The overall goal of this procedure is to transplant purified, quiescent adult neuron skeletal muscle satellite cells for the treatment of skeletal muscle injury. This is accomplished by first dissecting skeletal muscle from adult mice and enzymatically digesting skeletal muscle, and then sorting the quiescent satellite cells by magnetic bead separation. Next, the isolated cells are expanded on collagen coated culture dishes, and in the final step, the in vitro expanded cells are injected into the cardio toin treated skeletal muscles of adult mice.
Ultimately, the engraftment and contribution of in vitro expanded satellite cells to the regeneration of muscle fibers can be assessed by xal staining. Hi, my name is a sushi ura. I'm associate professor in the Stem Cell Institute, university of Minnesota.
The isolation of cent satellite cell, which is a mass stem cell population, is essential to the understanding of muscle stem cell biology and the regeneration as well as the stem cell transplantation for therapies in muscular dystrophies such as mass Duchenne muscular dystrophies. The main advantage of this technique over existing method like facts is that our method is a rapid economical and reliable purification method of quiescent satellite cells from the adult mouse scatter muscle. Demonstrating procedures will be performed by Dr.Rio Moto, Hashi postdoc and Yoko Asura junior scientist in my laboratories.
To isolate mononuclear cells from mouse skeletal muscle begin by pinching and then slitting the abdominal skin of each three to eight week old euthanized adult mouse. Peel the skin in opposite directions to completely expose the triceps and the hind leg muscle, and then trim along the leg bones to remove the tibialis anterior gastro quadriceps and triceps skeletal muscles. Transfer the muscles into ice cold, sterile PBS in a 10 centimeter plate and wash off the blood.
Then transfer the muscles to a new sterile six centimeter plate. Next, under a stereo microscope, remove the connective tissue, blood vessels, nerve bundles, and add epigenic tissue from the muscles. Then using ophthalmological scissors, cut and mince the tissue into a smooth pulp.
Trying not to leave large pieces. Transfer the minced muscles into a 50 milliliter tube and digest the tissue in five milliliters of collagenase solution at 37 degrees Celsius. After an hour, use a syringe equipped with an 18 gauge needle to tri the tissue slurry several times.
To homogenize the mixture, incubate the homogenate for another 15 minutes, and then tri the mixture again To dissociate the slurry into a single cell suspension, add up to 50 milliliters of media and mix the cell solution. Then filter the cell suspension through a 70 micron cell strainer into a new 50 milliliter falcon tube. Count the collected cells and then spin down the cell suspension after washing cells, resuspend the pellet in 200 microliters of media, and then transfer the cells into a 1.5 milliliter micro centrifuge tube to stain and separate the cells.
Place the micro centrifuge tube on ice and then incubate the cells with one microliter each of the listed antibodies bodies. After 30 minutes, wash the cells in one milliliter of media two times. Then resuspend the pellet in 200 microliters of media and incubate the cells 10 microliters of anti PE magnetic beads on ice.
After another 30 minutes, wash the cells in one milliliter of max buffer two times and resuspend the pellet in one milliliter of buffer. Then place an LD column in the magnet and rinse the column with 1.5 milliliters of max buffer. Dispense the cell suspension onto the column, collecting the PE negative flow through fraction in a 1.5 milliliter tube.
Then spin down the PE negative fraction and resuspend the pellet in 100 microliters of media. Now incubate the cells in five microliters of anti-US IgG magnetic beads on ice, and then after 30 minutes, wash the cells in one milliliter of max buffer two times Resus suspending the pellet in 500 microliters of buffer. Next, place an MS column onto the magnet and rinse it with one milliliter of max buffer.
Then dispense the cells onto the column, discarding the alpha seven negative flow through fraction. Rinse the column two times with one milliliter of max buffer, and then remove it from the magnet. Then flush one milliliter of max buffer through the column with a plunger to elute the magnetically labeled integrin seven positive cells into a fresh 1.5 milliliter micro centrifuge tube.
Then after spinning down the cell suspension, resuspend the purified cells in myoblast medium. Finally, plate the cells on a matrigel coated six centimeter plate containing five milliliters of myoblast medium. To maintain the cell culture, feed the cells every other day with myoblast medium growing myoblasts.
Exhibit a small round shape and express myo D in their nuclei. When ready to begin the cell fusion, rinse the cells once with PBS and then tryps anize them in a 5%CO2 incubator at 37 degrees Celsius. After three minutes, collect the dissociated cells in myoblast medium, spin down the cells resus, suspending the pellet in myoblast medium, and reflate the cells onto new matrigel coated plates.
To differentiate the cell culture, feed the cells every other day with differentiation medium. After one day, the myoblasts will exit the cell cycle and undergo differentiation into myosin heavy chain or MHC positive myocytes. By day three to five, the cells fuse with each other and generate multinucleated myo tubes.
24 hours before myoblast transplantation, shave the hair around the tibias anterior or TA muscle of a sedated two month old nod skid mouse. Then use a 31 gauge insulin syringe to inject 10 micromolar of cardio toin into the muscle. The next day, detach the proliferating myoblasts from the matrigel coated plates with trypsin as just demonstrated and spin down the dissociated cells, resuspend one times 10 to the sixth of the cells in 50 microliters of media.
Then load the cells into a 31 gauge insulin syringe and transplant the cells intramuscularly into the recipient animal for TBIs anterior muscle regeneration. One to four weeks after injection harvest the TA muscles for histological analysis. Freshly isolated quiescent satellite cells display a small round shape, and more than 90%of these cells express PAC seven as a definitive marker.
Ex vivo expanded myoblasts can be utilized for intramuscular cell injection experiments for the examination of myoblast contribution to muscle fiber regeneration and self-renewal of satellite cells. When cultured in differentiation medium, the myoblasts exit the cell cycle fuse with each other and become multinucleated myo tubes that express myosin heavy chain a few days to several months after transplantation. The beta galacto side positive donor derived cells, which include proliferating myoblasts, self-renewing satellite cells, and newly formed muscle fibers can be detected in the cardio toin injured muscle After each development.
This technique paved the way for the researchers in the field of muscle stem cell and muscle origination to explore thetic stem cell transplantation for muscular dystrophy, I'm Fushi. I'm Yoko. I'm Norio.Thank.
Thank you for watching.
