The isolation of key Megakaryocyte Progenitor populations, allows for the fine analysis of the lineage hierarchy in cell culture on molecular assays, up to the single cell level. This protocol combines a more ethical practice by minimizing the number of animals required, in an efficient process for the cell sorting of MEP and MKP populations. For bone, collection spray the body of an eight to 12 week old C57 black six mouse, with 70%ethanol.
Use scissors to make a point five to one centimeter incision of the skin, perpendicular to the spine. And tear the skin around the whole body, by pulling it down. Place the mouse face down on the dissection pad, then locate the pelvic bones by sliding fingers or scissors, along the exposed spine, from top to bottom.
To locate the iliac crest, identify the small bump in the lumbar region near the hind limbs. After placing the scissors parallel to the spine, against the vertebrae, and close to the iliac crest bump, proceed to cut the muscles along the side of the spine above the pelvic bone, by sliding the scissors along the vertebrae, down to the tail. Then cut between the vertebrae and the iliac crest, staying as close to the vertebrae as possible.
And cut the remaining muscles to detach the limb from the body. Transfer the detached limbs to a clean surface. After discarding the rest of the body in compliance with the institutional guidelines, use forceps and scalpels to expose the pelvic, femoral, and tibial bones, by removing surrounding tissue.
Use forceps to hold the distal end of the femur, and carefully dislocate the femoral head from the pelvic bone, by gently slicing the muscles around the articulation with a scalpel. Scrape off the remaining muscle from the pelvic bone, and cut in the middle of the cavity that held the femoral head. Keep the ilium and discard the triangular thin side of the bone.
Using a scalpel, remove the residual tissues around the ilium, and place the cleaned bone in sterile PBS supplemented with 2%Newborn Calf Serum. Then use scissors to cut off the foot from the leg at the ankle. Holding the lower part of the tibia with the forceps, scrape the muscle up toward the knee.
Discard the fibula. Then make a cut across the tibial plateau with the scalpel, and place the tibia in sterile PBS 2%NBCS. After removing the residual tissues around the femur, hold the upper side of the femur with forceps, and place the scalpel blade at the base of the kneecap.
Apply a force toward the kneecap, parallel to the femur to detach the kneecap. Then place the femur, in sterile PBS 2%NBCS. In a laminar flow cabinet, transfer the bones to a sterile Petri dish filled with sterile PBS 2%NBCS.
Use a scalpel to cut off the head of the femurs. Fill a one milliliter syringe with sterile PBS 2%NBCS, and attach a 21 gauge needle, to the outlet. Then fill a five milliliter polypropylene tube with two milliliters of sterile PBS 2%NBCS.
Holding the femur with forceps, insert the needle in the groove left, after the kneecap removal by applying rotation, ensuring that the needle is completely inserted into the bone, up to the bevel. After the needle insertion, transfer the bone with the needle into the tube containing two milliliters of PBS 2%NBCS. Then dispense and aspirate the PBS 2%NBCS, from the syringe until the bone is clear.
Remove the needle from the femur, and insert it into the hole at the opposite side, where the femur head was. After dispensing and aspirating the buffer, discard the bone. For the iliac crest and tibia, use forceps to hold the bone and gently insert the needle in the open side, by applying rotation.
Ensuring that the needle is completely inserted into the bone, up to the bevel. Then transfer the bone with the needle into the tube containing two milliliters of PBS 2%NBCS. Dispense and aspirate the PBS 2%NBCS from the syringe until the bone is clear.
Pass all the the cell suspension through a 40 micron cell strainer cap, placed onto a sterile five milliliter polystyrene tube, and add 500 microliters of PBS 2%NBCS. Set aside 100 microliters of the cell suspension as total bone marrow. Then store it on ice for the staining procedure.
Pellet the filtered suspension by centrifugation. After discarding the supernatant, resuspend the pellet in a freshly prepared primary antibody cocktail, with the incubation on ice, for 30 to 45 minutes. Set aside 10 microliters of the cell suspension into a sterile five milliliter polystyrene tube, labeled Lin Pores Fraction, and add 90 microliters of PBS 2%NBCS.
Meanwhile, prepare a beads for magnetic depletion, by resuspending them in the vial, with thorough vortexing for 30 seconds. Transfer a volume of beads corresponding to two beads per target cell, into a five milliliter polypropylene tube, and wash the beads twice, with PBS 2%NBCS, by placing tube on the magnet. After removing the washing buffer with a sterile glass pasteur pipette, resuspend the beads in 500 microliters of sterile PBS 2%NBCS.
For the first step of magnetic depletion, add 250 microliters of beads onto the pellet of washed cells, and incubate with gentle mixing for five minutes on ice. Then add two milliliters of sterile PBS 2%NBCS, with gentle mixing, and place the tube in the magnet for two minutes. Proceed to collect the non-magnetic fraction with a sterile glass pasteur pipette.
And for the second step of magnetic depletion, add it to the remaining 250 microliters of magnetic beads. Place the tube sealed with paraffin on a tube roller, for 20 minutes at four degrees Celsius. Then add two milliliters of sterile PBS 2%NBCS with gentle mixing.
And place the tube with the magnet for two minutes. Collect the non-magnetic fraction into a sterile five milliliter polypropylene tube, labeled, non-magnetic Lin Neg Fraction, with a sterile glass pastreur pipette, and proceed with cell sorting megakaryocytes progenitors, as described in the text manuscript. For the gating strategy for cell sorting, in the Lin Neg population, the cells expressing C-kit, and a low to no expression of Sca-1 or CD16/32 antigen are selected.
In this population, MEP are identified as CD150 positive and CD9 dim cells. The MKP as CD150 positive, and CD9 bright cells. In the representative Flow Cytometry Analysis, cells identified as MEP and Mkp, were labeled with fluorescence conjugated antibodies for CD41a, and CD42c classical markers, of the megakaryocytic and platelet lineages.
Both markers were expressed by the cells of the MKp population, and were not detected at the surface of the cells of the MEP population. The DNA content of the sorted megakaryocytes, demonstrated that the cells are mostly 2n for the MEP population. And a small portion of the MKp cells are foreign.
Higher ploidy cells were not significantly detected in these populations. In semi-solid clonogenic assays, CFU-MK was detected in both MEP and MKp populations. BFU-E was not detected in the MKp population, but detected in MEP and, the CD150 negative CD9 dim progenitor cell population.
Microscopic observation on the third day of differentiation, shows that MEP and MKp produced mainly megakaryocytes, identified as large cells. Megakaryocytes obtained after three days of culture, were identified using CD41 and CD42c expression, and represent 54, and 82%of the cells produced from MEP and MKp cell populations, respectively. The ploidy of the megakaryocytes produced, was greater from the megakaryocyte derived from the MKp population, compared it to the MEP population.
Only the cells derived from the MKp population, were capable of proplatelet emission, suggesting a more advanced maturation stage for the MKp population. The use of the pelvic bone, drastically increases to cell number, while two-step magnetic depletion improves the efficacy of the lineage depletion. This protocol allows for the subsequent culture of MEP and MKp population of single cells, which, together with transcriptomic and proteomic analysis, will certainly have to decipher the mechanisms involved, implemented biogenesis.
