In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.
Muscle stem cells remain associated with their endogenous niche and can be manipulated easily, for instance, through siRNA transfection. This method makes it possible to analyze muscle stem cells in culture in a setting which resembles the in-vivo situation more closely than standard 2D culture models by preserving the niche. Begin by cutting sterile pasteur pipettes with a diamond pen.
For each mouse, use one large bore pipette with an opening of about 0.3 centimeters and a length of about 10 to 12 centimeters and a second glass pipette with a small opening of about 0.1 centimeter and a length of approximately 22 centimeters. Smoothing the edges of both pipettes by holding the pipette tips in the flame of a Bunsen burner for five to 10 seconds with gentle movement. Immediately before use, coat both pipettes with sterile horse serum by filling the whole pipette with 2 milliliters of horse serum for five minutes, then injecting the horse serum and allowing the pipettes to dry for five minutes at room temperature.
Spray all equipment and the hind limbs of the mouse with 70%ethanol. Use hardened fine curved scissors and fine forceps to remove the skin and expose the underlying muscles. Remove the surrounding fascia with the fine curved forceps without damaging the underlying muscles.
To remove the tibialis anterior or TA, grab the distal TA tendon with forceps and cut it with fine scissors. While holding the TA at the tendon, pull it towards the knee and cut the muscle close to the knee to expose the EDL muscle. Lift the distal EDL tendon with curved forceps and cut with fine Vannas spring scissors.
Expose the proximal EDL tendon by carefully pulling the EDL towards the knee. Then cut the proximal tendon with scissors. Incubate the EDL muscles in the reaction tube at 37 degrees Celsius in a circulating water bath.
Stop the digestion when muscles loosen up and single mile fibers are visible. Working under a sterile binocular microscope equipped with a heating plate flushed the muscles with warm isolation medium using the large bore pipette. Dissociate the muscles with the large bore pipette until the desired number of myofibers are floating freely in the solution.
To wash the debris, use the small bore glass pipette to transfer non-contracted myofibers to the second well filled with isolation medium. Then transfer 50 to 100 non-contracted myofibers to one well of a 24 well plate filled with myofiber culture medium. Incubate the myofibers at 37 degrees Celsius and 5%carbon dioxide for 72 to 96 hours.
Transfect myofiber associated muscle stem cells four hours after myofiber isolation. Combine 25 microliters of Opti-MEM with the respective volume of siRNA with 25 microliters Opti-MEM containing 1.5 microliters of transfection reagent. Incubate the reaction mix for five minutes and add it to the cells in the 24 well plate.
Then incubate the plate at 37 degrees Celsius for the respect of time. Only critical steps of the immunofluorescent staining protocol are demonstrated here. Carefully, discard the myofiber culture medium while leaving some solution in the well.
Add 500 microliters of 2%paraformaldehyde to fix the myofibers with their adjacent muscle stem cells. Incubate the plate for five minutes at room temperature. Remove the supernatant carefully and wash the myofibers three times with PBS.
Cover the plate with tinfoil during incubation steps after the incubation with secondary antibodies. Use a hydrophobic pen to draw a circle on a microscopic glass slide. Then transfer the myofibers in the smallest volume possible to the slide and disperse them.
Remove the residual liquid with the small bore pasteur pipette or a 200 microliter pipette. Use two drops of aqueous mounting medium and cover the myofibers with a cover slip. Then allow the slides to dry and store them at four degrees celsius in the dark followed by microscopic analysis.
This protocol demonstrates derivation and culture of single myofibers from murine EDL muscles. Immunofluorescent staining for Pax7 was used to identify nuclei of muscle stem cells. The magnified area exposes the myofiber with its adjacent muscle stem cell and demonstrates the PAX7 immunofluorescence signal in the nucleus of a muscle stem cell.
Muscle stem cells myogenic progression can be analyzed by marker expression. Presence of Pax7 and absence of MyoD expression is associated with quiescent muscle stem cells of freshly isolated myofibers. MyoD expression can be observed in proliferating muscle stem cells.
After 72 hours, muscle stem cells form clusters of progenies with different myogenic states, which is paralleled by expression of different myogenic markers. Pax7 only cells are self-renewing stem cells. Pax7 and MyoD double positive cells are proliferating.
Whereas myo D only cells are differentiating cells. The siRNA transfection of muscle stem cells showed accumulation of cytoplasmic siRNA in a granular fashion indicating efficient uptake. Quantification of transfected Pax7 positive cells per myofiber revealed that the number of transfected cells increased up to 74%after 30 hours with no adverse effect on muscle stem cell numbers.
When attempting this protocol, it is of utmost importance to carefully dissect the EDL without causing any damage. In addition to siRNA transfection, the analysis of transgenic animals or the incubation with recombinant proteins is possible for further functional analyses of muscle stem cells on their adjacent myofibers.
