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Developmental Biology

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

Published: March 21st, 2017

DOI:

10.3791/54253

1Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, 2Indiana Center for Biological Microscopy, Department of Medicine, Indiana University School of Medicine, 3Department of Pediatrics, Indiana University School of Medicine
* These authors contributed equally

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.

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 "niche" 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.

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 μm11. 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 microen....

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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

  1. Cross Mx1-Cre+ mice with RBPJlox/lox mice10 to obtain Mx1-Cre positive RBPJlox/lox mic.......

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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,

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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.......

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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).

....

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Name Company Catalog Number Comments
Ketamine cocktail IU School of Medicine Ketamine 90-100mg/kg, Xylazine 2.5-5.0 mg/kg, Acepromazine 1.0-2.5 mg/kg
TRITC dextran Tdb Consultancy TD150-100mg Other color dextran may be used.
Andis hair trimmer Braintree Scientific CLP-323 75
Gauze sponge Med Vet International PK224 4-ply, 2X2
Nair depilatory cream Commercial store
Saline Med Vet International RXSAL-POD1LT 0.9% Sodium Chloride poly bottle
Insulin syringe Fisher Scientific 14-826-79 28g, 1/2cc
Fine Forceps Fine Science Tools 00108-11, 00109-11 straight forcep, angled forcep
Scissor Fine Science Tools 15018-10
Needle holder Fine Science Tools 12002-14
5-0 silk suture Fisher Scientific MV-682 Other non-absorbable suture may be used
WillCo- glass bottom dish WillCo GWSt-5040
Optical microscope oil Leica
Stereotaxic stage insert  IU School of Medicine Custom design
Olympus FV1000 confocal microscope system  Olympus
Olympus XLUMPLFL 20xW, NA 0.95 objective  Olympus
Small heating pad Commercial store Zoo Med reptile heating pad
Imaris 8.1 imaging software Bitplane 3/4 D Image Visualization and Analysis software

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