This protocol present the method to transitionally improve cardiac function in Duchenne's muscular dystrophy mice by transplanting exon derived from normal myogenic progenitor cells. The exosomal transplantation can improve heart function in DMD mice in association with increased expression of dystrophy in recipient hearts. Appropriate method for exon isolation, purification, and intramyocardial transplantation contribute to the success of the exon transplantation to improve cardiac function for mice with Duchenne's muscular dystrophy.
MPC-derived exosome therapy might improve skeletal muscle function by exosome-mediated normal dystrophin mRNA transfer. This video demonstrates the critical techniques for exosome purification, intramyocardial exosome delivery, and echocardiography post-transplantation. The demonstration of echocardiography will be done by Yan Shen, technician from our lab.
To begin this procedure, seed five million C2C12 cells into a 15 centimeter culture dish with 20 milliliters of complete DMEM containing 10%FBS and antibiotics. Incubate at 37 degrees Celsius with 5%carbon dioxide. Next, use a swinging bucket rotor to ultracentrifuge FBS at 100, 000 times G and at four degrees Celsius for 18 hours to prepare exosome-depleted medium.
Discard the pellet and collect the supernatant. When the monolayer cells reach 80%confluence in the culture dish, replace the complete DMEM with exosome-depleted medium. Use a transfer pipette to collect the supernatant from the cell culture dish every 48 hours.
Centrifuge the tubes at 150 times G for ten minutes to remove the remaining cells. Filter the supernatant through a 0.22 micrometer filter to eliminate any cell debris and transfer the filtered medium to ultra-clear tubes. Next, use a swing bucket rotor to ultracentrifuge the tubes at 100, 00 times G and at 4 degree Celsius for 120 minutes to precipitate the exosomes.
Resuspend the exosome-containing pellet in PBS and then fill the entire ultra-clear tube with PBS. Ultracentrifuge this suspension at 100, 00 times G and at 4 degrees Celsius for 120 minutes to eliminate contaminating proteins. Discard the supernatant and resuspend the exosome pellet in 100 microliters of PBS.
Store at minus 80 degrees Celsius for future use. First, fix each anesthetized mouse in the supine position on a surgical platform by securing each limb with tape. Place a 3-0 suture horizontally below the upper teeth to hold the upper jaw in place.
Next, apply depilatory cream to the left side of the mouse's chest to remove the fir from the skin. Using a 24 gauge catheter, perform endotracheal intubation via the oral cavity. And use a a rodent ventilator to ventilate the mouse with room air at a rate of 195 breaths per minute.
Disinfect the skin with 70%alcohol and 10%povidone iodine. Then make a 10 to 15 millimeter oblique incision from the left sternal edge to the left armpit. Use scissors to cut the pectoralis major and pectoralis minor.
And make a left thoracotamy through the fourth intercoastal space. Gently insert retractor bands to spread the thoracic cavity to a width of 10 millimeters taking care not to damage the left lung. After this, use two straight tweezers to remove the pericardium.
Pull them apart and place them behind the retractor tips to expose the heart. Use a 31 gauge insulin needle to inject either MPC-Exo or PBS intramyocardially into the anterior wall of the left ventricle at one site. Then, use 6-0 nylon sutures to close the thoracic cavity, the pectoralis muscles, and the skin, in sequence.
Two days after the PBS exosome transplantation, anesthetize the mice as outlined in the text protocol. Use tape to fix a mouse in the supine position and apply preheated acoustic gel on the left chest area. Next, use echocardiography to assess the left ventricular function.
Obtain the parasternal long axis view of the left ventricle in two dimensions and then rotate the ultrasound probe 90 degrees to obtain a left ventricle short axis view at the papillary muscle level. After this, record M-mode echocardiographic images and measure the left ventricular end-diastolic diameter, the left ventricular end-systolic volume, the left ventricular end-diastolic volume, and the left ventricular end-systolic volume. The heart rate of the mice should be controlled at least 400 bpm for the cardiac measurement function.
After exosomes are isolated and purified from C2C12 cells, their presence is confirmed using transmission electron microscopy analysis. The transmission electron microscopy image shows the morphology of the bright and round-shaped vesicles of C2C12-derived exosomes. Western blot analysis confirms the presence of exosome markers including CD63 and Tsg101.
A translucent edema area is observed after the intramyocardial injection into the anterior wall of the left ventricle of mdx mice which indicates that the injection into the myocardium is successful. Immunofluorescent staining for dystrophin is then performed to determine whether cardiac MPC-Exo delivery restores dystrophin-protein expression in mdx hearts. Partial restoration of dystrophin expression is observed with membrane localization in some of cardiomyocytes.
The cardiac function is measured by echocardiography two days after intramyocardial delivery to determine whether transplation of MPC-Exo improves the cardiac function in mdx mice. The MPC-Exo treatment is seen to improve interior wall movement compared with PBS, suggesting that MPC-Exo transplantation improved cardiac function in mdx mice. Using a 31 gauge insulin needle with the tip at about 20 degrees is critical for the success for delivery of most exosomes into the myocardium and maximizes exposure of injected exosomes to host cardiomyocytes.
Following the procedure, dystrophin expression can be detected after MPC-derived exosome injection by immunofluorescent staining indicating that cardiac MPC-derived exosome delivery restores dystrophin-protein expression. This technique will pave the way for cardiomyopathy treatment with exosome-carrying therapeutic genetic materials including DNA, mRNA, lncRNA, or microRNAs.
