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In Vivo Imaging of Hematopoietic Stem Cells in the Bone Marrow Niche

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Progenitor Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 916))

Abstract

Even though hematopoietic stem cells (HSC) are amongst the first somatic stem cells exploited for therapeutic purposes, their application is still limited by the inability to expand them ex vivo without impairing their function. Moreover, it has recently emerged that several types of leukemia develop and relapse through complex interactions with bone marrow (BM) components and may directly affect the HSC and their niche. Increasing attention has therefore been dedicated to the BM microenvironment the HSC reside in, with the view that a better understanding of the molecular regulators of HSC-niche interaction in vivo will allow improving HSC mobilization, collection and transplantation and provide clues for the development of innovative leukemia treatments. This chapter focuses on a recently established technique for the visualization of transplanted hematopoietic stem and progenitor cells (HSPC) within the calvarium bone marrow of live mice (Lo Celso et al. Nature 457:92–96, 2007). Intravital microscopy is a rapidly developing field, driven by constant improvement in both detection technologies (i.e., spatial resolution, depth of penetration, spectral definition) and probe availability (i.e., increasingly sophisticated genetic and chemical reporter systems). We therefore discuss the current limitations and challenges related to intravital microscopy of the HSC niche and introduce a number of potential imaging approaches, which could be promising candidates for future development of this technique.

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References

  1. 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 457:92–96

    Article  PubMed  CAS  Google Scholar 

  2. Xie Y, Yin T, Wiegraebe W, He XC, Miller D, Stark D, Perko K, Alexander R, Schwartz J, Grindley JC, Park J, Haug JS, Wunderlich JP, Li H, Zhang S, Johnson T, Feldman RA, Li L (2009) Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457:97–101

    Article  PubMed  CAS  Google Scholar 

  3. Lewandowski D, Barroca V, Duconge F, Bayer J, Van Nhieu JT, Pestourie C, Fouchet P, Tavitian B, Romeo PH (2010) In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution. Blood 115:443–452

    Article  PubMed  CAS  Google Scholar 

  4. 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 114:290–298

    Article  PubMed  Google Scholar 

  5. Lo Celso C, Wu JW, Lin CP (2009) In vivo imaging of hematopoietic stem cells and their microenvironment. J Biophotonics 2:619–631

    Article  PubMed  Google Scholar 

  6. Maslov K, Zhang HF, Hu S, Wang LV (2008) Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries. Opt Lett 33:929–931

    Article  PubMed  Google Scholar 

  7. Zhang HF, Maslov K, Stoica G, Wang LV (2006) Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nat Biotechnol 24:848–851

    Article  PubMed  CAS  Google Scholar 

  8. Branchini BR, Ablamsky DM, Davis AL, Southworth TL, Butler B, Fan F, Jathoul AP, Pule MA (2010) Red-emitting luciferases for bioluminescence reporter and imaging applications. Anal Biochem 396:290–297

    Article  PubMed  CAS  Google Scholar 

  9. Nakajima K, Komiyama Y, Hojo H, Ohba S, Yano F, Nishikawa N, Ihara S, Aburatani H, Takato T, Chung UI (2010) Enhancement of bone formation ex vivo and in vivo by a helioxanthin-derivative. Biochem Biophys Res Commun 395:502–508

    Article  PubMed  CAS  Google Scholar 

  10. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma’ayan A, Enikolopov GN, Frenette PS (2010) Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466:829–834

    Article  PubMed  CAS  Google Scholar 

  11. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90:1103–1163

    Article  PubMed  CAS  Google Scholar 

  12. Lin MZ, McKeown MR, Ng HL, Aguilera TA, Shaner NC, Campbell RE, Adams SR, Gross LA, Ma W, Alber T, Tsien RY (2009) Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem Biol 16:1169–1179

    Article  PubMed  CAS  Google Scholar 

  13. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572

    Article  PubMed  CAS  Google Scholar 

  14. Lange S, Katayama Y, Schmid M, Burkacky O, Brauchle C, Lamb DC, Jansen RP (2008) Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9:1256–1267

    Article  PubMed  CAS  Google Scholar 

  15. Ai HW, Hazelwood KL, Davidson MW, Campbell RE (2008) Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat Methods 5:401–403

    Article  PubMed  CAS  Google Scholar 

  16. Chu J, Zhang Z, Zheng Y, Yang J, Qin L, Lu J, Huang ZL, Zeng S, Luo Q (2009) A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions. Biosens Bioelectron 25:234–239

    Article  PubMed  CAS  Google Scholar 

  17. Shyu YJ, Suarez CD, Hu CD (2008) Visualization of ternary complexes in living cells by using a BiFC-based FRET assay. Nat Protoc 3:1693–1702

    Article  PubMed  CAS  Google Scholar 

  18. Tomosugi W, Matsuda T, Tani T, Nemoto T, Kotera I, Saito K, Horikawa K, Nagai T (2009) An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat Methods 6:351–353

    Article  PubMed  CAS  Google Scholar 

  19. Xu X, Gerard AL, Huang BC, Anderson DC, Payan DG, Luo Y (1998) Detection of programmed cell death using fluorescence energy transfer. Nucleic Acids Res 26:2034–2035

    Article  PubMed  CAS  Google Scholar 

  20. Itoh RE, Kurokawa K, Ohba Y, Yoshizaki H, Mochizuki N, Matsuda M (2002) Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol Cell Biol 22:6582–6591

    Article  PubMed  CAS  Google Scholar 

  21. Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M (2001) Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411:1065–1068

    Article  PubMed  CAS  Google Scholar 

  22. Sakaue-Sawano A, Ohtawa K, Hama H, Kawano M, Ogawa M, Miyawaki A (2008) Tracing the silhouette of individual cells in S/G2/M phases with fluorescence. Chem Biol 15:1243–1248

    Article  PubMed  CAS  Google Scholar 

  23. Palmer AE, Tsien RY (2006) Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1:1057–1065

    Article  PubMed  CAS  Google Scholar 

  24. Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279:22284–22293

    Article  PubMed  CAS  Google Scholar 

  25. Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053

    Article  PubMed  CAS  Google Scholar 

  26. Knopfel T, Tomita K, Shimazaki R, Sakai R (2003) Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. Methods 30:42–48

    Article  PubMed  CAS  Google Scholar 

  27. Jankowski A, Kim JH, Collins RF, Daneman R, Walton P, Grinstein S (2001) In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin. J Biol Chem 276:48748–48753

    Article  PubMed  CAS  Google Scholar 

  28. Medintz IL, Uyeda HT, Goldman ER, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4:435–446

    Article  PubMed  CAS  Google Scholar 

  29. Texier I, Josser V (2009) In vivo imaging of quantum dots. Methods Mol Biol 544:393–406

    Article  PubMed  CAS  Google Scholar 

  30. Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56–62

    Article  PubMed  CAS  Google Scholar 

  31. Phan TG, Bullen A (2010) Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper. Immunol Cell Biol 88:438–444

    Article  PubMed  Google Scholar 

  32. Lo Celso C, Scadden D (2007) Isolation and transplantation of hematopoietic stem cells (HSCs). J Vis Exp (2) 157

    Google Scholar 

  33. Bryder D, Rossi DJ, Weissman IL (2006) Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol 169:338–346

    Article  PubMed  CAS  Google Scholar 

  34. Sipkins DA, Wei X, Wu JW, Runnels JM, Cote D, Means TK, Luster AD, Scadden DT, Lin CP (2005) In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435:969–973

    Article  PubMed  CAS  Google Scholar 

  35. Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377

    Article  PubMed  CAS  Google Scholar 

  36. Lo Celso C, Lin CP, Scadden DT (2011) In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Nat Protoc 6:1–14

    Article  PubMed  CAS  Google Scholar 

  37. Foudi A, Hochedlinger K, Van Buren D, Schindler JW, Jaenisch R, Carey V, Hock H (2009) Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 27:84–90

    Article  PubMed  CAS  Google Scholar 

  38. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Mark Scott and Dr. Martin Spitaler for discussion and input on the setup and subsequent description of in vivo confocal and two-photon microscopy with a commercially available system.

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Authors and Affiliations

  1. Division of Cell and Molecular Biology, Imperial College, London, UK

    Oliver Barrett, Roberta Sottocornola & Cristina Lo Celso

Authors
  1. Oliver Barrett
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  2. Roberta Sottocornola
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  3. Cristina Lo Celso
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Corresponding author

Correspondence to Cristina Lo Celso .

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Editors and Affiliations

  1. Fac. Life Sciences, Healing Foundation Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

    Kimberly A. Mace

  2. School of Medicine & Dentistry, Centre for Cutaneous Research, Blizard I, Barts and The London, Newark Street 4, London, E1 2AT, United Kingdom

    Kristin M. Braun

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© 2012 Springer Science+Business Media New York

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Barrett, O., Sottocornola, R., Celso, C.L. (2012). In Vivo Imaging of Hematopoietic Stem Cells in the Bone Marrow Niche. In: Mace, K., Braun, K. (eds) Progenitor Cells. Methods in Molecular Biology, vol 916. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-980-8_18

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  • DOI: https://doi.org/10.1007/978-1-61779-980-8_18

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-979-2

  • Online ISBN: 978-1-61779-980-8

  • eBook Packages: Springer Protocols

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