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.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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 ...

Research

JoVE Journal

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 ...

Purification of Progenitors from Skeletal Muscle

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

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

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, ...

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 ...

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 ...

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 ...

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 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 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 ...