The overall goal of this experiment is to determine the fitness of bone marrow hematopoietic stem cells in vivo using a competitive transplant assay. So this method can help answer key questions in the field of stem cell biology and hematopoietic cell transplantation about the impact of various manipulations on stem cell self-renewal after transplant. The main advantages of this technique are that it requires a limited number of animals and that it does not involve cell sorting.
Though this method can provide insight into stem cell function, it can be also useful in other fields such as immunology, through the use of mixed bone marrow chimeras. The initial development of this technique in the 1960s paved the way for researchers in the field of stem cell biology to explore the identity and function of hematopoietic stem cells in humans and mice. Demonstrating the procedure will be Roxanne Hetu-Arbour, a Master's student in the lab, and Edward Owusu Kwarteng, a PhD student.
Begin by placing the mouse inside a biological safety cabinet on a surgical platform and wet the skin and fur with 70%ethanol. Next, take hold of a hind foot and cut the skin up along the leg. Proceed to pull away the skin and cut away the excess muscle.
Then, with the scissor blades against the pelvic bones as a counter-force, pull on the leg bone to dislocate the femur. Detach the tibia and the femur from the kneecap, then remove the foot, and remove the remaining bits of muscle and tendons. Then, place the bones in two to three milliliters of sterile PBS in one well of a six-well tissue culture plate.
When all of the bones have been harvested, grasp the bones one at a time with a forceps, and insert a 25-gauge needle attached to a one-milliliter syringe filled with sterile PBS into one end of the bone. Depress the plunger, collecting the cells in a 15-milliliter conical tube until the center of each bone is white. Using a 22-gauge needle, dissociate the cells into a single cell suspension by repeatedly passing them through the needle tip.
After all of the bone marrow has been collected, filter the cells through a 70-micron nylon strainer and count the resulting cell suspensions. Then, centrifuge the bone marrow cells, remove the supernatant, and dilute the samples to a one times ten to the eighth cells per milliliter concentration in fresh PBS, on ice. To establish donor hematopoietic stem cell equivalents, transfer 30 microliter aliquots of cells into five-milliliter round-bottomed polystyrene tubes and add an equal volume of unlabeled antibody against CD16 and CD32 to the samples for five minutes at room temperature to block any non-specific staining.
Next, incubate the cells in 90 microliters of the appropriate fluorochrome conjugated primary antibody master mix on ice, protected from light. After 30 minutes, add two milliliters of staining buffer to the cells and vortex the tubes. Follow this with centrifugation.
Decant the supernatants and resuspend the pellets by flicking. Then, label the cells with 10 microliters of fluorochrome conjugated streptavidin on ice for 20 minutes in the dark. At the end of the incubation, wash the cells in two more milliliters of buffer and analyze the samples by flow cytometry.
Using one sample as a baseline, establish the estimated hematopoietic stem cell equivalents for each of the samples. After calculating the number of hematopoietic stem cells per sample as outlined in the text, mix the competitor and test hematopoietic stem cells at a one-to-one ratio and adjust the final volume of the cells to 200 microliters per injection in sterile PBS. For adoptive transfer of the hematopoietic stem cells, warm up an irradiated recipient mouse with a heat lamp.
Then aspirate 750 microliters of cells into one one-milliliter tuberculin syringe equipped with a 27-gauge needle and remove the bubbles. Next, with the mouse in the appropriate restraining device, wipe the injection site with ethanol. Insert the needle into a lateral tail vein, bevel side up and parallel to the skin, and gently depress the plunger.
When all of the cells have been injected, remove the needle and compress the injection site with sterile gauze for a few seconds to stop the bleeding. Then place the mouse in a clean cage. To analyze the peripheral blood, pierce the facial skin near the hairless spot located under the jawbone and collect one to two drops of blood in an EDTA tube.
Place the tube in a rotator to prevent clotting while waiting to proceed. Next, mix one milliliter of freshly-prepared, room temperature, red blood cell lysis buffer with the blood and transfer the sample to a five-milliliter, round-bottomed polystyrene tube. After four minutes at room temperature, add four volumes of ice-cold PBS to each tube and cover the opening with a cap.
Mix the tube a few times by inversion and immediately place the cells on ice. After centrifugation, decant the clear red supernatants and resuspend the pellets by flicking. Then add two milliliters of staining buffer to the cells and briefly vortex them.
Follow this with a a second centrifugation. Resuspend the pellets by flicking and proceed with flow cytometry staining as detailed in the text. The competitor-derived cells should be present in all three of the myeloid, B-lymphoid, and T-lymphoid lineages.
The competitor and donor cells are also found within both lineage negative and hematopoietic stem cell bone marrow cell populations. When the donor hematopoietic stem cells are functionally equivalent to the competitor cells, the proportions of the donor and the competitor-derived cells are equal. The residual host cells are typically almost exclusively T lymphocytes, as these cells are more radioresistant.
When the donor cells are observed at a much lower proportion of peripheral blood leukocytes than the competitor cells, some aspects of hematopoietic stem cell function are defective, and warrant further investigation. When the donor and competitor cells are both present in low numbers and the host cells represent the majority of peripheral blood cells, the transplant is considered unsuccessful, and no conclusions can be drawn about the relative functionality of the donor versus the competitor cells. Once mastered, the bone marrow preparation and the transplantation steps can be completed in six to seven hours if they are performed properly.
While attempting this procedure, it is important to remember to maintain sterile conditions during the cell preparation and the animal manipulation steps. Following this procedure, other methods, like a limiting dilution assay, can be performed to answer additional questions regarding the functional stem cell numbers. After watching this video, you should have a good understanding of how to perform competitive bone marrow transplants.
Don't forget that working with irradiation can be extremely hazardous, and that the appropriate precautions should always be taken while performing this procedure.
