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.

<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>

Nationwide Children's Hospital has licensed the exon 2 skipping program described herein to Audentes Therapeutics. K.M.F. and N.W. have received royalty payments as a result of this license.

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 ...

direct-reprogramming-human-fibroblasts-into-myoblasts-to-investigate

Research

JoVE Journal

Genetics

6.1K Views.  The Research Institute at Nationwide Children’s Hospital. 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.

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

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

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. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

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

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 ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

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 routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

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

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

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

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

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 is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

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 differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

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 traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

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

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...