עבודה זו נתמכה על ידי קרן המדע הלאומית השוויצרית (מענק מס &#39;310030-166313), &quot;The Fondation Suisse pour la recherche sur les maladies musculaires&quot;, &quot;Foundation Marcel Levaillant&quot; ו- &quot;Fondation pour la recherche ostéo-articulaire&quot;.

<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>

למחברים אין אינטרסים פיננסיים מתחרים.

הבידוד והתרבות של myoblasts האדם מן שריר השלד המבוגר מציע מודל במבחנה ללמוד בידול שרירים התחדשות שרירים. במאמר זה, אנו מספקים פרוטוקול המאפשר טיהור של תשואות גבוהות של myoblasts האדם בצורה פשוטה וחסכונית. בנוסף, טכניקה זו מספקת תוצאות אמין לשחזור במונח?...

שרירי השלד האנושיים מורכבים מקבוצות של סיבי שריר רב-תכליתיים, סינתטיים, הנובעים מאיחוי של תאים מבשר מיוגניים. שרירי השלד יש את היכולת להתחדש לאחר הפציעה תודה לנוכחות של SCS, תאי גזע שריר השלד הממוקם בין קרום הפלזמה של myofibers (sarcolemma) ואת הלמידה הבסיסית. בשרירים ללא פגע, SC הם בעיקר נוכחים במצב שקט. בתגובה ללחץ מכני או לפציעה, SC הופכים לפעילים (myoblasts), מתרבים, ועוברים תהליך הבחנה כדי ליצור מיופיבים חדשים או חידוש עצמי כדי לחדש את מאגר ה- SC 1 , 2 . במהלך השנים, מספר טכניקות פותחו כדי לבודד SCs הצאצאים שלהם, myoblasts, מן ביופסיות שריר השלד. עם הבנה גדולה יותר של פני השטח סמנים תאים הביע על תאים אלה ואת המתודולוגיה של מינון תא מופעלת הקרינה (FACS) 3, 4 , 5 , עכשיו ניתן לבודד אוכלוסיות טהור של myoblasts האדם מדגימות שרירים. סידן איתות מסדיר את התפתחות שריר השלד, הומיאוסטזיס, התחדשות. בפרט, הכניסה Ca 2 + המופעל בעקבות דלדול החנות תאיים, הנקראים SOCE, הוא בעל חשיבות רבה 6 עבור תהליכים אלה. לאחר גירוי התא, רמת Ca 2 + ב reticulum endo / sarcoplasmic (ER / SR) פוחתת, אשר בתורו מפעילה ממברנה פלזמה Ca 2 + ערוצים המאפשרים Ca 2 + כניסה למלא את ER / SR 7 . שני חלבונים עיקריים המעורבים SOCE הם ER / SR Ca 2 + -חישה מולקולת אינטראקציה סטרומה 1 (STIM1) חלבון הממברנה פלזמה Ca 2 + ערוץ Orai1. שריר השלד מבטא בשפע את שני החלבונים הללו, כמו גם חלבונים אחרים של אותן משפחות (STIM2Ora Orai2-Orai3) 8 , 9 ו כמה קה 2 + ערוצי לערוץ של קולטן חולף פוטנציאלי (TRPC) משפחה 10 , 11 , 12 , 13 . Ca 2 + כניסה דרך מסלול SOCE הוא בעל חשיבות רבה עבור היווצרות שריר / התחדשות 14 . מוטציות של חלבונים STIM1 או Orai1 יש השפעות מזיקות על תפקוד השרירים, המוביל בעיקר לשריר היפוטוניה 15 . מודלים בעלי חיים עם ליקוי SOCE גם להציג מסת שרירים מופחת כוח, יחד עם עייפות משופרת 6 , 16 , 17 . כאמור, חלבונים אחרים של STIM ו- Orai, כמו גם ערוצי TRPC רבים, מתבטאים בשרירי השלד, ותפקידים אלה לא נקבעו עד כה. על ידי דפיקות דמשלו רמת הביטוי שלהם, ולכן ניתן לחקור את ההשלכות שלהם ב- SOCE ואת תפקידיהם במהלך בידול שריר השלד האנושי. המדידה SOCE ניתן לבצע באמצעות שתי גישות שונות: הקלטות הנוכחי אלקטרו ו Ca 2 + מדידות. Electrophysiology הוא בהחלט שיטה ישירה יותר, כפי שהיא מודדת את הנוכחי של עניין וחתימה אלקטרופיזיולוגי הקשורים אליו. עם זאת, טכניקה זו קשה מאוד להחיל על תאי השריר, בעיקר בגלל גודל גדול של תאי שריר וגודל קטן של הנוכחי SOCE אנדוגני. Cytosolic Ca 2 + הדמיה היא טכנית מאוד נגיש, אבל המדד הוא יותר עקיף, כמו Ca 2 + רמת נמדדת cytosol משקף הן את כניסת Ca 2 + ואת שאיבה מחדש מתוך התא או בחנויות הפנימיות. מתודולוגיה הכוללת בידוד myoblasts מ חתיכות קטנות של skel האדםשריר etal לאחר עיכול אנזימטי, הגברה, FACS מוסבר במאמר זה. תהליך של בידול שרירים ואת פרוטוקול immunofluorescence לעקוב אחר הביטוי של סמנים בידול לאורך זמן מתוארים 18 . לבסוף, המדידה של SOCE ואת התפקיד של ערוצי יון שונים Ca 2 + איתות והבחנה שריר השלד מוסברים.

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

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

תאי הלוויין (SC) הם תאי גזע שרירים הממוקמים בין קרום הפלזמה של סיבי השריר לבין הלמינה הבסיסית. הם חיוניים עבור התחדשות שריר. עם פגיעה, אשר מתרחשת לעתים קרובות בשרירי השלד, SCs מופעלים. הם מתרבים כמו myoblasts ולהבדיל לתקן נגעים שרירים. בין אירועים רבים המתרחשים במהלך בידול השריר, cytosolic Ca 2 + אותות הם בעלי חשיבות רבה. אלה Ca 2 + אותות נובעים Ca 2 + שחרור מ הפנימי Ca 2 + חנויות, כמו גם מ Ca 2 + כניסה מהחלל תאיים, במיוחד את החנות המופעלות Ca 2 + כניסה (SOCE). מאמר זה מתאר מתודולוגיה המשמשת כדי להשיג אוכלוסייה טהורה של myoblasts האדם מדגימות שריר שנאספו לאחר ניתוח אורתופדי. הרקמה היא מתעכל מכנית אנזימטית, ואת התאים הם מוגבר ולאחר מכן מיון על ידי cytometry הזרימה על פי נוכחות של speciסמני קרום. לאחר שהושג, myoblasts האדם מורחבים ומחויבים להבדיל על ידי הסרת גורמי גדילה מן המדיום התרבות. רמות הביטוי של גורמים שעתוק ספציפיים ב immunosluorescence חוץ גופית משמשים כדי להעריך את תהליך בידול myogenic בתנאי שליטה לאחר השתקת חלבונים המעורבים Ca 2 + איתות. לבסוף, אנו מפרטים את השימוש Fura-2 כמו ratiometric Ca 2 + בדיקה המספק מדידות אמין לשחזור של SOCE.

לאחר הדיסוציאציה האנזימטית של דגימת שריר אנושית, התאים גדלו ב- GM. מיובלסטים אנושיים, שהוגדרו כתאי CD56 + / CD146 + / CD45- / CD34- / CD144-, התקבלו לאחר FACS. Myoblasts ייצג יותר מ 60% של האוכלוסייה נותחו ( איור 1 ). ראשי myoblasts האדם הוחזרו, גדל המפגש, ותרבותי DM במשך 48 שעות. לאחר immunosta...

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

כאן, אנו מתארים מתודולוגיה כדי להשיג אוכלוסייה טהורה של myoblasts האדם מן רקמת שריר בוגר. תאים אלה משמשים ללמוד בידול שריר השלד במבחנה , בפרט, ללמוד חלבונים המעורבים Ca 2 + איתות.

בידוד של Myoblasts האדם, הערכה של בידול Myogenic, ו-מופעלות סידן מופעל מדידה

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

isolation-human-myoblasts-assessment-myogenic-differentiation-store

Research

JoVE Journal

Developmental Biology

10.0K Views.  Geneva University Hospitals & Faculty of Medicine.  כאן, אנו מתארים מתודולוגיה כדי להשיג אוכלוסייה טהורה של myoblasts האדם מן רקמת שריר בוגר. תאים אלה משמשים ללמוד בידול שריר השלד במבחנה , בפרט, ללמוד חלבונים המעורבים Ca 2 + איתות. 

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

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

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

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

בידוד וכמוני Immunocytochemical אפיון של תאים ראשוניים myogenic וFibroblasts משרירי שלד אדם

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

In Vitro Differentiation of Mature Myofibers for Live Imaging

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

In Vitro Differentiation of Mature Myofibers for Live Imaging

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

במבחנת הבידול של בוגרות Myofibers עבור הדמיה חיה

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

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

בידוד והבידול של מכשולים ראשוניים מתוך שריר השלד של העכבר Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

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

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

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

הקרינה מבוסס מדידה הכניסה חנות המופעל הסידן בתאים חיים: מתא הסרטן תרבותי כדי סיב שריר שלד

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

Biology

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

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

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

בידוד ותרבות של Myofibers פרט ותאי הלווין שלהם משרירי שלד למבוגרים

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

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

אפיון פונקציונלי של גרסאות RYR1 אנושיות מבוטאות אנדוגניות

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Medicine

DNA Electroporation, Isolation and Imaging of Myofibers

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

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

DNA Electroporation, בידוד והדמיה של Myofibers

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

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

בידוד מיו-בריאלס מבריות מביופסיות שריר השלד וקביעת תפקוד מתכווץ עם מתמר כוח רזולוציה ננו-ניוטון

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

בידוד מיוציטים פרוזדוריים וחדריים באיכות גבוהה למדידות בו זמנית של Ca2+ Transients וזרם סידן מסוג L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

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

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

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

תפילת מיופיבר יחיד לבדיקה להערכת פונקציונליות תאי גזע שריר למבוגרים Ex Vivo

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