Name: combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches
Uploaded: 2017-03-21T13:00:00
Description: Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Imaging was carried out in the Indiana Center for Biological Microscopy at Indiana University, directed by Dr. Ken Dunn. The stereotaxic device is a prototype designed and made by Mark Soonpaa, Wells Center for Pediatric Research. This work was supported by NIH/R01DK097837-09 (NC), NIH/R01HL068256-05 (NC), NIH/NIDDK1U54DK106846-01 (NC), the MPN research Foundation (NC) and the CTSI Collaborative project IUSM/Notre Dame (NC).

All procedures involving the use of animals were performed with authorization of the Animal Care and Use Committee of Indiana University School of Medicine. Ensure to adhere to the legislation on animal experimentation of the country where the work is performed.
1. Preparation of Mx1CreRBPJ-/- Recipient Mice
<ol>
	<li>Cross Mx1-Cre+ mice with RBPJlox/lox mice10 to obtain Mx1-Cre positive RBPJlox/lox mic...

<ol>
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This protocol describes an experimental design optimized to study the kinetics of hematopoietic cells engraftment by Intravital Florescent Microscopy. In this study, the expansion of myeloid progenitor cells in a WT BM or in a Notch signaling defective BM was tracked in the bone calvarium by following Lys-GFP positive myeloid cells after BMT into RBPJWT or RBPJKO recipients. This approach is proposed as a model that can be applied to address similar questions, for example: i) to determine the expansion and localization i...

Intravital multiphoton fluorescence microscopy (IVFM) is a powerful imaging technique that allows for the high-resolution, real-time imaging of tissues with depth up to 1mm, depending on the tissue. When applied to the mouse calvarium, it permits observing the behavior of the hematopoietic cells within the BM in a non-invasive manner up to 60-100 &#956;m11. This approach is used here to determine the kinetics of engraftment of normal myeloid progenitors in the BM of RBPJ knock-out mice lacking canonical Notch signaling.
Recent work from our group demonstrated that defective canonical Notch signaling in the BM microenvironment leads to a myeloproliferative-like disease12. Loss of Notch signaling was obtained by conditional deletion of the DNA binding domain of RBPJ, the critical transcription factor downstream of canonical Notch signaling, using Mx1-Cre induced recombination10. In this study, the Mx1-Cre/RBPJlox/lox mice model was used. Conditional deletion of the DNA-binding motif of RBPJ results in the loss of signaling from all Notch receptors. In the Mx1-Cre model, Cre expression is driven by the Mx1 promoter activated upon administration of polyI:C resulting in the induction of targeted gene deletion in blood cells as well as in stromal components of multiple organs, including BM, spleen and liver.
Mx1-Cre+/RBPJlox/lox and Mx1-Cre-/RBPJlox/lox mice induced with polyI:C (hereon indicated as RBPJKO and RBPJWT, respectively) were lethally irradiated and transplanted with normal, wild type hematopoietic cells. Starting from week 4 after transplantation, RBPJKO recipients developed significant leukocytosis followed by splenomegaly. Although RBPJKO mice presented increased percentage of myeloid progenitors in the BM at week 8 after transplant and at later time points, analysis of BM at weeks 4 and 6 did not reveal striking differences in their myeloid cell content compared to control RBPJWT recipients. This observation, together with the fact that Mx1-Cre is expressed in different hematopoietic organs, raised the question whether the BM microenvironment had direct impact on the initiation of the myeloproliferative phenotype.
To determine whether the BM was a critical initial site of disease development, IVFM of the mouse calvarium was used in combination with BM transplantation (BMT), the RBPJ knock-out model, and a lineage tracking system. Transgenic mice expressing EGFP under the control of the specific lysozyme promoter (Lys-GFP)9 were used to obtain donor cells that could be visualized during BM imaging after BMT. Lysozyme expression is specific to myeloid cells and Lys-GFP marks cells from the common myeloid progenitor (CMP) to the mature granulocyte13.
IVFM of the BM at different time points demonstrated that Lys-GFP cells homed similarly to the BM of RBPJWT and RBPJKO recipients, but expanded and engrafted faster in the BM of RBPJKO recipients. This difference was dramatic at the earlier time point (week 2) and decreased over time (weeks 4 and 6). However, at these later time points, evaluation of the hematopoietic compartment in the same recipient showed a steady increase in the number of myeloid cells circulating in the PB and localized in the spleen of RBPJKO mice, indicating an increased output of cells from the BM into the circulation. Analysis of Lys-GFP cells localization in the BM of transplanted mice at 6 weeks revealed that myeloid cells were residing further from the vasculature in the RBPJKO microenvironment than in the control.
Collectively, the combination of IVFM with these specific animal models provided insights in the engraftment dynamics of myeloid cells in the RBPJKO BM microenvironment. The experimental design and quantitative approach described here is proposed as a paradigm that can be applied to address similar questions. For example, the use of other cell specific lineage tracking models, such as RAG1-GFP14 or Gata1-GFP15 mice, may allow following the behavior of lymphoid or erythroid progenitors, respectively, in the BM.

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combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Cohorts of 2 RBPJKO and 2 RBPJWT recipients were imaged in an individual imaging session at different time points: 24 h and 2, 4 and 6 weeks after transplantation of BM Lys-GFP cells (workflow is illustrated in Figure 1A).
In each mouse, images were acquired from 6 standard regions of the BM calvarium, identified by their position in relation to the bifurcation of the central vein (Figure 2A, <s...

Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

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