Tang were partially supported by the American Heart Association: GRNT31430008, NIH-AR070029, NIH-HL086555, NIH-HL134354.

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
<ol>
	<li>Seed 5 x 106 C2C12 cells in a 15 cm cell culture dish with 20 mL complete Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin G and 100 &#956;g/mL streptomycin. Incub...

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+mdx+mouse+cardiomyopathy:+physiological+and+morphological+findings.">Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings.</a> Neuromuscular Disorders. 14 (8-9), 491-496 (2004).</li><li>Siemionow, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+Dystrophin+Expressing+Chimeric+Cells+of+Myoblast+Origin+as+a+Novel+Stem+Cell+Based+Therapy+for+Duchenne+Muscular+Dystrophy.">Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.</a> Stem Cell Reviews and Reports. 14 (2), 189-199 (2018).</li><li>Sienkiewicz, D., Kulak, W., Okurowska-Zawada, B., Paszko-Patej, G., Kawnik, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duchenne+muscular+dystrophy:+current+cell+therapies.">Duchenne muscular dystrophy: current cell therapies.</a> Therapeutic Advances in Neurological Disorders. 8 (4), 166-177 (2015).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+engraftment+of+myogenic+progenitors+from+adipose-derived+stem+cells+and+muscle+regeneration+in+dystrophic+mice.">Long-term engraftment of myogenic progenitors from adipose-derived stem cells and muscle regeneration in dystrophic mice.</a> Human Molecular Genetics. 24 (21), 6029-6040 (2015).</li><li>Ju, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Cardiac+Mesenchymal+Stem+Cell-Derived+Exosomes+for+Angiogenesis.">Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes for Angiogenesis.</a> Journal of Cardiovascular Translational Research. 11 (5), 429-437 (2018).</li><li>Ju, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Cardiac+Mesenchymal+Stem+Cell-Derived+Exosomes+Promotes+Repair+in+Ischemic+Myocardium.">Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes Promotes Repair in Ischemic Myocardium.</a> Journal of Cardiovascular Translational Research. 11 (5), 420-428 (2018).</li><li>Ruan, X. 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All authors declares that they have no conflicts of interest.

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 s...

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&#8217;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.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:424px;">0.22-&#956;m Filter</td>
			<td style="width:280px;">Fisherbrand</td>
			<td style="width:185px;">09-720-004</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm Cell Culture Dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>157150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-gauge catheter</td>
			<td style="width:280px;">TERUMO</td>
			<td>SR-OX2419CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">31-gauge insulin needle</td>
			<td style="width:280px;">BD</td>
			<td>328291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% paraformaldehyde&#160;</td>
			<td style="width:280px;">Affymetrix</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-0 suture</td>
			<td style="width:280px;">Pro Advantage by NDC</td>
			<td>P420697</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td style="width:280px;">Thermo Fisher Scientific</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic Antimycotic Solution</td>
			<td>Corning&#160;</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Dystrophin antibody</td>
			<td style="width:280px;">Abcam</td>
			<td>ab15277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antigen retriever&#160;</td>
			<td style="width:280px;">Aptum Biologics</td>
			<td>R2100-US</td>
			<td>Antigen recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autofluorescence Quenching Kit&#160;</td>
			<td style="width:280px;">Vector Laboratories</td>
			<td>SP-8400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C2C12 cell line</td>
			<td>ATCC</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifuge</td>
			<td>Unico</td>
			<td>C8606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Change-A-Tip High Temp Cauteries</td>
			<td>Bovie Medical Corporation</td>
			<td>HIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscopy</td>
			<td style="width:280px;">Zeiss</td>
			<td>Zeiss 780 Upright Confocal</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DBA/2J-mdx mice</td>
			<td>The Jackson Laboratory</td>
			<td>013141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Corning&#160;</td>
			<td>10-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td>Corning&#160;</td>
			<td>35-011-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum&#160;</td>
			<td style="width:280px;">MP Biomedicals, LLC</td>
			<td>191356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td style="width:280px;">Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:280px;">Henry Schein</td>
			<td>056344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting Medium with DAPI&#160;</td>
			<td style="width:280px;">Vector Laboratories</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Retractor Set</td>
			<td>Kent Scientific</td>
			<td>SURGI-5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol tert-octylphenyl ether</td>
			<td style="width:280px;">Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rodent ventilator</td>
			<td style="width:280px;">Harvard Apparatus</td>
			<td>55-7066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW-28 Ti rotor</td>
			<td>Beckman</td>
			<td>342207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The Vevo 2100 Imaging Platform</td>
			<td style="width:280px;">FUJIFILM VisualSonics</td>
			<td>Vevo 2100</td>
			<td>Ultrasound System&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge</td>
			<td>Beckman</td>
			<td>365672</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Clear Tubes</td>
			<td>Beckman</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (XylaMed)</td>
			<td style="width:280px;">Bimeda-MTC Animal Health Inc.</td>
			<td>1XYL003 8XYL006</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and transplantation of myogenic progenitor cell derived exosomes to improve cardiac function in duchenne muscular dystrophic mice

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.

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 (<strong class="xfig...

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Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

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, ...

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.

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JoVE Journal

Genetics

6.0K Görüntüleme.  Augusta University. Bu protokol, duchenne'nin kas distrofisi farelerinde normal miyojenik progenitör hücrelerinden elde edilen ekson nakli ile kardiyak fonksiyonu geçici olarak iyileştirme yöntemini sunar. Ekzozomal transplantasyon, dmd farelerde kalp fonksiyonlarını iyileştirebilir ve alıcı kalplerde distrofi ekspresyonunun artması yla ilişkili olarak. Ekson izolasyon, saflaştırma ve intramiyokardiyal transplantasyon için uygun yöntem, Duchenne kas distrofisi olan fareleriçin kardiyak fonksiyon...