Name: isolation, culture, and transplantation of muscle satellite cells
Uploaded: 2014-04-08T18:00:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Ringraziamo il Dott. Shahragim Tajbakhsh per la fornitura di topi Myf5 + / nLacZ. Ringraziamo anche Alexander Hron e Michael Baumrucker per la lettura critica di questo manoscritto. Questo lavoro è stato sostenuto da finanziamenti della Muscular Dystrophy Association (MDA) e Gregory Marzolf Jr. MD Centro Award.

Gli animali sono stati alloggiati in un ambiente SPF e sono stati monitorati dalla Direzione Risorse ricerca su animali (RAR) dell&#39;Università del Minnesota. . Gli animali sono stati sacrificati con mezzi adeguati (CO 2 inalazione o iniezione KCl dopo essere stati anestetizzati con iniezione IP di Avertin (250 mg / kg) Tutti i protocolli sono stati approvati dalla cura degli animali ed uso Comitato Istituzionale (IACUC, numero di codice: 1304-30.492 ) dell&#39;Università del Minnesota. <p class="jove...

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In questo protocollo, cellule satelliti quiescenti possono essere facilmente purificati da adulto muscolo scheletrico di topi da digestione con collagenasi e separazione MACS mediata da anticorpi superficie. Questo metodo richiede circa 6 ore e non necessita di apparecchiature costose come una macchina FACS. Inoltre, questo metodo è relativamente poco costoso rispetto alla superficie di separazione FACS mediata da anticorpi. Una maggiore resa di cellule satelliti quiescenti si aspetta anche in confronto a FACS per ques...

Cellule satelliti muscolari sono una piccola frazione di cellule staminali miogeniche situati sotto la lamina basale delle fibre muscolari scheletriche. Essi sono caratterizzati dall&#39;espressione di Pax7, Pax3, c-Met, M-caderina, CD34, Syndecan-3, e calcitonina 1 - 3. Le cellule satelliti hanno dimostrato di essere responsabile della rigenerazione muscolare come cellule staminali muscolari. Nel muscolo adulto, cellule satelliti sono normalmente mitoticamente riposo 4-8. A seguito di infortunio, le cellule satellite sono attivati, avviare espressione di MyoD, e entrare nel ciclo cellulare per espandere la loro progenie, chiamato cellule precursori miogeniche o mioblasti 3. Dopo diversi cicli di divisione cellulare, mioblasti escono dal ciclo cellulare e fusibile tra loro al fine di differenziamento in miotubi multi-nucleate, seguite da fibre muscolari mature. Mioblasti isolati dal muscolo adulto possono essere facilmente espanse ex vivo. La capacità di mioblasti per diventare fibre muscolari in rigenerazione muscolare e perfibre muscolari ectopiche forma nei tessuti non muscolari è utilizzata da trapianto dei mioblasti, un potenziale approccio terapeutico per la distrofia muscolare di Duchenne (DMD) 4, disfunzioni urologiche 9, e insufficienza cardiaca 10. Infatti, mioblasti sono stati trapiantati con successo nel muscolo di entrambe mdx (modello DMD), topi e pazienti DMD 11-14. I mioblasti normali iniettati fondono con le fibre muscolari ospitante per migliorare l&#39;istologia e la funzione del muscolo malato. Il lavoro precedente ha dimostrato che sottopopolazioni di mioblasti sono più Stem Cell-like e rimangono in uno stato indifferenziato più a lungo nel muscolo durante la rigenerazione muscolare 5. Studi recenti hanno dimostrato che le cellule appena isolate satellitari di muscolo adulto contengono una popolazione di cellule simil-staminali che presenta attecchimento più efficiente e attività di auto-rinnovamento nella rigenerazione del muscolo 5-8. Pertanto, la purificazione di una popolazione pura di cellule satelliti quiescenti da adulto mu scheletricosclerosi è essenziale per la comprensione della biologia delle cellule satelliti, mioblasti e rigenerazione muscolare, e per lo sviluppo di terapie cellulari. Tuttavia, attuali metodi di purificazione potenziali di cellule satelliti quiescenti richiedono l&#39;uso di un costoso fluorescenza-attivato cell sorting (FACS) 1,2,6-8 macchina. Inoltre, l&#39;esposizione laser FACS tende ad indurre la morte cellulare durante la separazione, che causa bassa resa di cellule satelliti quiescenti 15. Qui, presentiamo un nuovo metodo per la purificazione rapido, economico e affidabile di cellule satelliti quiescenti di topo adulto muscolo scheletrico. Questo metodo utilizza dissociazione enzimatica seguita da cellule magnetico attivato classificare (MACS). Dopo aver isolato cellule satelliti quiescenti puri, queste cellule possono essere coltivate avere un gran numero di mioblasti dopo vari passaggi. Mostriamo anche che l&#39;iniezione intramuscolare di questi appena isolate cellule satellite quiescenti o ex vimioblasti vo espansi possono essere trapiantate in cardiotossina (CTX) indotta rigenerazione del mouse muscolo scheletrico per esaminare il contributo delle cellule derivate dal donatore alla rigenerazione delle fibre muscolari, nonché ai compartimenti cellulari satellitari per l&#39;esame delle attività di auto-rinnovamento.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Collagenase Type 2</td>
			<td>Worthington</td>
			<td>CLS-2</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Marigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen</td>
			<td>10569010</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>100 mg (3-4 mg/ml)</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>bFGF, human, Recombinant</td>
			<td>Gibco-Invitrogen</td>
			<td>PHG0263</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A5611-1G</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F10 Medium</td>
			<td>Gibco-Invitrogen</td>
			<td>11550-043</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>3600511</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco-Invitrogen</td>
			<td>26050088</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycin</td>
			<td>Gibco-Invitrogen</td>
			<td>15640055</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco-Invitrogen</td>
			<td>14190144</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco-Invitrogen</td>
			<td>25200072</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>18G needle with 12cc Syringe</td>
			<td>Fisher Scientific</td>
			<td>22-256-563</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#956;m)</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 ml tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10 ml disposable pipet</td>
			<td>BD Biosciences</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody-PE</td>
			<td>eBiosciences</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45 antibody-PE</td>
			<td>eBiosciences</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sca1 antibody-PE</td>
			<td>eBiosciences</td>
			<td>Dec-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;7 antibody</td>
			<td>MBL International</td>
			<td>ABIN487462</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini &#38; MidiMACS Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>LD Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma Aldrich</td>
			<td>C9759-1MG</td>
			<td>Stock 10 &#956;M in PBS</td>
		</tr>
		<tr>
			<td>31G Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>328438</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge (Microfuge 22R)</td>
			<td>Beckman Coulter</td>
			<td>368826</td>
			<td></td>
		</tr>
		<tr>
			<td>S241.5 Swinging Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>368882</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (Allegra X-22R)</td>
			<td>Beckman Coulter</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr>
			<td>Nod/Scid immunodeficient mice</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code 394</td>
			<td>Use 2 months old mice</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Name of the reagent</td>
			<td></td>
			<td></td>
			<td>Recipie</td>
		</tr>
		<tr>
			<td>10% and 2% FBS DMEM</td>
			<td></td>
			<td></td>
			<td>DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>0.2% Collagenase solution</td>
			<td></td>
			<td></td>
			<td>Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM.</td>
		</tr>
		<tr>
			<td>10% Matrigel solution</td>
			<td></td>
			<td></td>
			<td>Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20&#176;C until use.</td>
		</tr>
		<tr>
			<td>Matrigel-coated plate</td>
			<td></td>
			<td></td>
			<td>Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37&#176;C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20&#176;C for reusing.</td>
		</tr>
		<tr>
			<td>0.01% Collagen solution</td>
			<td></td>
			<td></td>
			<td>Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O.</td>
		</tr>
		<tr>
			<td>Collagen-coated plate</td>
			<td></td>
			<td></td>
			<td>Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months.</td>
		</tr>
		<tr>
			<td>bFGF stock solution</td>
			<td></td>
			<td></td>
			<td>bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 &#956;l in 500 &#956;l microcentrifuge tubes and kept in -80&#176;C.</td>
		</tr>
		<tr>
			<td>Myoblast medium</td>
			<td></td>
			<td></td>
			<td>500 mL HAM&#8217;S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 &#956;g of bFGF (20 &#956;l of bFGF stock).</td>
		</tr>
		<tr>
			<td>Differentiation medium</td>
			<td></td>
			<td></td>
			<td>500 mL DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>10 &#956;M Cardiotoxin stock</td>
			<td></td>
			<td></td>
			<td>1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS.</td>
		</tr>
	</tbody>
</table>

isolation, culture, and transplantation of muscle satellite cells

Cellule satelliti del muscolo sono una popolazione di cellule staminali necessarie per lo sviluppo post-natale del muscolo scheletrico e la rigenerazione, pari al 2-5% dei nuclei sublaminal nelle fibre muscolari. Nel muscolo adulto, cellule satelliti sono normalmente mitoticamente riposo. Dopo la lesione, tuttavia, le cellule satelliti iniziano proliferazione cellulare per produrre mioblasti, loro progenie, per mediare la rigenerazione del muscolo. Il trapianto di mioblasti derivati ​​dalle cellule satellite è stato ampiamente studiato come una possibile terapia per varie malattie, tra cui rigenerativi distrofia muscolare, insufficienza cardiaca e disfunzione urologica. Trapianto di mioblasti in distrofico muscoli scheletrici, cuore infartuato, e le disfunzioni vie urinarie ha dimostrato che i mioblasti engrafted possono differenziarsi in fibre muscolari nei tessuti dell&#39;ospite e visualizzare miglioramento funzionale parziale in queste malattie. Pertanto, lo sviluppo di metodi di purificazione efficiente di cellule satelliti quiescenti da muscl scheletricoe, così come la creazione di cellule derivate da colture di mioblasti satellitari e metodi di trapianto di mioblasti, sono essenziali per la comprensione dei meccanismi molecolari alla base di cellule auto-rinnovamento satellitare, attivazione e differenziazione. Inoltre, lo sviluppo di terapie cellulari per la distrofia muscolare e altre malattie rigenerative dipendono anche da questi fattori. Tuttavia, attuali metodi di purificazione potenziali di cellule satelliti quiescenti richiedono l&#39;uso di costosi fluorescenza-attivato cell sorting (FACS) macchine. Qui, presentiamo un nuovo metodo per la purificazione rapido, economico e affidabile di cellule satelliti quiescenti di topo adulto muscolo scheletrico per dissociazione enzimatica seguita da cellule magnetico attivato classificare (MACS). Dopo aver isolato cellule satelliti quiescenti puri, queste cellule possono essere coltivate avere un gran numero di mioblasti dopo vari passaggi. Questi appena isolatocellule satelliti quiescenti o ex vivo mioblasti espansi possono essere trapiantate in cardiotossina (CTX) indotta rigenerante topo muscoli scheletrici per esaminare il contributo delle cellule derivate dal donatore alla rigenerazione delle fibre muscolari, nonché compartimenti cellulari satellite per l&#39;esame di auto-rinnovamento attività.

Cellule satelliti quiescenti appena isolate mostrano un piccolo, forma rotonda (Figura 1G), e Pax7 espresso come marcatore definitivo per le cellule satellite quiescenti. Più del 90% delle cellule appena isolate esprimono Pax7 (Figura 1H e 1I). Maggior parte delle cellule contaminate sono da cellule del sangue che non crescono in modo efficiente in vitro seguente condizioni di coltura mioblasti. Così, mioblasti derivati ​​dalle cellule satelliti dominano ...

Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Isolamento, Cultura e trapianto di cellule muscolari satellite

L&#39;isolamento e la coltura di una popolazione pura di cellule satelliti quiescenti, una popolazione di cellule staminali muscolari, è essenziale per la comprensione del muscolo biologia delle cellule staminali e rigenerazione, nonché trapianto di cellule staminali per terapie in distrofia muscolare e altre malattie degenerative.

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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Isolation and Transplantation of Hematopoietic Stem Cells HSCs

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Biologia

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

Isolamento di cellule umane ombelicale muscolatura liscia arteriosa (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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

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

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Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Primary Culture of Rat Hepatic Cells

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolamento e Cultura del mouse principale acinose pancreatiche Cellule

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Isolamento di polmonare cellule muscolari lisce di topi neonati

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolamento e colture primarie di cellule di topo aortica endoteliali

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...