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A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Abstract

We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 °C–5% CO2 incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 °C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

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Figure 1: Morphological features of mMSCs.
Figure 2: Cell surface markers and differentiation capacity of mouse mesenchymal stem cells (mMSCs).

References

  1. 1

    Fuchs, E. & Segre, J.A. Stem cells: a new lease on life. Cell 100, 143–155 (2000).

    CAS  Article  Google Scholar 

  2. 2

    Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Friedenstein, A.J., Gorskaja, J.F. & Kulagina, N.N. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp. Hematol. 4, 267–274 (1976).

    CAS  PubMed  Google Scholar 

  4. 4

    Bianco, P., Riminucci, M., Gronthos, S. & Robey, P.G. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19, 180–192 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Sun, S. et al. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 21, 527–535 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Baksh, D., Song, L. & Tuan, R.S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J. Cell Mol. Med. 8, 301–316 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Horwitz, E.M. et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc. Natl. Acad. Sci. USA 99, 8932–8937 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Koc, O.N. et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18, 307–316 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Petite, H. et al. Tissue-engineered bone regeneration. Nat. Biotechnol. 18, 959–963 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Quarto, R. et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N. Engl. J. Med. 344, 385–386 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Grinnemo, K.H. et al. Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium. J. Thorac. Cardiovasc. Surg. 127, 1293–1300 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Barry, F.P. Mesenchymal stem cell therapy in joint disease. Novartis. Found Symp. 249, 86–102, 170–4, 239–41 (2003).

    PubMed  Google Scholar 

  14. 14

    Peister, A. et al. 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 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Phinney, D.G., Kopen, G., Isaacson, R.L. & Prockop, D.J. 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 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Baddoo, M. et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J. Cell Biochem. 89, 1235–1249 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Tropel, P. et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp. Cell Res. 295, 395–406 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Eslaminejad, M.B., Nikmahzar, A., Taghiyar, L., Nadri, S. & Massumi, M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev. Growth Differ. 48, 361–370 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Nadri, S. & Soleimani, M. Isolation murine mesenchymal stem cells by positive selection. In Vitro Cell Dev. Biol. Anim. 43, 276–282 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Nadri, S. et al. An efficient method for isolation of murine bone marrow mesenchymal stem cells. Int. J. Dev. Biol. 51, 723–729 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Modderman, W.E., Vrijheid-Lammers, T., Lowik, C.W. & Nijweide, P.J. Removal of hematopoietic cells and macrophages from mouse bone marrow cultures: isolation of fibroblastlike stromal cells. Exp. Hematol. 22, 194–201 (1994).

    CAS  PubMed  Google Scholar 

  22. 22

    Falla, N. et al. Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow. Blood 82, 3580–3591 (1993).

    CAS  PubMed  Google Scholar 

  23. 23

    Van Vlasselaer, P., Falla, N., Snoeck, H. & Mathieu, E. Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin. Blood 84, 753–763 (1994).

    CAS  PubMed  Google Scholar 

  24. 24

    Wang, Q.R. & Wolf, N.S. Dissecting the hematopoietic microenvironment. VIII. Clonal isolation and identification of cell types in murine CFU-F colonies by limiting dilution. Exp. Hematol. 18, 355–359 (1990).

    CAS  PubMed  Google Scholar 

  25. 25

    Kitano, Y., Radu, A., Shaaban, A. & Flake, A.W. Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells. Exp. Hematol. 28, 1460–1469 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Muraglia, A., Cancedda, R. & Quarto, R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J. Cell Sci. 113 (Part 7): 1161–1166 (2000).

    CAS  PubMed  Google Scholar 

  27. 27

    Digirolamo, C.M. et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br. J. Haematol. 107, 275–281 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Keating, A., Horsfall, W., Hawley, R.G. & Toneguzzo, F. Effect of different promoters on expression of genes introduced into hematopoietic and marrow stromal cells by electroporation. Exp. Hematol. 18, 99–102 (1990).

    CAS  PubMed  Google Scholar 

  29. 29

    Deans, R.J. & Moseley, A.B. Mesenchymal stem cells: biology and potential clinical uses. Exp. Hematol. 28, 875–884 (2000).

    CAS  Article  Google Scholar 

  30. 30

    Otsuka, T. et al. Expression of the c-kit ligand and interleukin 6 genes in mouse bone marrow stromal cell lines. Stem Cells 12, 409–415 (1994).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This study was supported by Iran Stem Cell Technology Institute.

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Correspondence to Masoud Soleimani or Samad Nadri.

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Soleimani, M., Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc 4, 102–106 (2009). https://doi.org/10.1038/nprot.2008.221

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