This protocol is significant because lin-negative cells and SK cells isolated from the same donor mice had no apparent difference in the generation of MLL-AF9 induced AML. Also, IP injection of leukemic cells can successfully develop a mouse model of AML. Compared to conventional transplantation methods, this technique is more convenient and less expensive.
To begin, sterilize the 8 to 10-week-old CD45.1 female C57BL/6J mouse with 70%ethanol. Place the mouse onto a sterile surgical pad on a styrofoam board and pin the legs through the mouse paw pads. Cut the skin above the abdominal cavity at the midline and widen the subcutaneous space toward the hind legs with sharp-end sterile scissors.
Extend the incision from the abdominal midline down to the ankles and widen the subcutaneous space below the hind legs with the blades of sharp-end scissors. Cut the achilles tendon with sharp-end scissors. Hold the tendon using forceps with teeth and cut the other end attached to the femur to remove the gastrocnemius muscle.
Cut the quadriceps tendon attached to the knee with sharp-end scissors. Hold the tendon and cut the muscle head attached to the femur to remove the gastrocnemius muscle. Cut the other muscles surrounding the femur at the end attached to the tibia.
Next, cut the ankle with sharp-end scissors, ensuring that the tibia remains intact. Hold the distal end of the femur and cut the hip joint with sharp-end scissors, ensuring that the femoral head remains intact. Transfer the tibias and femurs to a flow buffer in a 15 milliliter tube.
Separate the tibia and femur by breaking the knee by hand. Remove the patella, cartilage, and femoral condyles to expose the tibial plateau and distal femur. Remove the muscles using sterile gauze and soak the bones in a flow buffer.
Cut the femoral neck and flush the bone marrow cells with flow buffer from both ends of the femur using a 10 milliliter syringe with a 23 gauge needle. Next, cut the tibial malleolus and flush the bone marrow cells with flow buffer from both ends of the tibia. Disperse the cells by pipetting up and down using a 10 milliliter syringe with an 18 gauge needle.
Centrifuge the single-cell suspension at four degrees Celsius and 400 G for three minutes. Discard the supernatant and resuspend the cells in five milliliters of red blood cell, or RBC, lysis buffer to lyse the RBCs for three minutes. Add five milliliters of flow buffer to stop the lysis.
Then centrifuge the cell suspension and resuspend the pellet in five milliliters of flow buffer. Place a 70 micron cell strainer onto a 50 milliliter tube and pass the suspension through the strainer to collect the cells. Adjust the cell concentration with flow buffer to one times 10 to the eight per milliliter in round bottom polypropylene tubes.
Select lineage-negative cells using a mouse hematopoietic cell isolation kit according to the manufacturer's instructions. Once done, centrifuge the sorted hematopoietic stem cells and unsorted lineage-negative cells and resuspend in three milliliters of 2X of cytokines, IMDM media, and three milliliters of viral supernatant in a retro-reductant coated dish. Incubate the dish in a humidified 5%carbon dioxide incubator at 37 degrees Celsius for 6 or 24 hours.
After transduction, harvest the cells by centrifugation. If needed, use trypsin to collect the cells attached to the dish bottom. Discard the supernatant and resuspend the pellet in pre-warmed PBS.
Inject cells into the primary recipient mouse retro-orbitally or intraperitoneally with a 27 gauge half needle. Monitor the mouse daily. After one month, collect blood weekly by retro-orbital bleeding to monitor leukocytosis by evaluating the complete blood count on a HEMAVET.
Proptose the eye with the thumb and index finger, then penetrate the venous sinus plexus with a sterile hematocrit capillary tube through the inner canthus. Collect 20 to 25 microliters of blood into an EDTA blood collection tube and close the eyelids to stop the bleeding. Apply one drop of gentamycin sulfate ophthalmic solution to the eye.
When the white blood cells reach four times 10 to the four cells per microliter, isolate the bone marrow cells by flushing the femurs and tibias with flow buffer, followed by RBC lysis as described earlier. Next, to harvest the splenocytes, place the mouse onto a sterile surgical pad on a styrofoam board and pin the legs through the mouse paw pads. Sterilize the mouse with 70%ethanol.
Cut the skin and muscle at the midline to expose the abdominal cavity with sharp-end sterile scissors. Isolate the spleen and put it in a flow buffer in a 15 milliliter tube. Mesh the spleen through a 70 micron strainer in a six centimeter dish with three milliliters of flow buffer.
Transfer the cells from the dish to a 15 milliliter tube, then centrifuge the single-cell suspension, discard the supernatant, and resuspend the pellet in five milliliters of RBC lysis buffer for three minutes. Add five milliliters of flow buffer to stop the lysis and centrifuge the cell suspension. Resuspend the pellet in five milliliters of flow buffer, mix and pass through a 70 micron cell strainer to collect cells into a 50 milliliter tube.
Identify primary acute myeloid leukemia, or AML, cells by stain the splenocytes and bone marrow cells with FITC conjugated anti-mouse CD45.1 antibody and detecting on a flow cytometer. The cells are first gated on FSC-A FSC-H and FSC-A SSC-A to acquire singlets. The CD45.1 positive population is gated on the FL1 plot by comparing it to unstained cells.
For secondary transplantation, resuspend CD45.1 AML splenic cells from primary retro-orbital recipients in PBS and inject them retro-orbitally into CD45.2 male mouse. The presence of aberrant leukocytosis and increased infiltration of leukemic cells in the bone marrow and spleen supports the feasibility of using bone marrow lineage-negative cells to generate primary AML. No significant differences were observed in primary recipients translated with LSK or lineage-negative cells.
The AML cells spread in the abdominal cavity of primary recipients by mixed lineage leukemia or MLL-AF9 transduced lineage-negative cells. These results also confirmed the establishment of primary AML via intraperitoneal injection of MLL-AF9 transduced bone marrow lineage-negative cells in the form of leukocytosis and the presence of AML cells in the bone marrow and spleen of recipient mice. However, primary transplantation via intraperitoneal injection took longer to develop AML than via retro-orbital injection despite the equal number of donor cells.
The secondary recipients showed leukocytosis and significant hepatosplenomegaly in less than one month post-transplantation. The AML cells were also detected in the peripheral blood, bone marrow, and spleen, as well as in the peritoneal cavity. The intraperitoneal injection of eight times 10 to the five AML cells achieved comparable engraftment to retro-orbital injection of eight times 10 to the five AML.
The tertiary recipient mice acutely exhibited AML signs including leukocytosis and hepatosplenomegaly, the presence of leukemic cells in the blood, bone marrow, and spleen, histological observation of the femur and spleen further demonstrated the infiltration of leukemic cells. Compared to the secondary and tertiary transplantations, the tertiary transplantation progressed much faster than the secondary transplantation. For the current model, sorting LSK cells is unnecessary.
Lin-negative cells can be transduced with AML virus for 6 hours or 24 hours. It won't affect the efficiency of the model establishment.
