The overall goal of this procedure is to track fluorescently marked hematopoietic clones in live tissues, including bone marrow using a novel confocal and two photon microscopy strategy. To do this, murn hematopoietic progenitor and stem cells are isolated and co transduced with lentiviral gene ontology or Lego vectors encoding five fluorescent proteins, Ian, EGFP, Venus, TD tomato, and m cherry. The transduced cells are then transplanted into myelo ablated recipient mice and after periods of time of up to 120 days, bone marrow and organs are harvested from the recipient mice and combined confocal and two photon microscopy is performed.
The resulting images are reconstructed in 3D to track the location of fluorescently marked transplanted clones and cells over time. This facilitates the study of clonal evolution during hematopoesis and the fate of bone marrow derived cells in other organs. The main advantage of this technique over physical sectioning of tissues is that it combines the benefits of high resolution imaging with optical sectioning via confocal microscopy and visualization of structural components via twofold on microscopy.
This method can help answer key questions in the hematology field, such as special and temporal clonal analysis of hematopoiesis during bone marrow regeneration from early engraftment with geographical maintenance of expanding clones over time, Very large volumes of intact dense tissue can be examined. For example, approximately 3000 images were computationally reconstructed to visualize color marked clones in a sternal fau. Although this method can provide insights into clonal architecture in normal and perturb hematopoiesis, it can also be applied to the study of organ regeneration, immune responses or tumor metastatic patterns.
In this procedure, lentiviral gene ontology or Lego vectors are encoding five fluorescent proteins, Ian EGFP, Venus, TD tomato, and M Cherry. Each fluorescent protein is expressed from a strong constitutive spleen focus forming virus or SFFV promoter. After producing Lego vectors encoding the fluorescent proteins and titrating the viral particles, harvest the bone marrow from the anterior and posterior limbs of sacrifice donor mice by dissecting hum eye femurs and tibia.
Use a scalpel to thoroughly remove all muscle ligaments and excess tissue from the bones. Next cut across the end of each bone as close as possible to the joint. Using a 27 gauge needle on a syringe containing I 10 medium, flush the bone marrow out into a 50 milliliter conical centrifuge tube centrifuge at 500 G and four degrees Celsius to pellet the cells.
After discarding the supernatant to lyce the red cells add 50 milliliters of a CK buffer centrifuge as before, then discard the supernatant and repeat after discarding the second supernatant. Resuspend the cells in 10 milliliters of I 10 medium. Then use a max lineage depletion kit to purify lineage negative progenitor cells according to the modified kit protocol detailed in the accompanying document.
Once the lineage negative cells have been purified, plate them in a T 75 flask at a density of five times 10 to the fifth cells per milliliter in stem span medium supplemented with murine IL three murine IL 11, human FL three ligand and murine stem cell factor incubate at 37 degrees Celsius 5%CO2 for 48 hours. After two days, use a cell scraper to resuspend the cells and transfer one to four times 10 to the five cells per well of a 12. Well plate add vectors at an MOI of six to seven each in stem span medium with cytokines and four micrograms per milliliter of protamine sulfate to the cells.
To bring the total volume in each well to one milliliter, be sure to prepare a single color control for each fluorescent protein. After 24 hours, resuspend the cells using a scraper and transfer them to a five milliliter centrifuge tube. Then centrifuge and resuspend them in stem span medium without cytokines centrifuge again and resuspend the cells in 100 microliters of stem span medium without cytokines per injection or for confocal microscopy or flow cytometry.
Transfer them to 12 well plates containing fresh medium with cytokines and culture for an additional 96 hours. In preparation for the bone marrow transplant, place irradiated recipient mice in a cage under a warming light while the mice are warming. Prepare syringes for injection with one to four times 10 to the fifth lineage negative cells in 100 microliters of stem span per mouse.
Prepare one syringe for each mouse. When the tail veins are dilated, the mice are ready to be injected. Place the mouse to be injected in a mouse restrainer and disinfect the tail with a pad soaked in 70%ethanol.
This step also allows for better visualization of the tail vein. Then inject the cells into the tail vein. Transplant an entire cohort of animals with the same population of transduced donor bone marrow cells at various times up to 120 days post transplantation.
Retrieve the tissues and organs for intact imaging without sectioning. Place individual organs in 35 millimeter number zero. Cover glass culture dishes in 50 to 100 microliters of cold PBS for time-lapse imaging.
Place the organs in DMEM 10%FBS containing 20 millimolar heaps at 37 degrees Celsius and image immediately. Here microscopy is performed using an inverted Leica SP five five channel confocal and multi photon system equipped with multi-line argon diode 561 nanometer helium neon 594 nanometer and helium neon 633 nanometer visible lasers. Place one of the single Lego transduced samples on the microscope stage and focus.
Collect lambda image stacks at bandwidth intervals of five nanometers spanning the whole light spectrum. Next, use the software to select a region of interest in the image and view the histogram of the reference spectrum. Save this data.
Then repeat this process for each fluorescent protein in the experiment. Next, use the spectra collected from individual fluorescent proteins to set the bandwidths in channel mode. To do this for each channel, click on the black slider and insert the bandwidth to be used.
The bandwidth chosen should encompass the peak region of each fluorescent proteins excitation spectrum while ensuring that there is no overlap between fluorescent channels. Set the gain and offset for each detector so that the dynamic range of the sample output is at a detectable level, similar between fluorescent proteins and without saturation. For example, the M cherry protein is about half as bright as EGFP, so the gain and offset should be adjusted to give a similar dynamic range for both.
Finally, in channel mode, capture an image of each single color control sample with all five channels and check to ensure that each fluorescent protein is visible only in the corresponding channel and absent from the rest. Once the spectra for all channels has been validated, save the settings. These can be imported and used in future experiments that utilize the same fluorescent proteins and sample tissue.
Here combined confocal and two photon microscopy is demonstrated using sternum bone marrow using a scalpel and tweezers by section the sternum along the sagittal plane. Then make a transversal cut at the joint between two sternal fossy. Place the bones cut face down in a 35 millimeter number zero cover glass culture dish containing 50 to 100 microliters of cold PBS imaging will take place through the cut face.
Check the position of the bones in a dissecting microscope. Position the sample on the microscope stage. Then set the imaging software to sequentially capture two photon with second harmonic generated signal.
Then five color confocal excitation. Set the boundaries of the Zack collection area with the five microns step size over 150 to 300 microns. Use the tile function to instruct the software to automatically generate stitched volumes comprising the entire sternum Fosse approximately 2.5 by 1.2 square millimeters in the XY direction.
Initiate the capture to sequentially collect X, Y, Z images in all five channels plus two photon microscopy with second harmonic generated signal from bone and fibrillar collagen for four D time-lapse imaging. Place freshly excised, uncut, popliteal lymph node, spleen, lung, or calvarial bone samples onto 35 millimeter number zero. Cover glass culture dishes in 50 to 100 microliters of DMEM with 10%FBS and 20 millimolar heaps at 37 degrees Celsius.
Place the sample on the heated microscope stage. Then in the software switch the capturing parameters to use two photon titanium sapphire excitation at 860 nanometers combined with confocal excitation and the Leica resonance scanner at 8, 000 hertz per second. Taking 80 micron Z stacks at 22nd intervals for up to one hour for simultaneous three color two photon imaging instruct the software to use the titanium sapphire tuned at 860 nanometers simultaneously with the OPO laser tuned at 1, 130 nanometers for TD tomato or 1, 140 nanometers for M cherry.
Finally initiate the capture to collect four D images. As before, once all of the images have been collected, perform 3D and four D reconstruction and analysis using Amer 64 bit version or similar software in the software. Open the Zack image, select surpass to display the 3D volume visualizer.
Then use Navigate to rotate the volume 360 degrees. Next select animation. Use key frames to add selected views, zoom and rotations of the volume.
Preview the movie and edit as necessary. Finally, save the file in a desired movie format to trace clonal history of fluorescently marked hematopoietic stem and progenitor cells. Donor cells cot transduced with five different Lego vectors were transplanted into a recipient mouse and the sternal bone marrow was imaged and analyzed as described in this video.
At four days post-transplant, clusters of cells marked in a wide variety of colors were visible at close proximity to the bone edge, which is shown in white. By day 31, large clone of unique green yellow color had expanded to occupy the entire Fosse as illustrated in the merged as well as the single channel images. FPS content analysis demonstrated homogeneous marking by two FPS variance EGFP and Venus.
This 3D reconstruction video demonstrates the spatial architecture of the marked clones encased in the sternal bone. This image shows an example of bone marrow derived cells in popliteal lymph node 120 days following transplantation. Scattered cells are mostly marked in yellow and red and are peripherally surrounded by collagen fibers shown in white in the heart viewed from the epicardial side.
Numerous individual cells are visible interspersed between the cardiomyocytes shown in white visualized by their two photon autofluorescence at 780 nanometers Once mastered. This technique can be done in several days if it is performed properly After its development. This technique paved the way for researchers in the field of organ regeneration and development and to more biology to explore spatial temporal arrangements of clonally complex cellular and structure elements via multicolor labeling and confocal and two photon imaging.
After watching this video, you should have a good understanding of how to develop fluorescence marking methodology for tracing the clonal marking of hematopoietic stem and progenitor cells in live organs.
