1. Tissue Collection: <ol><li> Verwenden Mäusen zwischen 7 und 12 Wochen alt. In der Regel werden mindestens zwei Tiere verwendet: Die eine ist die Einrichtung der FACS einfarbige Kontrollen für Entschädigung und für die Fluoreszenz-minus-eins Isotypkontrolle Proben erforderlich. Die andere ist die eigentliche Probe für die Sortierung. </li><li> Euthanize die Mäuse durch CO 2-Inhalation oder nach einer Ethik-Protokoll der Wahl. Legen Mäuse auf einem Papiertuch, nach oben und Spray mit 70% Ethanol gründlich, um eine Kontamination mit der Maus Fell zu verhindern. </li><li> Heben Sie die Haut in der Bauchregion mit einer Pinzette, dann machen Sie einen kleinen Längsschnitt mit einer scharfen Schere. </li><li> Peel die beiden Hautlappen in entgegengesetzte Richtungen (oben und unten) vollständig aussetzen Hinterbein darunter liegenden Muskeln. </li><li> Excise das Muskelgewebe durch das Einfügen und Erweiterung der Schere an beiden Seiten der Tibia. </li><li> Wiederholen Sie diesen Vorgang für den Oberschenkelknochen, um das Muskelgewebe zu extrahieren. </li><li> Legen Sie alle herausgeschnitten Muskelgewebe in 60mm Petrischalen, mit einer Schale für das Gewebe von jeder Maus. </li><li> Wiederholen Sie die oben für alle Mäuse. </li></ol> 2. Distanzieren Tissue in einzelne Zellen durch enzymatische Verdauung: In diesem Abschnitt des Protokolls, verwenden Sie sterile Reagenzien und Instrumente und Arbeiten unter sterilen Bedingungen. <ol start="9"><li> Geben Sie 2 ml Collagenase II (Sigma, Kat. Nr. 6885, 500U / ml) und 20 ul von 250mm CaCl 2 Lager zu jedem Petri-Schale. </li><li> Reißen Sie das Muskelgewebe in kleine Stücke (ca. 1 mm 3) mit zwei Pinzetten (eine gezackte, eins mit 2 Zähnen, von Fine Science Tools, cat # 11051-10, 11154-10), aufmerksam zu entfernen und zu entsorgen, wie viel kontaminiertes Nicht-Muskelgewebe wie möglich. </li><li> Decken Sie die Petrischalen und inkubieren sie bei 37 Grad Celsius für 30 Minuten. </li><li> Rufen Sie die Petri-Schalen und ein 3-ml-Spritze Kolben gründlich Maische die Gewebestücke. Verwenden Sie einen Stempel für jede Probe, um Kontaminationen zu vermeiden. </li><li> Fügen Sie einige (~ 5 ml) sterilen kalten PBS zu jeder Petrischale und Transfer des Gewebes Schlamm in einen 50 mL Falcon-Röhrchen (spülen Sie die Petri-Schale bei Bedarf alle Gewebe zu erholen) </li><li> Shake the Falcon Röhrchen kräftig, füllen Sie das Röhrchen mit PBS, Zentrifuge @ 800rpm x 5 Minuten (Eppendorf Tischgerät Modell: 5810R), und saugen Sie den Überstand (3 Mal wiederholen) </li><li> 1ml Mischung von Collagenase D (Roche, Kat. Nr. 11088882001, 1,5 HE / ml) / Dispase II (Roche, Kat. Nr.: 04942078001, 2.4u / ml) und 10 &amp;mgr; l von CaCl 2 (250mm) in jedes Röhrchen, bei 37 Grad Celsius mit Rotation für eine Stunde. </li><li> Fügen Sie 5-10 ml kalte, sterile PBS, Wirbel und Pipette gründlich, um das Gewebe zu homogenisieren. </li><li> Füllen Sie Rohre mit PBS und gut mischen. </li><li> Übertragen Sie die Flüssigkeit in einen 40 um Zellsieb auf einem 50 mL Falcon-Röhrchen platziert, um herauszufiltern, die übrigen große Stücke von Gewebe. Teilen Sie jede Probe gleichmäßig in drei 50 mL Falcon-Röhrchen. Füllen Sie jedes der Röhrchen mit PBS zentrifugieren @ 1600rpm für 5 Minuten bei 8 Grad Celsius. </li><li> Überstand verwerfen und sammeln Zellen für die Färbung. </li></ol> 3. Antikörper-Färbung und Sortierung: In diesem Abschnitt des Protokolls, mit sterilen FACS und Sammlung Puffer und Arbeit unter sterilen Bedingungen. Kommerzielle Antikörper sind in der Regel als steril, wenn sie richtig gehandhabt. <ol start="20"><li> Für einzelne Farb-und Isotyp-Kontroll-Proben, resuspendieren der Probe in 1 ml FACS-Puffer und teilen die suspendierten Zellen in neun 1,5 ml pre-markierten Eppendorf-Röhrchen (100 ul pro Röhre). <ol><li> Richten Sie einfarbige Färbung mischt nach der folgenden Tabelle: ACHTUNG: Die endgültige Konzentration von Isotyp-Kontroll-Antikörper in der Fleck sollte, dass der primäre Antikörper sind sie die Kontrolle für übereinstimmen. Zum Beispiel, wenn anti-Sca-1 für 1 μg/10 6 Zellen verwendet wird, muss der entsprechende Isotyp in der gleichen Konzentration eingesetzt werden. <table border="1"><tbody><tr><td> Tube # </td><td> Name der Röhre </td><td> Firma </td><td> Cat # </td><td> Klon </td><td> Isotyp </td><td> Verdünnung </td></tr><tr><td> 1 </td><td> Non-Fleck </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> sigma </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> sigma </td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31-FITC CD45-FITC </td><td> eBioscience </td><td> 11-0311-85 </td><td> 390 </td><td> Rat IgG2a </td><td> 500 </td></tr><tr><td> Haus gemacht </td><td></td><td> I3 / 2 </td><td> Rat IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1-PECY7 </td><td> eBioscience </td><td> 25-5981-82 </td><td> D7 </td><td> Rat IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α-7 APC </td><td> Haus gemacht </td><td></td><td> R2F2 </td><td> Rat IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Set up Isotypkontrolle Färbung mischt anhand der folgenden Tabelle: <table border="1"><tbody><tr><td> Tube # </td><td> Name von Rohr </td><td> Hoechst </td><td> PI </td><td> CD31- FITC </td><td> CD45- FITC </td><td> Sca- PE-CY7 </td><td> A-7- APC </td><td> Rat- IgG2b- APC </td><td> Rat- IgG2a- pecy7 </td><td> Rat- IgG2a- FITC </td><td> Rat- IgG2b- FITC </td></tr><tr><td> 7 </td><td> Iso- CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> Iso- Sca- pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> Iso- α-7-APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li> Pipettieren der einzelnen Farb-und Isotopen-Kontrollfärbung mischt (wir normalerweise passen Sie die Konzentration von Antikörpern, so dass zwischen 5 und 10 l Färbung Mix zu jeder Probe hinzugefügt werden) in die entsprechenden Eppendorf-Röhrchen aus Schritt 20. Gut mischen und auf Eis inkubieren für 1 Stunde. </li></ol></li><li> Stain Zellen von Probe-Maus mit 800μL der Antikörper-Cocktail (unten) für FACS cell sorting. Gut mischen und auf Eis inkubieren für 1 Stunde. Antikörper-Cocktail: Hoechst, PI, CD31-FITC, CD45-FITC, SCA1-PECY7, α-7 APC ACHTUNG: Das optimale Verhältnis von Antikörpern gegen Zellen müssen durch individuelle Titration jeder Antikörper, da erhebliche Unterschiede zwischen den Chargen gibt bestimmt werden kann. Die optimale Menge an Antikörper verwendet werden sollte in μgs Antikörper pro 10 6 Target-Zellen exprimiert und konstant gehalten werden, wenn mit der gleichen Charge des Antikörpers. Klar, wenn die Zahl der Zielzellen nicht signifikant ändert zwischen den Experimenten, oder wenn die Färbung Band mit der Anzahl der Zellen skaliert wird, eine feste Verdünnung von jeder Antikörper können ebenfalls verwendet werden. </li><li> Wash: In ca. 1 ml FACS-Puffer zu jedem der 9 Röhren, fügen Sie etwa 20 mL FACS-Puffer, um das Probenröhrchen. </li><li> Zentrifugieren Sie die Eppendorf-Röhrchen @ 3000rpm für 5 Minuten (Eppendorf Tischzentrifuge, Modell: 5417C). Centrifuge das Probenröhrchen @ 1600rpm für 5 Minuten (Eppendorf Tischzentrifuge, Modell: 5810R). </li><li> Absaugen und den Überstand verwerfen aus allen Rohren. </li><li> Die Zellen in der FACS-Puffer (1 ml für jede der Steuerungen, 4ml für die Sortierung Probe), um Pipette Zellen in &quot;FACS-Röhrchen&quot; durch die Zelle Sieb Kappe (BD Falcon cat # 352235, 40 um Porengröße) zu entfernen restlichen Klumpen, kann Einfluss auf die Durchflusszytometer. </li><li> Richten Sie die FACS-Sorter mit einfarbigem Kontrollen und Isotypkontrollen. </li><li> Sammeln sortierten Zellen nach der folgenden Tabelle in einer 3,5-ml-Collection-Puffer (95% DMEM, 5% FBS) getrennt. Normalerweise, wenn die Ernte beide Hinterbeine, könnte ein einzelner Wildtyp-Maus ungefähr 150.000 myogenen Vorläuferzellen oder 100.000 adipogenen Vorläuferzellen, die Lebensfähigkeit von über 95%, wie von reanalyzing sortierten Zellen durch FACS gemessen am Ende des Verfahrens (Abbildung 1 Ertrag ) Definition von myogenen und adipogenen Vorfahren auf Oberflächenbeschmutzung Basis <table border="1"><tbody><tr><td> Antikörper / Fleck </td><td> Myogenen Vorläuferzellen (MP) </td><td> Adipogene Vorläuferzellen (AP) </td></tr><tr><td> Hoechst </td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31-FITC </td><td> - </td><td> - </td></tr><tr><td> CD45-FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1-PECY7 </td><td> - </td><td> + </td></tr><tr><td> α-7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. Transplantation: <ol start="28"><li> Centrifuge sortierten Zellen @ 1500rpm für 5 Minuten, entfernen Sie den Überstand und Transfer Zellen autoklaviert Eppendorf-Röhrchen (1,5 ml), spülen Zellen mit 500 ul sterilem PBS (Serum-freien!), Dann Zentrifuge @ 3000rpm für 5 Minuten. Unter Beibehaltung aller Reagenzien unter sterilen Bedingungen und Arbeiten in einer Gewebekultur Kapuze, resuspendieren myogenen Vorläuferzellen in PBS oder adipogenen Vorläuferzellen in Matrigel (BD cat # 354234) bei ca. 10 6 Zellen / ml. </li><li> Transplant myogenen Vorläuferzellen oder adipogenen Vorläuferzellen zu Empfängermäuse. <ol><li> Betäuben die Maus mit isoflouran nach Ihrer Institution Politik. </li><li> Shave das Haar um die Injektion Region, dann sterilisieren der Haut durch Reiben mit 70% Ethanol </li><li> Inject 20 &amp;mgr; l von myogenen Vorläuferzellen oder adipogenen Vorläuferzellen (= 20k-Zellen) mit einem 3 / 10 CC Insulinspritze (BD cat # 309300). </li></ol></li><li> Nach einer angemessenen Zeit (3 Wochen funktioniert gut, aber Muskelfasern Ausdruck Spender stammenden transgenen Marker sind in der Regel innerhalb einer Woche nach der Transplantation deutlich), betäuben den Empfänger Mäuse aus Schritt 29 durch intraperitoneale Injektion von 400 ul Avertin (Sigma Cat # T04840-2, 25 mg / mL), dann transkardial perfuse mit 50 ml PBS (enthält 10 uM EDTA) zuerst, gefolgt von 15 ml 4% PFA gefolgt. Sammeln Zielgewebe, post-fix mit 4% PFA über Nacht, wenn erforderlich, dann Transfer bis 20% Saccharose über Nacht. Embed in OCT, Gefrier-und Gefrier. </li></ol> 5. Repräsentative Ergebnisse: Wenn MP in Maus-Skelettmuskel transplantiert werden, sie leicht mit bereits vorhandenen Muskelfasern verschmelzen. Daher ist jede genetische Label in der Spenderzellen wird leicht erkennbar in den Fasern, die sie erhalten haben. Abbildung 2 zeigt ein Beispiel, wo die transplantierten Zellen exprimierten humanen alkalischen phostphatase zeigte histochemisch. Wenn AP transplantiert sind das Ergebnis hängt stark von der Umgebung der Transplantation Ort: bei transplantierten subkutan diese Zellen Herkunft geben Adipozyten und Myofibroblasten (siehe Abbildung 2). In vielen anderen Standorten, einschließlich Muskeln, sie nicht überleben, wenn Verfettung hat 3 induziert worden. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Sorting-Strategie zur Isolierung von Vorläuferzellen Populationen aus Muskelfleisch (A) Sorting Strategie:. Lebende Zellen wurden auf der Grundlage Vorwärtsstreuung und side scatter identifiziert. Hoechst-Färbung wurde verwendet, um kernlosen Schutt und Propidiumiodid (PI)-Färbung auszuschließen, um tote Zellen auszuschließen. Hämatopoetischen (CD45) und Endothelzellen (CD31)-Zellen wurden aus der Sortierweichen ausgeschlossen. Die α7 + Hoechst + PI - CD45 - CD31 - Scal - Teilmenge enthält alle myogenen Vorläuferzellen (MP). Die Scal + Hoechst + PI - CD45 - CD31 - α7 - Bevölkerung enthält adipogenen Vorläuferzellen (AP). (B) Fluoreszenz-minus-eins (FMO) Isotypkontrollen bestätigen die Spezifität der Färbung. (C) Reinheit überprüft der MP-und AP-Teilmengen nach dem Sortieren. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Repräsentative Ergebnisse nach MP-und AP-Transplantation. AP wurden subkutan (oberes Feld) transplantiert und MP intramuskulär (unten). In beiden Fällen entstand Spenderzellen aus einer Maus zum Ausdruck transgene menschliche alkalische Phosphatase, durch die braune Färbung identifiziert.

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <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+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

Das Ziel dieses Protokolls ist es, eine vernünftige Balance zwischen hohen Ausbeuten und hoher Rentabilität des gereinigten Zellen zu schlagen. Der wichtigste Schritt bei der Sicherstellung, dass gesunde Zellen gewonnen werden, ist, wie vorherzusehen war, die enzymatische Spaltung des Ausgangsmaterials Gewebe. Die Handhabung des Gewebes sollte besonders schonende, das ist schwer zu beweisen oder zu schätzen, auch in ein audiovisuelles Format. Ein weiterer Schlüsselfaktor ist die Länge des Verfahrens. Je länger es dauert, bis aus dem Spender zum Empfänger Tier gehen, desto geringer die Rentabilität und damit das Anwachsen Effizienz. Sollte es irgendwelche Fragen zu Wirtschaftlichkeit, die nicht sofort, indem man die Häufigkeit der PI positives Ereignis in der gereinigten Zellproben nach der Sortierung beantwortet, empfehlen wir, dass ein Grenzverdünnung Assay clonogenicity Maßnahme 3 durchgeführt wird. Typische Arten aus unbeschädigten Muskel routinemäßig Ertrag etwa 1 in 15-20 Zellen in der Lage zu initiieren myogene oder fibro / adipogenen Kolonien in vitro. Während praktisch 100% der Kolonien von Abgeordneten initiiert differenzierte Myotuben enthalten, enthalten nur etwa ein Drittel der Bienenvölker aus FAPs erhalten Adipozyten neben smooth muscle actin positive Myofibroblasten.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Collagenase II </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 U / ml </td></tr><tr><td> Collagenase D </td><td> Roche </td><td> 11088882001 </td><td> 1,5 U / ml </td></tr><tr><td> Displace II </td><td> Roche </td><td> 04942078001 </td><td> 2,4 U / ml </td></tr><tr><td> Zellsieb </td><td> BD </td><td> 352340 </td><td> 40 mu m </td></tr><tr><td> Tube mit Zellsieb </td><td> BD </td><td> 352235 </td><td> 40 mu m </td></tr><tr><td> Hoechst33342 </td><td> Sigma-Aldrich </td><td> B2261 </td><td> 2-5 ug / ml </td></tr><tr><td> CD31-FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td> Klon 390 </td></tr><tr><td> CD45-FITC </td><td> Hausgemachte </td><td></td><td> Klon I3 / 2 </td></tr><tr><td> SCA1-PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> Klon D7 </td></tr><tr><td> α-7-Integrin-APC </td><td> Hausgemachte </td><td> Erkundigen Sie sich bei Rossi Labor </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

Skelettmuskulatur enthält mehrere Vorläuferzellen Populationen unterschiedlicher embryonaler Herkunft und Entwicklungspotenzial. Myogenen Vorläuferzellen, in der Regel mit Wohnsitz in einem &quot;Satelliten-Zell-Position&quot; zwischen den myofiber Plasmamembran und der Laminin-rich Basalmembran, dass ensheaths es sind selbst erneuernden Zellen, die sich ausschließlich auf die myogenen Linie 1,2 begangen werden. Wir haben kürzlich eine zweite Klasse des Schiffes verbundenen Vorläuferzellen, die Myofibroblasten und weiße Fettzellen, die durch die effiziente Eingabe Proliferation Schäden reagiert und bietet trophische Unterstützung myogenen Zellen während der Geweberegeneration 3,4 erzeugen kann beschrieben. Eines der am meisten vertraut Assays auf die Entwicklungs-und regenerative Potential einer gegebenen Zellpopulation bestimmen stützt sich auf ihre Isolation und Transplantation 5-7. Zu diesem Zweck haben wir Protokolle für deren Reinigung mittels Durchflusszytometrie aus enzymatisch gespalten Muskeln, die wir in diesem Artikel skizziert werden optimiert. Die Bevölkerung erhält mit dieser Methode enthält entweder myogene oder fibro / adipogenen Kolonie-bildenden Zellen: keine andere Zelltypen sind in der Lage die Expansion in vitro oder in vivo überleben Lieferung. Wenn jedoch diese Populationen unmittelbar nach der Art der molekularen Analyse (zB qRT-PCR) eine im Auge behalten müssen, dass die frisch sortiert Teilmengen können andere Schadstoffe Zellen, die die Fähigkeit zur Bildung von Kolonien oder Pfropfung Empfänger Mangel enthalten, verwendet.

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

Verfahren zur enzymatischen Dissoziation Oberfläche Kennzeichnung und Reinigung mittels Durchflusszytometrie von fibro / adipogene und myogenen Vorläuferzellen aus murinen Skelettmuskel.

Reinigung von Vorläufern aus Skelettmuskeln

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

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.2K Aufrufe.  University of British Columbia. Verfahren zur enzymatischen Dissoziation Oberfläche Kennzeichnung und Reinigung mittels Durchflusszytometrie von fibro / adipogene und myogenen Vorläuferzellen aus murinen Skelettmuskel.

Serie: Purification of Progenitors from Skeletal Muscle

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

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

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

Adulten und embryonalen Skelettmuskel Microexplant Kultur und Isolation der Skelettmuskulatur Stem Cells

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

Forschung

Biologie

Mitochondrial Isolation from Skeletal Muscle

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

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

Mitochondriale Isolation von der Skelettmuskulatur

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

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.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

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

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

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

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

Laser-Schädigung der Zebrafisch Embryonale Skeletal Muscle

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

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

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

Isolation und Kultur der einzelnen Muskelfasern und ihre Satelliten aus adulten Skelettmuskel

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

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

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

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

Isolierung von Blutgefäß abgeleiteten multi Vorläufer von menschlichen Skelettmuskel

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

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

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

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

Isolierung und Charakterisierung von Exosomen aus Skelettmuskelfibroblasten

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

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Identifizierung, Isolierung und Charakterisierung von fibro-adipogenen Vorläufern (FAPs) und myogenen Vorläufern (MPs) in der Skelettmuskulatur der Ratte

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

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

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

Isolierung von Mitochondrien aus der Skelettmuskulatur der Maus für respirometrische Assays

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

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.