Our protocol will help researchers understand the physiological effects of three different bone marrow transplantation methods and how they affect experimental outcomes in clonal hematopoiesis setting. Total body radiation bone marrow transplantation can negatively impact cardiovascular organs and alter disease pathogenesis. Thus, our lab has developed two alternative methods to minimize or avoid possible side-effects.
Demonstrating the procedures will be Eunbee Park a graduate student, and Megan Evans a postdoctoral fellow, both from my laboratory For thorax and abdomen shielding, place the adjustable tray and the x-ray irradiator at the correct distance to achieve uniform irradiation. And place the anesthetized mice on a flat led plate inverted to each other in the supine position. Secure the paws to the plate with tape, and place the led shielding so that the lower end aligns with the xiphisternum bone and the upper end of the lead shield sits near the thymus.
After shielding, place the mice into the irradiator and expose the animals to two, 5.5 gray fractions of irradiation separated by a four to 24 hour interval. For head shielding, carefully tape the four paws of the mouse to the abdomen. Place the mouse in a conical restrainer and slide the restrainer into the slot within the lead shield so that the mouse's head and ears are completely covered, leaving the rest of the mouse's body exposed for a radiation.
After shielding place the mice into the irradiator and expose the animals to, to 5.5 gray fractions of a radiation separated by a four to 24 hour interval. After each radiation treatment, place the anesthetized mice in a cage on a heated mat with monitoring until fully recovered. To isolate the bones, disinfect the skin of the donor mouse with 70%ethanol and make a small transverse skin incision below the rib cage.
Holding the skin tightly at either side of the incision, tear in opposite directions towards the head and feet and peel the skin from all of the limbs. Cut over the shoulders and elbow joints, and use a lab wipe to remove the attached muscles and connective tissues from the humeri. Carefully dislocate the joints between the femur and hip bones.
And use blunt scissors to cut along the femoral heads to detach the legs. Cut over the knee joint, to separate the femur and tibia. And use lab wipes to carefully remove the attached muscles and connective tissues from the bones.
Then pull the bones from mice of the same genotype into individual 50 milliliter conical tubes containing 20 milliliters of ice cold sterile PBS on ice. To isolate the bone marrow cells in a bio-safety class two cabinet, use an 18 gauge needle to make a small hole in the bottom of a sterile 500 microliter micro tube. And place the tube into an individual sterile 1.5 milliliter micro centrifuge tube containing 100 microliters of ice cold sterile PBS.
When all of the tubes have been prepared, remove the PBS from the tube containing the isolated bones and transfer the bones onto a sterile 100 millimeter cell culture dish. Use fine forceps and small scissors to carefully remove the epiphysis from the ends of each bone, and place up to six bones into each 500 microliter tube. When all of the bones have been cut, extract the bone marrow cells by centrifugation.
If all the marrow contents have been removed, the bones should appear white and translucent with a relatively large red pellet at the bottom of the 1.5 milliliter micro centrifuge tube. For bone marrow cell transplant, dilute the isolated bone marrow cells in serum free RPMI media, and load 200 microliters of the cell suspension into one 0.5 milliliter insulin syringe per mouse to be injected. After confirming a lack of response to pedal reflex, slowly inject the entire volume of cells into the retro orbital vein of each anesthetized recipient animal.
Then place a drop of proparacaine onto the eye for pain relief. And allow the mouse to regain consciousness while being monitored. To compare the effect of three BMT preconditioning methods on donor cell engraftment.
The fractions of donor cells in peripheral blood and heart tissue were analyzed by flow cytometry at one month post BMT. In this representative analysis, in the peripheral blood of recipient mice that received total body irradiation, monocytes, neutrophils and B cells were largely ablated and replaced by the progeny of donor bone marrow derived cells. In addition, the resident recipient cardiac monocyte and neutrophil populations were almost completely replaced by donor derived cells.
In the partially shielded irradiation group, the donor derived cardiac immune cell replacement was modest. The recipient mouse bone marrow cells within the shielded regions likely contributed to the lower level of peripheral blood reconstitution compared to mice that received total body irradiation. In the group without BMT preconditioning, donor derived cells were detectable in the peripheral blood and hearts of recipient mice at four weeks post BMT.
In addition, Tet2 deficient donor cells gradually expanded over time. In comparison, recipient mice engrafted with wild type donor cells showed minimal clonal expansion of donor cells. They optimization of these experimental models will enable more rigorous studies of the clonal hematopoiesis driver genes that contribute to all cause mortality such as cardio-metabolic disease and cancer.
We hope that our protocols will allow researchers to study clonal hematopoiesis to investigate how it contributes to cardiovascular disease and other disease states.
