Name: direct reprogramming of human fibroblasts into myoblasts to investigate therapies for neuromuscular disorders
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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.

<ol>
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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 is also to have viral production that has maximum purity and accurate quantification. For example, viral genome quantification using qPCR provides reasonable measurements, but quantification by ddPCR (digital droplet PCR) is more accurate.
In addition, the adequate confluence of fibroblasts for myoblast conversion is critical. If the cells are below 70% or above 80% confluent, the myogenic differentiation may be impaired. If cells are too confluent, there will be the superposition of layers of myotubes, which interfere with staining and imaging. The concentration of doxycycline is crucial for correct activation and sustained expression of the Myod gene. It is very critical to always add the doxycycline to the medium right before doing media changes, as it degrades quickly after diluted in medium and stored at 4 &#176;C. The stock should be stored at -20 &#176;C at a concentration of 1000X and protected from light. Do not re-freeze thawed aliquots. It is very important to follow these details to ensure reproducible experiments and discriminate an impaired differentiation due to a genetic mutation from technical issues. Nevertheless, depending on the mutation or the type of disease, a good differentiation may not be possible. To ensure trustful results, it is very important to replicate experiments at similar passage numbers.
In our experience, the differentiation capacity persists at least up to passage 25-27, especially in wild-type controls. The same may be valid for some diseased cell lines, but it depends on the cell line. Some DMD cell lines still retain the myogenic potential above P20. In opposition, a myotonic dystrophy type 1 (DM1) cell line presented reduced myogenicity after P8. However, in the case of DM1, this is not surprising as it has been demonstrated that mutations in DM1 indirectly play a role in muscle differentiation16. The retention of the myogenic differentiation capacity should be addressed individually, but generally, most of the cell lines retain it up to P20-25.
In summary, the conversion of fibroblasts into myoblasts is a powerful tool to study and test therapeutic strategies for neuromuscular disorders. It facilitates access to human cell models by avoiding the complicated obtention of muscle biopsies and reduce the inconvenience of a muscle biopsy for the patients.

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><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&#039; GCTGCTGAAGTTTGTTGG&lt;br/&gt; TTTCTC 3&#039;</td><td>Rv_5&#039; CTTTTGGCAGTTTTTGCC&lt;br/&gt; CTGT 3&#039;</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 &amp;micro;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 &amp;amp; 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. The fibroblasts should be passed to a new dish when confluence is reached (Figure 2B). Figure 3A shows the ideal confluence of fibroblasts before changing to myoblast growth medium supplemented with doxycycline. The cells should be around 70% confluent because they still proliferate during the conversion process. If cells are above 80% confluent, the differentiation may be compromised. The conversion into myoblasts takes two to four days, and it is confirmed by observation of the morphology. The cells become elongated and parallelly oriented, as shown in Figure 3B. After the addition of the differentiation medium, the myoblasts stop dividing and start to fuse to form multinucleated myotubes (Figure 3C). When the myotubes borders look white and bright, they are about to detach (Figure 4). At this point, collect or fix the cells.
The differentiation success will vary between different cell lines/mutations. Immunostaining of muscle proteins expressed by mature myotubes confirms the myogenic potential of converted fibroblasts (Figure 5). RNA-Seq analysis comparing FM myotubes and skeletal muscle showed high-level expression of transcripts from the embryonic (MYH3) and neonatal (MYH8) myosin chain genes and good overall transcriptome-wide correlation with muscle (Figure 6). Transcripts for the giant sarcomeric proteins titin (TTN), nebulin (NEB), and obscurin (OBSCN) are also expressed by FM myotubes, indicating upregulation of these large transcripts involved in myofibrillogenesis. Thus, FM cells have a muscle-specific expression profile, demonstrating that they are a useful and reliable surrogate for muscle-derived cell lines.
To illustrate exon skipping, we used this protocol in one of the most frequent exon duplications in the DMD gene. Duplication of exon 2 leads to disruption of the DMD reading frame, thus the restoration of the reading frame following exon skipping should lead to the expression of the full-length dystrophin. However, it is also possible that skipping of exon 2 is very efficient resulting in an out-of-frame transcript. Nevertheless, in this case, skipping of both copies of exon 2 induces the utilization of an alternative internal ribosome entry site (IRES) present in exon 5, thereby producing functional N-truncated dystrophin that was identified in patients still ambulant in their 70s12. Figure 7A shows representative results of RT-PCR of FM cells with exon 2 duplication. FM cells were treated either with AON or AAV1-U7 carrying an antisense sequence to skip exon 2. In Figure 7B, an immunoblot shows the detection of the N-truncated dystrophin in FM cells treated with AAV1-U7. In vitro treatment of FM cells serves as proof of concept for exon-skipping strategies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61991/61991fig01.jpg" /> 
Figure 1: Schematic representation of fibroblasts conversion into myogenic cells. A skin biopsy is obtained from human subjects. Skin fragments are placed on culture dishes. Within one week, fibroblasts start to emerge. Fibroblasts are first transduced with the hTERT gene, and then with the Myod gene, using lentiviral vectors. After antibiotic selection of infected cells, the conversion into myoblasts is induced by the addition of doxycycline to the myoblast growth medium. Within two to four days, the cells become elongated and parallelly oriented. After switching to differentiation medium, the myoblast fuse with each other and form multinucleated myotubes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig01large.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/61991/61991fig02.jpg" /> 
Figure 2: Skin biopsy fragments in culture. (A) First fibroblasts emerging from skin fragment. (B) Confluent fibroblasts emerged from the skin fragment. Scale bar: 50 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig02large.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/61991/61991fig03.jpg" /> 
Figure 3: Fibroblasts transdifferentiation. (A) Representative image of 70% confluent fibroblasts. (B) Converted myoblasts have elongated morphology and are parallelly organized. (C) Myotubes were differentiated for 7 days. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig03large.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/61991/61991fig04.jpg" /> 
Figure 4: Representative image of detaching myotubes. The arrows indicate the white and bright edges of myotubes starting to detach. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig04large.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/61991/61991fig05.jpg" /> 
Figure 5: Immunofluorescence of differentiated myotubes. Immunostaining of myosin heavy chain in myotubes derived from a healthy (A) individual and patients with neuromuscular disorders (B and C). In B are shown cells from myotonic dystrophy type 1 (DM1) carrying 230 CTG repeats, and in C are DM1 cells with 900 CTG repeats. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61991/61991fig06.jpg" /> 
Figure 6: Transcriptome pattern of FM myotubes compared to skeletal muscle. Transcriptome pattern of FM myotubes compared to skeletal muscle. The read counts per million mapped reads for 12,134 transcripts are shown for Illumina RNA-Seq libraries prepared from FM myotubes and a human skeletal muscle biopsy. Transcript levels between the two libraries had a Pearson correlation of 0.71 and a Spearman rank correlation of 0.73. Transcripts for the developmental myosin heavy chains and the large sarcomeric proteins are highlighted in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61991/61991fig07.jpg" /> 
Figure 7: Representative RT-PCR and Western blot showing DMD exon skipping in FM cells. (A) Expression DMD by RT-PCR. Fibroblasts from a patient harboring a duplication of DMD exon 2 were converted into FM cells. RNA extracted from muscle biopsy was used as the control, showing that FM untreated cells express the same duplicated transcript. FM cells treated with AON have a partial skipping of exon 2 duplication, while AAV1-U7 treated cells showed a predominance of transcripts with exon 2 duplication skipped. (B) Representative immunoblot of FM cells treated with AAV1-U7. Smaller N-truncated dystrophin was detected 14 days after treatment (indicated by the arrow). Data previously published in Wein et al. Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nature Medicine. 2014. 2020 Springer Nature Limited. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61991/61991fig07large.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>Fibroblast growth medium</td>
			<td>DMEM with 20% FBS, 1% antibiotic-antimicotic</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td>10% DMSO, 90% fibroblast medium</td>
		</tr>
		<tr>
			<td>Doxycycline stock solution 1000X</td>
			<td>8 mg of doxycycline in 1 mL ultra-pure water. Filter in 0.22 &#181;m syringe filter. Aliquot in PCR tubes. Store at -20 &#176;C, protected from light.</td>
		</tr>
		<tr>
			<td>Myoblast medium</td>
			<td>Skeletal muscle cell growth medium (see list above) with supplements, 8 &#181;g/mL doxycycline. For example: 100 &#181;L of 1000X stock solution in 100 mL.</td>
		</tr>
		<tr>
			<td>Differentiation medium</td>
			<td>Skeletal muscle cell differentiation medium with supplements (see list above), 8 &#181;g/mL doxycycline. For example: 100 &#181;L of 1000X stock solution in 100 mL.</td>
		</tr>
		<tr>
			<td>Blocking solution for IF staining</td>
			<td>10% goat serum (or serum of animal in which secondary antibody was raised) in 1X PBS</td>
		</tr>
		<tr>
			<td>Base buffer for protein extraction</td>
			<td>NaCl 150 mM, Tris 50 mM, 0.05 % NP-40. Adjust pH to 7.4. Store at 4 &#176;C.</td>
		</tr>
	</tbody>
</table>
Table 1: Medium recipes

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

Nanyang Technological University

Research

JoVE Journal

Genetics

6.1K Views.  The Research Institute at Nationwide Children’s Hospital. Hallo allemaal. Mijn naam is Nicolas Wein. Ik ben hoofdonderzoeker bij het Nationwide Children's Hospital in Columbus, Ohio.En vandaag laten we je een video zien over hoe je een huidbiopsie nemen en deze omzetten in myoblasten en myotube. Dit is een zeer nuttig hulpmiddel om neuromusculaire stoornis te bestuderen. Bovendien is het een veel snellere technologie in vergelijking met IPSC omdat we twee verschillende soorten virussen gebruiken om dit type cel direct te bestrijden.Dez...

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

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

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

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

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

Bioengineering

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Developmental Biology

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 ...

Medicine

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Biology

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

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

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