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In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow

Nature Protocols volume 6, pages 1–14 (2011)Cite this article

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Abstract

In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ∼1 week.

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Figure 1: Diagram representing a custom-made mouse imaging setup.
Figure 2: Diagram representing two options for scalp incision.
Figure 3: Imaged area and specific spatial reference points.
Figure 4: Examples of bone marrow calvarium intravital microscopy images obtained with lasers and filters listed in Table 2.
Figure 5: Validation of DiD signal through comparison with autofluorescence signal (measuring DiD:autofluorescence signal ratio).
Figure 6: Image analysis procedure.

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Acknowledgements

We thank F. Ferraro, S. Lane, S. Lymperi, E. Ozcivici, A. Sanchez-Aguilera and M. Spitaler for critical input on the manuscript. C.L.C. was funded by the European Molecular Biology Organization and Human Frontier Science Program and is currently funded by Imperial College London, Kay Kendall Leukaemia Foundation and Cancer Research UK. C.P.L. and D.T.S. are funded by multiple NIH grants.

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Authors and Affiliations

  1. Center for Regenerative Medicine, Boston, Massachusetts, USA

    Cristina Lo Celso & David T Scadden

  2. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Cristina Lo Celso, Charles P Lin & David T Scadden

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

    Cristina Lo Celso

  4. Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Charles P Lin

  5. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    David T Scadden

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  1. Cristina Lo Celso
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Correspondence to Cristina Lo Celso.

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CLC and CPL have no conflicting financial interests. DTS is a shareholder in Fate Therapeutics and consultant to Genzyme, Hospira and Fate Therapeutics.

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Lo Celso, C., Lin, C. & Scadden, D. In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Nat Protoc 6, 1–14 (2011). https://doi.org/10.1038/nprot.2010.168

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