Name: Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches (Video) | JoVE Journal
Uploaded: 2017-03-21T13:00:00.000+00:00
Duration: 11 min 6 s
Description: Learn intravital fluorescence microscopy to study hematopoietic cell engraftment dynamics in bone marrow niches using genetic models. This method enables real-time visualization of cellular homing and regeneration pathways.

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5 minutes

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 mice12 and Mx1-Cre negative RBPJlox/lox littermates to use as controls. Verify the genotype by PCR10.</li>
	<li>Use 6-8 week-old Mx1Cre+/RBPJlox/lox and Mx1Cre-/RBPJlox/lox mice to perform the polyI:C induction.</li>
	<li>Inject polyI:C 200 &#181;g i.p. in Cre+ and Cre- mice. Give one polyI:C injection every other day for 3 days the first week. Give one polyI:C injection the second week, 7 days after the previous injection (four injections in total).
	<ol>
		<li>Use RBPJKO (induced Mx1Cre+/RBPJlox/lox) and RBPJWT (induced Mx1Cre-/RBPJlox/lox) mice as recipients 3 weeks after the last polyI:C injection. 
		NOTE: It is recommended to use mice induced by pI:pC 3 weeks after injection. The IFN&#945; response triggered by polyI:C induces significant changes in the BM, resulting in the immunophenotypic expansion of HSC and decreased output of mature progenitors into the peripheral blood16,17. Representation of the hematopoietic subsets is normalized 3 weeks after injection and the mice can be utilized without the confounding effects of inflammation. This induction protocol has been optimized for RBPJ. If deleting a different gene, the induction protocol may vary depending on the construct, and deletion must be validated. We validated ~100% deletion of the RBPJ region between loxP sites by RT-PCR after a total of four polyI:C injections.</li>
	</ol>
	</li>
</ol>
2. Preparation of Lys-EGFP Donor Bone Marrow Cells for Transplantation
<ol>
	<li>Euthanize one Lys-EGFP mouse (Carbon dioxide followed by cervical dislocation) 1 or 2 h before the transplant.</li>
	<li>Spray the animal body surface with 70% ethanol.</li>
	<li>Use surgical scissors to make a skin incision on both legs around the ankle and with surgical forceps pull away skin and fur together to expose clean muscle tissue.</li>
	<li>Use surgical scissors to remove as much muscle from the legs as possible. Using a scissors, cut the bones (at the knee and ankle joint) and clean any remaining muscle tissue from the femurs and tibias using gauze sponges. Place the bones (two femurs and two tibias) into a 6-well plate containing DMEM 10% FBS.</li>
	<li>Crush the bones in a mortar with 10 mL cold 2mM EDTA PBS and pipet the bone marrow cells to bring the cells into single-cell suspension. Alternatively, flush the bones with 2 mM EDTA PBS 3 times from each side with a 1 mL syringe.</li>
	<li>Filter the bone marrow cells by using a 70-&#181;m filter into a 15 mL centrifuge tube. Rinse filter with 2-3 mL of PBS. Spin the cells down 10 min at 460 x g, resuspend the cells in 10 mL of fresh DMEM 10% FBS.</li>
	<li>Count bone marrow cells on a hemocytometer and adjust the concentration to 1.5 x 107 cells/mL in IMDM without serum. Use 3 x 106 cells per animal with a volume of 200 &#181;L. About 1/3 cells of total BM are myeloid GFP+ cells. Leave the cells on ice until ready for injection. Use 0.5 x 105 cells to determine GFP expression by FACS9.</li>
</ol>
3. Bone Marrow Transplantation of Lys-GFP Cells into RBPJKO Mice
<ol>
	<li>Restrain recipient mice in a pie cage. Irradiate mice with a lethal dose of gamma radiation (1,200 Rad) on a Cs 137 irradiator. Use a split dose protocol: 900 rads in the evening followed by 300 rads the next morning (16 h apart).</li>
	<li>Transplant the lethally irradiated RBPJWT and RBPJKO recipient mice 5-6 h after the second dose of radiation. Inject BM cells harvested from Lys-EGFP mice at a concentration of 3 x 106 cells per animal via tail vein injection (See details for harvesting cells in section 2).</li>
	<li>Image independent cohorts of transplanted mice by IVFM at different time points: 24 h, and at weeks 2, 4 and 6, as described below (See section 4 &#38; 5 for in vivo imaging procedure).</li>
</ol>
4. Surgical Preparation for Intravital Imaging
<ol>
	<li>Sterilize surgical instruments. Two fine forceps (one straight, one angled), one pair of fine scissors and one pair of needle holders. Prepare operative area with all supplies needed for procedure.</li>
	<li>Give the mouse an IP injection of ketamine cocktail anesthetic (Xylazine 2.5-5 mg/kg + acepromazine 1.0-2.5 mg/kg + ketamine 90-100 mg/kg) using a 26-28 G needle syringe. &#160;Animal will be monitored every 15 min during the procedure and anesthetic will be supplemented as necessary at &#188; of the original dose.</li>
	<li>Place the mouse on a proper heat source (37 &#176;C heating pad, animal protected from direct contact with heating pad) and visually monitor the respiratory rate.</li>
	<li>Check reflexes using the toe pinch response. Ensure that the animal is completely under anesthesia before beginning any surgical procedures.</li>
	<li>Use a 26-28 gauge needle syringe to give mice a tail vein injection of a fluorescent vascular marker (Dextran, 100 &#956;L of 20 mg/mL solution).</li>
	<li>Apply vet eye ointment to both eyes. Clip the dorsal surface of the animal&#39;s head with small electric clippers. Apply a hair removal cream for 5 min. Use gauze sponges to remove the cream and then rinse with saline. Prep the clean scalp with 70% alcohol using a cotton swab.</li>
	<li>Use fine forceps and scissor to make a small midline skin incision (10-20 mm) on the scalp to expose the underlying dorsal skull surface. Use 5-0 surgical silk to place two stay sutures in the skin on each side of the incision, creating a flap to expose the calvarium for imaging.</li>
	<li>Position the mice on their back and submerge the exposed scalp in a glass bottom dish filled with microscope oil. Transport the animal to the mutiphoton imaging room.</li>
	<li>Place the animal on the microscope stage with the calvarium positioned on the glass dish above the objective and then cover with a 37 &#176;C heating pad (animal must be protected from direct contact with heat).</li>
</ol>
5. In Vivo High Resolution Imaging of the Mouse Calvarium
<ol>
	<li>Use an inverted confocal system modified for multiphoton imaging (see Materials Table). Following manufacturer&#39;s instructions tune a 2-photon laser to 830 nm, place a 20X W, NA 0.95 objective lens in the microscope nose piece and check the laser beam alignment. 
	NOTE: Upright microscope systems are most commonly used for these studies, but an inverted multiphoton system may also be utilized. In this study, a custom designed atraumatic stereotaxic device was used. Although there are several commercially available stereotaxic devices for upright microscope systems, there is no commercially available stereotaxic device for an inverted microscope system aimed at securing the mouse skull. As alternative to a custom stereotaxic device, the skull can be secured in position above the objective utilizing various tape or glue methods for stability.</li>
	<li>Open an image acquisition software. In the &#34;Acquisition Settings&#34; panel check if one directional scanning mode is selected. Set up the speed of scanning to 4 &#956;s/pixel, frame rate to 512 x 512 pixels and zoom to 1.5. Select 20X W Na 0.95 objective from the list of available objective lenses to match the lens positioned in the nosepiece.</li>
	<li>Access the &#34;Dye List&#34; from the &#34;Image Acquisition Control&#34; panel and select &#34;Two Photon&#34;. Open the &#34;Light Path &#38; Dyes&#34; window and select DM690-980 excitation DM. Open the 2P laser shutter by checking the check box in the Laser Unit 2. In the &#34; Microscope Controller&#34; window, select RDM690 mirror.</li>
	<li>Select &#34; EPI LAMP&#34;, choose B/G epi-filter cube and focus the objective onto the specimen to visualize vascular flow and the calvarium bone marrow niche, using as reference the bifurcation of the central vein (a) and the coronary suture (b) (Figure 2A).</li>
	<li>Collect images using non-descanned mode. Select three external detectors: PMT detector1 to collect SHG signal of collagen (emission filter - 430/100 nm), GaAsP detector2 to collect GFP signal (emission filter - 525/50) and GaAsP detector3 to collect signal of TRITC-dextran (emission filter - 605/90 nm).
	<ol>
		<li>Perform imaging at a scan rate of 4&#956;s/pixel with no averaging to minimize phototoxicity. Collect images at a constant laser power and detector gain adjusted to utilize the full dynamic range of the detector with minimal saturation.</li>
		<li>Collect series of sections through the depth of tissue (60 x 1 &#956;m Z-stacks) from 6 regions of calvarium bone marrow. Use step size settings of 1 &#956;m, zoom 1.5 and 512 x 512 pixels frame size (423 &#181;m x 423 &#181;m). 
		Note: Overall total time required to image one mouse is 1-1.5 h.</li>
	</ol>
	</li>
</ol>
6. Quantitative Analysis
<ol>
	<li>Perform the image quantitation and 3D reconstructions using a dedicated 3D/4D image quantitation and visualization software as per manufacturer&#39;s instructions (see Materials Table). Visualize interactively Z-stacks in 3D utilizing Maximum Intensity Projection (MIP), alpha-blend or shadow projection volume rendering algorithms.</li>
	<li>Segment GFP cells using the &#34;Spot Object segmentation module&#34;. Apply stack arithmetic processing (channel subtraction) to eliminate false positive count of GFP cells (this eliminates signal of bone cells displaying strong fluorescence in green and red channels).</li>
	<li>Perform segmentation of vasculature and bone surface using the Surface segmentation module. If required, calculate distances of cells to any of the above surfaces by applying X-tension algorithms called &#34;distance of spot to surface&#34;.</li>
</ol>

<ol><li>Carlesso, N., Cardoso, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell+regulatory+niches+and+their+role+in+normal+and+malignant+hematopoiesis%5BTitle%5D)+AND+%22Curr+Opin+Hematol%22%5BJournal%5D)">Stem cell regulatory niches and their role in normal and malignant hematopoiesis.</a> Curr Opin Hematol. 17 (4), 281-286 (2010).</li>
<li>Lo Celso, C., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+haematopoietic+stem+cell+niche+at+a+glance%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124 (PT 21), 3529-3535 (2011).</li>
<li>Calvi, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li>
<li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121 (7), 1109-1121 (2005).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+haematopoietic+stem+cell+niche+and+control+of+the+niche+size%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425 (6960), 836-841 (2003).</li>
<li>Mendez-Ferrer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466 (7308), 829-834 (2010).</li>
<li>Naveiras, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone-marrow+adipocytes+as+negative+regulators+of+the+haematopoietic+microenvironment%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment.</a> Nature. 460 (7252), 259-263 (2009).</li>
<li>Scheiermann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adrenergic+nerves+govern+circadian+leukocyte+recruitment+to+tissues%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Adrenergic nerves govern circadian leukocyte recruitment to tissues.</a> Immunity. 37 (2), 290-301 (2012).</li>
<li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+fluorescent+granulocytes+and+macrophages%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 716-726 (2000).</li>
<li>Han, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inducible+gene+knockout+of+transcription+factor+recombination+signal+binding+protein-J+reveals+its+essential+role+in+T+versus+B+lineage+decision%5BTitle%5D)+AND+%22Int+Immunol%22%5BJournal%5D)">Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision.</a> Int Immunol. 14 (6), 637-645 (2002).</li>
<li>Lo Celso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live-animal+tracking+of+individual+haematopoietic+stem%2Fprogenitor+cells+in+their+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457 (7225), 92-96 (2009).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Notch-dependent+repression+of+miR-155+in+the+bone+marrow+niche+regulates+hematopoiesis+in+an+NF-kappaB-dependent+manner%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-kappaB-dependent manner.</a> Cell Stem Cell. 15 (1), 51-65 (2014).</li>
<li>Miyamoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloid+or+lymphoid+promiscuity+as+a+critical+step+in+hematopoietic+lineage+commitment%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment.</a> Dev Cell. 3 (1), 137-147 (2002).</li>
<li>Luc, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Down+Regulation+of+Mpl+marks+the+transition+to+lymphoid-primed+multipotent+progenitors+with+gradual+loss+of+granulocyte-monocyte+potential%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Down Regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.</a> Blood. 111 (7), 3424-3434 (2008).</li>
<li>Suzuki, M., Moriguchi, T., Ohneda, K., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+contribution+of+the+Gata1+gene+hematopoietic+enhancer+to+erythroid+differentiation%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation.</a> Mol Cell Biol. 29 (5), 1163-1175 (2009).</li>
<li>Essers, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458 (7240), 904-908 (2009).</li>
<li>Pietras, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Re-entry+into+quiescence+protects+hematopoietic+stem+cells+from+the+killing+effect+of+chronic+exposure+to+type+I+interferons%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.</a> J Exp Med. 211 (2), 245-262 (2014).</li>
<li>Scott, M. K., Akinduro, O., Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+4-dimensional+tracking+of+hematopoietic+stem+and+progenitor+cells+in+adult+mouse+calvarial+bone+marrow%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow.</a> J Vis Exp. (91), e51683(2014).</li>
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The authors have nothing to disclose.

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.

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

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

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 ...

combining-intravital-fluorescent-microscopy-ivfm-with-genetic-models

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Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.<br...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Tracking Mouse Bone Marrow Monocytes In Vivo

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice

Intravital microscopy allows the study of dynamic biological processes such as tissue regeneration and tumor development. The calvarial bone marrow, a highly dynamic tissue, offers insights into hematopoiesis and vascular function. Using a biocompatible 3D-printed head fixation implant allows for repetitive longitudinal imaging, enhancing our understanding of tissue dynamics and the tumor microenvironment.

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

Learn intravital fluorescence microscopy to study hematopoietic cell engraftment dynamics in bone marrow niches using genetic models. This method enables real-time visualization of cellular homing and regeneration pathways.

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

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

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