This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) [grant no. 18K07544 to Y.A.], Grants-in-Aid for Research on Nervous and Mental Disorders [grant no. 28-6 to Y.A.], and the Japan Agency for Medical Research and Development [grant nos. 18ek0109239h0002, 18lm0203066h0001, and 18lm0203069h0001 to Y.A.].

The Ethics Committee of the National Center of Neurology and Psychiatry approved this study (approval ID: A2017-018, A2018-029). All individuals gave informed consent before providing urine. All experiments were performed under the relevant guidelines and regulations.
1. Isolation and primary culture of UDCs
NOTE: UDCs were isolated according to a previously published protocol8,9,10 with some modifications.
<ol>
	<li>Collect urine samples during spontaneous micturition in sterilized plastic bottles. 
	NOTE: Sterilization of the external urethral orifice is not needed. Midstream urine is desirable to reduce the risk of viral contamination. If the stock time before the next procedure is &#62;1 h, urine samples should be transferred to 4 &#176;C to preserve the cell viability. However, a temperature of &#60;4 &#176;C should be avoided because insoluble precipitates may appear.</li>
	<li>Centrifuge the entire urine sample at 400 x g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant, leaving 1 mL in the tube.</li>
	<li>Resuspend the pellets individually in the remaining 1 mL of urine and then collect those in a single 50 mL tube.</li>
	<li>Add 10 mL of washing buffer consisting of 99 mL of PBS without calcium and magnesium, 1% penicillin/streptomycin (P/S), 0.5 &#181;g/mL amphotericin B, and centrifuge the samples at 200 x g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant, leaving 0.2 mL in the tube.</li>
	<li>Resuspend the cell pellets in 4.5 mL of primary medium composed of a 1:1 mixture of high glucose Dulbecco&#39;s modified Eagle medium (DMEM)without sodium pyruvate and Ham&#39;s F-12 nutrient mix supplemented with recombinant human epidermal growth factor (EGF), insulin, hydrocortisone, epinephrine, T3, transferrin, 10% tetracycline-free fetal bovine serum (FBS), 1% P/S, and 0.5 &#181;g/mL amphotericin B.</li>
	<li>Seed the cells in three wells of gelatine-coated six well plates (total volume of each well, 1.5 mL). Culture humidified at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Add 1.5 mL of the primary medium daily for the next 3 days.</li>
	<li>On day 4, replace the medium with 1.5 mL of growth medium supplemented with recombinant human EGF, insulin, hydrocortisone, epinephrine, T3, transferrin, 15% tetracycline-free FBS, 0.5% L-alanine-L-glutamine, 0.5% nonessential amino acids, and 2.5 ng/mL fibroblast growth factor-basic (bFGF), recombinant human platelet-derived growth factor (PDGF), EGF, and 1% P/S.</li>
	<li>Change the growth medium every other day. 
	NOTE: UDC colonies appear within a week.</li>
	<li>When the UDC culture becomes 80&#8722;90% confluent, remove the medium and wash cells with PBS, split all the cells using 0.25% trypsin-EDTA and seed at 3,000-5,000 cells/cm2 onto a new gelatine-coated 60 mm dish (passage 1). 
	NOTE: The UDCs can be stored in liquid nitrogen. UDCs are usually divided at 60&#8722;70% confluency in 60 mm culture dish into three stock tubes.</li>
</ol>
2. Retroviral construct
<ol>
	<li>Amplify the coding region of MYOD1 (NM_002478.4) plasmid by polymerase chain reaction (PCR). 
	NOTE: The mixture for MYOD1 amplification and conditions for the thermal cycler are shown in Table 1 and Table 2, respectively.</li>
	<li>Detect a single band of about 1,000 bp size by 0.7% agarose gel electrophoresis using 1 &#181;L of the amplified PCR product to confirm that the MYOD1 sequence is amplified successfully.</li>
	<li>Clean the PCR product using the clean-up kit and determine its concentration with a spectrophotometer.</li>
	<li>Incubate the mixture as shown in Table 3 at 37 &#176;C overnight to digest retroviral vector with a Tet-on system and puromycin resistant gene at restriction enzyme-targeted regions in the multiple cloning site.</li>
	<li>Detect a single band by 0.7% agarose gel electrophoresis using 1 &#181;L of the digested product to confirm that the retroviral vector was digested successfully.</li>
	<li>Clean the digested product using clean-up kit and determine its concentration by spectrophotometer.</li>
	<li>To clone the amplified MYOD1 fragment (produced by steps 2.1&#8722;2.3) into the digested retroviral vector (produced by steps 2.4&#8722;2.6), perform an in-fusion cloning reaction. Set up the reaction as shown in Table 4, incubate the reaction for 15 min at 50 &#176;C, and then place on ice.</li>
	<li>Perform transformation using E. coli competent cells according to the manufacturer&#39;s instructions (Table of Materials).</li>
	<li>Select the transformed competent cells by culturing on an LB culture plate consisting of 10 g/L bacto tryptone, 5 g/L bacto yeast extract, 5 g/L NaCl, 15 g/L bacto agar, and 50 mg/L ampicillin.</li>
	<li>Pick up the selected colony and culture in LB culture medium without bacto agar at 200 rpm at 37 &#176;C overnight.</li>
	<li>Purify the MYOD1-inserted retroviral vectors using a plasmid purification kit (Table of Materials) and quantify using a spectrophotometer.</li>
	<li>Confirm that MYOD1 is correctly inserted into the retroviral vector by direct sequencing of the PCR product amplified by the forward and reverse primers targeted respectively on both sides across the inserted MYOD1 sequence. 
	NOTE: The MYOD1 sequence can be detected sandwiched between retroviral vector sequences when in-fusion cloning is successful.</li>
	<li>For retroviral production, seed the packaging cells at 50,000 cells/cm2 on 10 cm collagen-coated plates and culture in DMEM with 10% FBS humidified at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>When the packaging cells proliferate to 80% confluency, mix 30 &#181;g of MYOD1-inserted retroviral vectors, 30 &#181;g of packaging vectors, and transfection reagent containing cell-penetrating peptide (Table of Materials) by vortexing, and incubate them for 10 min.</li>
	<li>Add the incubated mixture to the medium of packaging cells and culture humidified at 37 &#176;C and 5% CO2. 
	NOTE: Collagen coating is necessary. Transfection may be better at 24 h or more after seeding because packaging cells easily detach from the culture plate.</li>
	<li>After 4 h or overnight, change the medium to fresh growth medium.</li>
	<li>Collect the viral supernatant and replace with fresh medium at 24 and 48 h after cotransfection and combine the supernatant.</li>
	<li>To concentrate the viral supernatant, mix it with the concentrator reagent and incubate at 37 &#176;C overnight, then centrifuge at 1,500 x g for 45 min at 4 &#176;C.</li>
	<li>Filter the retrovirus supernatant through a PVDF filter with 0.45 &#181;m pores.</li>
	<li>Check the titer of the retroviral vector using a quantitative PCR kit and thermal cycler system according to the manufacturer&#39;s instructions.</li>
	<li>Divide the viral supernatant into small aliquots and stock them at -80 &#176;C.</li>
</ol>
3. Infection with MYOD1-retroviral vector in UDCs
<ol>
	<li>Seed the UDCs at 3,000-5,000 cells/cm2 on a gelatine-coated 60 mm dish.</li>
	<li>After 24 h of seeding, infect the thawed retrovirus (step 2.21) at a multiplicity of infection of 200 by adding hexadimethrine bromide at a concentration of 8 &#181;g/mL.</li>
	<li>After a 24 h incubation humidified at 37 &#176;C and 5% CO2, replace the culture medium with fresh growth medium containing 1 &#181;g/mL puromycin to select the MYOD1-transduced cells. Change the medium every other day. 
	NOTE: When selecting for MYOD1-positive cells, 1 &#181;g/mL puromycin is usually used for selection. The appropriate dose should be determined. Use a plate containing untransfected cells and choose the dose that kills all the cells in 3&#8722;5 days. MYOD1-positive cells should be selected within 7&#8722;10 days after adding puromycin. The MYOD1-transduced UDCs can be stored in liquid nitrogen.</li>
</ol>
4. Myogenic differentiation of MYOD1-transduced UDCs treated with 3-deazaneplanocin A hydrochloride (DZNep)
NOTE: Recently, it has been reported that DZNep, a histone methyltransferase inhibitor, could significantly promote the expression of MYOGENIN, one of the late muscle regulatory factors, and also lead to myotube differentiation7.
<ol>
	<li>Plate MYOD1-transduced UDCs in the collagen-coated wells at a density of 3.5 x 104 cells/cm2. Culture humidified at 37 &#176;C and 5% CO2.</li>
	<li>After 24 h, change the growth medium to differentiation medium composed of high glucose DMEM with L-alanine-L-glutamine, 5% horse serum, ITS supplement, 1 &#181;g/mL doxycycline, and 5 &#181;M DZNep. 
	NOTE: The 10 mM DZNep solution can be stored at -80 &#176;C for 3 months. Use a defrost freezer and avoid repeated freeze-thaw cycles. Both MYOD1 activated by doxycycline and DZNep suppress proliferation and promote the myogenic differentiation of UDCs. Therefore, it is recommended that doxycycline and DZNep are added after the induction of myogenic differentiation. DZNep promotes myogenic differentiation of the MYOD1-UDCs in a dose-dependent manner. On the other hand, it shows cytotoxicity at a high concentration. Therefore, determine the appropriate concentration of DZNep, ranging from 1&#8722;10 &#181;M depending on its effects on myogenic differentiation and cellular bioavailability.</li>
	<li>After 3 days, change the differentiation medium to fresh differentiation medium without DZNep. Then, change the medium every 3 days. 
	NOTE: UDCs fuse to each other and form myotubes within 1&#8722;2 weeks after differentiation.</li>
</ol>
5. Exon skipping in MYOD1-converted UDCs
NOTE: Here, three protocols are described to evaluate exon skipping in patient-derived cells: 1) reverse transcriptase polymerase chain reaction (RT-PCR) of dystrophin mRNA; 2) semiquantification of restored dystrophin protein signal by Western blot; and 3) semiquantification of restored dystrophin fluorescence signal by immunocytochemistry. All the methods can detect exon skipping in a dose-dependent manner.
<ol>
	<li>Evaluation of exon skipping efficiency by RT-PCR
	<ol>
		<li>To transfect antisense oligonucleotide (ASO) into MYOD1-converted UDCs obtained from DMD patients on the day 7 after differentiation, mix ASO, transfection reagent (Table of Materials), and differentiation medium to a final concentration of 1&#8722;10 &#181;M. Culture humidified at 37 &#176;C and 5% CO2.</li>
		<li>After 72 h of incubation with ASO, change the medium to fresh differentiation medium without ASO.</li>
		<li>From 3&#8722;7 days after ASO transfection, remove differentiation medium and wash 1x with PBS. Add cell lysis buffer, lyse the UDCs and harvest the total RNA using an RNA extraction kit.</li>
		<li>Measure the RNA concentration with a spectrophotometer.</li>
		<li>Combine the required reagents for one-step RT-PCR reaction in PCR tubes according to Table 5.</li>
		<li>Place the PCR tubes with the mixture in a thermocycler. Run the thermocycler, according to Table 6.</li>
		<li>Perform microchip electrophoresis and calculate the exon skipping efficiency using the molar concentration as below. 
		Exon skipping efficiency (%) = skipped band / (skipped band + non-skipped band) x 100 
		NOTE: Store the PCR product in a refrigerator at 4 &#176;C for short-term storage or -20 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>Detection of dystrophin after exon skipping by Western blotting
	<ol>
		<li>Transfect ASO and culture MYOD1-UDCs according to steps 5.1.1 and 5.1.2.</li>
		<li>Change the medium every 3 days.</li>
		<li>After 2 weeks of differentiation, extract the total protein from the cultured cells using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors.</li>
		<li>Sonicate the lysates on ice and centrifuge at 14,000 x g for 15 min at 4 &#176;C.</li>
		<li>Collect the supernatant and determine protein concentrations using a BCA protein assay kit.</li>
		<li>Add 15 &#181;g of total protein in a 0.5 mL tube and dilute by adding the RIPA buffer containing protease inhibitors to a total volume of 10 &#181;L. Run 15 &#181;g of total protein per lane.</li>
		<li>Add sample buffer, reducing agent, and deionized water as shown in Table 7. Denature the cell lysates at 70 &#176;C for 10 min.</li>
		<li>Prepare the tris-acetate running buffer containing 8.95 g/L tricine, 6.06 g/L tris base, 1.0 g/L sodium dodecyl sulfate (SDS).</li>
		<li>Load the sample (20 &#181;L) onto tris-acetate 3&#8722;8% gel and perform electrophoresis at 150 V for 75 min.</li>
		<li>Prepare the blotting buffer without methanol.</li>
		<li>Soak the PVDF membrane for 20 s in methanol and then in the blotting buffer until use (at least 10 min). Cut the PVDF membrane to a size of 6 x 8 cm using mini gels and 8 x 12 cm using midi gels.</li>
		<li>Cut the blotting papers to the same size as that of the PVDF membrane and soak those in the blotting buffer until use.</li>
		<li>After electrophoresis, cut the gel to the same size as that of the PVDF membrane and soak the gel in distilled water.</li>
		<li>Place the blotting papers, PVDF membrane, and gel on the semidry transfer apparatus (Figure 1). Transfer at 4 mA/cm2 for 30 min.</li>
		<li>Rinse the membrane 2x with distilled water.</li>
		<li>Prepare anti-dystrophin (1:500) and anti-&#945;-tubulin (1:1,200) antibody as the primary antibodies.</li>
		<li>Prepare HRP-conjugated anti-mouse antibody (1:100) as a secondary antibody.</li>
		<li>Incubate the membranes with the primary antibodies, wash with wash buffer, then incubate with the secondary antibody using an automated Western-processing device (Table of Materials) at room temperature. 
		NOTE: Anti-&#945;-tubulin antibody is usually used as a loading control. The mixed primary antibody solutions, including 1:500 anti-dystrophin and 1:1,200 anti-&#945;-tubulin antibodies work well when the antibody reaction is performed simultaneously.</li>
		<li>Rinse the membrane in distilled water.</li>
		<li>Detect the proteins using chemiluminescent detection reagent and a charge-coupled device (CCD) camera-based imager.</li>
		<li>Analyse the data using appropriate software.</li>
	</ol>
	</li>
	<li>Detection of dystrophin after exon skipping by immunocytochemistry
	<ol>
		<li>Directly reprogram the UDCs into the myotubes in collagen-coated 96 well plate according to steps 4.1&#8722;4.3.</li>
		<li>Transfect the ASO and culture MYOD1-UDCs according to steps 5.1.2 and 5.1.3.</li>
		<li>After 2 weeks of differentiation, wash the cells with PBS and fix them in 4% paraformaldehyde for 10 min at 4 &#176;C.</li>
		<li>Permeabilize MYOD1-UDCs in 0.1% nonionic detergent for 10 min at room temperature and block those with 10% goat serum for 15 min at 37 &#176;C.</li>
		<li>Incubate the cells with a primary antibody overnight at 4 &#176;C.</li>
		<li>Wash the cells with PBS and incubate those with the secondary antibody for 30 min at room temperature. 
		NOTE: Here, mouse anti-dystrophin (1:30) is used as the primary antibody, anti-mouse IgG is used as the secondary antibody, and Hoechst (1:10,000) is used for nuclei staining.</li>
		<li>Image the plates using a fluorescent microscope and use an analyzer to semiquantify the fluorescence signal automatically in every well under the same condition.</li>
	</ol>
	</li>
</ol>

<ol><li>Hoffman, E. P., Brown, R. H., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dystrophin%3A+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51, 919-928 (1987).</li>
<li>Cirak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exon+skipping+and+dystrophin+restoration+in+patients+with+Duchenne+muscular+dystrophy+after+systemic+phosphorodiamidate+morpholino+oligomer+treatment%3A+an+open-label%2C+phase+2%2C+dose-escalation+study%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.</a> Lancet. 378, 595-605 (2011).</li>
<li>Komaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+administration+of+the+antisense+oligonucleotide+NS-065%2FNCNP-01+for+skipping+of+exon+53+in+patients+with+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy.</a> Science Translational Medicine. 10 (437), (2018).</li>
<li>Antoury, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+extracellular+mRNA+in+human+urine+reveals+splice+variant+biomarkers+of+muscular+dystrophies%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies.</a> Nature Communications. 9, 3906(2018).</li>
<li>Rahmoune, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+transporters+in+human+renal+proximal+tubular+cells+isolated+from+the+urine+of+patients+with+non-insulin-dependent+diabetes%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes.</a> Diabetes. 54, 3427-3434 (2005).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Urine+derived+cells+are+a+potential+source+for+urological+tissue+reconstruction%5BTitle%5D)+AND+%22The+Journal+of+Urology%22%5BJournal%5D)">Urine derived cells are a potential source for urological tissue reconstruction.</a> The Journal of Urology. 180, 2226-2233 (2008).</li>
<li>Takizawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modelling+Duchenne+muscular+dystrophy+in+MYOD1-converted+urine-derived+cells+treated+with+3-deazaneplanocin+A+hydrochloride%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride.</a> Scientific Reports. 9, 3807(2019).</li>
<li>Zhou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human+induced+pluripotent+stem+cells+from+urine+samples%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Generation of human induced pluripotent stem cells from urine samples.</a> Nature Protocols. 7, 2080-2089 (2012).</li>
<li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+myogenic+differentiation+of+human+urine-derived+cells+as+a+potential+source+for+skeletal+muscle+regeneration%5BTitle%5D)+AND+%22Journal+of+Tissue+Engineering+and+Regenerative+Medicine%22%5BJournal%5D)">Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.</a> Journal of Tissue Engineering and Regenerative Medicine. 11, 334-341 (2017).</li>
<li>Kim, E. Y., Page, P., Dellefave-Castillo, L. M., McNally, E. M., Wyatt, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+urine-derived+cells+with+inducible+MyoD+for+modeling+human+muscle+disease%5BTitle%5D)+AND+%22Skeletal+Muscle%22%5BJournal%5D)">Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease.</a> Skeletal Muscle. 6, 32(2016).</li>
</ol>

The National Center of Neurology and Psychiatry is now developing NS-065/NCNP-01, an exon 53 skipping drug for DMD, with Nippon Shinyaku Co., Ltd.

Here, we describe a detailed protocol of exon skipping in MYOD1-converted UDCs obtained from DMD patients. Using the assay system, we screened optimal antisense sequences efficiently. We assume that MYOD1-converted UDCs can be useful for the investigation of the pathophysiology of the disease.
Evaluation of exon skipping using patient-derived cells at the mRNA level is indispensable for screening new drugs and assessing patient eligibility before undertaking clinical trials. Calculation of exon skipping efficiency can be evaluated only at an mRNA level.
Evaluation of exon skipping at the protein level is also important because dystrophin restoration is important as a surrogate biomarker to predict the benefits of exon skipping. To date, screening of antisense oligonucleotide sequences is often performed using primary muscle cell lines or immortalized myoblast cell lines including human rhabdomyosarcoma (RD) cells, but we cannot measure the recovery of dystrophin levels using muscle cell lines or RD cell lines because they express this protein endogenously. We can clearly detect the restoration of dystrophin in DMD patient-derived MYOD1-UDCs in a dose-dependent manner. In our new assay, we consider that the evaluation of restored protein by Western blotting is superior in quantifiability. On the other hand, evaluation by immunocytochemistry using 96 well plates is ideal for screening many candidate compounds simultaneously.
In this article, we describe a detailed protocol for an efficient modelling DMD muscle using MYOD1-converted UDCs along with RT-PCR, Western blotting, and immunocytochemistry to evaluate the restored dystrophin at the mRNA and protein levels after exon skipping. UDCs can be collected noninvasively and easily. Therefore, we assume that the brand-new in vitro assay can be applied to a wide range of basic and translational studies regardless of the type of muscular disorders.

Duchenne muscular dystrophy (DMD), a progressive, fatal muscle disease, is caused by frame-shift mutations in the DMD gene that result in the absence of dystrophin protein1. Antisense oligonucleotide-based exon skipping therapy is thought to be promising for DMD. This therapy is based on the conversion of the severer DMD phenotype to the milder Becker muscular dystrophy-like phenotype by altering pre-mRNA splicing to restore the DMD reading frame2. We have recently completed a first-in-human study based on repeated intravenous administration of the phosphorodiamidate morpholino oligomer (PMO) viltolarsen, which can induce exon 53 skipping in DMD, and demonstrated an excellent safety profile, promising efficacy, and acceptable pharmacokinetic parameters (registered as UMIN: 000010964 and ClinicalTrials.gov: NCT02081625)3.
However, to develop cost-effective and efficient treatments for the disease, in vitro tests using primary muscle cells obtained from DMD patients are essential for drug screening and patient eligibility verification before undertaking clinical trials, as well as biomarkers that reflect the efficacy of exon skipping therapies during human trials4. Very recently, we reported a novel technology to develop patient-specific MYOD1-converted urine-derived cells (UDCs)5,6 as a primary myoblast model of DMD7. Thus, to generate the myoblasts, only the collection of urine from patients is required and no invasive procedure is needed. In this article, we describe a detailed protocol for efficient modelling of DMD muscle using MYOD1-converted UDCs treated with 3-deazaneplanocin A hydrochloride to evaluate the restored dystrophin mRNA and protein after exon skipping.

<table><tbody><tr><td>1% P/S Solution Stabilized</td><td>Thermo Fisher</td><td>15070-063</td><td>Cell culture</td></tr><tr><td>Amphotericin B</td><td>Sigma Aldrich</td><td>A2942</td><td></td></tr><tr><td>Anti-dystrophin</td><td>Abcam</td><td>ab15277</td><td>Western blot (WB)</td></tr><tr><td>Anti-dystrophin</td><td>Leica</td><td>NCL-DYS1</td><td>Immunocytochemistry(ICC)</td></tr><tr><td>Anti-mouse IgG, Dylight 488</td><td>Vector Laboratories</td><td>DK-2488</td><td>ICC</td></tr><tr><td>Anti-&amp;alpha;-tubulin</td><td>Sigma</td><td>T6199</td><td>Western bot and ICC</td></tr><tr><td>BZ-X800</td><td>KEYENCE</td><td>BZ-800</td><td>Fluorescent microscope</td></tr><tr><td>CELLBANKER</td><td>ZENOAQ</td><td>CB011</td><td>Cell stock in liquid nitrogen</td></tr><tr><td>ChemiDoc MP Imaging System</td><td>Bio-Rad</td><td>170-8280J1</td><td>WB</td></tr><tr><td>CloneAmp HiFi PCR premix</td><td>Clontech</td><td>639298</td><td>Retroviral production</td></tr><tr><td>cOmplete Protease Inhibitor Cocktail</td><td>Roche</td><td>4693116001</td><td>Protein extraction for WB</td></tr><tr><td>E.coli DH5 &amp;alpha; Competent Cells</td><td>TAKARA</td><td>9057</td><td></td></tr><tr><td>ECL Prime Western Blotting Detection Reagent</td><td>GE healthcare</td><td>RPN2232</td><td>WB</td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-15</td><td>Cell culture</td></tr><tr><td>Endo-Porter</td><td>GeneTools</td><td>2922498000</td><td>ASO transfection</td></tr><tr><td>Extra Thick Blot Filter Paper</td><td>Bio-Rad</td><td>1703965</td><td>WB</td></tr><tr><td>EzFastBlot HMW</td><td>Atto</td><td>AE-1460</td><td>WB</td></tr><tr><td>fibroblast growth factor-basic</td><td>Sigma-Aldrich</td><td>F0291</td><td>Cell culture</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td>Cell culture</td></tr><tr><td>GP2-293 packaging cells</td><td>Clontech</td><td>631458</td><td>Retroviral production</td></tr><tr><td>Ham&#039;s F-12 Nutrient Mix</td><td>Thermo Fisher Scientific</td><td>11765-054</td><td>Cell culture</td></tr><tr><td>High glucose DMEM with GlutaMAX-I</td><td>Thermo Fisher Scientific</td><td>10569-010</td><td>Cell culture</td></tr><tr><td>High glucose DMEM without sodium pyruvate</td><td>GE Healthcare</td><td>SH30022.01</td><td>Cell culture</td></tr><tr><td>HiSpeed Plasmid Purification Kit</td><td>QIAGEN</td><td>12643</td><td>Retroviral production</td></tr><tr><td>Histofine Simple Stain MAX PO</td><td>NICHIREI BIOSCIENCE INC.</td><td>424151</td><td>WB</td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific</td><td>H3570</td><td>ICC</td></tr><tr><td>Human PDGF-AB</td><td>Peprotech</td><td>100-00AB-10UG</td><td>Cell culture</td></tr><tr><td>iBind Flex Solution</td><td>Thermo Fisher Scientific</td><td>SLF2020</td><td>WB</td></tr><tr><td>iBind Flex Western Device</td><td>Thermo Fisher Scientific</td><td>SLF2000</td><td>WB (Automated mestern-processing device)</td></tr><tr><td>Immobilon-P Transfer Membrane (PVDF)</td><td>MERCK</td><td>IPVH304F0</td><td>WB</td></tr><tr><td>In-Fusion HD cloning Kit</td><td>Clontech</td><td>639648</td><td>Retroviral production</td></tr><tr><td>ITS Liquid Media Supplement</td><td>Sigma-Aldrich</td><td>I3146</td><td>Cell culture</td></tr><tr><td>MILTEX HV 0.45 &amp;mu;m filter</td><td>MERCK</td><td>SLHV033RS</td><td>Retroviral production</td></tr><tr><td>MultiNA</td><td>SHIMADZU</td><td>MCE-202</td><td>Microchip electrophoresis</td></tr><tr><td>MYOD1 (GFP-tagged)</td><td>ORIGENE</td><td>RG209108</td><td>Retroviral production</td></tr><tr><td>NanoDrop</td><td>Thermo Fisher</td><td>ND-ONE-W</td><td>Spectrophotometer</td></tr><tr><td>Nonessential amino acids</td><td>Thermo Fisher</td><td>11140-050</td><td>Cell culture</td></tr><tr><td>NucleoSpin Gel and PCR Clean-Up Kit</td><td>Clontech</td><td>740986.20</td><td>PCR clean up</td></tr><tr><td>NuPAGE 3-8% Tris-Acetate Protein Gels</td><td>Invitrogen</td><td>EA03785BOX</td><td>WB</td></tr><tr><td>NuPAGE Antioxidant</td><td>Invitrogen</td><td>NP0005</td><td>WB</td></tr><tr><td>NuPAGE LDS Sample Buffer</td><td>Invitrogen</td><td>NP0007</td><td>WB</td></tr><tr><td>NuPAGE Sample Reducing Agent</td><td>Invitrogen</td><td>NP0009</td><td>WB</td></tr><tr><td>NuPAGE Tris-Acetate SDS Running Buffer</td><td>Invitrogen</td><td>LA0041</td><td>WB</td></tr><tr><td>One Step TB Green PrimeScript RT-PCR Kit</td><td>TAKARA</td><td>RR066A</td><td>Titer check of retroviral vector</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>14190-250</td><td>Cell culture</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23227</td><td>WB</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>Sigma-Aldrich</td><td>TR-1003</td><td>Retroviral infection</td></tr><tr><td>pRetroX-TetOne-Puro Vector</td><td>Clontech</td><td>634307</td><td>Retroviral vecor</td></tr><tr><td>Puromycin</td><td>Clontech</td><td>631305</td><td>Cell culture</td></tr><tr><td>QIAGEN OneStep RT-PCR Kit</td><td>Qiagen</td><td>210212</td><td>PCR</td></tr><tr><td>REGM Bullet Kit</td><td>Lonza</td><td>CC-3190</td><td>Material for growth medium of UDCs</td></tr><tr><td>REGM SingleQuots</td><td>Lonza</td><td>CC-4127</td><td>Material for primary medium of UDCs</td></tr><tr><td>Retrovirus Titer Set</td><td>TAKARA</td><td>6166</td><td>Titer check of retroviral vector</td></tr><tr><td>Retro-X Concentrator</td><td>Clontech</td><td>631455</td><td>Retroviral production</td></tr><tr><td>RIPA buffer</td><td>Thermo Fisher Scientific</td><td>89901</td><td>WB</td></tr><tr><td>RNeasy kit</td><td>Qiagen</td><td>74104</td><td>RNA extraction for PCR</td></tr><tr><td>Tetracycline-free foetal bovine serum</td><td>Clontech</td><td>631106</td><td>Cell culture</td></tr><tr><td>Triton-X</td><td>MP Biomedicals</td><td>9002-93-1</td><td>ICC</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Gibco</td><td>25300054</td><td>Cell culture</td></tr><tr><td>XCell SureLock Mini-Cell</td><td>Invitrogen</td><td>EI0001</td><td>WB</td></tr><tr><td>Xfect transfection reagent</td><td>Clontech</td><td>631317</td><td>Transfection of plasmids into packaging cells</td></tr></tbody></table>

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.

We could collect the UDCs easily and non-invasively. UDCs formed colonies within a week after starting primary cell culture we observed a marked proliferative ability. The culture of UDCs was straightforward, and bacterial or fungal contamination was rare when the procedure was performed correctly.
Figure 2 shows representative phase-contrast images of the UDC colony a week after primary culture (Figure 2A) and MYOD1-UDCs a week after differentiation (Figure 2B). Figure 3 shows the successful detection of exon skipping in UDCs obtained from DMD patients by RT-PCR. Figure 3A shows RT-PCR analysis of dystrophin after antisense oligonucleotide treatment in DZNep-treated MYOD1-UDCs derived from a 6-year-old male with an exon 45-54 deletion in the DMD gene. The open reading frame was restored by exon 44 skipping. On day 14 following differentiation, we confirmed the induction of exon skipping in a dose-dependent manner (Figure 3B). The upper bands denote native products, and the lower bands denote exon 44-skipped products that restored the open reading frame.
Figure 4 shows the successful detection of dystrophin after exon skipping in the UDCs obtained from DMD patients by Western blotting in a dose-dependent manner. We also detected the restored dystrophin expression using immunocytochemistry (Figure 5). We measured the intensities of dystrophin with a fluorescent microscope 1 week after the antisense oligonucleotide (ASO) transfection on a 96 well plate (Figure 5A). Markedly higher fluorescent signals were observed in MYOD1-UDCs treated with ASO than in MYOD1-UDCs treated with control ASO (Figure 5B).
These results suggest that our new assay can evaluate exon skipping efficiently in MYOD1-UDCs obtained from DMD patients at the mRNA and protein level.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60840/60840fig1.jpg" /> 
Figure 1: Schematic representation of the transfer stack for semidry Western blot. Two papers soaked in the blotting buffer were laid down at the negative terminal, and two papers soaked in the buffer were stacked on top of this. The gel, which has been soaked in the buffer, was laid gently over the PVDF membrane. <a href="https://www.jove.com/files/ftp_upload/60840/60840fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60840/60840fig2.jpg" /> 
Figure 2: Representative images of the UDCs. (A) Phase-contrast image of UDCs a week after primary culture. Scale bar = 200 &#181;m. Inset: A magnified image of the area in the white rectangle. (B) Phase-contrast image of MYOD1-UDCs a week after differentiation. Scale bar = 50 &#181;m. This figure has been modified from Takizawa et al.7. <a href="https://www.jove.com/files/ftp_upload/60840/60840fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60840/60840fig3.jpg" /> 
Figure 3: Successful evaluation of exon skipping in urine-derived cells (UDCs) obtained from DMD patients by RT-PCR. (A) RT-PCR analysis of dystrophin after antisense oligonucleotide treatment in 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1-UDCs derived from Duchenne muscular dystrophy (DMD) patient with an exon 45-54 deletion. DZNep-treated MYOD1-UDCs were also treated with the control antisense at 1-10 &#181;M concentration as controls. The upper bands were unskipped products (Ex 45-54 deletion) that remained out of the reading frame. The lower bands were the exon 44-skipped products (Ex 44-54 deletion and Ex 44 skipped) that restored the open reading frame. (B) Skipping efficiency was calculated as (exon 44-skipped transcript molarity)/(native + exon 44-skipped transcript molarity [marked with arrows]) x 100% using a microchip electrophoresis system. One-way ANOVA followed by Bonferroni&#39;s post hoc test was used to compare the skipping efficiencies (n = 3 for each group, ****P &#60; 0.0001). The data are expressed as the mean &#177; SEM. This figure has been modified from Takizawa et al.7. <a href="https://www.jove.com/files/ftp_upload/60840/60840fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60840/60840fig4.jpg" /> 
Figure 4: Successful evaluation of exon skipping in urine-derived cells (UDCs) from DMD patients by Western blot. (A) Representative Western blot for dystrophin in DZNep-treated MYOD1-UDCs from DMD patient with an exon 45-54 deletion after exon 44 skipping. For dystrophin detection, anti-dystrophin (against C-terminal) was used. (B) The relative intensities of the bands normalized to &#945;-tubulin expression were compared in patient-derived cells with and without antisense oligonucleotide treatment by performing one-way ANOVA followed by Bonferroni&#39;s post hoc test (n = 3 for each group, **P &#60; 0.01, ***P &#60; 0.001, HI = healthy individual). This figure has been modified from Takizawa et al.7. <a href="https://www.jove.com/files/ftp_upload/60840/60840fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60840/60840fig5.jpg" /> 
Figure 5: Heatmaps of immunocytochemistry for dystrophin after antisense oligonucleotide treatment in DZNep-treated MYOD1-UDCs obtained from DMD patient with exon 45-54 deletion. (A) Deletion of exon 45-54 restored the open reading frame based on the exon skipping of exon 44. (B) The signal intensity was quantified using a fluorescent microscope after 1 week of antisense oligonucleotide transfection on a 96 well plate. One-way ANOVA followed by Bonferroni&#39;s post hoc test was used for the comparison (n = 3-4 for each group, ****P &#60; 0.0001). This figure has been modified from Takizawa et al.7. <a href="https://www.jove.com/files/ftp_upload/60840/60840fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>2x PCR premix</td>
			<td>12.5 &#956;L</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Forward primer</td>
			<td>5 pmol</td>
			<td>0.2 &#956;M</td>
		</tr>
		<tr>
			<td>Reverse primer</td>
			<td>5 pmol</td>
			<td>0.2 &#956;M</td>
		</tr>
		<tr>
			<td>Template</td>
			<td>80 ng</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilized distilled water</td>
			<td>up to 25 &#956;L</td>
			<td></td>
		</tr>
		<tr>
			<td>Total volume per reaction</td>
			<td>25 &#956;L</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Mixture for MYOD1 amplification by RT-PCR.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>98 &#176;C</td>
			<td>10 s</td>
			<td></td>
		</tr>
		<tr>
			<td>55 &#176;C</td>
			<td>10 s</td>
			<td>} 35 cycles</td>
		</tr>
		<tr>
			<td>72 &#176;C</td>
			<td>10 s</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Conditions for the thermal cycler for MYOD1 amplification.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>10x K buffer</td>
			<td>2 &#956;L</td>
		</tr>
		<tr>
			<td>Retroviral vector (500 ng/&#956;L)</td>
			<td>2 &#956;L</td>
		</tr>
		<tr>
			<td>Restriction enzyme 1 (2&#8722;15 U)</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td>Restriction enzyme 2 (2&#8722;15 U)</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td>Sterilized distilled water</td>
			<td>14 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>20 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 3: Mixture for a tube to digest retroviral vector.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Purified MYOD1 fragment</td>
			<td>100 ng</td>
		</tr>
		<tr>
			<td>Digested retroviral vector</td>
			<td>100 ng</td>
		</tr>
		<tr>
			<td>5x Enzyme premix</td>
			<td>4 &#956;L</td>
		</tr>
		<tr>
			<td>Sterilized distilled water</td>
			<td>up to 20 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>20 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 4: Mixture for the in-fusion cloning reaction.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Volume/Reaction (&#956;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Variable</td>
			<td>-</td>
		</tr>
		<tr>
			<td>One-step RT-PCR buffer</td>
			<td>4</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>dNTP mix (containing 10 mM of each dNTP)</td>
			<td>0.8</td>
			<td>400 mM of each dNTP</td>
		</tr>
		<tr>
			<td>Forward primer (10 mM)</td>
			<td>1.2</td>
			<td>0.6 mM</td>
		</tr>
		<tr>
			<td>Reverse primer (10 mM)</td>
			<td>1.2</td>
			<td>0.6 mM</td>
		</tr>
		<tr>
			<td>One-step RT-PCR enzyme mix</td>
			<td>0.8</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RNase inhibitor (optional)</td>
			<td>Variable</td>
			<td>5&#8722;10 units/reaction</td>
		</tr>
		<tr>
			<td>Template RNA</td>
			<td>50&#8722;400 ng</td>
			<td></td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>20</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 5: Necessary compounds for one reaction of the one-step RT-PCR.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 cycle</td>
			<td>Reverse transcription</td>
			<td>30 min</td>
			<td>50 &#176;C</td>
		</tr>
		<tr>
			<td>1 cycle</td>
			<td>Initial PCR activation step</td>
			<td>15 min</td>
			<td>95 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="3">1 cycle</td>
			<td>Denaturation</td>
			<td>1 min</td>
			<td>94 &#176;C</td>
		</tr>
		<tr>
			<td>Annealing</td>
			<td>1 min</td>
			<td>60 &#176;C</td>
		</tr>
		<tr>
			<td>Extension</td>
			<td>1 min</td>
			<td>72 &#176;C</td>
		</tr>
		<tr>
			<td>1 cycle</td>
			<td>Final extension</td>
			<td>7 min</td>
			<td>72 &#176;C</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td></td>
			<td>&#8734;</td>
			<td>4 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 6: Thermal cycler condition for one-step RT-PCR.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Protein (15 &#956;g)</td>
			<td>10 &#956;L</td>
		</tr>
		<tr>
			<td>Sample buffer (4x)</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td>Reducing agent (10x)</td>
			<td>2 &#956;L</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>3 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>20 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 7: Preparation of samples for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Watch this Scientific Journal Video about Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells at JoVE.com

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

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 ...

exon-skipping-directly-reprogrammed-myotubes-obtained-from-human

Research

JoVE Journal

Genetics

7.0K Views.  National Center of Neurology and Psychiatry. We hebben een nieuw protocol opgesteld voor de directe herprogrammering van menselijke urine-afgeleide cellen in myobuizen. Deze in vitro modellering stelt ons in staat om exon overslaan in de myotubes afgeleid van patiënt met Duchenne spierdystrofie te evalueren. We ontdekten dat 3-deazaneplanocin A hydrochloride, DZnep, histon methyltransferase remmer aanzienlijk kan bevorderen directe screening van UDC's in myotube in een korte tijd.Deze nieuwe autologe UDC gebaseerde ziekte modell...

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

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

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Developmental Biology

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 ...

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 ...

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype&#160;is&#160;more similar to human DMD patients, was&#160;used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Exon skipping is currently a&#160;most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations often cause a distorted reading frame and premature stop codons, resulting in an almost total lack of dystrophin protein. The reading frame can be corrected using antisense oligonucleotides (AONs) that induce exon skipping. The morpholino AON viltolarsen (code name: NS-065/NCNP-01) has been shown to induce exon 53 skipping, restoring the reading frame for patients with exon 52 deletions. We recently administered NS-065/NCNP-01 intravenously to DMD patients in an exploratory investigator-initiated, first-in-human trial of NS-065/NCNP-01. In this methods article, we present the molecular characterization of dystrophin expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial. The characterization of dystrophin expression was fundamental in the study for showing the efficacy since no functional outcome tests were performed.

Here, we present the molecular characterization of dystrophin 38 expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

Neuroscience

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 &#176;C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...

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

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