Name: generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection
Uploaded: 2015-02-17T15:00:00
Description: Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

1. Anbefalinger Inden start <ol><li> Brug ikke kvikskranker forurenet med mycoplasma, fordi de ikke vil differentiere ordentligt ind i sinus node celler. Test for mycoplasma forurening, før du starter protokollen. Gør dette ved hjælp af en PCR kit til hurtig, meget følsom påvisning af mycoplasma og følg manufacturer`s protokol. </li><li> For hver petriskål (trin 2.3.4) Coat en 10 cm2 cellekultur skål med sterilt 7 ml 0,1% gelatine fra koldt vand fiskeskind i 1 time ved 37 ° C. Fjern gelatine og lad...

<ol>
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Evnen til at producere stamceller afledt pacemaker-celler kan tillade rekonstituering af korrekt hjerterytme i betydningen &quot;biologiske pacemakere&quot;. Ligeledes vil narkotikatests in vitro drage fordel af deres tilgængelighed. Kvikskranker kan give anledning til en celle type pattedyrs krop, herunder kardiomyocytter med pacemaker celleegenskaber 8,9,10,11,12,13. Men typisk cellepopulationerne inden &quot;embryoide legemer&quot; er meget heterogene, hvilket uundgåeligt fører til krav om pålidelige u...

Udtrykket &quot;syg sinus-syndrom&quot; opsummerer flere sygdomme, der fører til en forringelse af pacemaker-systemet. Det består patologisk, symptomatisk sinus bradykardi, sinoatrialt blok, sinusstop samt takykardi-bradykardi syndrom. Derved en &quot;syg sinus-syndrom&quot; ofte ledsaget af generelle hjertesygdomme såsom en iskæmisk hjertesygdom, kardiomyopati eller myocarditis. På nuværende tidspunkt er terapeutiske fremgangsmåder baseret på implantation af elektriske pacemakere. Men går det sammen med en række risici, såsom infektioner og batteri fiasko. Samlet er forekomsten af ​​komplikationer er stadig meget højt i patienter, der har implanteret en kunstig pacemaker. Endvidere, i modsætning til den endogene pacemaker, disse enheder ikke reagerer på hormon stimulation. En fremtidig alternativ kan påberåbe sig tilgængeligheden af ​​&quot;biologiske pacemakere&quot;, hvortil kvikskranker kan tjenesom en passende cellulær kilde, og som også ville være særdeles værdifuldt til in vitro test af lægemidler. Men et stort problem ligger i meget sjældne udseende sinus nodal celler i embryoide organer (EBS) - det typisk ikke overstiger ~ 0,5% 1. Tidligere blev det vist, at &quot;fremad programmering&quot; til specifikke cardiomyocyte undertyper er muligt via overekspression af forskellige tidlige kardiovaskulære transkriptionsfaktorer såsom Mesoderm-specifik-posterior 1 (MesP1) og NK2 transskription faktor relateret, locus 5 (Nkx2.5) 2, 3. For normal størrelse og funktion sinusknude (SAN), T-box transkriptionsfaktor Tbx3 er afgørende, som har vist sig at indlede pacemaker genet program og til at styre differentieringen af SAN 4. Mens dette forbedrede udseende af funktionelle pacemaker-celler, indholdet stadig ikke overstiger ~ 40% inden for hele cardiomyocytic cellepopulation. Derfor blev yderligere Myh6-promotoren baseret antibiotisk selektion trin 5 indføres af os. Dette fører i sidste ende til endnu uobserverede cardiomyocyte aggregater (&quot;inducerede sinoatrial organer&quot; iSABs &quot;), der udviser stærkt forøgede slå frekvenser (&gt; 400 bpm) in vitro, for første gang at tilnærme dem af en muse hjerte og kan sammenlignes med in vitro dyrkede sinus nodal celler isoleret fra et muse hjerte 6. Under Isoprenaline administration opnås endog slå frekvenser på 550 bpm. Især iSABs består af over 80% funktionelle nodal celler som det fremgår af en omfattende fysiologiske analyser 7. For nylig, at flere tilgange generere sinus nodal celler ved hjælp af direkte omprogrammering 19, overflademarkører 14 eller farmakologisk behandling med små molekyler 16,17 blev beskrevet. Men ingen af ​​disse metoder har ført til sådan en høj renhed af pacemaker-celler og slå frekvenser tæt på murine hankunst som observeret i iSABs. Desuden i en ex vivo model for dyrkede voksne mus ventrikulære skiver, der har mistet deres spontane bankende aktivitet, de iSABs er i stand til at integrere ind i skive væv og derved forbliver spontant aktiv og håndfast pacing hjertet skiver til sammentrækninger 7. En detaljeret protokol for generering af Disse udvalg er beskrevet i dette dokument.

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			<td>Phoenix Europe GmbH</td>
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			<td>SigmaAldrich</td>
			<td>M3148&#160;</td>
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			<td height="20" style="height:20px;">1-Thioglycerol</td>
			<td>SigmaAldrich</td>
			<td>M6145</td>
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			<td>TPP</td>
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			<td>TPP</td>
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generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

Behandling af &quot;syg sinus-syndrom&quot; er baseret på kunstige pacemakere. Disse er forsynet farer som fiasko og infektioner batteri. Desuden mangler de hormon reaktionsevne og den samlede procedure er omkostningskrævende. &quot;Biologiske pacemakere&quot; genereret fra kvikskranker kan blive et alternativ, men det typiske indhold af pacemaker celler i embryoide legemer (EB&#39;er) er meget lav. Den beskrevne protokol kombinerer &quot;fremad programmering&quot; af murine kvikskranker via sinusknuden inducer TBX3 med Myh6-promotor baseret antibiotikaselektion. Dette giver cardiomyocyte aggregater konsistente på&gt; 80% fysiologisk funktionelle pacemaker celler. Disse &quot;inducerede-sinoatriale-organer&quot; (&quot;iSABs&quot;), er spontant kontraherende ved endnu unåede frekvenser (400-500 bpm), som svarer til nodal celler isoleret fra mus hjerter og er i stand til at pace murine myocardium ex vivo. Ved hjælp af beskrevne protokol yderst ren sinusnodal enkelte celler kan genereres som f.eks kan anvendes til in vitro test af lægemidler. Endvidere kan iSABs genereret ifølge denne protokol blive et afgørende skridt i retning af hjerte tissue engineering.

Den beskrevne protokol tillader generation af iSABs med et bankende frekvens på omkring 450 bpm fra kvikskranker (vist i filmen), som er tæt på musen hjerte slå frekvens. Efter dissociation af iSABs (trin 2.8.8) de observerede enkelte celler viser den typiske form af celler fra sinusknuden (spindel og spider celler) som vist i figur 1. Disse celler stærkt udtrykkelige proteiner, der er kendt for at være af afgørende betydning for den funktion, af sinusknuden som hyperpolarisationsaktiveret cyklis...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Denne protokol beskriver, hvordan man producere funktionel sinus nodal væv fra murine pluripotente stamceller (PSC). T-BOX3 (TBX3) overekspression plus kardial myosin-tung-kæde (Myh6) promoter antibiotisk selektion fører til meget rene pacemaker celleaggregater. Disse &quot;induceret sinoatriale-organer&quot; (&quot;iSABs&quot;) indeholder over 80% pacemaker celler, viser stærkt forøgede bankende satser og er i stand til at pace myocardium ex vivo.

Generation af murine pacemaker celleaggregater Baseret på ES-celle-programmering i kombination med Myh6-Promotor-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

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Generation of Mosaic Mammary Organoids by Differential Trypsinization

Generation af Mosaic Mammary Organoids af Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Generation af en menneskelig iPSC-baseret blod-hjerne barrierchip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...