Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.
Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMDMDX mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMDMDX mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMDMDX mutant mice.
Duchenne muscular dystrophy (DMD) is an X-linked recessive, progressive neuromuscular disease caused by a mutation in DMD gene and the loss of functional dystrophin1. Dystrophin is expressed primarily in skeletal muscle and myocardium, and is less expressed in smooth muscle, endocrine glands and neurons2,3. DMD is the most common type of muscular dystrophy with an incidence of one per 3,500 to 5,000 newborn boys worldwide4,5. Individuals typically develop progressive muscle necrosis, loss of independent walking by early adolescence, and death in the second to third decades of their lives due to heart failure and respiratory failure6.
Dilated cardiomyopathy, arrhythmias and congestive heart failure are common cardiovascular manifestations of DMD7,8. The disease can’t be cured, supportive treatment may improve symptoms or delay the progression of heart failure, but it is very difficult to improve the heart function9,10.
Similar to DMD patients, X-linked muscular dystrophy (MDX) mice are deficient in dystrophin protein and present symptoms of cardiomyopathy11, and are therefore widely used in DMD associated cardiomyopathy research. In order to restore dystrophin in affected muscles, allogeneic stem cell therapy has proven to be an effective treatment for DMD12,13,14. Exosomes, 30-150 nm membrane vesicles secreted by various cell types, play a key role in cell-to-cell communication through genetic material transport, such as messenger RNA (mRNA) and non-coding RNAs15,16,17,18,19,20,21.
Our previous studies have shown that exosomes derived from myogenic progenitor cells (MPC), such as C2C12 cell line, can transfer dystrophin mRNA to host cardiomyocytes after direct cardiac injection22, indicating that allogeneic delivery of MPC-derived exosomes (MPC-Exo) can transiently restore DMD gene expression in MDX mice. This article focuses on MPC-Exo purification and transplantation techniques.
Animals were handled according to approved protocols and animal welfare regulations of the Institutional Animal Care and Use Committee of the Medical College of Georgia at Augusta University.
1. Isolation and Purification of MPC-derived Exosomes
2. Intramyocardial Exosome Delivery
NOTE: We used six mice in each group.
3. Echocardiography
NOTE: A single observer blinded to the experimental groups performs echocardiography and data analysis.
4. Immunohistochemistry
A flow chart for isolating and purifying exosomes from C2C12 cells is shown in Figure 1A. To confirm the presence of exosomes, we performed transmission electron microscopy analysis. Transmission electron microscopy image (Figure 1B) shows the morphology of the bright and round shape vesicles of C2C12 derived exosomes. Western blot analysis confirmed the presence of exosome markers, including CD63 and TSG101 (Figure 1C).
We observed a translucent edema area after intramyocardial PBS/MPC-Exo injection into the anterior wall of the left ventricle of MDX mice (Figure 2), indicating the successful injection into the myocardium.
To determine whether cardiac MPC-Exo delivery restores dystrophin protein expression in MDX hearts, we performed immunofluorescent staining for dystrophin, and imaging with a confocal microscope. We observed partial restoration of dystrophin expression with membrane localization in some of cardiomyocytes (Figure 3A). To determine whether transplantation of MPC-Exo improves the cardiac function in MDX mice, we measured cardiac function by echocardiography 2 days after intramyocardial PBS / MPC-Exo delivery. As shown in Figure 3B, MPC-Exo treatment improved anterior wall movement compared with PBS, suggesting that MPC-Exo transplantation improved cardiac function in MDX mice.
Figure 1: Exosome purification and characterization by electron microscopy. (A) The diagram shows each step of isolation and purification of MPC-Exo. (B) The image shows round and cup-shaped vesicles under electron microscopy. Scale bar = 100 nm. (C) Western blot analysis confirmed the presence of exosome markers CD63 and TSG101. Please click here to view a larger version of this figure.
Figure 2: Intramyocardial injection of PBS or MPC-Exo. A translucent edema was observed in the left ventricular anterior wall after intramyocardial PBS/exosome delivery. (A) PBS injection. (B) MPC-Exo injection. Please click here to view a larger version of this figure.
Figure 3: Intramyocardial delivery of MPC-Exo partially recovers dystrophin expression in MDX hearts, and improves heart function measured by echocardiography. (A) Confocal immunofluorescent staining the heart sections of MDX mice 2 days after intramyocardial PBS/ MPC-Exo delivery. Blue = DAPI, Green = anti-dystrophin antibody. (B) Echocardiographic measurements of cardiac function after 2 days of PBS/MPC-Exo treatment. Please click here to view a larger version of this figure.
The method of isolating pure exosomes is essential for studying the function of exosomes. One of the common techniques for exosome isolation is polyethylene glycols (PEGs) mediated precipitation17,18,25. Exosomes can be precipitated in PEGs, and pelleted by low-speed centrifugation. PEG-mediated purification is very convenient, low-cost, it does not need any advanced equipment, but there is concern about the purity of exosomes since other lipoproteins may be precipitated together and are difficult to remove. Ultrafiltration is a routine method for exosome separation, based upon the molecular weight and exclusion size limits26. Ultrafiltration isolation is faster than ultracentrifugation based separation, but it can cause structural damage to large vesicles. The presence of various epitopes on the membrane of exosomes, such as CD9, CD63 and CD8127, provides an alternative method of isolating exosomes by immunoaffinity interactions between these epitopes and antibodies bound to magnetic beads. Although immunoisolation has a high specificity28, this technique has disadvantages of only isolating a subset of the total exosome population and may distort the results of the experiment. Therefore, ultracentrifugation seems to be more suitable for this study on exosome function.
In this paper, we present a method for exosomes purification by sequential ultracentrifugation. After removing the remaining cells from the supernatant by centrifugation at 150 x g, the cell debris, apoptotic bodies and vesicles larger than 220 nm were removed by passing the supernatant through a 0.22 μm filter. Exosomes were then pelleted at 100,000 x g by initial ultracentrifugation. To remove possible protein contamination, we performed a second 100,000 x g ultracentrifugation after resuspending the exosome pellet in PBS. As per our experience, the advantage of sequential ultracentrifugation is the production of exosomes with high purity and cost-efficiency, however, it has disadvantage of low yield.
In order to allow the injected exosomes to cover most of the left ventricle and avoid leakage, we performed one intramyocardial injection using a 31 G insulin needle with the tip bent at about 20°. This technique is critical for the successful delivery of most exosomes into the myocardium and maximizes exposure of injected exosomes to host cardiomyocytes. In addition, we recommend injecting exosomes into the central region of the anterior wall of the left ventricle. After the injection is completed, we usually hold the needle at the injection site for 1 min to prevent liquid leakage. Successful injections were confirmed by the presence of a translucent edema around the injection site.
In our study, we found that allogeneic MPC-Exo transplantation can transiently restore dystrophin expression in heart and improve cardiac function in MDX mice22, which may provide new strategies for symptom relief in DMD patients. Since we observed recovered dystrophin protein expression in MDX mouse hearts, we assume that this is the mechanism for improved heart function, however, we cannot exclude other mechanisms, such as anti-inflammation; a recent report demonstrated that mesenchymal stem cell-derived exosomes improve the microenvironment of infarcted myocardium contributing to angiogenesis and anti-inflammation29, moreover, Aminzadeh et al.30 recently reported that cardiosphere-derived cells (CDCs) and their exosomes could transiently restore the expression of full length dystrophin in DMD mice. Considering that DMD is a systemic disease involving multiple organs, the local myocardial delivery of exosome is not suitable for treating respiratory failure due to diaphragm myopathy. Thus, systemic administration of exosomes, such as intravenous injection, has therapeutic potential, however, the major challenge is to develop an effective strategy for targeting exosomes to multiple muscle tissues. More importantly, exosome treatment has only a transient effect on partially restoring dystrophin expression, and improving heart function. More effective, long-term DMD treatment is needed.
The authors have nothing to disclose.
Tang were partially supported by the American Heart Association: GRNT31430008, NIH-AR070029, NIH-HL086555, NIH-HL134354.
0.22-μm Filter | Fisherbrand | 09-720-004 | |
15-cm Cell Culture Dish | Thermo Fisher Scientific | 157150 | |
24-gauge catheter | TERUMO | SR-OX2419CA | |
31-gauge insulin needle | BD | 328291 | |
4% paraformaldehyde | Affymetrix | AAJ19943K2 | |
50 mL Centrifuge Tubes | Thermo Fisher Scientific | 339652 | |
6-0 suture | Pro Advantage by NDC | P420697 | |
Alexa Fluor 488 goat anti-rabbit IgG | Thermo Fisher Scientific | A-11008 | |
Antibiotic Antimycotic Solution | Corning | 30-004-CI | |
Anti-Dystrophin antibody | Abcam | ab15277 | |
Antigen retriever | Aptum Biologics | R2100-US | Antigen recovery |
Autofluorescence Quenching Kit | Vector Laboratories | SP-8400 | |
C2C12 cell line | ATCC | CRL-1772 | |
Centrifuge | Unico | C8606 | |
Change-A-Tip High Temp Cauteries | Bovie Medical Corporation | HIT | |
Confocal microscopy | Zeiss | Zeiss 780 Upright Confocal | |
DBA/2J-mdx mice | The Jackson Laboratory | 013141 | |
DMEM | Corning | 10-013-CM | |
Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
Goat serum | MP Biomedicals, LLC | 191356 | |
Isoflurane | Patterson Veterinary | 07-893-1389 | |
Ketamine | Henry Schein | 056344 | |
Mounting Medium with DAPI | Vector Laboratories | H-1500 | |
Mouse Retractor Set | Kent Scientific | SURGI-5001 | |
Polyethylene glycol tert-octylphenyl ether | Fisher Scientific | BP151-100 | |
Rodent ventilator | Harvard Apparatus | 55-7066 | |
SW-28 Ti rotor | Beckman | 342207 | |
The Vevo 2100 Imaging Platform | FUJIFILM VisualSonics | Vevo 2100 | Ultrasound System |
Ultracentrifuge | Beckman | 365672 | |
Ultra-Clear Tubes | Beckman | 344058 | |
Xylazine (XylaMed) | Bimeda-MTC Animal Health Inc. | 1XYL003 8XYL006 |