Isolation, Culture, and Differentiation Potential of Mouse Marrow Stromal Cells

Fernando Anjos‐Afonso1, Dominique Bonnet1

1 Haematopoietic Stem Cell Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.3
DOI:  10.1002/9780470151808.sc02b03s7
Online Posting Date:  October, 2008
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Abstract

This unit describes how to isolate and expand mesenchymal stromal cells (MSCs) from mouse bone marrow. For reasons that are not clear, it has been difficult to isolate these cells (also known as mesenchymal stem cells). Furthermore, different mouse strains seem to have specific requirements for successful extraction and culture of these cells. A general and easy protocol is presented here for isolating stromal cells from different inbred and transgenic mice commonly used in the stem cell biology field. Curr. Protoc. Stem Cell Biol. 7:2B.3.1‐2B.3.11. © 2008 by John Wiley & Sons, Inc.

Keywords: mouse mesenchymal stromal cells; isolation; expansion; differentiation

     
 
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Table of Contents

  • Introduction
  • Support Protocol 1: Oil Red O Staining
  • Support Protocol 2: Safranin O Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 6‐ to 10‐week‐old mice of appropriate strain(s)
  • CMF‐PBS/2% FBS: phosphate‐buffered saline, without Ca2+ and Mg2+ (CMF‐PBS; GIBCO‐Invitrogen, cat. no. 20012) containing 2% (v/v) heat‐inactivated fetal bovine serum (FBS; StemCell Technologies, cat. no. 06471)
  • Complete MSC expansion medium (see recipe)
  • 0.4% (w/v) trypan blue (Sigma, cat. no. T6140) solution in CMF‐PBS (GIBCO‐Invitrogen, cat. no. 20012)
  • Phosphate‐buffered saline without Ca2+ and Mg2+ (CMF‐PBS)
  • 0.25% (w/v) trypsin/1 mM EDTA solution (ethylenediaminetetraacetic acid in Hanks' balanced salt solution; GIBCO‐Invitrogen, cat. no. 25200)
  • Fetal bovine serum (FBS; StemCell Technologies, cat. no. 06471): low endotoxin; batch preselected for MSC growth; do not heat inactivate the serum; store at −20°C and thaw before use overnight at 4°C (do not thaw at 37°C)
  • Fluorphore‐conjugated antibodies (BD Bioscience Pharmingen; choice of fluorophore at the discretion of the researcher):
    • Anti‐mouse CD45 (clone 30‐F11)
    • Anti‐mouse CD11b (clone M1/70)
    • Anti‐mouse CD31 (clone 390)
    • Anti‐mouse CD105 (clone MJ7/18)
    • Anti‐mouse CD73 (clone TY/23)
    • Anti‐mouse Sca1 (clone E13‐161.7)
    • Anti‐mouse CD44 (clone IM7)
    • Rat‐IgG2a (clone R35‐95) isotype control
    • Rat‐IgG2b (clone A95‐1) isotype controls
  • Live/dead discriminative dye stock solution, e.g.:
    • 200 mg/ml of 4′,6‐diamidino‐2‐phenylindole (DAPI; Invitrogen, cat. no. D1306)
    • 40 mg/ml of 7‐amino‐actinomycin D (7‐AAD; Invitrogen, cat. no. A1310)
  • Adipogenic induction medium (see recipe), 37°C
  • Osteogenic induction medium (see recipe), 37°C
  • Alkaline phosphatase detection kit (Sigma, Kit 86)
  • Chondrogenic induction medium (see recipe), 37°C
  • Sterile dissecting scissors
  • Insulin syringe or 1‐ml syringe with a 27‐ to 29‐G needle
  • 5‐ and 15‐ml polyprene tubes
  • Refrigerated centrifuge, 4°C
  • 25‐cm2 tissue culture–treated culture flasks/dishes (or other appropriate size)
  • Humidified cell culture incubator (37°C, 5% CO 2)
  • 30°C and 37°C water bath
  • 0.22‐µm filter
  • Cell sorter (e.g., MoFlow, Dako‐Cytomation)
  • Additional reagents and equipment for assessing cell number and viability (unit 1.3)

Support Protocol 1: Oil Red O Staining

  Materials
  • Mouse MSC cells cultured for adipogenic differentiation ( protocol 1, step 34a)
  • Phosphate‐buffered saline, without Ca2+ and Mg2+ (CMF‐PBS; GIBCO‐Invitrogen, cat. no. 20012)
  • Oil Red O stain working solution (see recipe)
  • 10% (v/v) neutral buffered formalin (NBF; see recipe)
  • 60% (v/v) isopropanol
  • Gill's hematoxylin solution (Vector Laboratories, cat. no. H‐3401)
  • Phase‐contrast microscope (100× to 400× magnification recommended)

Support Protocol 2: Safranin O Staining

  Materials
  • Mouse MSC cells cultured for chondrogenic differentiation ( protocol 1, step 34b)
  • 10% (v/v) neutral buffered formalin (NBF; see recipe)
  • 50%, 70%, 75%, 95%, 100% (v/v) ethanol
  • Xylene
  • Weigert's iron hematoxylin solution kit (Sigma, cat. no. HT1079), containing stock solutions A and B
  • Fast Green staining solution (see recipe)
  • 1% (v/v) acetic acid: prepare by diluting glacial acetic acid (Sigma, cat. no. A9967)
  • 0.1% (w/v) Safranin O (Sigma, cat. no. 84120) staining solution: filter through filter paper before use
  • Resinous mounting medium (Vector Laboratories, cat. no H‐5000)
  • Light microscope (100× to 400× magnification recommended)
  • Additional reagents and equipment for embedding and sectioning cells (supplied by histology service)
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Figures

Videos

Literature Cited

Literature Cited
   Anjos‐Afonso, F., Siapati, E.K., and Bonnet, D. 2004. In vivo contribution of murine mesenchymal stem cells into multiple cell‐types under minimal damage conditions. J. Cell Sci. 117:5655‐5664.
   Colter, D.C., Class, R., DiGirolamo, C.M., and Prockop, D.J. 2000. Rapid expansion of recycling stem cells in cultures of plastic‐adherent cells from human bone marrow. Proc. Natl. Acad. Sci. U.S.A. 97:3213‐3218.
   Colter, D.C., Sekiya, I., and Prockop, D.J. 2001. Identification of a subpopulation of rapidly self‐renewing and multipotential adults stem cells in colonies of human marrow stromal cells. Proc. Natl. Acad. Sci. U.S.A. 98:7841‐7845.
   Javazon, E.H., Colter, D.C., Schwarz, E.J., and Prockop, D.J. 2001. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single‐cell‐derived colonies than human marrow stromal cells. Stem Cells 19:219‐225.
   Peister, A., Mellad, J.A., Larson, B.L., Hall, B.M., Gibson, L.F., and Prockop, D.J. 2004. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103:1662‐1668.
   Phinney, D.G., Kopen, G., Isaacson, R.L., and Prockop, D.J. 1999. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J. Cell. Biochem. 72:570‐585.
   Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Graig, S., and Marshak, D.R. 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284:143‐147.
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