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 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.
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.
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.
2. Retroviral construct
3. Infection with MYOD1-retroviral vector in UDCs
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.
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.
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.
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. Please click here to view a larger version of this figure.
Figure 2: Representative images of the UDCs. (A) Phase-contrast image of UDCs a week after primary culture. Scale bar = 200 µm. 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 µm. This figure has been modified from Takizawa et al.7. Please click here to view a larger version of this figure.
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 µM 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's post hoc test was used to compare the skipping efficiencies (n = 3 for each group, ****P < 0.0001). The data are expressed as the mean ± SEM. This figure has been modified from Takizawa et al.7. Please click here to view a larger version of this figure.
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 α-tubulin expression were compared in patient-derived cells with and without antisense oligonucleotide treatment by performing one-way ANOVA followed by Bonferroni's post hoc test (n = 3 for each group, **P < 0.01, ***P < 0.001, HI = healthy individual). This figure has been modified from Takizawa et al.7. Please click here to view a larger version of this figure.
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's post hoc test was used for the comparison (n = 3-4 for each group, ****P < 0.0001). This figure has been modified from Takizawa et al.7. Please click here to view a larger version of this figure.
Reagent | Volume | Final concentration |
2x PCR premix | 12.5 μL | 1x |
Forward primer | 5 pmol | 0.2 μM |
Reverse primer | 5 pmol | 0.2 μM |
Template | 80 ng | |
Sterilized distilled water | up to 25 μL | |
Total volume per reaction | 25 μL |
Table 1: Mixture for MYOD1 amplification by RT-PCR.
98 °C | 10 s | |
55 °C | 10 s | } 35 cycles |
72 °C | 10 s |
Table 2: Conditions for the thermal cycler for MYOD1 amplification.
Reagent | Volume |
10x K buffer | 2 μL |
Retroviral vector (500 ng/μL) | 2 μL |
Restriction enzyme 1 (2−15 U) | 1 μL |
Restriction enzyme 2 (2−15 U) | 1 μL |
Sterilized distilled water | 14 μL |
Total volume | 20 μL |
Table 3: Mixture for a tube to digest retroviral vector.
Reagent | Volume |
Purified MYOD1 fragment | 100 ng |
Digested retroviral vector | 100 ng |
5x Enzyme premix | 4 μL |
Sterilized distilled water | up to 20 μL |
Total volume | 20 μL |
Table 4: Mixture for the in-fusion cloning reaction.
Solution | Volume/Reaction (μL) | Final concentration |
RNase-free water | Variable | – |
One-step RT-PCR buffer | 4 | 1x |
dNTP mix (containing 10 mM of each dNTP) | 0.8 | 400 mM of each dNTP |
Forward primer (10 mM) | 1.2 | 0.6 mM |
Reverse primer (10 mM) | 1.2 | 0.6 mM |
One-step RT-PCR enzyme mix | 0.8 | – |
RNase inhibitor (optional) | Variable | 5−10 units/reaction |
Template RNA | 50−400 ng | |
Total volume | 20 |
Table 5: Necessary compounds for one reaction of the one-step RT-PCR.
1 cycle | Reverse transcription | 30 min | 50 °C |
1 cycle | Initial PCR activation step | 15 min | 95 °C |
1 cycle | Denaturation | 1 min | 94 °C |
Annealing | 1 min | 60 °C | |
Extension | 1 min | 72 °C | |
1 cycle | Final extension | 7 min | 72 °C |
Hold | ∞ | 4 °C |
Table 6: Thermal cycler condition for one-step RT-PCR.
Reagent | Volume |
Protein (15 μg) | 10 μL |
Sample buffer (4x) | 5 μL |
Reducing agent (10x) | 2 μL |
Deionized water | 3 μL |
Total volume | 20 μL |
Table 7: Preparation of samples for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
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.
The authors have nothing to disclose.
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.].
1% P/S Solution Stabilized | Thermo Fisher | 15070-063 | Cell culture |
Amphotericin B | Sigma Aldrich | A2942 | |
Anti-dystrophin | Abcam | ab15277 | Western blot (WB) |
Anti-dystrophin | Leica | NCL-DYS1 | Immunocytochemistry(ICC) |
Anti-mouse IgG, Dylight 488 | Vector Laboratories | DK-2488 | ICC |
Anti-α-tubulin | Sigma | T6199 | Western bot and ICC |
BZ-X800 | KEYENCE | BZ-800 | Fluorescent microscope |
CELLBANKER | ZENOAQ | CB011 | Cell stock in liquid nitrogen |
ChemiDoc MP Imaging System | Bio-Rad | 170-8280J1 | WB |
CloneAmp HiFi PCR premix | Clontech | 639298 | Retroviral production |
cOmplete Protease Inhibitor Cocktail | Roche | 4693116001 | Protein extraction for WB |
E.coli DH5 α Competent Cells | TAKARA | 9057 | |
ECL Prime Western Blotting Detection Reagent | GE healthcare | RPN2232 | WB |
EGF | Peprotech | AF-100-15 | Cell culture |
Endo-Porter | GeneTools | 2922498000 | ASO transfection |
Extra Thick Blot Filter Paper | Bio-Rad | 1703965 | WB |
EzFastBlot HMW | Atto | AE-1460 | WB |
fibroblast growth factor-basic | Sigma-Aldrich | F0291 | Cell culture |
Glutamax | Thermo Fisher Scientific | 35050-061 | Cell culture |
GP2-293 packaging cells | Clontech | 631458 | Retroviral production |
Ham's F-12 Nutrient Mix | Thermo Fisher Scientific | 11765-054 | Cell culture |
High glucose DMEM with GlutaMAX-I | Thermo Fisher Scientific | 10569-010 | Cell culture |
High glucose DMEM without sodium pyruvate | GE Healthcare | SH30022.01 | Cell culture |
HiSpeed Plasmid Purification Kit | QIAGEN | 12643 | Retroviral production |
Histofine Simple Stain MAX PO | NICHIREI BIOSCIENCE INC. | 424151 | WB |
Hoechst 33342 | Thermo Fisher Scientific | H3570 | ICC |
Human PDGF-AB | Peprotech | 100-00AB-10UG | Cell culture |
iBind Flex Solution | Thermo Fisher Scientific | SLF2020 | WB |
iBind Flex Western Device | Thermo Fisher Scientific | SLF2000 | WB (Automated mestern-processing device) |
Immobilon-P Transfer Membrane (PVDF) | MERCK | IPVH304F0 | WB |
In-Fusion HD cloning Kit | Clontech | 639648 | Retroviral production |
ITS Liquid Media Supplement | Sigma-Aldrich | I3146 | Cell culture |
MILTEX HV 0.45 μm filter | MERCK | SLHV033RS | Retroviral production |
MultiNA | SHIMADZU | MCE-202 | Microchip electrophoresis |
MYOD1 (GFP-tagged) | ORIGENE | RG209108 | Retroviral production |
NanoDrop | Thermo Fisher | ND-ONE-W | Spectrophotometer |
Nonessential amino acids | Thermo Fisher | 11140-050 | Cell culture |
NucleoSpin Gel and PCR Clean-Up Kit | Clontech | 740986.20 | PCR clean up |
NuPAGE 3-8% Tris-Acetate Protein Gels | Invitrogen | EA03785BOX | WB |
NuPAGE Antioxidant | Invitrogen | NP0005 | WB |
NuPAGE LDS Sample Buffer | Invitrogen | NP0007 | WB |
NuPAGE Sample Reducing Agent | Invitrogen | NP0009 | WB |
NuPAGE Tris-Acetate SDS Running Buffer | Invitrogen | LA0041 | WB |
One Step TB Green PrimeScript RT-PCR Kit | TAKARA | RR066A | Titer check of retroviral vector |
PBS | Thermo Fisher Scientific | 14190-250 | Cell culture |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23227 | WB |
Polybrene Infection / Transfection Reagent | Sigma-Aldrich | TR-1003 | Retroviral infection |
pRetroX-TetOne-Puro Vector | Clontech | 634307 | Retroviral vecor |
Puromycin | Clontech | 631305 | Cell culture |
QIAGEN OneStep RT-PCR Kit | Qiagen | 210212 | PCR |
REGM Bullet Kit | Lonza | CC-3190 | Material for growth medium of UDCs |
REGM SingleQuots | Lonza | CC-4127 | Material for primary medium of UDCs |
Retrovirus Titer Set | TAKARA | 6166 | Titer check of retroviral vector |
Retro-X Concentrator | Clontech | 631455 | Retroviral production |
RIPA buffer | Thermo Fisher Scientific | 89901 | WB |
RNeasy kit | Qiagen | 74104 | RNA extraction for PCR |
Tetracycline-free foetal bovine serum | Clontech | 631106 | Cell culture |
Triton-X | MP Biomedicals | 9002-93-1 | ICC |
Trypsin-EDTA (0.05%) | Gibco | 25300054 | Cell culture |
XCell SureLock Mini-Cell | Invitrogen | EI0001 | WB |
Xfect transfection reagent | Clontech | 631317 | Transfection of plasmids into packaging cells |