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Intravital Imaging of Blood Flow and HSPC Homing in Bone Marrow Microvessels

  • Jonas Stewen
  • Maria Gabriele BixelEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2017)

Abstract

Two-photon intravital microscopy (2P-IVM) is an advanced imaging technique that allows the visualization of dynamic cellular behavior deeply inside tissues and organs of living animals. Due to the deep tissue penetration, imaging of highly light-scattering tissue as the bone becomes feasible at subcellular resolution.

To better understand the influence of blood flow on hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM) microvasculature of the calvarial bone, we analyzed blood flow dynamics and the influence of flow on the early homing behavior of HSPCs during their passage through BM microvessels. Here, we describe a 2P-IVM approach for direct measurements of red blood cell (RBC) velocities in the BM microvasculature using repetitive centerline scans at the level of individual arterial vessels and sinusoidal capillaries to obtain a detailed flow profile map. Furthermore, we explain the isolation and enrichment of HSPCs from long bones and the transplantation of these cells to study the early homing behavior of HSPCs in BM sinusoids at cellular resolution. This is achieved by high-resolution spatiotemporal imaging through a chronic cranial window using transgenic reporter mice.

Key words

Intravital imaging Bone marrow microvessels Blood flow velocities Hematopoietic stem cells Stem cell homing Two-photon microscopy 

Notes

Acknowledgments

We thank F. Winkler for sharing his expertise on the imaging setup for head immobilization. This work was supported by the Max Planck Society, the University of Münster, the DFG cluster of excellence “Cell in Motion,” and the European Research Council (AdG 339409 AngioBone).

References

  1. 1.
    Weigert R, Sramkova M, Parente L, Amornphimoltham P, Masedunskas A (2010) Intravital microscopy: a novel tool to study cell biology in living animals. Histochem Cell Biol 133(5):481–491.  https://doi.org/10.1007/s00418-010-0692-zCrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J, Cote D, Rowe DW, Lin CP, Scadden DT (2009) Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457(7225):92–96.  https://doi.org/10.1038/nature07434CrossRefGoogle Scholar
  3. 3.
    Bixel MG, Kusumbe AP, Ramasamy SK, Sivaraj KK, Butz S, Vestweber D, Adams RH (2017) Flow dynamics and HSPC homing in bone marrow microvessels. Cell Rep 18(7):1804–1816.  https://doi.org/10.1016/j.celrep.2017.01.042CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zhao Y, Bower AJ, Graf BW, Boppart MD, Boppart SA (2013) Imaging and tracking of bone marrow-derived immune and stem cells. Methods Mol Biol 1052:57–76.  https://doi.org/10.1007/7651_2013_28CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76CrossRefGoogle Scholar
  6. 6.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940.  https://doi.org/10.1038/nmeth818CrossRefPubMedGoogle Scholar
  7. 7.
    Andresen V, Alexander S, Heupel WM, Hirschberg M, Hoffman RM, Friedl P (2009) Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging. Curr Opin Biotechnol 20(1):54–62.  https://doi.org/10.1016/j.copbio.2009.02.008CrossRefPubMedGoogle Scholar
  8. 8.
    LaComb R, Nadiarnykh O, Carey S, Campagnola PJ (2008) Quantitative second harmonic generation imaging and modeling of the optical clearing mechanism in striated muscle and tendon. J Biomed Opt 13(2):021109.  https://doi.org/10.1117/1.2907207CrossRefPubMedGoogle Scholar
  9. 9.
    Genthial R, Beaurepaire E, Schanne-Klein MC, Peyrin F, Farlay D, Olivier C, Bala Y, Boivin G, Vial JC, Debarre D, Gourrier A (2017) Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy. Sci Rep 7(1):3419.  https://doi.org/10.1038/s41598-017-03548-5CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L (2003) Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425(6960):836–841.  https://doi.org/10.1038/nature02041CrossRefGoogle Scholar
  11. 11.
    Morrison SJ, Scadden DT (2014) The bone marrow niche for haematopoietic stem cells. Nature 505(7483):327–334.  https://doi.org/10.1038/nature12984CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ding L, Saunders TL, Enikolopov G, Morrison SJ (2012) Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481(7382):457–462.  https://doi.org/10.1038/nature10783CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gao X, Xu C, Asada N, Frenette PS (2018) The hematopoietic stem cell niche: from embryo to adult. Development 145(2).  https://doi.org/10.1242/dev.139691CrossRefGoogle Scholar
  14. 14.
    Lassailly F, Foster K, Lopez-Onieva L, Currie E, Bonnet D (2013) Multimodal imaging reveals structural and functional heterogeneity in different bone marrow compartments: functional implications on hematopoietic stem cells. Blood 122(10):1730–1740.  https://doi.org/10.1182/blood-2012-11-467498CrossRefGoogle Scholar
  15. 15.
    Kusumbe AP, Ramasamy SK, Adams RH (2014) Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507(7492):323–328.  https://doi.org/10.1038/nature13145CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Abboud CN (1995) Human bone marrow microvascular endothelial cells: elusive cells with unique structural and functional properties. Exp Hematol 23(1):1–3PubMedGoogle Scholar
  17. 17.
    Pries AR, Secomb TW, Gaehtgens P, Gross JF (1990) Blood flow in microvascular networks. Experiments and simulation. Circ Res 67(4):826–834CrossRefGoogle Scholar
  18. 18.
    Pries AR, Secomb TW, Gaehtgens P (1995) Structure and hemodynamics of microvascular networks: heterogeneity and correlations. Am J Phys 269(5 Pt 2):H1713–H1722Google Scholar
  19. 19.
    Kohler A, Schmithorst V, Filippi MD, Ryan MA, Daria D, Gunzer M, Geiger H (2009) Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones. Blood 114(2):290–298.  https://doi.org/10.1182/blood-2008-12-195644CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Tissue MorphogenesisMax Planck Institute for Molecular BiomedicineMünsterGermany

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