Name: adult and embryonic skeletal muscle microexplant culture and isolation of skeletal muscle stem cells
Uploaded: 2010-09-21T17:10:00
Duration: 14 min 36 s
Description: Watch this Scientific Journal Video about Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells at JoVE.com

We thank Patrick Paddison for his gift of the shRNAi shuttle vector. Angela Sloan generated the GFP RNAi image in Figure 3. We also thank the following funding bodies for their support: 
Muscular Dystrophy Campaign grant number RA2/592/2; SPARKS grant number 02BHM04, The Royal Society grant number 574006.G503/1948./JE and BBSRC grant number 6/SAG10077.

<ol>
	<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, 725-727 (1997).</li><li>Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+program+for+investigating+adult+human+skeletal+muscle+grown+aneurally+in+tissue+culture.">A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.</a> Neurology. 25, 58-67 (1975).</li><li>Smith, J., Schofield, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+fibroblast+growth+factors+in+long+term+primary+culture+of+dystrophic+(mdx)+mouse+muscle+myoblasts.">The effects of fibroblast growth factors in long term primary culture of dystrophic (mdx) mouse muscle myoblasts.</a> Exp Cell Res. 210, 86-93 (1994).</li><li>Fell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+in+culture.">The cell in culture.</a> J Clin Pathol. 11, 489-495 (1958).</li><li>Stewart, D. C., Kirk, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+simultaneous+measurement+of+several+parameters+of+embryo+heart+explants+growth+in+vitro.">The simultaneous measurement of several parameters of embryo heart explants growth in vitro.</a> J Cell Physiol. 40, 183-198 (1952).</li><li>Harvey, A. L., Robertson, J. G., Witkowski, 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=Maturation+of+human+skeletal+muscle+fibres+in+explant+tissue+culture.">Maturation of human skeletal muscle fibres in explant tissue culture.</a> J Neurol Sci. 41, 115-122 (1979).</li><li>Zammit, P. S., Heslop, L., Hudon, V., Rosenblatt, J. D., Tajbakhsh, S., Buckingham, M. E., Beauchamp, J. R., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+myoblast+proliferation+show+that+resident+satellite+cells+are+competent+to+fully+regenerate+skeletal+muscle+fibers.">Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers.</a> Exp Cell Res. 281, 39-49 (2002).</li><li>Konigsberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The embryonic origin of muscle. , (1986).</li><li>Trainor, P. A., Tan, S. S., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm:+regionalization+of+cell+fate+and+impact+on+craniofacial+development+in+mouse+embryos.">Cranial paraxial mesoderm: regionalization of cell fate and impact on craniofacial development in mouse embryos.</a> Development. 120, 2397-2408 (1994).</li><li>Tajbakhsh, S., Rocancourt, D., Cossu, G., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+genetic+hierarchies+controlling+skeletal+myogenesis+Pax-3+and+Myf-5+act+upstream+of+MyoD.">Redefining the genetic hierarchies controlling skeletal myogenesis Pax-3 and Myf-5 act upstream of MyoD.</a> Cell. 89, 127-138 (1997).</li><li>Tremblay, P., Dietrich, S., Mericskay, M., Schubert, F. R., Li, Z., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+for+Pax3+in+the+development+of+the+hypaxial+musculature+and+the+longrange+migration+of+muscle+precursors.">A crucial role for Pax3 in the development of the hypaxial musculature and the longrange migration of muscle precursors.</a> Dev Biol. 203, 49-61 (1998).</li><li>Tajbakhsh, S., Bober, E., Babinet, C., Pournin, S., Arnold, H., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+the+myf-5+locus+with+nlacZ+reveals+expression+of+this+myogenic+factor+in+mature+skeletal+muscle+fibres+as+well+as+early+embryonic+muscle.">Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle.</a> Dev Dyn. 206, 291-300 (1996).</li><li>Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+pattern+of+prospective+somites+in+the+presomitic+mesoderm+of+mouse+embryos.">A study of the pattern of prospective somites in the presomitic mesoderm of mouse embryos.</a> J Embryol Exp Morphol. 92, 269-285 (1986).</li><li>Cossu, G., Kelly, R., Donna, S. D. i., Vivarelli, E., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+differentiation+during+mammalian+somitogenesis+is+dependent+upon+a+community+effect.">Myoblast differentiation during mammalian somitogenesis is dependent upon a community effect.</a> Proc Natl Acad Sci USA. 92, 2254-2258 (1995).</li><li>Shainberg, A., Yagil, G., Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+myogenesis+in+vitro+by+Ca2++concentration+in+nutritional+medium.">Control of myogenesis in vitro by Ca2+ concentration in nutritional medium.</a> Exp Cell Res. 58, 163-167 (1969).</li><li>Dodson, M. V., Martin, E. L., Brannon, M. A., Mathison, B. A., McFarland, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+bovine+satellite+cell-derived+myotube+formation+in+vitro.">Optimization of bovine satellite cell-derived myotube formation in vitro.</a> Tissue Cell. 19, 159-166 (1987).</li><li>Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+Growth+factors,+ubiquitin+and+apoptosis+in+dystrophic+muscle:+Apoptosis+declines+with+age+in+the+mdx+mouse.">Muscle Growth factors, ubiquitin and apoptosis in dystrophic muscle: Apoptosis declines with age in the mdx mouse.</a> B Appl Myol. 6, 279-284 (1996).</li><li>Smith, J., Fowke, G., Schofield, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+in+dystrophic+(mdx)+muscle+is+inhibited+by+IGF-II.">Programmed cell death in dystrophic (mdx) muscle is inhibited by IGF-II.</a> Cell Death Differ. 2, 243-251 (1995).</li><li>Smith, J., Schofield, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+integration+of+an+mdx+skeletal+muscle+cell+line+into+dystrophic+(mdx)+skeletal+muscle:+evidence+for+stem+cell+status.">Stable integration of an mdx skeletal muscle cell line into dystrophic (mdx) skeletal muscle: evidence for stem cell status.</a> Cell Growth Differ. 8, 927-934 (1997).</li><li>O Shea, L., Johnson, C., Rooney, M., Gleeson, R., Woods, K., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipogenesis+and+skeletal+muscle+ageing.">Adipogenesis and skeletal muscle ageing.</a> Mech Ageing Dev. 122, 1354-1355 (2001).</li><li>Merrick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A role for Igf-2 in fast skeletal muscle specification during myogenesis in dystrophic and wild type embryos [dissertation]. , (2006).</li><li>Stadler, L. K. J., Merrick, D., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+stem+cell+and+fast+myosin+abnormalities+in+the+cav-3+/+and+mdx+dystrophic+embryo+reveal+an+embryonic+basis+for+muscular+dystrophy.">Morphological stem cell and fast myosin abnormalities in the cav-3 / and mdx dystrophic embryo reveal an embryonic basis for muscular dystrophy.</a> Abstr. Genet.Res. 90, 281-289 (2008).</li><li>Merrick, D., Stadler, L. K. J., Larner, D. P., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+stem+cell+and+fast+myosin+abnormalities+in+the+cav-3+/+and+mdx+dystrophic+embryo+reveal+an+embryonic+basis+for+muscular+dystrophy.">Morphological stem cell and fast myosin abnormalities in the cav-3 / and mdx dystrophic embryo reveal an embryonic basis for muscular dystrophy.</a> Dis Model Mech. 2, 374-388 (2009).</li><li>Matschak, T., Stickland, N. C., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explants+of+embryonic+Atlantic+salmon+muscle+in+culture.">Explants of embryonic Atlantic salmon muscle in culture.</a> Proc Soc Exp Biol A. 1, 23-23 (1997).</li><li>Smith, J., Hooper, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominance+and+independent+segregation+of+metabolic+cooperation+competence+and+pluripotency+in+an+embryonal+carcinoma+cell+hybrid.">Dominance and independent segregation of metabolic cooperation competence and pluripotency in an embryonal carcinoma cell hybrid.</a> Exp Cell Res. 181, 40-50 (1989).</li><li>Bulfield, G., Siller, W. G., Wight, P. A., Moore, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+linked+muscular+dystrophy+(mdx)+in+the+mouse.">X chromosome linked muscular dystrophy (mdx) in the mouse.</a> Proc Natl Acad Sci USA. 81, 1189-1192 (1984).</li><li>Hagiwara, Y., Sasaoka, T., Araishi, K., Imamura, M., Yorifuji, H., Nonaka, I., Ozawa, E., Kikuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveolin-3+deficiency+causes+muscle+degeneration+in+mice.">Caveolin-3 deficiency causes muscle degeneration in mice.</a> Hum Mol Genet. 9, 3047-3054 (2000).</li><li>Westbury, J., Watkins, M., Ferguson-Smith, A. C., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+temporal+and+spatial+regulation+of+the+cdk+inhibitor+p57+(kip2)+during+embryo+morphogenesis.">Dynamic temporal and spatial regulation of the cdk inhibitor p57 (kip2) during embryo morphogenesis.</a> Mech Dev. 109, 83-89 (2001).</li><li>Merrick, D., Ting, T., Stadler, L. K. J., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Insulin-like+growth+factor+2+in+specification+of+the+fast+skeletal+muscle+fibre.">A role for Insulin-like growth factor 2 in specification of the fast skeletal muscle fibre.</a> BMC Dev Biol. 7, 65-65 (2007).</li><li>Paddison, P. J., Caudy, A. A., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+suppression+of+gene+expression+by+RNAi+in+mammalian+cells+AQ4.">Stable suppression of gene expression by RNAi in mammalian cells AQ4.</a> Proc Natl Acad Sci USA. 99, 1443-1448 (2002).</li><li>Smith, J., Merrick, D. <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+Microexplant+Culture+and+Isolation+of+Skeletal+Muscle+Stem+Cells.">Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells.</a> Methods in Molecular Biology. 633, (2010).</li></ol>

No conflicts of interest declared.

Microdissected explant cultures can be used to reliably and reproducibly isolate cell populations containing a very high proportion (~85%) of proliferative Myf-5 positive skeletal muscle stem cells (SMSc). Under the rigorously controlled culture conditions described here primary explant cultures can be used to characterize the growth behaviours of genetically mutant mouse SMSc and can be used as a means of generating myotubes for detailed in vitro analysis of differentiation processes. Careful maintenance and manipulation of these cultures enables long-term culture and expansion. Using the methods described here it is also possible to derive clonal skeletal muscle stem cell lines from explant cultures by means of single cell dilution. To achieve the proliferation of isolated single cells during the cloning procedure, "conditioned medium" is used to mimic the normal requirement of these cells for high-density culture. The method is applicable (with modification) to embryonic, adult and aged-adult tissues and in addition to mouse can be used to isolate cells from the skeletal muscles of other species including human (Rao and Smith, unpublished), chick embryo and fish (salmon) 24. Clonally derived SMSc can be analysed in vivo by intramuscular transplantation and under these conditions injected SMSc will combine with host myotubes to form hybrid muscle fibres. Intramuscularly injected SMSc do not form tumours and have been found in host muscles in the satellite cell position more than a year after injection, suggesting that they are subject to endogenous control by the satellite stem cell niche.These cells can be re-isolated from injected hosts as proliferative SMSc more than 12 months after host injection 19.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM/F12 1:1 mix</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich Company</td>
<td>&nbsp;</td>
<td>Liquid medium: (Dulbecco's Modified Eagles's medium and Ham's F12 medium, 1:1 v/v)</td>
</tr>
<tr>
<td>100&#215; Glutamine (200mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich Company</td>
<td>&nbsp;</td>
<td>Diluted in medium to a 1&#215; concentration of 2 mM (Glutamine HYBRI-MAX R)
</td>
</tr>
<tr>
<td>Fetal calf serum (FCS)</td>
<td>&nbsp;</td>
<td>From a number of different companies</td>
<td>&nbsp;</td>
<td>Batch tested on primary cultures and skeletal muscle cell lines. 10-20% supplement to liquid media</td>
</tr>
<tr>
<td>Heraeus Labofuge 300</td>
<td>&nbsp;</td>
<td>Heraeus and distributors of Heraeus equipment</td>
<td>&nbsp;</td>
<td>Lab centrifuge capable of reaching 1,000 rpm</td>
</tr>
<tr>
<td>15 ml Falcon centrifuge tubes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>Must fit lab centrifuge</td>
</tr>
<tr>
<td>Tissue culture plasticware (25, 75 or 175 mm2 tissue culture vessels; 96-well tissue culture plates. 60 mm Petri dishes).</td>
<td>&nbsp;</td>
<td>Nunc (available from Fisher Scientific in the UK)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Humidified CO2 incubator (Heraeus)</td>
<td>&nbsp;</td>
<td>Heraeus and distributors of Heraeus equipment</td>
<td>&nbsp;</td>
<td>Ours is copper lined, recommended for reducing contamination</td>
</tr>
<tr>
<td>Sterile hood with laminar air flow (Heraeus)</td>
<td>&nbsp;</td>
<td>Heraeus and distributors of Heraeus equipment</td>
<td>&nbsp;</td>
<td>Ours is a Class II hood &#x2013; suitable for use with Human tissues</td>
</tr>
<tr>
<td>Water Bath</td>
<td>&nbsp;</td>
<td>Grant Equipment</td>
<td>&nbsp;</td>
<td>Maintained always at 37&deg;C</td>
</tr>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Leica DMIRB, Leica Instruments, UK)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For sterilization (animals, dissection instruments) and swabbing benches, hood, etc. </td>
</tr>
<tr>
<td>Calcium- and magnesium-free phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich Company, UK</td>
<td>&nbsp;</td>
<td>Cell culture-tested PBS (Dulbecco&#x2019;s formula) is purchased as a ready-mixed powder or in tablet form and made up with doubledistilled water before sterilization by autoclave. PBS consists of 2.68 mM potassium chloride (KCl); 1.47 mM potassium phosphate monobasic (KH2PO4); 0.137 M sodium chloride (NaCl); and 8.1 mM sodium phosphate
dibasic (Na2HPO4). PBS can be prepared from scratch as follows: 200 mg KCl, 200 mg KH2PO4, 8 g NaCl and 1.15 g Na2HPO4/l of double-distilled water followed by
sterilization by autoclave.
</td>
</tr>
<tr>
<td>CryoTube vials</td>
<td>&nbsp;</td>
<td>Nunc (see above)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A Neubauer haematocytometer and coverslips</td>
<td>&nbsp;</td>
<td>Fisher Scientific, UK</td>
<td>&nbsp;</td>
<td>For estimating cell counting</td>
</tr>
<tr>
<td>Hand counter</td>
<td>&nbsp;</td>
<td>Fisher Scientific, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope, Zeiss Stemi 1000</td>
<td>&nbsp;</td>
<td>Zeiss, UK</td>
<td>&nbsp;</td>
<td>For preparation of explants</td>
</tr>
<tr>
<td>Small sterile hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile dissection instruments (including Jeweler&#x2019;s forceps)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Sterilised by autoclave</td>
</tr>
<tr>
<td>Sterile plastic collecting vessels (7 ml bijou tubes or 20ml universals)</td>
<td>&nbsp;</td>
<td>Nunc (Fisher Scientific)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Warm PECM</td>
<td>&nbsp;</td>
<td>Constituents purchased from Sigma-Aldrich, UK as above</td>
<td>&nbsp;</td>
<td>Made up in the sterile hood and warmed to 37&deg;C in the tissue culture waterbath. DMEM:F-12 supplemented with 20% FCS, 1% glutamine and + 1% penicillin &amp; streptomycin solution</td>
</tr>
<tr>
<td>Dispase (50 &mu;g/ml, equivalent to 6 units/mg) </td>
<td>&nbsp;</td>
<td>Available from MP Biomedicals, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;g/ml 4_,6 Diamidino-2-phenylindole, dihydrochloride (DAPI).</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, UK</td>
<td>&nbsp;</td>
<td>For microscopic visualization of apoptosis and mitosis</td>
</tr>
<tr>
<td>Vectashield fluorescent mounting fluid</td>
<td>&nbsp;</td>
<td>Vector Laboratories, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent upright microscope with ultraviolet filter (we use a Nikon Eclipse E600)</td>
<td>&nbsp;</td>
<td>Nikon, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Digital camera and imaging software (we use a Nikon Coolpix 995 camera; A Nikon D3 camera</td>
<td>&nbsp;</td>
<td>Nikon, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>OpenLab4.0a software</td>
<td>&nbsp;</td>
<td>Improvision, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Photoshop CS4</td>
<td>&nbsp;</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

adult and embryonic skeletal muscle microexplant culture and isolation of skeletal muscle stem cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

Watch this Scientific Journal Video about Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells at JoVE.com

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

adult-embryonic-skeletal-muscle-microexplant-culture-isolation

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Biology

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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 ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...