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. Incubate at 37 &#176;C and 5% CO2.</li>
	<li>Prepare exosome-depleted medium by ultracentrifugation of FBS at 100,000 x g for 18 h at 4 &#176;C using a swinging bucket rotor. Discard the pellet.</li>
	<li>Replace the complete DMEM with exosome-depleted medium when monolayer cells reach 80% confluence in the culture dish.</li>
	<li>Use a transfer pipette to collect the supernatant from the cell culture dish every 48 h for a total of three times. Collect the supernatant in 50 mL centrifuge tubes, and centrifuge at 150 x g for 10 min to remove the remaining cells.</li>
	<li>Filter the supernatant through a 0.22 &#956;m filter to eliminate cell debris. Ultracentrifuge the filtered medium in ultra-clear tubes using the swinging bucket rotor at 100,000 x g for 120 min at 4 &#176;C to precipitate exosomes.</li>
	<li>Resuspend the exosome-containing pellet in phosphate buffered saline (PBS) and fill the entire ultra-clear tube with PBS. Perform ultracentrifugation of this suspension at 100,000 x g for 120 min at 4 &#176;C to eliminate contaminating proteins.</li>
	<li>Discard the supernatant and resuspend the exosome pellet in 100 &#181;L PBS. Store at -80 &#176;C for future use.</li>
	<li>For electron microscopy analysis, place 3 &#181;L of exosomal suspension on a 200 mesh formvar-coated copper electron microscope grid for 5 min at room temperature. Continue with standard uranyl acetate staining23. Examine the semi-dry grid by transmission electron microscopy.</li>
</ol>
2. Intramyocardial Exosome Delivery
NOTE: We used six mice in each group.
<ol>
	<li>Anesthetize DBA/2J-DMDMDX mice (male; age &#62; 10 months; body weight (BW) &#62; 18 g) with intraperitoneal (i.p.) injection of 100 mg/kg BW of ketamine combined with 10 mg/kg BW of xylazine, and confirm the depth of anesthesia by toe pinch.</li>
	<li>Fix each mouse in supine position on a surgical platform by using tape to secure each limb and place a 3-0 suture horizontally below the upper teeth to hold the upper jaw in place.</li>
	<li>Apply the depilatory cream to the left side of the mouse chest to remove the fur from the skin.</li>
	<li>Perform endotracheal intubation via oral cavity using a 24 G catheter, and ventilate the mouse with room air at a rate of 195 breaths/min using a rodent ventilator (see the Table of Materials).</li>
	<li>Disinfect the skin with 75% alcohol and 10% povidone iodine.</li>
	<li>Make a 10&#8210;15 mm oblique incision from the left sternal edge to the left armpit, cut the pectoralis major and pectoralis minor by a scissor, then make a left thoracotomy through the fourth intercostal space. Gently insert the retractor bands to spread the thoracic cavity to a width of 10 mm. Take care to avoid damage to the left lung.</li>
	<li>Use two straight tweezers to remove the pericardium, pull them apart, and place them behind the retractor tips to expose the heart. Inject MPC-Exo (50 &#956;g in 30 &#181;L PBS) or 30 &#956;L PBS (as a control) intramyocardially into the anterior wall of the left ventricle using a 31 G insulin needle at one site.</li>
	<li>Use 6-0 nylon sutures to close the thoracic cavity, pectoralis muscles and skin in sequence.</li>
	<li>Recover mice. Once rhythmic, spontaneous breathing is present, remove the mice from the ventilator, and extubate. Observe and record the mice condition after surgery.</li>
</ol>
3. Echocardiography
NOTE: A single observer blinded to the experimental groups performs echocardiography and data analysis.
<ol>
	<li>Two days after PBS/exosome transplantation, anesthetize the mice with 1&#8210;2% isoflurane.</li>
	<li>Fix a mouse in supine position using tape, and apply the preheated acoustic gel on the left chest area.</li>
	<li>Assess left ventricular function by echocardiography as previously described16,24.
	<ol>
		<li>Obtain the parasternal long axis view of the left ventricle (LV) in two dimensions (B mode), and then rotate the ultrasound probe 90&#176; to obtain a LV short-axis view at papillary muscle level.</li>
		<li>Record M-mode echocardiographic images and measure left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESD), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV). 
		NOTE: Fractional shortening (FS%) = [(LVEDD &#8722; LVESD) / LVEDD] x 100, and left ventricular ejection fraction (LVEF%) = [(LVEDV &#8722; LVESV) / LVEDV] x&#160;100.</li>
	</ol>
	</li>
</ol>
4. Immunohistochemistry
<ol>
	<li>Two days after PBS/exosome transplantation, anesthetize mice (see step 2.1) and cut the sternum open to expose the thoracic cavity. Dissect the inferior vena cava, and perfuse the heart through the left ventricle apex with 3 mL PBS, followed by 3 mL of 4% paraformaldehyde at room temperature, using a butterfly catheter with a 25 G needle attached to a 5 mL syringe.</li>
	<li>Embed fixed hearts into paraffin, and cut into 5 &#956;m thick sections.</li>
	<li>Deparaffinize slides in xylene and rehydrate in ethanol solutions in following order: 100% xylene (3 min), 100% xylene (3 min), 100% ethanol (1 min), 100% ethanol (1 min), 95% ethanol (1 min), 80% ethanol (1 min), H2O (1 min).</li>
	<li>Fix tissue sections with 4% paraformaldehyde for 8 min at room temperature.</li>
	<li>Immerse slides in sodium citrate (0.01 M, pH 6.0) and perform antigen retrieval using an antigen retriever.</li>
	<li>Permeabilize sections for 1 h at room temperature with 1% polyethylene glycol tert-octylphenyl ether in PBS.</li>
	<li>Block sections with 5% goat serum in PBS for 1 h at room temperature.</li>
	<li>Incubate sections with diluted primary antibody (anti-dystrophin antibody) in PBS (1:100) overnight at 4 &#176;C, then wash three times with PBS for 5 min.</li>
	<li>Incubate sections with diluted secondary antibody (Alexa Fluor 488 conjugated goat anti-rabbit IgG) in PBS (1:400) for 45 min at room temperature.</li>
	<li>Control autofluorescence using an autofluorescence quenching kit (see the Table of Materials).</li>
	<li>Use mounting medium containing DAPI (see the Table of Materials) to mount the slides.</li>
	<li>Observe slides under a confocal microscope.</li>
</ol>

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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.

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

Watch this Scientific Journal Video about Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice at JoVE.com

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, ...

purification-transplantation-myogenic-progenitor-cell-derived

Research

JoVE Journal

Genetics

6.0K Views.  Augusta University. Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

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

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Bioengineering

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Developmental Biology

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Medicine

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

In this article, we describe a detailed protocol for efficient modelling of Duchenne muscular dystrophy muscle using MYOD1-converted urine-derived cells to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...