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

Questo lavoro è stato sostenuto dalla Fondazione nazionale svizzera della scienza (numero di sovvenzione 310030-166313), dalla &quot;Fondazione Suisse pour la recherche sur les maladies musculaires&quot;, dalla &quot;Fondazione Marcel Levaillant&quot; e dalla Fondazione per la recherche ostéo-articulaire.

I campioni muscolari umani (tessuti ottenuti da muscoli semitendinosi) sono stati raccolti durante la chirurgia ortopedica su pazienti sani come rifiuti chirurgici. Tutti i metodi relativi allo studio umano sono stati eseguiti secondo le linee guida e le regolamentazioni delle autorità svizzere di regolamentazione sanitarie e approvate dalla Commissione Cantonale d&#39;Etichetta de la Recherche delle autorità cantonali di Ginevra (protocollo CER n. 12-259) . Consensi informati e scritti sono stati ottenuti da tutti i ...

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	<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;isolamento e la cultura dei mioblasti umani dal muscolo scheletrico adulto offre un modello in vitro per studiare la differenziazione muscolare e la rigenerazione muscolare. In questo documento, forniamo un protocollo che consente la purificazione di elevate rese di mioblasti umane in modo semplice e costoso. Inoltre, questa tecnica fornisce risultati affidabili e riproducibili in termini di percentuale di mioblasti isolati e della loro efficacia di differenziazione myogen...

I muscoli scheletrici umani sono composti da gruppi di fibre muscolari contrattili e multinucleate derivanti dalla fusione di cellule precursori miogeniche. I muscoli scheletrici hanno la capacità di rigenerarsi dopo lesioni grazie alla presenza di SC, le cellule staminali del muscolo scheletrico situate tra la membrana plasmatica delle miofibere (sarcolemma) e la lamina basale. Nel muscolo non sinistro, gli SC sono prevalentemente presenti in uno stato quiescente. In risposta a stress meccanici o lesioni, le SC si attivano (myoblasti), si proliferano e subiscono differenze per formare nuove miofibere o auto-rinnovamento per ricostituire il pool SC 1 , 2 . Nel corso degli anni sono state sviluppate diverse tecniche per isolare SC e la loro progenie, i mioblasti, dalle biopsie muscolari dello scheletro. Con la maggiore comprensione dei marker di superficie delle cellule espressi su queste cellule e la metodologia della classificazione delle cellule attivata con fluorescenza (FACS) 3, 4 , 5 , è ora possibile isolare popolazioni puro di mioblasti umane dai campioni muscolari. La segnalazione del calcio regola lo sviluppo muscolare scheletrico, l&#39;omeostasi e la rigenerazione. In particolare, la voce Ca 2+ attivata dopo l&#39;esaurimento intracellulare del negozio, chiamata SOCE, è di grande importanza 6 per questi processi. Con la stimolazione cellulare, il livello Ca 2+ nel reticolo endo / sarcoplasmatico (ER / SR) diminuisce, che a sua volta attiva il canale Ca 2+ delle membrane plasmatiche che consentono l&#39;ingresso di Ca 2+ per riempire l&#39;ER / SR 7 . Le due principali proteine ​​coinvolte nella SOCE sono la proteina di interazione stromale 1 (STIM1) ER / SR Ca 2+ e la membrana plasmatica Ca 2+ canale Orai1. Il muscolo scheletrico esprime abbondantemente queste due proteine, così come altre proteine ​​delle stesse famiglie (STIM2 aNd Orai2-Orai3) 8 , 9 e diversi canali permeabili Ca 2+ della famiglia canonica potenziale recettoriale transiente (TRPC) 10 , 11 , 12 , 13 . La penetrazione Ca 2+ attraverso il percorso SOCE è di grande importanza per la formazione / rigenerazione muscolare 14 . Le mutazioni delle proteine ​​STIM1 o Orai1 hanno effetti deleteri sulla funzione muscolare, determinando principalmente l&#39;ipotonia muscolare 15 . Modelli animali con insufficienza SOCE mostrano anche ridotta massa muscolare e della forza, insieme con una maggiore affaticabilità 6, 16, 17. Come già accennato, altre proteine ​​di STIM e Orai, così come molti canali TRPC, sono espresse in muscoli scheletrici, ei loro rispettivi ruoli non sono stati ancora determinati. Colpendo dPossiede il loro livello di espressione, è quindi possibile esaminare le loro implicazioni nella SOCE e nei loro ruoli durante la differenziazione muscolare scheletrica umana. La misurazione SOCE può essere effettuata utilizzando due approcci diversi: registrazioni elettrofisiologiche di corrente e misurazioni Ca 2+ . L&#39;elettrofisiologia è sicuramente un metodo più diretto, in quanto misura la corrente dell&#39;interesse e la relativa firma elettrofisiologica. Tuttavia, questa tecnica è molto difficile applicarsi alle cellule muscolari, soprattutto a causa della grande dimensione delle cellule muscolari e della piccola dimensione della corrente SOCE endogena. L&#39;imaging Cytosolic Ca 2+ è tecnicamente molto accessibile, ma la misura è più indiretta, poiché il livello Ca 2+ misurato nel citosol riflette sia l&#39;ingresso di Ca 2+ che il riapprocco della cellula o nei negozi interni. Una metodologia che prevede l&#39;isolamento di mioblasti da piccoli pezzi di scafo umanoIl muscolo etale dopo la digestione enzimatica, l&#39;amplificazione e la FACS è spiegato in questo articolo. Il processo di differenziamento muscolare e il protocollo immunofluorescenza per seguire l&#39;espressione dei marcatori di differenziazione nel tempo sono descritti 18. Infine, si spiega la misurazione del SOCE e il ruolo dei differenti canali ionici nella distinzione del segnale di Ca 2+ e della differenziazione muscolare scheletrica.

<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

Le cellule satellitari (SC) sono cellule staminali muscolari situate tra la membrana plasmatica delle fibre muscolari e la lamina basale circostante. Sono essenziali per la rigenerazione muscolare. A seguito di lesioni, che si verificano spesso nei muscoli scheletrici, vengono attivati ​​SC. Si proliferano come mioblasti e si differenziano per riparare le lesioni muscolari. Tra molti eventi che si verificano durante la differenziazione muscolare, i segnali citocholic Ca 2+ sono di grande importanza. Questi segnali Ca 2+ derivano da Ca 2+ rilascio dalla interne Ca 2 + negozi, nonché da Ca 2+ voce dallo spazio extracellulare, in particolare il negozio azionato Ca 2+ entrata (Soče). Questo documento descrive una metodologia utilizzata per ottenere una pura popolazione di mioblasti umani provenienti da campioni muscolari raccolti dopo la chirurgia ortopedica. Il tessuto viene digerito meccanicamente ed enzimatico e le cellule vengono amplificate e poi ordinate mediante citometria a flusso in funzione della presenza di speciMarcatori a membrana fic. Una volta ottenuto, i mioblasti umani si espandono e si impegnano a differenziarsi rimuovendo i fattori di crescita dal mezzo di coltura. I livelli di espressione di fattori specifici di trascrizione e di immunofluorescenza in vitro vengono utilizzati per valutare il processo di differenziazione miogenica nelle condizioni di controllo e dopo la silenziazione delle proteine ​​coinvolte nella segnalazione Ca 2+ . Infine, abbiamo dettagliato l&#39;uso di Fura-2 come una sonda ratiometrica Ca 2+ che fornisce misure affidabili e riproducibili di SOCE.

Dopo la dissociazione enzimatica di un campione muscolare umano, le cellule sono state amplificate in GM. I mioblasti umani, definiti come cellule CD56 + / CD146 + / CD45- / CD34- / CD144-, sono stati ottenuti dopo FACS. Myoblasti rappresentavano più del 60% della popolazione analizzata ( Figura 1 ). I mioblasti umani primari sono stati riversati, coltivati ​​alla confluenza e coltivati ​​in DM per 48 h. Dopo l&#39;immunostaining per l&#39;espressione del fattore di trascrizione ME...

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

Qui descriviamo una metodologia per ottenere una popolazione pura di mioblasti umani dal tessuto muscolare adulto. Queste cellule sono utilizzate per studiare la differenziazione muscolare scheletrica in vitro e, in particolare, per studiare le proteine ​​coinvolte nella segnalazione Ca 2+ .

Isolamento dei mioblasti umani, valutazione della differenziazione myogenica e misurazione dell&#39;entrata del calcio a livello di deposito

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.

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