Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells

Hideo Ema1, Yohei Morita1, Hiromitsu Nakauchi1, Yumi Matsuzaki2

1 The University of Tokyo, Tokyo, null, 2 Keio University School of Medicine, Tokyo, null
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22B.1
DOI:  10.1002/0471142735.im22b01s67
Online Posting Date:  July, 2005
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Abstract

Protocols for the purification of CD34KSL cells, CD34+KSL cells, SP cells, and Tip‐SP CD34KSL cells are described. Mouse hematopoietic stem cells are highly enriched in the CD34KSL and SP fractions. These two populations overlap one another. A nearly homogenous population of HSCs can be obtained by isolating Tip‐SP CD34KSL cells. One of the earliest progenitor cell populations is CD34+KSL cells.

Keywords: hematopoietic stem cells; hematopoietic progenitor cells; SP cells

     
 
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Table of Contents

  • Basic Protocol 1: Purification of CD34−KSL cells and CD34+KSL Cells from Adult Bone Marrow
  • Basic Protocol 2: Purification of SP Cells from Adult Bone Marrow Cells
  • Alternate Protocol 1: Purification of TIP‐SP CD34−KSL Cells from Adult Bone Marrow
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of CD34−KSL cells and CD34+KSL Cells from Adult Bone Marrow

  Materials
  • C57BL/6 (B6) mice, 8 to 10 weeks of age (e.g., The Jackson Laboratory)
  • Phosphate‐buffered saline (PBS; Sigma cat. no. D‐565; also see appendix 2A)
  • Türk solution (Wako Chemicals)
  • Staining medium (SM; see recipe)
  • Ficoll‐Paque PLUS (density 1.077 ± 0.001 g/ml, Amersham Bioscience)
  • Monoclonal antibodies (Table 22.1.1)
  • Dynabeads M‐280 streptavidin (Dynal)
  • Streptavidin–Texas Red working solution (see recipe)
  • Dissecting instruments including small scissors and forceps
  • 5‐ml syringe with 25‐G needle
  • 50‐µm nylon mesh (e.g., Millipore), sterilized by autoclaving
  • 15‐ml centrifuge tubes (e.g., TPP AG)
  • Refrigerated centrifuge
  • Magnetic separation apparatus: MPC‐1 (Dynal)
  • FACSVantage SE (BD Biosciences) equipped with argon laser and dye laser and appropriate filters (Table 22.1.2), or equivalent flow sorting apparatus
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), counting cells using a hemacytometer ( appendix 3A), fractionation of cells using immunomagnetic beads (unit 3.5), and counting of viable cells ( appendix 3B)
    Table 2.0.1   Materials   Monoclonal Antibody List for Purification of CD34KSL Cells and CD34+KSL Cells from Adult Bone Marrow a   Monoclonal Antibody List for Purification of CD34KSL Cells and CD34+KSL Cells from Adult Bone Marrow   FACSVantage SE Settings c   FACSVantage SE Settings

    Antibody Stock conc. Clone Labeling Working dilution
    Anti–mouse Mac‐1 0.5 mg/ml M1/70 Biotinylated b
    Anti–mouse Gr‐1 0.5 mg/ml RB6‐8C5 Biotinylated b
    Anti–mouse B220 0.5 mg/ml RA3‐6B2 Biotinylated b
    Anti–mouse CD4 0.5 mg/ml L3T4 Biotinylated b
    Anti–mouse CD8a 0.5 mg/ml 53‐6.7 Biotinylated b
    Anti–mouse Ter‐119 0.5 mg/ml TER‐119 Biotinylated b
    Anti–mouse IL‐7R 0.5 mg/ml A7R34 Biotinylated b
    Anti–mouse Sca‐1 0.2 mg/ml E13‐161.7 PE 1:10
    Anti–mouse c‐kit 0.2 mg/ml 2B8 APC 1:10
    Anti–mouse CD34 0.5 mg/ml RAM34 FITC No dilution
    Fluorochrome PMT Excitation (nm) Emission (nm) Optical filter (nm)
    FITC FL1 488 525 530/30
    PE FL2 488 575 575/26
    PE‐Cy5.5 FL3 488 694 695/40 d
    APC FL4 600 660 660/20
    Texas Red FL5 600 615 630/22
    Hoechst 33342 FL6 FL3 350 450/BP20 (blue) d 660/BP20 (red) d

     aThe table shows rough estimates of working dilution for each antibody; however, precise titration is required. All of these reagents are available from either PharMingen or e‐Bioscience (see appendix 55). Abbreviations: PE, phycoerythrin; APC, allophycocyanin; FITC, fluorescein isothiocyanate.
     bEqual volumes of the stock solutions of each of these antibodies are usually mixed to prepare a lineage marker cocktail. However, because there is batch‐to‐batch variation among antibodies, it is highly recommended that each antibody be titrated before use.
    Table 2.0.2   Materials   Monoclonal Antibody List for Purification of CD34KSL Cells and CD34+KSL Cells from Adult Bone Marrow a   Monoclonal Antibody List for Purification of CD34KSL Cells and CD34+KSL Cells from Adult Bone Marrow   FACSVantage SE Settings c   FACSVantage SE Settings

    Antibody Stock conc. Clone Labeling Working dilution
    Anti–mouse Mac‐1 0.5 mg/ml M1/70 Biotinylated b
    Anti–mouse Gr‐1 0.5 mg/ml RB6‐8C5 Biotinylated b
    Anti–mouse B220 0.5 mg/ml RA3‐6B2 Biotinylated b
    Anti–mouse CD4 0.5 mg/ml L3T4 Biotinylated b
    Anti–mouse CD8a 0.5 mg/ml 53‐6.7 Biotinylated b
    Anti–mouse Ter‐119 0.5 mg/ml TER‐119 Biotinylated b
    Anti–mouse IL‐7R 0.5 mg/ml A7R34 Biotinylated b
    Anti–mouse Sca‐1 0.2 mg/ml E13‐161.7 PE 1:10
    Anti–mouse c‐kit 0.2 mg/ml 2B8 APC 1:10
    Anti–mouse CD34 0.5 mg/ml RAM34 FITC No dilution
    Fluorochrome PMT Excitation (nm) Emission (nm) Optical filter (nm)
    FITC FL1 488 525 530/30
    PE FL2 488 575 575/26
    PE‐Cy5.5 FL3 488 694 695/40 d
    APC FL4 600 660 660/20
    Texas Red FL5 600 615 630/22
    Hoechst 33342 FL6 FL3 350 450/BP20 (blue) d 660/BP20 (red) d

     cAbbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin.
     dFilters available from Omega Optical.

Basic Protocol 2: Purification of SP Cells from Adult Bone Marrow Cells

  Materials
  • Staining buffer 1 (SB1; see recipe)
  • 1 mg/ml (200×) Hoechst 33342 stock solution (Sigma cat. no. B‐2261; store ∼1 year at −20°C)
  • SB1 (see recipe) containing 1 µg/ml propidium iodide (PI; add fresh from 200 µg/ml working PI stock, see recipe)
  • FACS Vantage (BD Bioscience) or equivalent flow sorting apparatus equipped with UV laser capable of excitation at 350 nm and detection with 450/BP20 and 660/BP20 or equivalent optical filters (Table 22.1.2)
  • Additional reagents and equipment for purification of CD34KSL cells and CD34+KSL cells (see protocol 1)

Alternate Protocol 1: Purification of TIP‐SP CD34−KSL Cells from Adult Bone Marrow

  Materials
  • C57BL/6 (B6) mice, 8 to 10 weeks of age (e.g., The Jackson Laboratory)
  • Staining buffer 2 (SB2; see recipe)
  • Phosphate‐buffered saline (PBS; Sigma cat. no. D‐565; also see appendix 2A)
  • Türk solution (Wako Chemicals)
  • 1 mg/ml (200×) Hoechst 33342 stock solution (Sigma cat. no. B‐2261; store ∼1 year at −20°C)
  • 50 mM (100×) verapamil (Sigma) stock solution in 10% ethanol
  • Ficoll‐Paque PLUS (density 1.077 ± 0.001 g/ml, Amersham Bioscience)
  • Monoclonal antibodies (Table 22.1.3)
  • 200 µg/ml streptavidin‐APC‐Cy7 (Pharmingen); store up to 3 months at 4°C
  • SB2 (see recipe) containing 2 µg/ml propidium iodide (PI; add fresh from 200 µg/ml working PI stock, see recipe)
  • Mortar and pestle, sterile
  • Falcon 2350 cell strainer
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes, sterile (e.g., Falcon)
  • Refrigerated centrifuge
  • 250‐ml tissue culture flask or conical tube (Corning)
  • 60‐µm nylon mesh (e.g., Millipore), sterilized by autoclaving
  • MoFlo flow sorter (Cytomation) equipped with argon, UV, and helium‐neon laser, appropriate filters (Table 22.1.4), and 510‐nm long‐pass dichroic mirror (Omega Optical)
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), counting cells using a hemacytometer ( appendix 3A), and flow sorting (Chapter 5)
    Table 2.0.3   Materials   Monoclonal Antibody List for Purification of Tip‐SP CD34KSL Cells e   Monoclonal Antibody List for Purification of Tip‐SP CD34KSL Cells   MoFlo Settings g   MoFlo Settings

    Antibody Stock conc. Clone Labeling Working dilution
    Anti–mouse Mac‐1 0.5 mg/ml M1/70 Biotinylated f
    Anti–mouse Gr‐1 0.5 mg/ml RB6‐8C5 Biotinylated f
    Anti–mouse B220 0.5 mg/ml RA3‐6B2 Biotinylated f
    Anti–mouse CD4 0.5 mg/ml L3T4 Biotinylated f
    Anti–mouse CD8a 0.5 mg/ml 53‐6.7 Biotinylated f
    Anti–mouse Ter‐119 0.5 mg/ml TER‐119 Biotinylated f
    Anti–mouse Sca‐1 0.2 mg/ml E13‐161.7 PE No dilution
    Anti–mouse c‐kit 0.2 mg/ml 2B8 APC No dilution
    Anti–mouse CD34 0.5 mg/ml RAM34 FITC No dilution
    Fluorochrome PMT Excitation (nm) Emission (nm) Optical filter (nm) h
    FITC FL1 488 525 530/40
    PE FL2 488 575 580/30
    PI FL3 488 694 630/30
    PE‐Cy5 FL4 488 694 670/30
    Hoechst Red FL5 350 600 570/20
    Hoechst Blue FL6 350 450 405/30
    APC FL7 660 670 670/20
    APC‐Cy7 FL8 660 780 740ELP

     eThe table shows rough estimates of working dilution for each antibody; however, precise titration is required. All of these reagents are available from either PharMingen or e‐Bioscience (see appendix 55). Abbreviations: PE, phycoerythrin; APC, allophycocyanin; FITC, fluorescein isothiocyanate.
     fEqual volumes of the stock solutions of each of these antibodies are usually mixed to prepare a lineage marker cocktail. However, because there is batch‐to‐batch variation among antibodies, it is highly recommended that each antibody be titrated before use.
    Table 2.0.4   Materials   Monoclonal Antibody List for Purification of Tip‐SP CD34KSL Cells e   Monoclonal Antibody List for Purification of Tip‐SP CD34KSL Cells   MoFlo Settings g   MoFlo Settings

    Antibody Stock conc. Clone Labeling Working dilution
    Anti–mouse Mac‐1 0.5 mg/ml M1/70 Biotinylated f
    Anti–mouse Gr‐1 0.5 mg/ml RB6‐8C5 Biotinylated f
    Anti–mouse B220 0.5 mg/ml RA3‐6B2 Biotinylated f
    Anti–mouse CD4 0.5 mg/ml L3T4 Biotinylated f
    Anti–mouse CD8a 0.5 mg/ml 53‐6.7 Biotinylated f
    Anti–mouse Ter‐119 0.5 mg/ml TER‐119 Biotinylated f
    Anti–mouse Sca‐1 0.2 mg/ml E13‐161.7 PE No dilution
    Anti–mouse c‐kit 0.2 mg/ml 2B8 APC No dilution
    Anti–mouse CD34 0.5 mg/ml RAM34 FITC No dilution
    Fluorochrome PMT Excitation (nm) Emission (nm) Optical filter (nm) h
    FITC FL1 488 525 530/40
    PE FL2 488 575 580/30
    PI FL3 488 694 630/30
    PE‐Cy5 FL4 488 694 670/30
    Hoechst Red FL5 350 600 570/20
    Hoechst Blue FL6 350 450 405/30
    APC FL7 660 670 670/20
    APC‐Cy7 FL8 660 780 740ELP

     gAbbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PI, propidium iodide; APC, allophycocyanin.
     hFilters available from Omega Optical.
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Figures

  •   FigureFigure 22.B0.1 Sequential gating for CD34KSL cells. (A) FSC and SSC gate, (B) Lin gate, (C) CD34−/low gate, and (D) Sca‐1+ and c‐kit+ gate are demonstrated.
  •   FigureFigure 22.B0.2 Sequential gating for Tip‐SP CD34KSL cells (AC) Flow cytometric analysis of Hoechst dye efflux in mouse bone marrow cells. The SP cells are gated with polygons at the same position in each panel. Bone marrow cells were stained with the Hoechst dye in the presence of verapamil (A) or absence of verapamil (B). Mononucleated bone marrow cells were strained with the Hoechst dye (C). Indicated numbers are the mean percentages of SP cells in live cell gating (PI‐negative cells). (DF) Immunophenotype of mouse bone marrow cells stained with antibodies against c‐Kit, lineage mixture (Lin), Sca‐1, and CD34. (D) c‐kit+ and Lin cells (KL) are gated. (E) Sca‐1 and CD34 expression profile of KL cells is shown. (F) Hoechst Blue versus Red profile of CD34KSL population is shown. A significant portion of CD34KSL cells fall into the Tip of the SP cell region. (G) Tip‐SP cells are first gated on using the same region indicated in (F). (H) c‐kit and Lin profile of Tip‐SP cells. (I) Sca‐1 and CD34 profile of Tip‐SP, c‐KL cells. The gates from D to F are preferred for Tip‐SP CD34KSL cells.

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Literature Cited

Literature Cited
   Colvin, G.A., Lambert, J.F., Abedi, M., Hsieh, C.C., Carlson, J.E., Stewart, F.M., and Quesenberry, P.J. 2004. Murine marrow cellularity and the concept of stem cell competition: Geographic and quantitative determinants in stem cell biology. Leukemia 18:575‐583.
   Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183:1797‐1806.
   Matsuzaki, Y., Kinjo, K., Mulligan, R.C., and Okano, H. 2004. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 20:87‐93.
   Osawa, M., Hanada, K.‐I., Hamada, H., and Nakauchi, H. 1996. Long‐term lymphohematopoietic reconstitution by a single CD34‐low/negative hematopoietic stem cell. Science 273:242‐245.
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