrifuge, 2°C to 8°C
  • Additional reagents and equipment for counting cells (see appendix 3A)

Support Protocol 3: Isolation of Day 14.5 Post‐Coitus Fetal Liver Cells

  Materials
  • Male and female mice
  • 70% (v/v) isopropanol
  • Phosphate‐buffered saline ( appendix 2A) containing 2% (v/v) fetal bovine serum (PBS/2% FBS)
  • Iscove's modified Dulbecco's medium ( appendix 2A) containing 2% (v/v) fetal bovine serum (IMDM/2% FBS)
  • Forceps and scissors (for bulk dissection), nonsterile
  • Sharp scissors and forceps (for tissue isolation), sterile
  • 100‐mm petri dishes, sterile
  • Fine forceps, sterile
  • 14‐ml tube
  • 21‐G needle

Support Protocol 4: Harvesting Colonies from Methylcellulose‐Based Cultures

  Materials
  • Medium (e.g., culture medium, fixative, lysis solution)
  • Colonies in methylcellulose ( protocol 6)
  • 96‐well flat or round bottom plate or microcentrifuge tubes
  • Micropipettor with 200‐µl (or hand‐drawn Pasteur pipet) or 1‐ml tip
  • 4‐ and 50‐ml test tubes
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Figures

Videos

Literature Cited

Literature Cited
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