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

Vi takker Dr. Shahragim Tajbakhsh for å gi Myf5 + / nLacZ mus. Vi takker også Alexander Hron og Michael Baumrucker for kritisk lesning av dette manuskriptet. Dette arbeidet ble støttet med tilskudd fra muskeldystrofi Association (MDA) og Gregory Marzolf Jr MD senter Award.

Dyrene ble plassert i en SPF miljø og ble overvåket av Forsknings Animal Resources (RAR) ved University of Minnesota. . Dyrene ble avlivet av egnede midler (CO 2 innånding eller KCl injeksjon etter å ha blitt bedøvet med IP injeksjon av Avertin (250 mg / kg) Alle protokoller ble godkjent av Institutional Animal Care og bruk Committee (IACUC, kodenummer: 1304-30492 ) ved University of Minnesota. En. Isolering av mononukleære celler fra mus Skeletal Muscle <ol><li> Riktig of...

<ol>
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I denne protokollen, kan hvil satellitt celler lett bli renset fra voksen skjelettmuskulatur av mus ved collagenase fordøyelsen og overflate antistoff-mediert MACS separasjon. Denne metoden tar ca 6 timer, og trenger ikke noen kostbart utstyr som for eksempel en FACS-maskin. I tillegg er denne fremgangsmåte forholdsvis billig sammenlignet med overflate-antistoff-mediert FACS separasjon. Et høyere utbytte av quiescent satellittceller er også forventet i forhold til FACS for denne metoden fra FACS lasereksponering har...

Muskelsatellittceller er en liten bestand av myogeniske stamceller som ligger under basal lamina av skjelettmuskelfibre. De er preget av uttrykket av Pax7, Pax3, c-Met, M-cadherin, CD34, Syndecan-3, og calcitonin 1 - 3. Satellittceller har vist seg å være ansvarlig for muskelregenerasjonenen som muskel stamceller. I voksen muskel, satellittceller er normalt mitotisk sovende 4-8. Etter skade, er satellitt celler aktiveres, initiere uttrykk for MyoD, og gå inn i cellesyklus for å utvide sitt avkom, betegnes myogeniske forløper celler eller myoblasts tre. Etter flere runder med celledeling, myoblaster gå ut av cellesyklusen og sikring til hverandre for å gjennomgå differensiering til multi-nukleære myotubes, etterfulgt av moden muskelfibre. Myoblaster isolert fra voksen muskel kan lett utvides ex vivo. Kapasiteten for myoblasts å bli muskelfibre i regenererende muskler og tilskjema ektopiske muskelfibre i nonmuscle vev blir utnyttet av myoblast transplantasjon, en potensiell terapeutisk tilnærming for Duchenne muskeldystrofi (DMD) 4, urologisk dysfunksjon 9, og hjertesvikt 10. Faktisk myoblasts har blitt transplantert i muskel av begge MDX (DMD-modellen) muse og DMD pasienter 11-14. De injiserte normale myoblaster smelter med vertsmuskelfibre for å forbedre histologi og funksjon av den syke muskler. Tidligere arbeid har vist at subpopulasjoner av myoblasts er mer stamcellelignende og forbli i en udifferensiert tilstand lenger i muskelen under muskelregenerasjonenen fem. Nyere arbeider har vist at nylig isolerte satellittceller fra voksne muskel inneholde en stamcelle-liknende befolkning som viser mer effektiv engraftment og selvfornyelse aktivitet i regenererende muskel 5-8. Derfor, rensing av en ren populasjon av hvil satellitt celler fra voksne skjelett muscle er avgjørende for å forstå biologien til satellittceller, myoblasts og muskel gjenfødelse, og for utvikling av cellebasert terapi. Imidlertid, nåværende potensielle rensemetoder quiescent satellittceller krever bruk av en kostbar fluorescens-aktivert cellesortering (FACS) maskin 1,2,6-8. I tillegg tenderer FACS lasereksponering for å indusere celledød under separasjonen, noe som fører til lavere utbytte av quiescent satellittceller 15. Her presenterer vi en ny metode for rask, økonomisk og pålitelig rensing av hvil satellitt celler fra voksne mus skjelettmuskulatur. Denne metoden benytter enzymatisk dissosiasjon etterfulgt av magnetisk aktivert cellesortering (MACS). Etter isolering av rene quiescent satellittceller, kan disse cellene bli dyrket for å oppnå store mengder av myoblaster etter flere passeringer. Vi viser også at intramuskulær injeksjon av disse nylig isolerte hvilsatellittceller eller ex vivo utvidet myoblasts kan transplanteres inn Cardiotoxin (CTX)-indusert regenererende mus skjelettmuskulatur for å undersøke bidraget fra donor-deriverte celler til å regenerere muskelfibre, så vel som til satellittcelle avdelinger for undersøkelse av selvfornyelsesarbeid.

<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

Muskelsatellittceller er en stamcelle befolkningen nødvendig for postnatal skjelettmuskelutvikling og regenerasjon, sto for 2-5% av sublaminal kjerner i muskelfibrene. I voksen muskel, satellittceller er normalt mitotisk sovende. Etter skade, men satellittceller initiere cellulære proliferasjon for å produsere myoblaster, deres progenies, for å formidle til regenerering av muskelen. Transplantasjon av satellitt celle-avledet myoblasts har vært mye studert som en mulig behandling for flere regenerative sykdommer, inkludert muskeldystrofi, hjertesvikt, og urologisk dysfunksjon. Myoblast transplantasjon i dystrofe skjelettmuskulatur, infarcted hjerte, og dysfunctioning urinkanalene har vist at innpodet myoblasts kan differensiere i muskelfibre i verts vev og vise delvis funksjonell forbedring i disse sykdommene. Derfor er utviklingen av effektive rensemetoder quiescent satellittceller fra skjelett muscle, så vel som etablering av satellitt celle-stammer myoblast kulturer og transplantasjonsmetoder for myoblaster, er essensielle for å forstå de molekylære mekanismer som ligger til satellittceller selvfornyelse, aktivering og differensiering. I tillegg til utvikling av celle-baserte terapier for muskeldystrofi og andre regenererende sykdommer er også avhengig av disse faktorene. Imidlertid, nåværende potensielle rensemetoder quiescent satellittceller krever bruk av dyre fluorescens-aktivert cellesortering (FACS) maskiner. Her presenterer vi en ny metode for rask, økonomisk og pålitelig rensing av hvil satellitt celler fra voksne mus skjelettmuskulatur ved enzymatisk dissosiasjon fulgt av magnetisk-aktivert celle sortering (MACS). Etter isolering av rene quiescent satellittceller, kan disse cellene bli dyrket for å oppnå store mengder av myoblaster etter flere passeringer. Disse ferske isolerthvilsatellittceller eller ex vivo utvidede myoblasts kan transplanteres inn Cardiotoxin (CTX)-indusert regenererende mus skjelettmuskulatur for å undersøke bidraget fra donor-deriverte celler til å regenerere muskelfibre, samt satellitt celle avdelinger for undersøkelse av selvfornyelse aktiviteter.

Nylig isolerte hvilsatellittceller vise en liten, rund form (figur 1G), og uttrykte Pax7 som en definitiv markør for hvilsatellittceller. Mer enn 90% av nylig isolerte celler uttrykker Pax7 (figur 1H og 1I). De kontaminerte celler er fra blodceller som ikke effektivt vokse in vitro etter myoblast dyrkningsbetingelser. Således satellitt celle-stammer myoblaster dominerer i kulturen. Eventuelt kan vi gjenta trinnene MS kolonnerensing for (trinn 2,11 til 2,14) f...

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

Isolasjon, Kultur, og Transplantasjon av Muscle satellitt celler

Isoleringen og kulturen i en ren populasjon av hvilende satellittceller, en muskel stamcelle befolkning, er avgjørende for forståelsen av muskelstamcellebiologi og regenerering, samt stamcelletransplantasjon for terapi i muskeldystrofi og andre degenerative sykdommer.

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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