This work was supported by the European Research Council (ERG) and EMBO installation (ERG) and by a PhD fellowship from the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (MP).

NOTE: One mouse yields sufficient myoblasts for approximately two 35 mm dishes or two live-imaging dishes, so plan mattings, dissection, and coating (step 2.6) accordingly. Since myoblasts are isolated through sequential centrifugations and preplating, the protocol should be done in batches of 5 - 10 animals.
All procedures involving animal subjects were approved by the Animal Ethics Committee at Instituto de Medicina Molecular and University Pierre et Marie Curie&#160;
1. Dissection of Neonatal Mice Hind-limb Muscles
<ol>
	<li>Prepare all solutions in advance (Materials Table) and sterilize by filtration (0.22 &#181;m filter). Make sure all media are at 37 &#176;C before addition to the cells, except the formulations containing basement membrane matrix (e.g., Matrigel).</li>
	<li>Sterilize the dissection material (one each of: curved scissors, straight scissors, regular forceps, and fine-tip forceps) and the work bench by wiping them with 70% ethanol.</li>
	<li>Prepare a 100 mm Petri dish with 5 mL of Dulbecco&#39;s Phosphate Buffered Saline (DPBS) for muscle collection and keep it on ice until the mincing step.</li>
	<li>Decapitate P6 - P8 mice with straight scissors and sterilize the skin with 70% ethanol.</li>
	<li>Make an incision in the back skin and pull it gently towards the hind limbs until it is removed, completely exposing the hind-limb musculature.</li>
	<li>Use the forceps to remove fat tissue without damaging the muscles.</li>
	<li>To remove the dorsal hind-limb muscles, keep the limb stretched and bend the paw to expose the heel tendons. Use the curved scissors to separate muscle from bone, starting from the tendons, by gently sliding and cutting upwards. Excise the muscles and place them in iced DPBS.</li>
	<li>Isolate the quadriceps by pinching the muscle with fine-tip forceps and cutting around it without damaging the femur or the knee joint.</li>
	<li>After dissecting all animals, proceed to a sterile laminar flow cell culture hood, where all the following steps should be performed.</li>
</ol>
2. Myoblast Isolation
<ol>
	<li>Remove the excess of DPBS. Mince the tissue with sterilized curved scissors in order to obtain a uniform mass.</li>
	<li>Collect the minced tissue in a 50 mL conical centrifuge tube using 5 mL of digestion mix and incubate it with agitation at 37 &#176;C for 90 min.</li>
	<li>Stop the digestion by adding 6 mL of dissection medium and centrifuge the suspension for 5 min at 75 x g to pellet the remaining tissue.</li>
	<li>Carefully collect the supernatant. Make sure to not collect tissue debris. Centrifuge it at 350 x g for 5 min; resuspend it in 5 mL of dissection medium.</li>
	<li>Filter the cell suspension through a 40 &#181;m cell strainer. Add 25 mL of dissection medium and preplate it in a 150 mm dish for 4 h in a cell culture incubator (37 &#176;C and 5% CO2) to allow the fibroblasts to adhere.</li>
	<li>While preplating, coat dishes with 500 &#181;L of basement membrane matrix diluted 1:100 in cold IMDM for 1 h at RT. Wash once with DPBS and plate the cells immediately (step 2.8) or leave with growth medium until plating.</li>
	<li>After preplating, collect the supernatant and centrifuge it at 350 x g for 10 min.</li>
	<li>Resuspend it in growth medium and count the cells on a hemocytometer. Adjust the volume so that between 150,000 and 250,000 cells are plated per basement membrane matrix-coated dish. Keep the cells in a cell culture incubator.</li>
</ol>
3. Myofiber Differentiation
NOTE: After 3 d, the cells should start to fuse and form myotubes at around 70% confluency (Figure 1B).
<ol>
	<li>At this point, transfect the cells, if desired, with a siRNA or DNA of interest. If the cells are not to be transfected, change directly to differentiation medium and skip to step 3.4.</li>
	<li>Transfect with transfection reagents following the manufacturer&#39;s instructions. Incubate the cells for 5 h with siRNA-lipid complexes (20 nM + 1 &#181;L of reagent) or DNA-lipid complexes (1 &#181;g + 1 &#181;L of reagent). Optimize the siRNA and DNA concentrations if necessary.</li>
	<li>Wash them once with differentiation medium and then switch to new differentiation medium.</li>
	<li>The following day, dilute the basement membrane matrix 1:2 in ice-cold differentiation medium. Remove the existing medium and add 200 &#181;L of ice-cold matrix to each dish.</li>
	<li>Incubate for 30 min in a cell culture incubator.</li>
	<li>Supplement the differentiation medium with agrin (100 ng/mL) and carefully add 2 mL to the cells.</li>
	<li>Carefully change half of the medium every 2 d, always supplementing with agrin to a final concentration of 100 ng/&#181;L.</li>
	<li>Monitor cell differentiation and viability. Depending on a variety of factors (such as FBS and chicken embryo extract origins), the cells might take between 5 -&#160;10 differentiation d&#160;to reach full maturation (Figure 2).</li>
</ol>
4. Immunostaining in Glass-bottom Dishes
<ol>
	<li>For immunostaining, at any time-point of interest, wash the cells once with DPBS and fix them with 4% PFA at RT for 10 min.</li>
	<li>Wash them twice with DPBS. At this point, the cells can be stored at 4 &#176;C.</li>
	<li>Permeabilize them with 0.5% Triton X-100 for 5 min at RT.</li>
	<li>Wash them twice with PBS and block with blocking solution for 30 min at RT.</li>
	<li>Incubate them with primary antibody diluted in blocking solution O/N at 4 &#176;C.</li>
	<li>Wash 3x with DPBS for 5 min at RT.</li>
	<li>Incubate them with the secondary antibody and 0.2 &#181;g/mL of DAPI for 1 h at RT.</li>
	<li>Wash 3x with DPBS for 5 min at RT.</li>
	<li>Add 200 &#181;L of mounting medium and proceed to imaging.</li>
</ol>

<ol>
	<li>Janssen, I., Heymsfield, S. B., Wang, Z., Ross, R. <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+mass+and+distribution+in+468+men+and+women+aged+18-88+yr.">Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr.</a> J Appl Physiol. 89 (1), 81-88 (2000).</li><li>Manring, H., Abreu, E., Brotto, L., Weisleder, N., Brotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+excitation-contraction+coupling+related+genes+reveal+aspects+of+muscle+weakness+beyond+atrophy-new+hopes+for+treatment+of+musculoskeletal+diseases.">Novel excitation-contraction coupling related genes reveal aspects of muscle weakness beyond atrophy-new hopes for treatment of musculoskeletal diseases.</a> Front Physiol. 5, 37 (2014).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270 (5639), 725-727 (1977).</li><li>Pasut, A., Jones, A. E., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Individual+Myofibers+and+their+Satellite+Cells+from+Adult+Skeletal+Muscle.">Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle.</a> J Vis Exp. (73), (2013).</li><li>Meng, H., Janssen, P. M. L., 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=Tissue+Triage+and+Freezing+for+Models+of+Skeletal+Muscle+Disease.">Tissue Triage and Freezing for Models of Skeletal Muscle Disease.</a> J Vis Exp. (89), (2014).</li><li>Demonbreun, A. R., McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Electroporation,+Isolation+and+Imaging+of+Myofibers.">DNA Electroporation, Isolation and Imaging of Myofibers.</a> J Vis Exp. (106), (2015).</li><li>Falcone, S., Roman, W., 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=N-WASP+is+required+for+Amphiphysin-2/BIN1-dependent+nuclear+positioning+and+triad+organization+in+skeletal+muscle+and+is+involved+in+the+pathophysiology+of+centronuclear+myopathy.">N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.</a> EMBO Mol Med. 6 (11), 1455-1475 (2014).</li><li>Flucher, B. E., Phillips, J. L., Powell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dihydropyridine+receptor+alpha+subunits+in+normal+and+dysgenic+muscle+in+vitro:+expression+of+alpha+1+is+required+for+proper+targeting+and+distribution+of+alpha+2.">Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2.</a> J Cell Biol. 115 (5), 1345-1356 (1991).</li><li>Cooper, S. T., Maxwell, A. L., 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=C2C12+co-culture+on+a+fibroblast+substratum+enables+sustained+survival+of+contractile,+highly+differentiated+myotubes+with+peripheral+nuclei+and+adult+fast+myosin+expression.">C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.</a> Cell Motil Cytoskeleton. 58 (3), 200-211 (2004).</li><li>Al-Qusairi, L., Laporte, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-tubule+biogenesis+and+triad+formation+in+skeletal+muscle+and+implication+in+human+diseases.">T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases.</a> Skelet Muscle. 1, 26 (2011).</li><li>Vilmont, V., Cadot, B., Ouanounou, G., Gomes, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+to+study+mechanisms+of+neuromuscular+junction+development+and+maintenance.">A system to study mechanisms of neuromuscular junction development and maintenance.</a> Development. , (2016).</li><li>Vilmont, V., Cadot, B., Vezin, E., Le Grand, F., Gomes, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+disruption+perturbs+post-synaptic+components+and+contributes+to+impaired+MuSK+clustering+at+the+NMJ:+implication+in+ALS.">Dynein disruption perturbs post-synaptic components and contributes to impaired MuSK clustering at the NMJ: implication in ALS.</a> Sci Rep. 6, 27804 (2016).</li></ol>

The authors have nothing to disclose.

The use of this protocol for the cultivation of primary myoblasts gives rise to a special niche that greatly nurtures the development of myofibers. This is partially due to other cell types that are also present in very small numbers. A balance between myoblast concentration and culture purity must be achieved. A good cell culture also depends on the quality of the products used for the medium formulation. All products derived from animal sources should be thoroughly tested. In our experience, the digestion conditions should also be monitored.
As usual for primary cultures, experimental variability can be higher than in studies with isolated fibers or immortalized myoblasts. This variability can be diminished by the standardization of medium and digestion components, mice age and size, and the time points for culture manipulation and results collection. Nevertheless, the advantage of scrutinizing in real time the intricate mechanisms necessary for myofiber development greatly surpasses the variability drawback.
This protocol confers the advantages of in vitro approaches without compromising cell differentiation. Myofibers mature until triads are formed and contractions are coupled to calcium sparks. These functional outputs can be accessed in different experimental conditions. Furthermore, there can be many technical variations made to the protocol. Myoblasts can be harvested from neonatal mice with mutations of interest relating to muscle development. Cells can be lysed for biochemical analysis at different differentiation time points. Calcium indicators can be added to the culture to follow its dynamics. Optogenetic constructs can be used to enforce certain signaling pathways or to induce specific local responses. Finally, the myofibers can be cocultured with other cells types to study their interactions.

Skeletal muscle composes up to 40% of the human body weight1. Muscle-associated disorders represent an immense health and economic burden2. How this highly complex and organized tissue is formed, maintained, and regenerated constitutes an extensive and well-established research field. Depending on the specific scientific interest, the most suited approach can range from simple myotube cultures to complex in vivo models3-6.
The goal of this protocol is to provide an in vitro system that allows for the monitoring of myogenesis through live imaging and immunofluorescence. Compared to traditional approaches, this system offers a very complete and dynamic insight into the mouse myogenic process. Cells can be followed from the myoblast stage to the mature, multinucleated myofiber displaying transversal triads and peripheral nuclei7. This maturation level can be achieved using regular cell culture equipment, without the need for complex stimulatory or mechanical apparatuses. Although some successful in vitro systems have been reported8,9, to our knowledge, this is the only protocol generating mature mouse myofibers with T-tubules transversally paired with Sarcoplasmic Reticulum (SR). Thus, this in vitro system can be used to study the molecular mechanisms of triad formation, which are still poorly understood10.
A further advantage of using this system is the availability of validated mouse-targeted resources, such as antibodies, drugs, and RNAi tools. The relatively simple protocol does not require laborious steps, highly skilled manipulation, or expensive and dedicated equipment. Matured myofibers start appearing after 5 d of culture differentiation7, displaying contractility coupled with calcium sparks (unpublished data). In one week, the different developmental stages of one of the most complex cells in the mammalian body can be studied in combination with a variety of in vitro assays.

<table><tbody><tr><td>Dispase II</td><td>Gibco</td><td>17105041</td><td></td></tr><tr><td>Collagenase&amp;nbsp;type V</td><td>Sigma-Aldrich-Aldrich</td><td>C9263</td><td></td></tr><tr><td>IMDM, Glutamax supplemented</td><td>Gibco</td><td>31980022</td><td></td></tr><tr><td>Matrigel Growth reduced factor</td><td>Corning</td><td>354230</td><td>protein concentration of the lot should be around 10 mg/mL and endotoxin result should be &amp;lt;1.5</td></tr><tr><td>Chicken Embryo Extract</td><td>MP biomedical</td><td>2850145</td><td>it is also possible to prepare in the lab (Danoviz ME, Yablonka-Reuveni Z. Methods Mol Biol (2012))</td></tr><tr><td>Recombinant rat agrin</td><td>R&amp;amp;D systems</td><td>550-AG-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Horse Serum</td><td>GE Healthcare&amp;nbsp;</td><td>11581831</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778-150</td><td>used to transfect siRNA</td></tr><tr><td>Lipofectamine LTX</td><td>Invitrogen</td><td>15338-100</td><td>used to transfect DNA</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668030</td><td>used to transfect siRNA plus DNA</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190094&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Eurobio</td><td>CVFSVF0001</td><td></td></tr><tr><td>Cell strainer</td><td>Corning</td><td>21008-949</td><td></td></tr><tr><td>Fluorodishes</td><td>World precision instruments</td><td>FD35-100</td><td>dishes used to cultivate cells for live imaging</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>16% PFA (Paraformaldehyde)</td><td>Science Services GmbH</td><td>E15710</td><td></td></tr><tr><td>Goat Serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>BSA (Bovine Serum Albumine)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Saponine</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>use at 200 ng/&amp;micro;L</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions and Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Digestion Mix</td><td></td><td></td><td>in DPBS&lt;br/&gt; 5 mg/mL collagenase&lt;br/&gt; 3.5 mg/mL dispase&lt;br/&gt; sterile filtered, can be stored in working aliquotes for 2 weeks at -20 &amp;deg;C</td></tr><tr><td>Dissection Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 10% FBS&lt;br/&gt; 1% Penicillin/Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Growth Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 20% FBS&lt;br/&gt; 1% Chicken Embryo Extract&lt;br/&gt; 1% Penicillin-Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Differentiation Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 2% Horse Serum&lt;br/&gt; 1% Penicillin-Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Blocking Solution</td><td></td><td></td><td>in DPBS&lt;br/&gt; 10% Goat Serum&lt;br/&gt; 5% BSA&lt;br/&gt; add 0.1% saponine when incubating with primary and secondary antibodies</td></tr></tbody></table>

in vitro differentiation of mature myofibers for live imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

The extent of myofiber development is mostly determined by the purity and viability of the isolated myoblasts. The adhesion, proliferation, and fusion capacity can be used to empirically access those parameters (Figure 1 A, B). At proliferation D2, myoblasts should have adhered and should display the typical fusiform shape. Proliferation is expected to happen extensively at this stage, leading to spontaneous myotube formation the following day (Figure 1B).
Cell confluency might need slight adjustments. It should be increased if myoblasts take more than 3 d to proliferate and fuse. It should be decreased if myofibers are not allowed to grow and elongate relatively straight due to their density. Confluency typically decreases from the center to the periphery of the dish, so the best myofibers should be found towards the outer regions.
Myotubes will quickly elongate and display multiple centrally aligned nuclei (Figure 1C). By D5, some cells start acquiring striations and moving their nuclei to the periphery. The number of myofibers with mature characteristics will increase with time as well as with cell thickness (Figure 1D).
The degree of differentiation can be further observed by immunofluorescence. Myofibers fixed at differentiation D8 present transversal triads. This can be confirmed by imaging components of the T-tubules (DPHR) and the SR (triadin), which are expected to colocalize at the triads (Figure 2).
The functionality of myofibers can be addressed by live imaging. From differentiation D3 onwards, the cells display spontaneous twitching. By transfecting a calcium sensor (e.g., GCaMP6f11), it is possible to observe that the contractions are coupled with calcium peaks (Figure 3).
Using this system, we were able to identify a novel molecular pathway that is disrupted in centronuclear myopathies and myotonic dystrophies, which can therefore be a novel target for innovative molecular therapies7. We have also adapted this method to study the development of the neuromuscular junction (NMJ)12. Through the coculture with rat spinal cord explants, we have described a role for dynein in NMJ formation13.
<img alt="Figure 1" src="/files/ftp_upload/55141/55141fig1.jpg" /> 
Figure 1: Developmental Stages of the Myoblast Culture. A) At proliferation D2, myoblasts have adhered and started proliferating. B) At proliferation D3, a confluency of 60 - 80% is reached, and myoblasts start fusing spontaneously. C) At differentiation D3, myotubes containing centrally located nuclei are predominant. D) From differentiation D5 onwards (e.g., day 8), myofibers start exhibiting striations and peripheral nuclei and begin to thicken. Scale bar: 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55141/55141fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55141/55141fig2.jpg" /> 
Figure 2: Representative Confocal Image of a D8 Myofiber Immunostain. A) Immunostaining for dihydropyridine receptor (DHPR, top panel) and triadin (TRDN, middle panel). An overlay of the DHPR, TRDN, and DAPI channels shows colocalization of the triad components. B) An intensity profile of the yellow line drawn in A. C) A 3D image of volume rendering of myofibers stained for &#945;-actinin (green) and DAPI (blue). Scale bar and grid width: 5 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55141/55141fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55141/55141fig3.jpg" /> 
Figure 3: Live Imaging of Calcium Levels in Myofibers with Spontaneous Twitching. A) High-speed time-lapse (20 ms frames) microscopy of a calcium spark in a twitching myofiber. Calcium was detected through the expression of GCaMP6f (Addgene plasmid #40755). B) Quantification of the fluorescence intensity over time for the calcium sensor in panel A. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55141/55141fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Differentiation of Mature Myofibers for Live Imaging at JoVE.com

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

in-vitro-differentiation-of-mature-myofibers-for-live-imaging

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.2K Views.  Universidade de Lisboa. Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Video: In Vitro Differentiation of Mature Myofibers for Live Imaging

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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

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

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

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

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

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Genetics

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Biology

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 ...

Medicine

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

In Vitro Differentiation of Mature Myofibers for Live Imaging

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