Name: isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement
Uploaded: 2017-07-26T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

Ce travail a été soutenu par la Fondation nationale suisse de la science (numéro 310030-166313), la Fondation Suisse pour la recherche sur les maladies musculaires, la Fondation Marcel Levaillant et la Fondation pour la recherche ostéo-articulaire.

Des échantillons musculaires humains (tissus obtenus à partir de muscles semi-finissants) ont été recueillis lors de la chirurgie orthopédique chez des patients en bonne santé en tant que déchets chirurgicaux. Toutes les méthodes relatives à l&#39;étude humaine ont été réalisées conformément aux directives et règlements des Autorités suisses de réglementation de la santé et approuvées par la Commission Cantonale d&#39;Ethique de Recherche des Autorités cantonales de Genève en Suisse (protocole CER ...

<ol>
	<li>Dumont, N. A., Bentzinger, C. F., Sincennes, M. C., 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=Satellite+Cells+and+Skeletal+Muscle+Regeneration.">Satellite Cells and Skeletal Muscle Regeneration.</a> Compr Physiol. 5 (3), 1027-1059 (2015).</li><li>Sambasivan, R., Tajbakhsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+skeletal+muscle+stem+cells.">Adult skeletal muscle stem cells.</a> Results Probl Cell Differ. 56, 191-213 (2015).</li><li>Chen, W. C., 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=Isolation+of+blood-vessel-derived+multipotent+precursors+from+human+skeletal+muscle.">Isolation of blood-vessel-derived multipotent precursors from human skeletal muscle.</a> J Vis Exp. (90), e51195 (2014).</li><li>Zheng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+stem+cells+from+cultures+of+cryopreserved+human+skeletal+muscle.">Isolation of myogenic stem cells from cultures of cryopreserved human skeletal muscle.</a> Cell Transplant. 21 (6), 1087-1093 (2012).</li><li>Charville, G. 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=Ex+Vivo+Expansion+and+In+Vivo+Self-Renewal+of+Human+Muscle+Stem+Cells.">Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells.</a> Stem Cell Reports. 5 (4), 621-632 (2015).</li><li>Stiber, J., 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=STIM1+signalling+controls+store-operated+calcium+entry+required+for+development+and+contractile+function+in+skeletal+muscle.">STIM1 signalling controls store-operated calcium entry required for development and contractile function in skeletal muscle.</a> Nat Cell Biol. 10 (6), 688-697 (2008).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-Operated+Calcium+Channels.">Store-Operated Calcium Channels.</a> Physiol Rev. 95 (4), 1383-1436 (2015).</li><li>Darbellay, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIM1-+and+Orai1-dependent+store-operated+calcium+entry+regulates+human+myoblast+differentiation.">STIM1- and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation.</a> J Biol Chem. 284 (8), 5370-5380 (2009).</li><li>Darbellay, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+muscle+economy+myoblast+differentiation+and+excitation-contraction+coupling+use+the+same+molecular+partners,+STIM1+and+STIM2.">Human muscle economy myoblast differentiation and excitation-contraction coupling use the same molecular partners, STIM1 and STIM2.</a> J Biol Chem. 285 (29), 22437-22447 (2010).</li><li>Nilius, B., Owsianik, G., Voets, T., Peters, 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=Transient+receptor+potential+cation+channels+in+disease.">Transient receptor potential cation channels in disease.</a> Physiol Rev. 87 (1), 165-217 (2007).</li><li>Vandebrouck, C., Martin, D., Colson-Van Schoor, M., Debaix, H., Gailly, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+TRPC+in+the+abnormal+calcium+influx+observed+in+dystrophic+(mdx)+mouse+skeletal+muscle+fibers.">Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers.</a> J Cell Bio. 158 (6), 1089-1096 (2002).</li><li>Antigny, F., Koenig, S., Bernheim, L., Frieden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=During+post-natal+human+myogenesis,+normal+myotube+size+requires+TRPC1-+and+TRPC4-mediated+Ca(2)(+)+entry.">During post-natal human myogenesis, normal myotube size requires TRPC1- and TRPC4-mediated Ca(2)(+) entry.</a> J Cell Sci. 126 (11), 2525-2533 (2013).</li><li>Brinkmeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP+channels+in+skeletal+muscle:+gene+expression,+function+and+implications+for+disease.">TRP channels in skeletal muscle: gene expression, function and implications for disease.</a> Adv Exp Med Biol. 704, 749-758 (2011).</li><li>Pan, Z., Brotto, M., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+Ca2++entry+in+muscle+physiology+and+diseases.">Store-operated Ca2+ entry in muscle physiology and diseases.</a> BMB Rep. 47 (2), 69-79 (2014).</li><li>Lacruz, R. S., Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+caused+by+mutations+in+ORAI1+and+STIM1.">Diseases caused by mutations in ORAI1 and STIM1.</a> Ann N Y Acad Sci. 1356, 45-79 (2015).</li><li>Li, T., 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=STIM1-Ca(2+)+signaling+is+required+for+the+hypertrophic+growth+of+skeletal+muscle+in+mice.">STIM1-Ca(2+) signaling is required for the hypertrophic growth of skeletal muscle in mice.</a> Mol Cell Biol. 32 (15), 3009-3017 (2012).</li><li>Wei-Lapierre, L., Carrell, E. M., Boncompagni, S., Protasi, F., Dirksen, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orai1-dependent+calcium+entry+promotes+skeletal+muscle+growth+and+limits+fatigue.">Orai1-dependent calcium entry promotes skeletal muscle growth and limits fatigue.</a> Nat Commun. 4, 2805 (2013).</li><li>Konig, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+hyperpolarization+triggers+myogenin+and+myocyte+enhancer+factor-2+expression+during+human+myoblast+differentiation.">Membrane hyperpolarization triggers myogenin and myocyte enhancer factor-2 expression during human myoblast differentiation.</a> J Biol Chem. 279 (27), 28187-28196 (2004).</li><li>Crisan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3 (3), 301-313 (2008).</li><li>Bigot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Associated+Methylation+Suppresses+SPRY1,+Leading+to+a+Failure+of+Re-quiescence+and+Loss+of+the+Reserve+Stem+Cell+Pool+in+Elderly+Muscle.">Age-Associated Methylation Suppresses SPRY1, Leading to a Failure of Re-quiescence and Loss of the Reserve Stem Cell Pool in Elderly Muscle.</a> Cell Rep. 13 (6), 1172-1182 (2015).</li><li>Bettadapur, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+Culture+of+Aligned+Skeletal+Myotubes+on+Micromolded+Gelatin+Hydrogels.">Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels.</a> Sci Rep. 6, 28855 (2016).</li><li>Darbellay, B., Arnaudeau, S., Bader, C. R., Konig, S., Bernheim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIM1L+is+a+new+actin-binding+splice+variant+involved+in+fast+repetitive+Ca2++release.">STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release.</a> J Cell Biol. 194 (2), 335-346 (2011).</li><li>Sauc, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIM1L+traps+and+gates+Orai1+channels+without+remodeling+the+cortical+ER.">STIM1L traps and gates Orai1 channels without remodeling the cortical ER.</a> J Cell Sci. 128 (8), 1568-1579 (2015).</li><li>Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+measurements+in+organelles+with+Ca2+-sensitive+fluorescent+proteins.">Calcium measurements in organelles with Ca2+-sensitive fluorescent proteins.</a> Cell Calcium. 38 (3-4), 213-222 (2005).</li></ol>

L&#39;isolement et la culture des myoblastes humains à partir du muscle squelettique adulte offrent un modèle in vitro pour étudier la différenciation musculaire et la régénération musculaire. Dans cet article, nous fournissons un protocole qui permet de purifier les rendements élevés de myoblastes humains d&#39;une manière simple et limitée en termes de coûts. En outre, cette technique fournit des résultats fiables et reproductibles en termes de pourcentage de myob...

Les muscles squelettiques humains sont composés de groupes de fibres musculaires contractiles et multinucléées résultant de la fusion des cellules précurseurs myogéniques. Les muscles squelettiques ont la capacité de se régénérer après une blessure grâce à la présence de SC, les cellules souches du muscle squelettique situées entre la membrane plasmatique des myofibres (sarcolemma) et la lame basale. Dans les muscles non blessés, les SC sont principalement présents dans un état de repos. En réponse au stress mécanique ou aux blessures, les SC sont activés (myoblastes), prolifèrent et subissent soit une différenciation pour former de nouvelles myofibres, soit un auto-renouvellement pour reconstituer le pool SC 1 , 2 . Au cours des années, plusieurs techniques ont été développées pour isoler les SC et leur descendance, les myoblastes, à partir des biopsies des muscles squelettiques. Avec une meilleure compréhension des marqueurs de surface cellulaire exprimés sur ces cellules et de la méthodologie du tri cellulaire activé par fluorescence (FACS) 3, 4 , 5 , il est maintenant possible d&#39;isoler des populations pures de myoblastes humains à partir d&#39;échantillons musculaires. La signalisation de calcium régule le développement du muscle squelettique, l&#39;homéostasie et la régénération. En particulier, l&#39;entrée Ca 2+ qui est activé suite à l&#39; épuisement du magasin intracellulaire, appelé SOCE, est d&#39; une grande importance pour 6 ces processus. Lors de la stimulation cellulaire, le niveau de Ca 2+ dans le réticulum endo / sarcoplasmique (ER / SR) diminue, ce qui active les canaux Ca 2+ de la membrane plasmatique qui permettent l&#39;entrée de Ca 2+ pour remplir le ER / SR 7 . Les deux principales protéines impliquées dans SOCE sont la protéine de l&#39;interaction ERR / SR Ca 2+ -sensing stromal interaction molécule 1 (STIM1) et la chaîne plasma Ca 2+ canal Orai1. Le muscle squelettique exprime abondamment ces deux protéines, ainsi que d&#39;autres protéines des mêmes familles (STIM2 aNd Orai2-Orai3) 8 , 9 et plusieurs canaux perméables au Ca 2+ de la famille canonique canonique (TRPC) du récepteur transitoire 10 , 11 , 12 , 13 . L&#39;entrée de Ca 2+ à travers la voie SOCE revêt une grande importance pour la formation / régénération musculaire 14 . Les mutations des protéines STIM1 ou Orai1 ont des effets délétères sur la fonction musculaire, conduisant principalement à l&#39;hypotonie musculaire 15 . Les modèles animaux avec déficience de SOCE présentent également une masse musculaire et une force réduites, ainsi qu&#39;une fatigabilité accrue 6 , 16 , 17 . Comme mentionné, d&#39;autres protéines STIM et Orai, ainsi que de nombreux canaux TRPC, sont exprimés dans le muscle squelettique, et leurs rôles respectifs n&#39;ont pas encore été déterminés jusqu&#39;à présent. En battant dPossèdent leur niveau d&#39;expression, il est ainsi possible d&#39;étudier leurs implications dans SOCE et leurs rôles lors de la différenciation des muscles squelettiques humains. La mesure SOCE peut être effectuée en utilisant deux approches différentes: enregistrements électrophysiologiques actuels et mesures Ca 2+ . L&#39;électrophysiologie est certainement une méthode plus directe, car elle mesure le courant d&#39;intérêt et sa signature électrophysiologique associée. Cependant, cette technique est très difficile à appliquer aux cellules musculaires, principalement en raison de la grande taille des cellules musculaires et de la petite taille du courant endogène de la SOCE. L&#39;imagerie cytosolique Ca 2+ est techniquement très accessible, mais la mesure est plus indirecte, car le niveau de Ca 2+ mesuré dans le cytosol reflète à la fois l&#39;entrée de Ca 2+ et le refoulement de la cellule ou dans les magasins internes. Une méthodologie qui comprend l&#39;isolement des myoblastes à partir de petits morceaux de skel humainEtal après digestion enzymatique, amplification et FACS est expliqué dans cet article. Le processus de différenciation musculaire et le protocole d&#39;immunofluorescence pour suivre l&#39;expression de marqueurs de différenciation dans le temps sont décrits 18 . Enfin, la mesure de SOCE et le rôle de différents canaux ioniques dans la signalisation Ca 2 + et la différenciation musculaire squelettique sont expliqués.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Ham&#8217;s F10</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31550-023</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">FCS</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">10270-106</td>
			<td style="width:201px;">Lot 41G5532K</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">O5482</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">fetuin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">F3385</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">EGF</td>
			<td style="width:235px;">Corning</td>
			<td style="width:187px;">354001</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Dexamethansone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D1756</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Insulin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">I9278</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Creatine</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">27900</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pyruvate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">P4562</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Uridine</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">U3003</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Gentamycin</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">15710-049</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Trypsin-EDTA 0.05%</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">25300-062</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">70 &#956;m cell strainer</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352350</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">40 &#956;m cell strainer</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352340</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 50&#160;ml tube</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352070</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 15&#160;ml tube</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352096</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 5&#160;ml tube (FACS)</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352058</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Culture medium: OPTI-MEM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31985-047</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Transfectant reagnt: RNAiMax</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">1756064</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PBS</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">14190-094</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mounting medium</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">10981</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti-MyHC *</td>
			<td style="width:235px;">DSHB</td>
			<td style="width:187px;">MF20&#160;</td>
			<td style="width:201px;">concentrate</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti-myogenin</td>
			<td style="width:235px;">BD Pharmigen</td>
			<td style="width:187px;">556358</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Rabbit anti-MEF2</td>
			<td style="width:235px;">Santa Cruz Biotech</td>
			<td style="width:187px;">C-21</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Goat anti-mouse Alexa488</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">A11029</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Goat anti-rabbit Alexa 546</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">A11035</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD56-Alexa488</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557699</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD146-PECy7</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562135</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD45-PE-CF594</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562279</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD34-APC</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">345804</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD144-PE</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">560410</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Alexa Fluor 488 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557702</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PECy7 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557872</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PE-CF594 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562292</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">APC mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">550854</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PE mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">556650</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">DAPI</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D9542</td>
			<td style="width:201px;">CAUTION:&#160; toxic compound</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Paraformaldehyde</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">762400</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Fura-2 AM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">F1201</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pluronic F-127</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">P3000MP</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Thapsigargin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">T9033</td>
			<td style="width:201px;">CAUTION&#160;: toxic compound&#160;</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Ratio imaging acquisition software: Metafluor 6.3</td>
			<td style="width:235px;">Molecular Device</td>
			<td style="width:187px;"></td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="24">
			<td colspan="2" height="24" style="height:24px;">*&#160;: MF 20 was deposited to the DSHB by Fischman, D.A.&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement

Les cellules satellites (SC) sont des cellules souches musculaires situées entre la membrane plasmique des fibres musculaires et la lame basale environnante. Ils sont essentiels pour la régénération musculaire. En cas de blessure, qui se produit fréquemment dans les muscles squelettiques, les SC sont activés. Ils prolifèrent comme myoblastes et se différencient pour réparer les lésions musculaires. Parmi les nombreux événements qui se déroulent lors de la différenciation musculaire, les signaux cytosoliques de Ca 2+ sont d&#39;une grande importance. Ces signaux Ca 2+ proviennent de la libération de Ca de Ca 2+ magasins internes, ainsi que de Ca 2+ entrée de l&#39;espace extracellulaire, en particulier l&#39;entrée Ca 2+ magasin fonctionnant (SOCE). Cet article décrit une méthodologie utilisée pour obtenir une population pure de myoblastes humains à partir d&#39;échantillons musculaires recueillis après la chirurgie orthopédique. Le tissu est digéré mécaniquement et enzymatiquement, et les cellules sont amplifiées puis triées par cytométrie en flux selon la présence de spécimensMarqueurs de membrane de fic. Une fois obtenu, les myoblastes humains sont élargis et s&#39;engagent à se différencier en éliminant les facteurs de croissance du milieu de culture. Les niveaux d&#39;expression des facteurs de transcription spécifiques et de l&#39; immunofluorescence in vitro sont utilisés pour évaluer le processus de différenciation myogénique dans les conditions de contrôle et après le silence des protéines impliquées dans la signalisation du Ca 2+ . Enfin, nous détaillons l&#39;utilisation de Fura-2 comme une sonde ratiométrique Ca 2+ qui fournit des mesures fiables et reproductibles de SOCE.

Après la dissociation enzymatique d&#39;un échantillon de muscle humain, les cellules ont été amplifiées dans GM. Les myoblastes humains, définis comme CD56 + / CD146 + / CD45- / CD34- / CD144-, ont été obtenus après FACS. Les myoblastes représentaient plus de 60% de la population analysée ( figure 1 ). Les myoblastes humains primaires ont été reconstitués, cultivés en confluence et cultivés en DM pendant 48 h. Après immunocoloration pour l&#39;expression du facteur de tran...

Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

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

Ici, nous décrivons une méthodologie pour obtenir une population pure de myoblastes humains à partir d&#39;un tissu musculaire adulte. Ces cellules sont utilisées pour étudier la différenciation in vitro des muscles squelettiques et, en particulier, pour étudier les protéines impliquées dans la signalisation du Ca 2+ .

L&#39;isolement des myoblastes humains, l&#39;évaluation de la différenciation myogénique et la mesure de l&#39;entrée de calcium par magasin

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

The overall goal of this procedure is to obtain a pure population of human myoblasts from adult skeletal muscle. These cells are used for functional studies such as assessment of myogenic differentiation and measurement of store-operated calcium entry. The main advantage of this technique is that it allows a rapid, simple and reproducible isolation of human primary myoblasts.These myoblasts are the new ex-vivo to study the calcium signaling during muscle regeneration. This method can help answer key questions in the muscle field such as, identifying the ionic channels and the transcription factors involved during muscle differentiation. For this method can provide insight into gene functions during human myogenic differentiation.It can also be applied to other studies such as, potential for using myogenic stem cells to treat muscular dystrophies. Transfer the muscle sample into a sterile six centimeter plate, wash the sample with PBS twice. Remove any remaining adipose tissue and weigh the sample.Next, add trypsin and mince the muscle into pieces smaller than two millimeters in size. Once minced, use a 10 milliliter pipette to transfer the tissue into a 200 milliliter dissociation bottle containing a stir bar, then continue collecting minced tissue in trypsin solution until the dissociation bottle is filled to 90 milliliters. Now, incubate the tissue in a water bath at 37 degrees celsius for 60 minutes with gentle stirring.After an hour, add 10 milliliters of FCS to stop the reaction and homogenize the mixture using trituration. Now, load 70 micron strainers onto two 50 milliliter tubes and pass half of the cell suspension through each strainer by pipetting the suspension into the filters. Next, make a suspension of the cells in growth medium and plate 200, 000 cells onto six centimeter plates with four milliliters of medium.Incubate the plates, changing the medium every third day. After five to seven days, the cells should be 70%to 80%confluent, then, stain them. To begin, rinse the plate of confluent cells with PBS.Then, add one milliliter of trypsin solution and let the enzyme work for three minutes. Stop the reaction by adding two milliliters of medium and collect the cells in a 15 milliliter tube. Then, spin the cells down and re-suspend them in one milliliter of medium.Next, take a cell count while storing the cells on ice. For the facts analysis, load seven tubes with 100, 000 cells each. Load the remaining cells into an eighth tube for sorting.Wash the cells with one milliliter of cold fax buffer, then, centrifuge the tubes and aspirate and discard the supernatant. Add back 200 microliters of fax buffer with the appropriate antibodies and let the cells incubate on ice for 30 minutes. After 30 minutes, wash the cells again and re-suspend them in 500 microliters of fax buffer, then sort the cells using a flow cytometer.After excluding the CD45 positive hematopoietic cells, separate the CD45 negative cells based on CD56 expression. Then, gate the CD56 positive population for CD34, CD144 and CD146. Re-analyse a fraction of the sorted population, to check the purity.Now, pull the human myoblasts and wash them with five milliliters of medium. Then re-suspend them in one milliliter of medium. Transfer the suspension to a six centimeter plate with four milliliters of medium and incubate the plate at 37 degrees celsius with 7%carbon dioxide.For this procedure, have cultured myoblasts and make fresh transvection medium. Incubate the medium at room temperature for 15 to 20 minutes. During the medium incubation, prepare the myoblasts.First, rinse them once with PBS. Then, trypsinise the adherent cells. Stop the reaction by adding two milliliters of medium.Then, collection the cells into a 50 milliliter tube. Spin them down and re-suspend them in one milliliter of medium. For each transvection, prepare 500, 000 cells in 200 microliters of medium.To each 500 microliters of transvection medium, add a 200 microliter aliquot of cells and then mix using only two strokes of the pipette gun. Then, let the reactions proceed for five minutes at room temperature. After five minutes, transfer the reactions to 3.5 centimeter culture dishes, each containing one glass cover slip.Next, add 1.3 milliliters of medium for a final volume of two milliliters and incubate the dishes. After 48 hours of differentiation, the cells can be immunostained or analyzed using calcium imaging. For either option, first carefully wash the cells twice using one milliliter of room temperature PBS to wash.Try to avoid detaching the myotubes. For a calcium imaging, cover the washed cells with two micromolar fura-2 AM plus one micromolar threonic acid in calcium containing medium. Then, let them incubate for about 30 minutes before mixing.Next, wash the cells twice with unmodified calcium containing medium and then, incubate them for 10 to 15 minutes for the esterification. Now, remove the cover slip from a culture plate and install it into the experimental chamber. Load the chamber with calcium containing medium, place it under a microscope, then, start the data recording.Fura-2 is excited alternately at 340 and 380 nanometers. The emission light is collected at 510 nanometers. Diving the 340 nanometer nanometer images by the 380 images, provides the image ratios as the fluctuations and calcium concentration are relatively low, acquire one image ratio every two seconds.After two or three minutes, replace the medium with calcium free medium. Then, for one or two minutes, monitor the activity of the store operated calcium in the cells. Next, add one micromolar thapsigargin to the cells to deplete their internal calcium stores.Wait eight to 10 minutes, then, rapidly replace the medium with the calcium containing medium and record for five minutes while calcium enters the cells via the suc e channels. After terminating the experiment, analyze the images. Select an area where there are no cells for region one, which defines the background, then, select an area in the cytoplasm for region two.The two regions can be drawn on any of the images in the series. Next, go to the run experiments tab and select reference images for the 340 and 380 channels, select region one, then click on subtract background. Now, run the entire experiment to remove the background from all the images.Once completed, click log data option to open a spreadsheet in which the time and the ratio data are logged. Muscle cells were grown up, expanded and sorted using fax. Myoblasts represented more than 60%of the analyzed population.The primary human myoblasts were then cultured in differentiation medium and immunostained for the transcription factor of MEF2 and myosin heavy chain proteins. A majority of cells expressed myosin heavy chain and formed myotubes with fusion index values of about 60%the other 40%of the cells remained undifferentiated mononucleated cells, also known as reserve cells. The described store operated calcium entry experiment were successfully performed on the differentiated cells.Performing the same experiment to on cells transvected with small interfering RNA, directed against calcium permeable channels of the TRPC family, shows that the knockdown had a measurable impact on store operated calcium entry. After watching this video, you should have a good understanding of how to isolate human myoblasts from adult skeletal muscle. We use these cells to study the successive steps of muscle differentiation and in particular, deactivation of the transcription factors and calcium channels.Once mastered, this technique can be done in less than three hours if it is performed properly. It is a rapid, simple, reproducible method that obtains the yield of human myoblasts. While attempting this procedure, it is important to prevent the myoblast cultures from reaching full confidence, as there will be unwanted spontaneous differentiation.

Research

JoVE Journal

Developmental Biology

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

Isolement et quantitative immunocytochimique caractérisation des cellules myogéniques primaires et fibroblastes de muscle squelettique humain

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

Recherche

Biologie du développement

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse des Muscle intégrité poisson zèbre larves squelettique avec colorant bleu Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

La différenciation de la SH-SY5Y neuroblastome humain Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Isolement et différenciation des cellules souches adipeuses de porcines sous-cutanée des tissus adipeux

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Préparation et culture de cellules Myogenic Précurseurs / myoblastes primaires de muscle squelettique des adultes et les humains âgés

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Réalisé la différenciation des progéniteurs hématopoïétiques primitives et définitif des cellules souches pluripotentes humaines

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Isolement, Culture et différenciation des cellules stromales de la moelle osseuse et les ostéoclastes progéniteurs de souris

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Différenciation efficace des cellules souches pluripotentes humaines en cellules hépatiques

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Isolement, Propagation et Expression de la protéine Prion au cours de la différenciation neuronale de cellules souches humaines pulpe dentaire

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...