Name: direct reprogramming of human fibroblasts into myoblasts to investigate therapies for neuromuscular disorders
Uploaded: 2021-04-03T14:00:00
Description: Watch this Scientific Journal Video about Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders at JoVE.com

We would like to thank Dr. Vincent Mouly for sharing his knowledge in the past regarding the model. This work has been supported by the US National Institutes of Health National Institute of Neurological Disorders and Stroke (R01 NS043264 (K.M.F., and R.B.W.)), the US National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) (P50 AR070604-01 (K.M.F., K.M., R.N., and N.W.). N.W. has received fellowship support from the Ohio State University/Nationwide Children&#39;s Hospital Muscle Group and the Philippe Foundation. This work was also supported by internal discretionary funds and part of the exon 2 skipping work has been supported also by CureDuchenne (K.M.F.) and Association Francaise Contre Les Myopathies. IRB number: IRB #: IRB10-00358/ CR00005138 and IBCSC#: IBS00000123.

All experiments and biopsies were carried out following the ethical rules of the institutions involved under the approval of the Nationwide Children&#39;s Hospital Institutional Review Board.
1. Initiation of dermal fibroblasts culture
<ol>
	<li>Establishment of fibroblasts culture
	<ol>
		<li>Aliquot 10 mL of fibroblast medium (Table 1) in 15 mL conical tubes. The skin biopsy should be placed and transported in this medium. The biopsy can be stored at 4 &#176;C until it is ...

<ol>
	<li>Bigot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+down-regulates+the+myogenic+regulatory+factors+in+human+myoblasts.">Replicative aging down-regulates the myogenic regulatory factors in human myoblasts.</a> Biology of the Cell. 100 (3), 189-199 (2008).</li><li>Mamchaoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+pathological+human+myoblasts:+Towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: Towards a universal tool for the study of neuromuscular disorders.</a> Skeletal Muscle. 1 (34), (2011).</li><li>Pantic, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+and+versatile+immortal+muscle+cell+models+from+healthy+and+myotonic+dystrophy+type+1+primary+human+myoblasts.">Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.</a> Experimental Cell Research. 342 (1), 39-51 (2016).</li><li>Thorley, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+characteristics+are+preserved+in+hTERT/cdk4+human+myogenic+cell+lines.">Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines.</a> Skeletal Muscle. 6 (1), 43 (2016).</li><li>Chaouch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+skin+fibroblasts+expressing+conditional+MyoD+as+a+renewable+and+reliable+source+of+converted+human+muscle+cells+to+assess+therapeutic+strategies+for+muscular+dystrophies:+Validation+of+an+exon-skipping+approach+to+restore+dystrophin+in+duchen.">Immortalized skin fibroblasts expressing conditional MyoD as a renewable and reliable source of converted human muscle cells to assess therapeutic strategies for muscular dystrophies: Validation of an exon-skipping approach to restore dystrophin in duchen.</a> Human Gene Therapy. 20 (7), 784-790 (2009).</li><li>Zarei, A., Razban, V., Hosseini, S. E., Tabei, S. M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+cell+and+animal+models+of+human+disease+by+genome+editing+using+CRISPR/Cas9.">Creating cell and animal models of human disease by genome editing using CRISPR/Cas9.</a> The Journal of Gene Medicine. 21 (4), 3082 (2019).</li><li>Echevarr&#237;a, L., Aupy, P., Goyenvalle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon-skipping+advances+for+Duchenne+muscular+dystrophy.">Exon-skipping advances for Duchenne muscular dystrophy.</a> Human molecular genetics. 27 (2), 163-172 (2018).</li><li>Hwang, J., Yokota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+exon-skipping+therapies+using+antisense+oligonucleotides+and+genome+editing+for+the+treatment+of+various+muscular+dystrophies.">Recent advancements in exon-skipping therapies using antisense oligonucleotides and genome editing for the treatment of various muscular dystrophies.</a> Expert reviews in molecular medicine. 21, 5 (2019).</li><li>Wein, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Skipping+of+Single+Exon+Duplications+in+DMD+Patient-Derived+Cell+Lines+Using+an+Antisense+Oligonucleotide+Approach.">Efficient Skipping of Single Exon Duplications in DMD Patient-Derived Cell Lines Using an Antisense Oligonucleotide Approach.</a> Journal of Neuromuscular Diseases. 4 (3), 199-207 (2017).</li><li>De Angelis, F. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+snRNA+molecules+carrying+antisense+sequences+against+the+splice+junctions+of+exon+51+of+the+dystrophin+pre-mRNA+induce+exon+skipping+and+restoration+of+a+dystrophin+synthesis+in+&#916;48-50+DMD+cells.">Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in &#916;48-50 DMD cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (14), 9456-9461 (2002).</li><li>Gorman, L., Suter, D., Emerick, V., Sch&#252;mperli, D., Kole, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+alteration+of+pre-mRNA+splicing+patterns+by+modified+U7+small+nuclear+RNAs.">Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (9), 4929-4934 (1998).</li><li>Wein, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+from+a+DMD+exon+5+IRES+results+in+a+functional+dystrophin+isoform+that+attenuates+dystrophinopathy+in+humans+and+mice.">Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.</a> Nature Medicine. 20 (9), 992-1000 (2014).</li><li>Aur&#233;, K., Mamchaoui, K., Frachon, P., Butler-Browne, G. S., Lomb&#232;s, A., Mouly, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+oxidative+phosphorylation+of+immortalization+with+the+telomerase+gene.">Impact on oxidative phosphorylation of immortalization with the telomerase gene.</a> Neuromuscular Disorders. 17 (5), 368-375 (2007).</li><li>Barde, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+control+of+gene+expression+in+the+hematopoietic+system+using+a+single+Tet-on+inducible+lentiviral+vector.">Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector.</a> Molecular Therapy. 13 (2), 382-390 (2006).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> Journal of Visualized Experiments. (32), e1499 (2009).</li><li>Amack, J. D., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+defects+in+myotonic+dystrophy.">Myogenic defects in myotonic dystrophy.</a> Developmental Biology. 265 (2), 294-301 (2004).</li></ol>

To obtain FM cell lines with good quality, some steps are critical. The sooner the skin biopsy is processed, the greater the chances are to obtain healthy fibroblasts. The passage number of fibroblasts cultures is also important. Primary cells have limited proliferative capacity and after many passages, they enter in replicative senescence. Thus, it is better to have a stock of fibroblasts at a low passage number and transform cells at the earliest passage as possible. 
Another important step ...

Cellular models obtained directly from human tissues are useful to model many human genetic disorders, with the advantage of having the original genomic context and, in many cases, reproducing the same molecular and cellular hallmarks observed in the patients. In the field of neuromuscular disorders, muscle biopsies have been a great source of human myoblasts and have helped in the elucidation of pathological mechanisms. Additionally, they are an important tool for in vivo testing of drugs and gene therapies. On one hand, the derivation of myoblasts from muscle fragments is relatively easy. On the other hand, the culture and maintenance of primary myoblasts are challenging, because of their limited proliferation rate and replicative senescence in vitro1. An alternative for these limitations is to immortalize myoblasts with the insertion of the human telomerase (hTERT) and/or cyclin-dependent kinase 4 (CDK4) genes2,3, with preservation of skeletal muscle features4. Nevertheless, the obtention of primary myoblasts is still dependent on muscle biopsy, a surgical procedure with disadvantages to the patients, which, in many cases, have their muscles in advanced degeneration. Thus, the muscle of these patients is composed of a significant proportion of fibrotic and/or adipose tissue and yields fewer muscle cells, requiring the purification of the cells previously to the immortalization.
In contrast to muscle biopsies, skin biopsies are more accessible and are less harmful to patients. Primary fibroblasts can be derived from skin fragments in vitro. Although fibroblasts are not primarily affected by mutations causing neuromuscular disorders, they can be transdifferentiated into myoblasts. This can be achieved by the insertion of the Myod gene, a myogenic regulatory transcription factor5. In this manuscript, we describe the protocol to obtain transdifferentiated myoblasts, from the establishment of fibroblasts cultures to the obtention of differentiated myotubes (a representative summary of the method is depicted in Figure 1).
Pre-clinical testing of therapeutic strategies is dependent on cellular and animal models carrying mutations similar to the mutations found in human patients. Although the development of animal models has become more feasible with the advance of gene-editing technologies such as CRISPR/Cas96, it is still challenging and costly. Thus, patient-derived cell lines are an accessible option to have models, covering the large spectrum of mutations of disease such as Duchenne muscular dystrophy (DMD). Obtention and creation of cell models are crucial to the development of personalized therapies for such pathologies.
Among personalized therapies that have been investigated, exon skipping strategies is one of the promising ones for different muscular dystrophies7,8. This strategy consists of producing a shorter but functional protein. This is performed by hiding the exon definition to the spliceosome, therefore excluding the mutated exon from the final messenger. This is a very promising technology that has been approved by the FDA for DMD. Thus, we also describe in this protocol, methods to transfect myoblasts with two different exon skipping related technologies: antisense oligonucleotides (AON) and U7snRNA-adeno-associated virus (AAV). AON transfection is a good tool for the initial screening of several sequences designed to promote exon skipping9. However, the activity of AONs is transient. To obtain a sustained expression of antisense sequences, we also explored small nuclear RNAs (snRNAs) combined with AAV, allowing nuclear localization and inclusion in the splicing machinery10. U7 is an snRNA involved in the processing of histone mRNA that can be engineered to bind proteins that will redirect it to the spliceosome and deliver antisense sequences11. The use of modified U7 snRNAs in combination with AAV vectors overcomes limitations of AONs resulting in a continued expression of the AONs and better transduction of tissues of interest12. We use cells derived from DMD patients for this protocol to illustrate the exon-skipping strategy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA, phenol red</td>
			<td>Thermo Fisher</td>
			<td>2500056</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>20X Transfer buffer</td>
			<td>Thermo Fisher</td>
			<td>NP00061</td>
			<td></td>
		</tr>
		<tr>
			<td>20X Tris-acetate SDS running buffer</td>
			<td>Thermo Fisher</td>
			<td>LA0041</td>
			<td></td>
		</tr>
		<tr>
			<td>3-8% Tris-Acetate gel</td>
			<td>Thermo Fisher</td>
			<td>EA0378BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 flask</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimicotic 100X</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-myosin heavy chain, sarcomere antibody</td>
			<td>Developmental Studies Hybridome Bank</td>
			<td>MF20 supernatant</td>
			<td>Dilution 1:50</td>
		</tr>
		<tr>
			<td>Antioxidant</td>
			<td>Thermo Fisher</td>
			<td>NP0005</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay</td>
			<td>Thermo Fisher</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher</td>
			<td>D3571</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Millipore Sigma</td>
			<td>300410250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High glucose, GlutaMAX supplement, Pyruvate</td>
			<td>Thermo Fisher</td>
			<td>10569044</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I set (250U)</td>
			<td>Zymo Research Corporation</td>
			<td>E1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP2653-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dup2 human primers</td>
			<td>Fw_5&#39; GCTGCTGAAGTTTGTTGG 
			TTTCTC 3&#39;</td>
			<td>Rv_5&#39; CTTTTGGCAGTTTTTGCC 
			CTGT 3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dystrophin antibody</td>
			<td>Abcam</td>
			<td>ab15277</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>16000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Halt Protease inhibitor cocktail 100X</td>
			<td>Thermo Fisher</td>
			<td>78430</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Thermo Fisher</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680RD goat anti-Rabbit IgG (H+L)</td>
			<td>Li-Cor</td>
			<td>926-68071</td>
			<td>Dilution 1:5000</td>
		</tr>
		<tr>
			<td>Lab-Tek II CC2 chamber slide system</td>
			<td>Thermo Fisher</td>
			<td>15852</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli</td>
			<td>Bioworld</td>
			<td>105700201</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher</td>
			<td>L3000008</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel GFR membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412P-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher</td>
			<td>51000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane 0.45 &#181;m</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10600002</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat serum control</td>
			<td>Thermo Fisher</td>
			<td>10000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (PBS)</td>
			<td>Li-Cor</td>
			<td>927-40003</td>
			<td>Blocking buffer for Western blot</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR master mix</td>
			<td>Thermo Fisher</td>
			<td>K0172</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitor</td>
			<td>Thermo Fisher</td>
			<td>A32957</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standards</td>
			<td>Bio Rad</td>
			<td>1610374</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Thermo Fisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>Revert 700 Total Protein Stain for Western Blot Normalization</td>
			<td>Li-Cor</td>
			<td>926-11021</td>
			<td></td>
		</tr>
		<tr>
			<td>RevertAid kit</td>
			<td>Thermo Fisher</td>
			<td>K1691</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator-25</td>
			<td>Zymo Research Corporation</td>
			<td>R1018</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Aspen Surgical</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>Skeletal Muscle Cell Differentiation medium</td>
			<td>Promocell</td>
			<td>C23061</td>
			<td></td>
		</tr>
		<tr>
			<td>Skeletal Muscle Cell Growth medium</td>
			<td>Promocell</td>
			<td>C23060</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros Organics</td>
			<td>215682500</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Thermo Fisher</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra low temperature freezer</td>
			<td>Thermo Scientific</td>
			<td>7402</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Thermo Fisher</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield antifade mounting medium</td>
			<td>Vector Labs</td>
			<td>H1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 shows how to establish human skin-derived fibroblast cultures and convert them into myoblasts and then into differentiated myotubes. This type of cell line is extremely useful for the study of neuromuscular disorders and in vitro testing of potential therapies.
A schematic representation of the fibroblast conversion is shown in Figure 1. Figure 2A shows a fragment of skin and the fibroblasts emerging from it. Th...

Watch this Scientific Journal Video about Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders at JoVE.com

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies 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 ...

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Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...