Die Autoren möchten sich Alison Logar für ihre hervorragende technische Unterstützung bei der Durchflusszytometrie bedanken. Diese Arbeit wurde durch Zuschüsse aus dem Department of Defense (JH), der Henry J. Mankin Stiftungslehrstuhl (JH) und des Ministeriums für Bildung und Wissenschaft der Republik Kasachstan (AS) unterstützt. CWC wurde zum Teil von der American Heart Association Promotionsstipendium (11PRE7490001) unterstützt. M.Corselli wurde von der California Institute for Regenerative Medicine Ausbildungsförderung (TG2-01169) unterstützt.

1. Muskelbiopsie Verarbeitung <ol><li> Erhaltung menschlichen Skelettmuskelbiopsie auf Eis in Dulbecco modifiziertem Eagle Medium (DMEM) mit 5% fötalem Rinderserum (FBS) ergänzt und 1% Penicillin-Streptomycin (P / S) während des Transports. </li><li> Nach dem Erhalt der Muskelbiopsie, entfernen Sie die Probe aus dem Transportbehälter und waschen Sie es zweimal in Phosphat-gepufferte Kochsalzlösung (PBS) mit 2% Antibiotikum-Antimykotikum-Lösung (A / A) unter sterilen Bedingungen zu ergänzen. </li><li> Entfernen Sie den beigefügten Fettgewebe und Bindegewebe mit steriler Schere und Pinzette in DMEM mit 2% A / A ergänzt. </li><li> Entfernen Sie den großen Blutgefäße unter Dissektionsmikroskop und anschließend schneiden die Muskelprobe in kleine Stücke (&lt;1 cm 2 groß). </li><li> Wahren Schnittmuskelstücke (&lt;5 g) in 20 ml Erhaltungsmedium (PM, DMEM, ergänzt mit 10% FBS und 1% P / S) bei 4 ℃ für bis zu 5 Tage. </li></ol> 2. Muskel DissociatIonen-und Zellisolation <ol><li> Am Tag der Zellisolation entfernen Muskelstücke aus PM und wasche zweimal in PBS, ergänzt mit 2% P / S. Um die Aufschlusslösung zu machen, fügen Typ-I, Typ-II-und Typ-IV Kollagenasen (100 mg / ml) frisch in Uhr. </li><li> Fein hacken und Muskelstücke mit steriler Schere und Pinzette mechanisch Hackfleisch in einer Petrischale mit einer kleinen Menge von PM, bis die Lösung durchläuft 10 ml serologische Pipette ohne Blutgerinnung. Entfernen der Rest Fettgewebe und Bindegewebe während dieses Prozesses. Verwenden Sie mindestens 8-10 Gramm erwachsenen Muskel-oder 2 Gramm fetalen Muskel für jede Zelle Isolation. </li><li> Übertragen 4-5 Gramm Hackfleisch erwachsenen Muskel zu 20 ml (oder 2 Gramm Hackfleisch fötalen Muskel in 10 ml) OFTHE Aufschlusslösung mit 10 ml serologische Pipette. Verdauen für 50-60 min bei 37 ℃ auf einem Orbitalschüttler bei 70 bis 80 Umdrehungen pro Minute. Optimierung der Zellausbeute und Oberflächenantigen Konservierung durch Einstellen der Kochzeit und / oder Bewegungsgeschwindigkeit entsprechend der Menge basierendGewebe. Beachten Sie die Verdauung Status alle 15-20 min, bis die Trübung fast löscht. </li><li> Kräftig pipettieren Die verdaute Gewebe 3-5 mal mit einer 10 ml serologische Pipette. Hinzufügen gleiche Menge PM um die Reaktion abzubrechen, und dann bei 400 × g zentrifugieren für 4 min. </li><li> Entfernen Sie vorsichtig den Überstand; das Pellet mit 10 ml PM zu waschen, und dann durch ein 100 um Sieb filtriert Zelle. </li><li> Zentrifuge bei 400 g für 4 min und entfernen Sie vorsichtig den Überstand. Resuspendieren Pellets in Erythrozyten Lysepuffer (155 mM NH 4 Cl, 10 mM KHCO 3, 0,1 mm EDTA), filtriert durch ein 70 um Sieb Zelle um eine Einzelzellsuspension zu erhalten, und dann für 10 min bei RT inkubiert. Filtriert durch ein 70-m Zelle Sieb wieder, wenn eine Fällung beobachtet. </li><li> Zentrifuge bei 400 × g für 4 min und Resuspendieren des Zellpellets in 0,5 ml PBS. Die Anzahl der Zellen. Abrufen einer Gesamtmenge von mindestens 5 Millionen Zellen für die Zellsortierung. Verdünnen Sie die einze Zellsuspension auf weniger als 5 Millionen Zellen pro ml mit PBS-Färbung. Nehmen Sie 50-100 ul der Zellsuspension und aufgeteilt in 11 Rohre für Steuer Färbungen (ungefärbten Kontrolle, Negativkontrollen und einfarbige positive Kontrollen). </li></ol> 3. Zellmarkierung und Sortieren <ol><li> Inkubieren der Einzelzellsuspension für 10 min bei 4 ℃ in Mausserum (verdünnt 1:10 in PBS) zur Blockierung Zweck, falls erforderlich. </li><li> Fügen CD34-APC, CD45-APC-Cy7, CD56-PE-Cy7, CD144-PE, und CD146-FITC (alle 1: 100) in den Einzelzellsuspension und Inkubation für 20 min bei 4 ℃. Für negative Kontrolle, fügen äquivalenten Konzentrationen von APC-APC-Cy7-, PE-Cy7-, PE-und FITC-konjugierten Isotyp IgG-Antikörper und Inkubation für 20 min bei 4 ℃. Für einfarbige positive Kontrollen, fügen äquivalenten Konzentrationen von CD34-APC, CD45-APC-Cy7, CD56-PE-Cy7, CD144-PE, und CD146-FITC individuell in jedes Röhrchen und Inkubation für 20 min bei 4 ℃. </li><li> Nach Inkubation, Zentrifuge bei 400 g für 4 min zu waschen. Zellpellet in werden 1 - 2 ml DMEM mit 5% FBS und 1% P / S ergänzt. Die Endkonzentration der Zellsuspension sollte weniger als 5 Millionen Zellen pro ml sein. Hinzufügen 7-AAD (1: 100) und die Proben für 15 min bei RT tote Zelle Ausgrenzung. Für Negativkontrolle und Single-Farbpositivkontrollen, resuspendieren Zellpellets in 0,5 ml DMEM mit 5% FBS und 1% P / S ergänzt. </li><li> Übertragen Sie alle Zellsuspensionen auf Polystyrol-Rundröhrchen für die Durchflusszytometrie. Bereiten Zellröhrchen (jedes Rohr ist vorgefüllt mit 500 ul der geeigneten Kulturmedium: MPM für MEC; EGM-2 für PC, AC-Medium für ACS). Transportzellsuspensionen auf Eis auf die Zellsortierer. </li><li> Führen Zellsuspensionen auf der Zellsortierer in der Reihenfolge der ungefärbten Kontrolle, Negativkontrollen, einfarbig Positivkontrollen, und der Hauptzellsuspension während der Anpassung der Laserintensität, Kanalkompensation und Zellpopulation Gating stringent zu Zelle p maximierenurity (siehe Artikel beim Zeus und andere Fachzeitschriften für Details der Durchflusszytometrie veröffentlicht wurde). </li><li> Sammeln gewünschten Zellpopulationen in den entsprechenden Sammelröhrchen. Shop gesammelten Zellen bei 4 ℃, wenn nicht sofort der Aussaat. </li></ol> 4. Post-Sortierzellkultur <ol><li> Saatgut frisch sortiert MEC (P0) auf &lt;10.000 Zellen / cm 2 in MPM auf Platten mit vorge beschichtet Typ-I-Kollagen. Für die anschließende Passage von MEC, Saatgut 3500 - 4000 Zellen / cm 2 in MPM auf Platten / Flaschen mit Pre-beschichtet Typ-I-Kollagen. </li><li> Saatgut neu geordnet PCs (P0) in &lt;20.000 Zellen / cm 2 in EGM-2 auf Platten mit frisch 0,2% Gelatine beschichtet. 3-Verhältnis im PC-Medium (DMEM mit 20% FBS und 1% P / S ergänzt) auf regelmäßige Polystyrol-Kulturplatten bis P2: Für die anschließende Passage von PCs, bei 1 aufgeteilt Zellen. Von P3 Weiter, Samenzellen bei 6.500 - 7.000 Zellen / cm 2 in PC Medium auf regelmäßige Polystyrol-Kulturplatten / Kolben. </li><li> Saatgut neu geordnet ACs (P0) in &lt;20.000 Zellen / cm 2 in AC-Medium (DMEM mit 20% FBS und 1% P / S ergänzt) auf regelmäßige Polystyrol-Kulturplatten. Für die anschließende Passage von ACS, Samenzellen bei 6.500 - 7.000 Zellen / cm 2 in AC-Medium auf regelmäßige Polystyrol-Kulturplatten / Kolben. </li></ol>

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J.H. received remuneration from Cook MyoSite, Inc. for consulting services and for royalties received from technology licensing during the period that the above research was performed. All other authors have no conflict of interest to disclose.

Identifizierung und Reinigung von hBVSC Subpopulationen stellen einen wesentlichen Fortschritt in das Verständnis der MSC Ontogenese. Es gibt zunehmend Hinweise, die den Ursprung der perivaskulären MSCs und die Verbindung zwischen Gewebe-spezifischen Vorläuferzellen und Blutgefäße 22-25. Darüber hinaus ist die Fähigkeit, homogene Subpopulationen von hBVSCs weiter hilft dem Verständnis der MSC Heterogenität und Vaskuläre Zellbiologie 26 zu isolieren. In den ver...

Menschlichen Skelettmuskel wurde als klinisch attraktive Quelle für Stammzellen / Vorläuferzellen. Skelettmuskulatur enthält nicht nur verpflichtet myogenen Vorläuferzellen, Skelett-Myoblasten, aber auch primitive myogenen Stammzellen, einschließlich Satellitenzellen und Muskelstammzellen (MDSCs) 1. Die Verwendung von menschlichen Muskel-abgeleitete Stammzellen / Vorläuferzellen, autologen oder allogenen, in der regenerativen Medizin ist weitgehend in präklinischen Tiermodellen und klinischen Studien untersucht. Die regenerative Anwendungen von Muskelstammzellen / Vorläuferzellen reichen von Regeneration des dystrophischen Muskel in Duchenne-Muskeldystrophie (DMD) Patienten auf die Reparatur des verletzten Herzens bei Patienten mit Herzinfarkt. Seit der Entdeckung von mesenchymalen Stammzellen / Stromazellen (MSCs) und anderen multiVorläuferZellPopulationen, einschließlich Knochenmark stammenden multipotente adulte Vorläuferzellen (MAPCs) und Fettgewebe gewonnene Stammzellen (ADSCs), adulte Stammzellen /Vorläuferzellen sind umfassend auf den neuesten Stand 1-9 untersucht. Dennoch haben ihre Mutter Identität und Lokalisation in situ durch die Retrospektive Isolationsverfahren verdunkelt. Kürzlich wurde unter Verwendung von Fluoreszenz-aktivierter Zellsortierung (FACS), haben wir und andere Gruppen haben prospektiv identifiziert und gereinigt drei multiVorläuferZellPopulationen aus den Blutgefäßen im menschlichen Skelettmuskel und verschiedene andere Organe: myogenen Endothelzellen (MEC), Perizyten (PCs), und Adventitia-Zellen (ACS) 10. Intima, Tunica media und Adventitia: Diese drei Subpopulationen von menschlichen Blutgefäßstammzellen (hBVSCs) jeweils in den drei Strukturschichten der Blutgefäße gefunden werden. Genauer gesagt, MEC und PCs sind in Mikrogefäßen und Kapillaren nachgewiesen während ACS sind in der Adventitia Schicht von größeren Arterien und Venen lokalisiert. Jeder Vorläuferzellenteilsatz drückt eine einzigartige Kombination von Zelloberflächenantigenen: MEC (CD34 + / 56 + / 144 + / 45 -), PC (CD146 + / 34 - / 45 - / 56 -), und ACS (CD34 + / 31 - / 45 - / 56 - / 146 -). Weitere Charakterisierung dieser hBVSC Untergruppen zeigte, dass alle drei Vorläuferzellpopulationen besitzen mesodermalen Entwicklungspotentiale ähnlich typischen MSCs, einschließlich Skelett myogenesis, Osteogenesis, Chondrogenese und Adipogenese. Alle hBVSC Teilmengen weisen auch klassische MSC Marker, einschließlich CD44, CD73, CD90, CD105 und frisch und in der Kultur. Gemeinsam diese Beweisstücke unterstützte die vaskulären Ursprungs von MSCs. Darüber hinaus haben die therapeutischen Kapazitäten von MEC, PCs, und ACS kürzlich in verschiedenen Studien nachgewiesen. MEC aus erwachsenen menschlichen Muskelbiopsien sortiert wurden gezeigt, um verletzte und dystrophischen Skelettmuskeln und Reparatur verletzt Myokard effizienter als Skelettmyoblasten und Gefäß endo regenerierenthelial Zellen (ECs). Gereinigten PCs aus verschiedenen menschlichen Organen haben auch gezeigt, um zu reparieren / regenerieren verletzten und dystrophischen Skelettmuskulatur und zur Satelliten-Zellpool 13-16. Erst vor kurzem haben wir gezeigt, dass PCs aus menschlichen Skelettmuskel abgeleitet das Myokard durch indirekte und direkte parakrine Wirkung zellulären Interaktionen 17 effektiv zu reparieren. ACs, auf der anderen Seite, entweder direkt aus explantierten Blutgefäße oder durch FACS isoliert aus humanem Fettgewebe und Skelettmuskel gereinigt. Eine bemerkenswerte pro-angiogene Wirkung von ACS wurde in einem Maus-Hinter Extremitäten-Ischämie-Modell 19 demonstriert. Weiterhin haben ACs auch gezeigt, Myokard effizienter als herkömmliche MSCs reparieren, was die stabile therapeutische Potenzial von ACS in ischämischen Gewebereparatur 20. Die aktuellen Reinigungsprotokoll Zuschüsse gleichzeitige, Interessenten Reinigung von MEC, PCs undACs aus dem Gefäßsystem eines einzelnen menschlichen Skelettmuskelbiopsie. Dies ermöglicht es uns, zu studieren und / oder wählen Sie die optimale hBVSC Subpopulation für verschiedene therapeutische Zwecke. Darüber hinaus bietet diese neue Technik erweitert das Repertoire von Stammzellen / Vorläuferzellen, die aus menschlichem Skelettmuskel abgeleitet werden kann, so dass es eine ideale Quelle für multipotente Vorläuferzellen für die regenerative Medizin.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 1</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C5894</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 2</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1764</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 4</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1889</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD34 APC</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">555824</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD45 APC-Cy7</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">557833</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD56 PE-Cy7</td>
			<td style="width:99px;">BD Pharmingen&#160;</td>
			<td style="width:148px;">557747</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD144 PE</td>
			<td style="width:99px;">Beckman Coulter</td>
			<td style="width:148px;">A07481</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Anti-human CD146 FITC</td>
			<td style="width:99px;">AbD Serotec</td>
			<td style="width:148px;">MCA2141F</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">FACSAria II Flow Cytometer</td>
			<td style="width:99px;">Becton-Dickinson</td>
			<td style="width:148px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">EGM-2 Complete Medium</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:148px;">CC-3162</td>
			<td style="width:193px;">For culturing PCs (only P0)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11965</td>
			<td style="width:193px;">For culturing PCs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, with sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11995</td>
			<td style="width:193px;">For culturing MECs and ACs</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Fetal Bovine Serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">10437-028</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Heat-inactivated horse serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">26050-088</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15140-122</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15240-062</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Trypsin-EDTA 0.5%(10X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15400-054</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Dulbecco&#8217;s PBS without calcium and magnesium</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Chick embryo extract</td>
			<td style="width:99px;">Accurate Chemical</td>
			<td style="width:148px;">CE650T-10</td>
			<td style="width:193px;">Filter before use</td>
		</tr>
	</tbody>
</table>

isolation of blood-vessel-derived multipotent precursors from human skeletal muscle

Seit der Entdeckung von mesenchymalen Stammzellen / Stromazellen (MSCs), haben die einheimische Identität und Lokalisation von MSCs durch ihre Retrospektive Isolation in Kultur verdeckt worden. Kürzlich wurde unter Verwendung von Fluoreszenz-aktivierter Zellsortierung (FACS), haben wir und andere Forscher prospektiv identifiziert und gereinigt drei Subpopulationen von multipotenten Vorläuferzellen mit dem Gefäßsystem des menschlichen Skelettmuskel verbunden. Diese drei Zellpopulationen: Intima, Media und Adventitia: myogenen Endothelzellen (MEC), Perizyten (PCs) und Adventitia-Zellen (ACS), sind jeweils mit den drei Strukturschichten von Blutgefäßen lokalisiert. Alle diese menschliche Blutgefäßstammzellen (hBVSC) Populationen nicht nur klassische MSC Marker auszudrücken, sondern auch Entwicklungspotenziale mesodermalen ähnlich typischen MSCs besitzen. Bisher haben MEC, PCs und ACs durch verschiedene Protokolle isoliert und anschließend in separaten Studien aus. Die aktuelle Isolations ProtOcol, durch Änderungen an der Isolierungsverfahren und Anpassungen in den selektiven Zelloberflächenmarker, ermöglicht es uns, gleichzeitig reinigen alle drei Subpopulationen hBVSC durch FACS aus einer menschlichen Muskelbiopsie. Dieses neue Verfahren wird nicht nur rationalisieren die Isolierung von mehreren BVSC Subpopulationen sondern erleichtern auch die zukünftige klinische Anwendungen der hBVSCs für verschiedene therapeutische Zwecke.

FACS-Parameter werden zunächst auf der Grundlage der von den nicht markierten Kontrolle, Negativkontrollen und Single-Farbpositivkontrollen gewonnenen Daten korrigiert. Nach Ausschluß von toten Zellen wird die Fluoreszenz-markierte Zellsuspension auf eine Reihe von negativen und positiven Zelloberflächenmarker Auswahl unterworfen. Zuerst werden die CD45 +-Zellen vor CD56 + und CD56 gated - Zellen aus CD45 getrennt - Fraktion. Die CD56 +-Fraktion wird weiter auf C...

Watch this Scientific Journal Video about Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle at JoVE.com

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

Blutgefäße innerhalb der menschlichen Skelettmuskulatur Hafen mehrere Multi-Linie Vorläuferpopulationen, die sich ideal für die regenerative Anwendungen sind. Dieses Isolierungsverfahren ermöglicht die gleichzeitige Reinigung von drei multiVorläuferZellPopulationen jeweils drei Strukturschichten der Blutgefäße: myogenen Endothelzellen aus Intima, Perizyten von Medien und Adventitia-Zellen von 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-blood-vessel-derived-multipotent-precursors-from-human

Research

JoVE Journal

Biology

11.6K Aufrufe.  University of Pittsburgh.  Blutgefäße innerhalb der menschlichen Skelettmuskulatur Hafen mehrere Multi-Linie Vorläuferpopulationen, die sich ideal für die regenerative Anwendungen sind. Dieses Isolierungsverfahren ermöglicht die gleichzeitige Reinigung von drei multiVorläuferZellPopulationen jeweils drei Strukturschichten der Blutgefäße: myogenen Endothelzellen aus Intima, Perizyten von Medien und Adventitia-Zellen von Adventitia. 

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenzierung von embryonalen Stammzellen in Oligodendrozyten Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Forschung

Biologie

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 ...

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 ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Reinigung von Vorläufern aus Skelettmuskeln

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

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 Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Isolierung von Mitochondrien von minimalen Mengen an Maus-Skelettmuskel für die Hochdurchsatz-Mikroplatten-Atemmessungen

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Isolation of Intact Mitochondria von Skeletal Muscle durch differentielle Zentrifugation für hochauflösende Messungen Respirationstest

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolierung, Reinigung und Differenzierung von Osteoclast-Laufläufern aus der Rattenmarkierung

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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 ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolierung menschlicher ventrikulärer Kardiomyozyten aus Vibratome-Cut Myokardscheiben

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...