Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α

Nature Protocols volume 7, pages 2103–2111 (2012)Cite this article

Subjects

Abstract

Platelet-derived growth factor receptor α (PDGFR-α) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-α+Sca-1+ (PαS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PαS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PαS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 °C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45+Ter-119+)-positive cells. Approximately 10,000 PαS MSCs may then be isolated per mouse. The total protocol takes ∼7 h to complete.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Sequential gating strategy that allows for the identification and isolation of PαS cells.
Figure 2: Typical results seen when culturing PαS cells.
Figure 3: Surface marker expression of PαS cells after one passage.
Figure 4: Tri-lineage differentiation of PαS cells to bone, cartilage and fat.

Similar content being viewed by others

References

  1. Caplan, A.I. Mesenchymal stem cells. J. Orthop. Res. 9, 641–650 (1991).

    Article  CAS  Google Scholar 

  2. Sacchetti, B. et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131, 324–336 (2007).

    Article  CAS  Google Scholar 

  3. Friedenstein, A.J., Chailakhjan, R.K. & Lalykina, K.S. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 3, 393–403 (1970).

    CAS  PubMed  Google Scholar 

  4. Stappenbeck, T.S. & Miyoshi, H. The role of stromal stem cells in tissue regeneration and wound repair. Science 324, 1666–1669 (2009).

    Article  CAS  Google Scholar 

  5. Uccelli, A., Moretta, L. & Pistoia, V. Mesenchymal stem cells in health and disease. Nat. Rev. Immunol. 8, 726–736 (2008).

    Article  CAS  Google Scholar 

  6. Ren, G. et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2, 141–150 (2008).

    Article  CAS  Google Scholar 

  7. Németh, K. et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat. Med. 15, 42–49 (2009).

    Article  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Peister, A., Mellad, J.A., Larson, B.L., Hall, B.M. & Gibson, L.F. 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).

    Article  CAS  Google Scholar 

  10. Soleimani, M. & Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat. Protoc. 4, 102–106 (2009).

    Article  CAS  Google Scholar 

  11. Zhu, H. et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat. Protoc. 5, 550–560 (2010).

    Article  CAS  Google Scholar 

  12. Wagner, W. et al. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS ONE 3, e2213 (2008).

    Article  Google Scholar 

  13. Rombouts, W.J. & Ploemacher, R.E. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia 17, 160–170 (2003).

    Article  CAS  Google Scholar 

  14. da Silva Meirelles, L., Caplan, A.I. & Nardi, N.B. In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26, 2287–2299 (2008).

    Article  Google Scholar 

  15. Koide, Y. et al. Two distinct stem cell lineages in murine bone marrow. Stem Cells 25, 1213–1221 (2007).

    Article  CAS  Google Scholar 

  16. Morikawa, S. et al. Development of mesenchymal stem cells partially originate from the neural crest. Biochem. Biophys. Res. Commun. 379, 1114–1119 (2009).

    Article  CAS  Google Scholar 

  17. Morikawa, S. et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J. Exp. Med. 206, 2483–2496 (2009).

    Article  CAS  Google Scholar 

  18. Nombela-Arrieta, C., Ritz, J. & Silberstein, L.E. The elusive nature and function of mesenchymal stem cells. Nat. Rev. Mol. Cell Biol. 12, 126–131 (2011).

    Article  CAS  Google Scholar 

  19. Niibe, K. et al. Purified mesenchymal stem cells are an efficient source for iPS cell induction. PLoS ONE 6, e17610 (2011).

    Article  CAS  Google Scholar 

  20. Baddoo, M., Hill, K., Wilkinson, R., Gaupp, D. & Hughes, C. et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J. Cell Biochem. 89, 1235–1249 (2003).

    Article  CAS  Google Scholar 

  21. Falla, N., Vlasselaer, V., Bierkens, J., Borremans, B. & Schoeters, G. et al. Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow. Blood 82, 3580–3591 (1993).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  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. Muguruma, Y. et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 107, 1878–1887 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Project for Realization of Regenerative Medicine and Support for the Core Institutes for Induced Pluripotent Stem (iPS) Cell Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT; to H.O. and Y. Matsuzaki). This study was also supported, in part, by a grant-in-aid for Encouragement of Young Medical Scientists from Keio University (to Y. Mabuchi), a grant-in-aid for Scientific Research (KAKENHI; to Y. Mabuchi) and a grant-in-aid from the Global Century COE program of the MEXT, Japan, to Keio University. D.D.H. is funded by the Medical Research Council, UK.

Author information

Author notes

  1. Diarmaid D Houlihan and Yo Mabuchi: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Liver Research, National Institute for Health Research Biomedical Research Unit, University of Birmingham, Birmingham, UK

    Diarmaid D Houlihan

  2. Department of Physiology, Keio University School of Medicine, Tokyo, Japan

    Yo Mabuchi, Satoru Morikawa, Kunimichi Niibe, Daisuke Araki, Sadafumi Suzuki, Hideyuki Okano & Yumi Matsuzaki

  3. Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Yo Mabuchi

  4. Department of Dentistry and Oral Surgery, Keio University School of Medicine, Tokyo, Japan

    Satoru Morikawa, Kunimichi Niibe & Daisuke Araki

Authors
  1. Diarmaid D Houlihan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yo Mabuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satoru Morikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kunimichi Niibe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daisuke Araki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sadafumi Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hideyuki Okano
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yumi Matsuzaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y. Mabuchi, D.D.H. and Y. Matsuzaki designed the study. Y. Mabuchi and D.D.H. performed the experiments. H.O. and Y. Matsuzaki supervised all the experiments. Y. Mabuchi, D.D.H., S.M., K.N., D.A. and S.S. analyzed the data. Y. Mabuchi, D.D.H. and Y. Matsuzaki contributed to writing the manuscript.

Corresponding author

Correspondence to Yumi Matsuzaki.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Houlihan, D., Mabuchi, Y., Morikawa, S. et al. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α. Nat Protoc 7, 2103–2111 (2012). https://doi.org/10.1038/nprot.2012.125

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.125

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing