Skip to main content
SpringerLink
Log in

Analysis of Murine Hematopoietic Stem Cell Proliferation During Inflammation

  • Protocol
  • First Online:
Cellular Quiescence

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1686))

Abstract

Normally, quiescent hematopoietic stem cells (HSC) rapidly enter the cell cycle following exposure to inflammatory stimuli. The analysis of HSC cell cycle activity in murine bone marrow during inflammation is often complicated by the relative rarity of HSCs and shifts in Sca-1, a key cell surface marker used to identify HSCs. Here, we report a method to analyze HSC proliferation and cell cycle distribution under inflammatory conditions. Our approach uses EdU incorporation and Ki67 staining coupled with DNA content quantification by DAPI. We also incorporate the surface marker ESAM to help minimize the potential for contaminating events that may confound analysis in the HSC compartment.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132:631–644. doi:10.1016/j.cell.2008.01.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pietras EM, Warr MR, Passegué E (2011) Cell cycle regulation in hematopoietic stem cells. J Cell Biol 195:709–720. doi:10.1083/jcb.201102131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Schepers K, Campbell TB, Passegué E (2015) Normal and leukemic stem cell niches: insights and therapeutic opportunities. Cell Stem Cell 16:254–267. doi:10.1016/j.stem.2015.02.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rossi L, Lin KK, Boles NC et al (2012) Less is more: unveiling the functional core of hematopoietic stem cells through knockout mice. Cell Stem Cell 11:302–317. doi:10.1016/j.stem.2012.08.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kiel MJ, Yilmaz ÖH, Iwashita T et al (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121. doi:10.1016/j.cell.2005.05.026

    Article  CAS  PubMed  Google Scholar 

  6. Wilson A, Laurenti E, Oser G et al (2008) Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135:1118–1129. doi:10.1016/j.cell.2008.10.048

    Article  CAS  PubMed  Google Scholar 

  7. Cabezas-Wallscheid N, Klimmeck D, Hansson J et al (2014) Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 15:507–522. doi:10.1016/j.stem.2014.07.005

    Article  CAS  PubMed  Google Scholar 

  8. Foudi A, Hochedlinger K, Van Buren D et al (2008) Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 27:84–90. doi:10.1038/nbt.1517

    Article  PubMed  PubMed Central  Google Scholar 

  9. Winkler IG, Barbier V, Wadley R et al (2010) Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches. Blood 116:375–385. doi:10.1182/blood-2009-07-233437

    Article  CAS  PubMed  Google Scholar 

  10. Sun J, Ramos A, Chapman B et al (2014) Clonal dynamics of native haematopoiesis. Nature 514:322–327. doi:10.1038/nature13824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Busch K, Klapproth K, Barile M et al (2015) Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518:542–546. doi:10.1038/nature14242

    Article  CAS  PubMed  Google Scholar 

  12. Sawai CM, Babovic S, Upadhaya S et al (2016) Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals. Immunity 45:597–609. doi:10.1016/j.immuni.2016.08.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pietras EM, Reynaud D, Kang Y-A et al (2015) Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions. Cell Stem Cell 17:35–46. doi:10.1016/j.stem.2015.05.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. King KY, Goodell MA (2011) Inflammatory modulation of HSCs: viewing the HSC as a foundation for the immune response. Nat Rev Immunol 11:685–692. doi:10.1038/nri3062

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Takizawa H, Boettcher S, Manz MG (2012) Demand-adapted regulation of early hematopoiesis in infection and inflammation. Blood 119:2991–3002. doi:10.1182/blood-2011-12-380113

    Article  CAS  PubMed  Google Scholar 

  16. Pietras EM, Mirantes-Barbeito C, Fong S et al (2016) Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal. Nat Cell Biol 18(6):607–618. doi:10.1038/ncb3346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Essers MAG, Offner S, Blanco-Bose WE et al (2009) IFNα activates dormant haematopoietic stem cells in vivo. Nature 458:904–908. doi:10.1038/nature07815

    Article  CAS  PubMed  Google Scholar 

  18. Ueda Y, Cain DW, Cain DW et al (2009) IL-1R type I-dependent hemopoietic stem cell proliferation is necessary for inflammatory granulopoiesis and reactive neutrophilia. J Immunol 182:6477–6484. doi:10.4049/jimmunol.0803961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Baldridge MT, King KY, Boles NC et al (2010) Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infection. Nature 465:793–797. doi:10.1038/nature09135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pietras EM, Lakshminarasimhan R, Techner J-M et al (2014) Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons. J Exp Med 211:245–262. doi:10.1084/jem.20131043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Haas S, Hansson J, Klimmeck D et al (2015) Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors. Cell Stem Cell 17:422–434. doi:10.1016/j.stem.2015.07.007

    Article  CAS  PubMed  Google Scholar 

  22. Matatall KA, Shen C-C, Challen GA, King KY (2014) Type II interferon promotes differentiation of myeloid-biased hematopoietic stem cells. Stem Cells 32:3023–3030. doi:10.1002/stem.1799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ooi AGL, Karsunky H, Majeti R et al (2009) The adhesion molecule Esam1 is a novel hematopoietic stem cell marker. Stem Cells 27:653–661. doi:10.1634/stemcells.2008-0824

    Article  CAS  PubMed  Google Scholar 

  24. Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105:2415–2420. doi:10.1073/pnas.0712168105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Barbier V, Nowlan B, Lévesque J-P, Winkler IG (2012) Flow cytometry analysis of cell cycling and proliferation in mouse hematopoietic stem and progenitor cells. Methods Mol Biol 844:31–43. doi:10.1007/978-1-61779-527-5_3

    Article  CAS  PubMed  Google Scholar 

  26. Oguro H, Ding L, Morrison SJ (2013) SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13:102–116. doi:10.1016/j.stem.2013.05.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pronk CJH, Rossi DJ, Månsson R et al (2007) Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell 1:428–442. doi:10.1016/j.stem.2007.07.005

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by NIH grant K01DK98315 to E.M.P. We thank Biniam Adane and Brett Stevens for reagents and advice during the preparation of this chapter.

Author information

Authors and Affiliations

  1. Division of Hematology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Emilie Jalbert

  2. Division of Hematology, Department of Medicine, University of Colorado Anschutz Medical Campus, 12700 East 19th Ave, Research Complex 2, Mail Stop F754, Aurora, CO, 80045, USA

    Eric M. Pietras

Authors
  1. Emilie Jalbert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric M. Pietras
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eric M. Pietras .

Editor information

Editors and Affiliations

  1. Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA

    H. Daniel Lacorazza

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Jalbert, E., Pietras, E.M. (2018). Analysis of Murine Hematopoietic Stem Cell Proliferation During Inflammation. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7371-2_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7370-5

  • Online ISBN: 978-1-4939-7371-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation