The overall goal of this procedure is to assess the myogenic potential of induced pluripotent stem cell derived muscle progenitors in mouse models of acute and chronic muscle regeneration. This is accomplished by first assessing the myogenic potential of the cells in vitro. The second step is to transplant the cells via intramuscular injection.
Alternatively, cells are injected in the femoral artery by a microsurgical procedure. The final step is to evaluate the functional amelioration of the dystrophic phenotype of the treated animals. Ultimately, immunofluorescence and histology assays are used to show engraftment and functional contribution of the donor cells to the host muscles.
This method can help answer key questions in the field of skeletal muscle regeneration, such as investigation of cell therapy approaches. The implications of this technique extend towards therapy of muscular dystrophies as it could pave the way to autologous cell therapy strategies using IPS cells DER progenitors. Though this method can provide insight into stem cell based therapies, it can be applied to other systems such as delivery of molecules, intramuscularly, or intra artily, and subsequent analysis of the effect on muscle function and morphology.
Well first had the idea for this method when we realized that IPSL culture could provide a method to overcome some of the limitations of adult stem cells, such as the availability from tissue biopsies and their expansion potential. Begin by generating a stable cell line of items that are transduced with a tamoxifen inducible vector that overexpresses the myogenesis regulator myo D.After coating with 1%matri gel and washing with medium seed, a plate with the transduced cells and incubate at 37 degrees Celsius in growth medium. The cells should reach confluence in one or two days.
When they are ready, add one micromolar for hydroxy tamoxifen into the growth medium. After 24 hours, replace the growth medium with differentiation. Medium supplemented with one micromolar for hydroxy tamoxifen.
Examine the cultures daily for myo tube formation and replace half of the medium with fresh differentiation. Medium every other day when the cultures are ready. They're used in transplantation assays.
Here, dystrophic and immunodeficient mice are used to determine the extent of engraftment for intramuscular transplantation. Pretreat the animals with an injection of tamoxifen 24 hours before transplantation. The cells should also be pretreated with tamoxifen added into the growth medium the night before.
On the day of transplantation, detached by trypsin, count and centrifuge, the cells wash the cells once in PBS before Resus, suspending in calcium and magnesium free PBS to a concentration of tend to the six cells per 30 microliters. After anesthetizing the animal and sanitizing the skin, locate the tibialis anterior muscle. Identify the insertion point of the proximal tendon and insert the needle.
Tip two millimeters below this point. Insert five millimeters of the needle at a 15 degree incline relative to the tibia and slowly inject the cell suspension while retracting the needle. Empty the syringe with two millimeters of the needle still inside the muscle.
Take care when removing the needle from the muscle in order to avoid spilling the cell suspension through the needle's track. Following these same procedures, gastro anemia injections are performed two millimeters above the myo tendonous junction of the Achilles tendon and quadriceps. Femme injections are performed two millimeters above the distal tendon for intra arterial transplantation.
Pretreat the animals and cells with tamoxifen as demonstrated earlier. When ready for transplantation, the cells are prepared as previously demonstrated, except that they're filtered before centrifugation. Re suspend the pellet in calcium and magnesium free PBS and add 10%patent blue dye to allow for the visualization of the cell suspension distribution.
Carefully resuspend the cells in the syringe and remove all air bubbles before injecting, after anesthesia, shave and disinfect the area as shown, make a five to seven millimeter incision and localize the femoral bundle. Gently remove the connective fascia that covers the bundle with forceps. Next, carefully separate the femoral vein and nerve from the artery by gently introducing the tip of a forceps in between them and by progressively enlarging the hole.
The femoral vein is fragile. Take care not to pinch it during its detachment from the femoral artery. Once separated, lift the artery with one tip of the forceps and clamp the artery with the other tip.
With a 30 G needle, puncture the artery and inject 50 microliters of the cell suspension downstream of the clamped area at an infusion speed of approximately five microliters per second. Slowly remove the needle and the forceps from the artery to restore blood flow to the limb. Apply pressure with sterile gauze to avoid bleeding and or cauterize as required.
The patent blue dye allows immediate recognition of a correctly placed injection, if correctly injected the whole limb will rapidly turn light blue, suture the wound and monitor the animals until they recover. Administer analgesia for three days and inspect the wound Daily changes in the motor capacity of treated mice are evaluated by the treadmill test. Baseline measurements begin approximately one month before treatment and are used to evaluate the improvement of each animal tested.
The first step is to acclimate the animals to the exercise before testing. To do this, set the treadmill at a speed of six meters per minute for 10 minutes. Repeat this procedure every other day for one week.
Once they are familiar with the device, set the incline to 10 degrees and place the animals into the treadmill lanes. Turn the treadmill on with a starting speed of six meters per minute. Gently nudge any animal not willing to start the exercise.
Next, start the timer and increase the speed two meters per minute every two minutes as soon as an animal lies in the resting area for more than five seconds. Without attempting to reengage the treadmill, gently touch it with a stick to stimulate the restart of the exercise. A mouse is considered fatigued when it lies in the resting area for more than five seconds without attempting to reengage the treadmill.
After a series of three consecutive mechanical stimuli, one every five seconds, record the performance of each animal and repeat the measurements weekly or every 10 days for at least three times. Starting two weeks after cell transplantation. Repeat the same procedure in the treated mice after testing.
Use comparisons to baseline to evaluate changes in performance. 48 hours after four hydroxy tamoxifen administration, myo de positive nuclei are identifiable within myo DER transduced items in culture. As seen here.
After a week, the cells fuse and differentiate into multinucleated myo tubes. When transplanted intramuscularly into a mirroring model of acute muscle injury, items contribute to tissue regeneration results from the treadmill. Exercise tolerance test show amelioration in the motor capacity of treated mice after transplantation.
Ex vivo analyses of transplanted muscles show GFP positive areas representing the extent of colonization of IMS into the host tissue, thus demonstrating that donor cells eng graft into dystrophic muscle. Importantly, transplanted cells are able to differentiate in vivo forming new skeletal myo fibers. This example shows alphas Sarco glycan expression from genetically corrected ims into alphas Sarco glycan null skid beige mice.
Massen tri chrome staining revealed a decrease in the amount of fibrotic tissue in treated muscle. Following with this procedure at the methods like the FreeWheel test, Evan Blue Diop assay and single fiber or whole muscle mechanics can be performed in order to answer additional questions about the improvement in voluntary motor capacity, fiber fragility, and specific force. After watching this video, you should have a good understanding of how to evaluate the muscle differentiation ability and graftman potential and functional efficacy of IPS cell derived myogenic progenitors using in vitro, in vivo and ex vivo assays.
