骨髄ニッチに造血細胞の生着のダイナミクスを研究する遺伝的モデルと生体内蛍光顕微鏡 (IVFM) を組み合わせること

The overall goal of this procedure is to visualize and measure the in vivo engraftment of transplanted blood cells to recipient bone marrow niches. The main advantage of this technique is that it allows visualization of cellular processes like homing, expansion, and localization to different bone marrow niches of donor hematopoietic cells following transplantation. Used in combination with the engineered animal models, this approach can help in defining molecular pathways critical for hematopoietic cell regeneration.

Unlike histology or other imaging techniques, intravital microscopy allows capturing cell-to-cell interactions in a live animal on a cellular level and in real time. We decided to use this method to determine whether the bone marrow microenvironment was the initial site of the development of the myeloproliferative disease caused by loss of Notch signaling. Challenges for individuals new to this technique include minimizing breathing artifact, optimally positioning and stabilizing the head and avoiding image saturation.

One to two hours before the transplant, spray a lysed EGFP mouse with 70%ethanol. Next, use surgical scissors to make an incision in the skin of each leg around the ankle and surgical forceps to remove the skin and fur together to expose the clean muscle tissue. Remove as much muscle from the legs as possible.

Then cut the bones at the knee and ankle joints and use gauze sponges to remove any remaining muscle tissue from the femurs and tibias. Rinse the cleaned bones in a six-well plate containing DMEM supplemented with 10%FBS. Then transfer all four hind leg bones into 10 milliliters of cold EDTA in PBS into a mortar and crush the bones with a pestle.

Triturate the bone marrow cells to obtain a single-cell suspension and strain the slurry through a 70 micron filter into a 15 milliliter centrifuge tube. Rinse the filter with two to three milliliters of PBS. Then centrifuge the cells and resuspend the pellet in 10 milliliters of fresh DMEM plus 10%FBS.

For the injection, centrifuge the cell suspension then count and adjust the concentration of cells to 1.5 times 10 to the seventh cells per milliliter in IMDM without serum. Place the cells on ice. Five to six hours after the second dose of radiation, inject three times 10 to the sixth bone marrow cells into the tail vein of each radiated recipient animal.

Add the appropriate time point post-transplantation. Place the anesthetized recipient mouse onto a 37 degrees Celsius heating pad and confirm a lack of response to toe pinch. Apply ointment to the animal's eyes and administer 100 microliters of the appropriate fluorescent marker via the tail vein.

Next, use small electric clippers to remove the hair from the dorsal surface of the animal's head, followed by the application of a depilatory cream. After five minutes, remove the cream with gauze sponges and rinse off any remaining solution with saline. Disinfect the scalp with a 70%ethanol soaked cotton swab.

Then use fine forceps and scissors to make a 10 to 20 millimeter midline skin incision on the scalp to expose the underlying dorsal skull surface. Using 5-0 surgical silk, place two stay sutures on each side of the incision creating a flap to expose the calvarium for imaging. Apply a droplet of oil on the exposed skull.

With the mouse in the supine position, submerge the exposed scalp in a glass bottom dish filled with microscope oil. Then place the animal on a multiphoton imaging stage with the calvarium above the objective and cover the animal with a 37 degrees Celsius heating pad. It is essential that the head remain stabilized in the correct position during the multiphoton image acquisition.

Use a stereotactic device or secure the mouse with tape as appropriate. Using an inverted confocal system modified for multiphoton imaging, tune the two photon laser to 830 nanometers. Next, open the appropriate image acquisition software.

Under the acquisition settings menu, confirm that the one directional scanning mode is selected and set the scanning speed to four microseconds per pixel, the frame rate to 512 by 512 pixels and the zoom to 1.5. Select the 20X watt in a 0.95 objective. Open the dye list window and select two photon.

Then select the RDM690 mirror in the microscope controller window. In the light path and dice window, select the laser unit two box to check if the two photon laser shutter is open. Select EPI Lamp and choose the B/G EPI filter cube.

Then focus the objective on the specimen to visualize the vascular flow in the calvarium bone marrow niche, using the bifurcation of the central vein and the coronary suture as reference points. Using the non-descanned mode, select three external detectors and image the specimen at a scan rate of four microseconds per pixel with no averaging and a constant laser power and detector gain to utilize the full dynamic range of the detector with a minimal saturation. After collecting a series of sections through the depth of the tissue, open the appropriate dedicated 3 to 4D image quantitation and visualization software.

Interactively visualize the Z stacks using the appropriate 3D rendering algorithm according to the manufacturer's instructions. Then use the spot object segmentation module to segment the GFP cells and apply stack arithmetic processing to eliminate the false positive GFP cells in the green and red channels. Finally, use the surface segmentation module to reform the segmentation of the vasculature and bone surface, applying X tension distance of spot to surface algorithms to calculate the distances of the cells to any of the distal surfaces as necessary.

Here, Z stacks collected from each of the six standard regions of the bone marrow calvarium were rendered as 3D maximal intensity projection, 3D shadow projection including the bone component, and 3D segmented images, allowing quantitation of the cells and the measurement of their distances from the bone and vessels. In this experiment, in vivo imaging of myeloid cells in the Notch-defective bone marrow microenvironment revealed that donor lysed GFP cells have a similar homing efficiency in the wild type and knockout recipients at 24 hours after transplant while they exhibit different engraftment at later time points. Analysis of the dynamics of myeloid progenitor expansion showed that adaptively bone marrow transplanted cells expanded more rapidly and in greater numbers in knockout recipients at two weeks after transplant.

The donor bone marrow cells in wild type recipients expanded later but in two-fold higher numbers than the donor cells and knockout recipients at four weeks, a time where donor cells in the knockout bone marrow are decreased because it's going rapidly into circulation. And that by six weeks, the differences in the number of donor cells in the bone marrow calvarium level out. Further, 3D segmentation analysis of the lower middle region of knockout and wild type calvaria at six weeks post-bone marrow transplant indicates that the lysed GFP cells localized at the same distance from the bone in both types of recipient animals, but interestingly, they reside more distantly from the vasculature in the knockout mice.

Once mastered, the intravital bone marrow imaging can be completed in one hour and 30 minutes per mouse if performed properly. While using this approach, it's important to remember to sample multiple areas of the bone marrow of the calvarium per mouse and to analyze at least three mice per experiment to address any intra and inter sample variations. After watching this video, you should have a good understanding of how to combine bone marrow transplantation and the intravital imaging to determine the kinetics of the engraftment of specific blood subpopulation to the bone marrow microenvironment.