This protocol is significant because it enables the study of stem cell function across different skeletal muscles, deepening our understanding of how they contribute to homeostasis, regeneration, and disease progression. Using this protocol, we can isolate two populations of live stem cells from specific muscles and study how they work. To begin muscle isolation, spray down the euthanized mouse with ethanol and place it with the abdomen facing up.
Using scissors, make a 0.5 centimeter long horizontal incision into the abdominal skin. Using both hands, pull the skin to expose the torso as well as the lower extremities up to the hind limbs. Locate the gracilis or the inner thigh muscle and using a pair of curved forceps grab onto it and slightly lift the muscle.
Using scissors, make a 0.5 centimeter incision to cut out the gracilis muscle. Repeat the procedure on the other leg. Using a scalpel, cut the fascia by making a 0.5 centimeter incision along the lateral side of the tibia.
Then grab the fascia using curved forceps and remove it by pulling, exposing the tendons at the distal end of the hind limb. Next, insert a pair of straight forceps with super fine tips in between the distal tendon of the TA and the EDL muscle. Separate the muscles by sliding the forceps toward the proximal end of the muscles.
Bring the forceps back to the distal end and cut the distal tendon. Gently grab the distal tendon of the TA with curved forceps and lift the muscle up and over its proximal attachment. To carefully cut the proximal tendon, cut the other end at the closest possible to its attachment and transfer the TA to a Petri dish.
Then use the straight forceps with super fine tips to go underneath the distal tendon of the EDL and slide the forceps toward the proximal end of the muscle to separate the muscles from each other. Bring the forceps back to the distal end and cut the distal tendon without damaging the muscle. By gently grabbing the distal tendon of EDL with curved forceps, lift the muscle up and over its proximal attachment to carefully cut the proximal tendon as close to its attachment as possible.
Cut the other end and transfer the EDL muscle to a Petri dish. Repeat the procedure for the other hind limb. Use straight forceps with super fine tips to reach in between the achilles tendon and the lower hind limb bones.
Slide the forceps towards the proximal end of the muscle to separate the muscle from the bones. Bring the forceps back to the distal end and cut the distal tendon. Pull the gastrocnemius or GA muscle up and over the fibula.
To locate the proximal soleus tendon. Insert the straight forceps in between the soleus and GA muscles and move the forceps toward the distal end of the muscles to separate the soleus from the GA.Now cut the proximal soleus tendon. Grab it with curved forceps and carefully lift the sous to access its distal tendon.
Cut the distal tendon to isolate the soleus from the GA and place the soleus in the Petri dish containing the wash medium. Then cut the GA and place it in the Petri dish. To isolate the triceps muscle, use straight forceps having super fine tips to reach in between the triceps and the humerus bone and slide the forceps toward the proximal end of the muscle to separate the muscle from the bone.
Next, cut the distal end of the triceps muscle and using curved forceps pull it up and over the elbow to access the proximal tendon. Cut the proximal tendon of the triceps and transfer the muscle into a Petri dish. Repeat the procedure for the other four limb.
To remove the fur and the skin from the jaw, use scissors to make a 0.5 centimeter incision below the eye, cutting in the caudal direction. Then using the thumbs and the index fingers of both hands, pinch onto each side of the incision and remove the skin by pulling it upward and downward. Locate the major tendon of the masseter in the caudal side below the eye and insert the flat scalpel blade in between the bone and the muscle to cut the tendon.
Grab the major masseter tendon with curved forceps and cut it with a scalpel blade or scissors in the rostral direction to separate the masseter muscle from the jawbone. Place the isolated masseter muscle in the Petri dish and repeat the procedure for the second masseter muscle. Use scissors to make a thoracotomy in the middle of the sternum and cut through the sternum.
Expose the diaphragm by cutting 360 degrees through the rib cage. Then to separate the upper body from the abdomen use scissors to cut through the trachea, esophagus, vena cava, and abdominal aorta. Next, using scissors, preform a laparotomy one centimeter below the sternum and make a cut around 360 degrees.
Place the closed scissors between the rib cage and the abdominal organs and press down. Gently pull on the rib cage to separate it from the abdominal organs. To separate the diaphragm from the rib cage, loosely hold the diaphragm between two fingers and cut through the rib cage with scissors.
Cut the diaphragm as close to the ribs as possible around 360 degrees and place the isolated diaphragm in a Petri dish. Mince the isolated muscles one by one by cutting them into pieces of roughly one millimeter. Transfer the minced muscles to a 15 liter conical tube containing five liters of dissociation buffer, and incubate the tube at 37 degrees Celsius for 35 minutes in a shaking water bath at a speed of 60 rotations per minute.
After enzymatic digestion of the minced muscles, transfer the suspension from the 15 milliliter tube to a 50 milliliter tube. Using a 10 milliliter syringe fitted with a 20 gauge needle, re-suspend the sample by pulling it up and down through the needle five times. Draw the cell suspension into the syringe and strain the sample into a new 50 milliliter conical tube fitted with a cell strainer on top.
To retrieve all mono nucleated cells, wash the empty conical tube with 20 milliliters of wash media and pour it through the strainer to combine with the strained sample. Retrieve the remaining volume under the cell strainer with a P1000 pipette. Following tissue dissociation and antibody staining, the skeletal muscle stem cells or MuSCs and fibro adipogenic progenitors or FAPs from individual muscles were purified by fluorescence activated cell sorting.
After initial gating to identify the cells and separate the singlets from doublets, subsequent gates were set using FMO controls to identify staining thresholds. The stain sample was gated and the SCA1 positive CD31 negative and CD45 negative population corresponding to FAPs was sorted into a separate collection tube. The double negative population was further gated to sort the VCA positive population corresponding to the MuSCs.
The diaphragm and triceps muscle single cell suspensions, unlike the others, had a higher relative abundance of MuSCs than FAPs. EDU staining, though robust, indicated a difference in fractions of EDU positive cells for the two stem cell types and across the different muscles. For all tissues, however, the mean fraction of EDU positive MuSCs was higher than that of EDU positive FAPs.
MuSCs isolated from EDL and GA showed significantly lower EDU incorporation compared to those from TA, diaphragm, gracilis or triceps. Similarly, significant variation in EDU incorporation was also observed in FAPs isolated from different muscles. Finally, cell purity was confirmed with immunofluorescent staining, indicating the specificity of the stem cell isolation protocol.
The most critical step is the mechanical digestion of the tissue. When the tissue is cut too much, the viability declines. When the tissue is cut too little, the EEL declines.
The protocol can be combined with assays that measure cell behavior, such as engraftment following transplantation to better understand stem cell functions in vivo. This technique may answer whether differences in stem cell behavior within individual muscles can contribute to disease phenotypes and diseases characterized by unique patterns of affected muscles.
