We are grateful for expert technical assistance from the UNC Flow Cytometry Core and UNC Microscopy Core. We thank members of the Qian lab and the Liu lab for helpful discussions and critical reviews of the manuscript. This study was supported by NIH/NHLBI R00 HL109079 grant to Dr. Liu and American Heart Association (AHA) Scientist Development Grant 13SDG17060010 and the Ellison Medical Foundation (EMF) New Scholar Grant AG-NS-1064-13 to Dr. Qian.

Il protocollo descritto qui utilizza topi neonati. La cura degli animali e gli esperimenti sono eseguiti in conformità con le linee guida stabilite dalla Divisione di Medicina di Laboratorio animali (àlam) presso l&#39;Università del North Carolina, Chapel Hill. 1. Preparazione di buffer e Media <ol><li> Preparare fibroblasti (FB) mezzo: Supplemento 500 ml supporti IMDM con 100 ml di siero fetale bovino (FBS) e 6 ml di penicillina / streptomicina. </li><li> Preparare ICM medio: Supplemento 400 ml supporti DMEM con 100 ml mezzi M199 e 50 ml di FBS. </li><li> Preparare media B27: Supplemento 490 ml RPMI1640 multimediali con 10 ml B27 media. </li><li> Preparare Plat-E terreno di coltura: Supplemento 500 ml supporti DMEM con 50 ml di FBS, 5 ml di penicillina / streptomicina e 5 ml aminoacidi non essenziali. </li><li> Preparare Plat-E medio trasfezione: Supplemento 500 ml supporti DMEM con 50 ml di FBS e 5 ml aminoacidi non essenziali. </li><li> Prepare gelatina rivestito 24 pozzetti: Aggiungere soluzione di gelatina 0,5 ml di 0,1% ad un pozzo di 24 pozzetti, incubare a 37 ° C per almeno 10 minuti e aspirare a destra prima dell&#39;uso. </li><li> Preparare tampone etichettatura FACS: Supplemento 500 ml DPBS con 10 ml di FBS e 2 ml di EDTA (0,5 M) per raggiungere una concentrazione finale di 2 mm. Passare attraverso un filtro di 0,22 micron e conservare a 4 ° C. </li><li> Preparare MACS ordinamento buffer: Supplemento 500 ml DPBS con 2,5 g BSA e 2 ml di EDTA (0,5 M) per ottenere una concentrazione finale di 2 mM. Passare attraverso un filtro di 0,22 micron e conservare a 4 ° C. </li><li> Preparare tampone HBS 2x per trasfezione: integrare 500 ml di tampone HEPES (50 mM) con 280 mM NaCl (8,1816 g), KCl 10 mM (0,37,275 mila g), 1,5 mM Na 2 HPO 4 (0,10,647 mila g), glucosio 12 mM (1,08,096 mila g ). Aggiungere circa 650 ml NaOH 10 N per aggiustare il pH a 7,05-7,12. Filtrare attraverso un filtro di 0,22 micron pori e conservare a 4 ° C. </li><li> Preparare 2,5 M CaCl 2 soluzione per la trasfezione: Aggiungere 18,376 g CaCl 2 a 50 ml DDH 2 O. Filtrare attraverso un filtro di 0,22 micron pori e conservare a 4 ° C. </li><li> Preparare ICC (immunoistochimica) tampone fissazione: Aggiungere 5 ml di paraformaldeide (PFA, 32%) a 35 ml DPBS per raggiungere una concentrazione finale del 4%. </li><li> Preparare tampone permeabilizzazione ICC: Aggiungere 0,1 ml di Triton a 100 ml DPBS per raggiungere una concentrazione finale di 0,1%. </li><li> Preparare ICC tampone bloccante: Aggiungere 5 g di albumina di siero bovino (BSA) per 100 ml DPBS per raggiungere una concentrazione finale del 5%. </li><li> Preparare tampone colorazione ICC: Aggiungere 1 g BSA a 100 ml DPBS per raggiungere una concentrazione finale di 1%. </li></ol> 2. Generazione di neonatali mouse fibroblasti cardiaci <ol style="margin-left: 40px;"><li> Generare fibroblasti cardiaci dal metodo di coltura espianto <ol><li> Clean neonatale αMHC-GFP topo transgenico (P1 a P2) con il 75% di etanolo. Tagliare la testa con le forbici sterili. Poi fare una incisione orizzontale da sotto un&#39;ascella all&#39;altra. Utilizzare uno sterileforcipe smussato e piegate a sezionare il cuore e collocarlo in un pozzetto della piastra 24 pozzetti contenenti tampone PBS ghiacciata. <ol><li> In alternativa, spremere il cuore dopo l&#39;incisione orizzontale. </li></ol></li><li> Controllare espressione GFP nel cuore al microscopio a fluorescenza. Poiché l&#39;espressione GFP è guidato da αMHC promotore che è un promotore specifico cardiaco, cuore di topo transgenico dovrebbe essere in verde 15, mentre il cuore negativo è scarsa in alcuna fluorescenza. </li><li> Prendere 3-4 GFP cuori positivi in un centro ben piatto di coltura 60 mm e tritateli pezzetti meno di 1 mm 3 dimensioni di forbici sterili e pinze. </li><li> Mettere 3-4 cuori tritato in un piatto 10 centimetri con 2 ml di mezzi FB. Lasciate che i tessuti stabilirsi per 3 ore. </li><li> Lentamente aggiungere 8 ml supporti FB pre-riscaldato per i piatti contenenti tessuti cardiaci. Non disturbare i tessuti per tre giorni. </li><li> Sostituire i media ogni tre giorni. </li><li> Il giorno 7, aspirmangiato il terreno di coltura e lavare le cellule con DPBS. Aggiungere 3 ml 0,05% tripsina per ciascuna piastra e digerire a 37 ° C per 5 min. </li><li> Aggiungere 5 ml supporto FB per placare la tripsina. Staccare delicatamente le cellule con un raschietto cellulare. Pipettare media su e giù per dissociare ulteriormente meccanicamente il tessuto. </li><li> Raccogliere le cellule e filtrare attraverso 40 micron filtri cellulari per evitare la contaminazione dei frammenti di tessuto cardiaco, e poi pellet cellule facendo girare a 200 xg per 5 min. </li><li> Lavare le cellule una volta con MACS tampone e le cellule sono pronte per l&#39;ordinamento. </li></ol></li><li> Generare fibroblasti cardiaci dal metodo di digestione enzimatica <ol><li> Harvest GFP cuori positivi come 2.1.1. Trasferire tutti i cuori in un piatto 10 centimetri con 10 ml di DPBS ghiacciate. </li><li> Spremere ventricoli con pinza sterile per rimuovere il sangue e lavare una volta con DPBS gelide. </li><li> Tagliare i cuori di essere libero di altri tessuti e grasso. </li><li> Tagliare ogni cuore in circa 4 pezzi che sono ancora collegate liberamente. </li><li> Se meno di 20 cuori, trasferire i cuori in un tubo da 15 ml con 8 ml caldo 0,05% tripsina-EDTA, e incubare a 37 ° C per 15 min. <ol><li> Se ci sono 20-30 cuori, trasferire i cuori in una provetta conica da 50 ml con 10 ml caldo 0,05% tripsina-EDTA, e incubare a 37 ° C per 15 min. </li></ol></li><li> Eliminare la tripsina e aggiungere 5 ml (per meno di 20 cuori) o 10 ml (per 20-30 cuori) caldo collagenasi di tipo II (0,5 mg / ml) in HBSS. </li><li> Vortex tubo sul vortex per 1 min a livello velocità intorno 4 a 6 (se il liquido non ottiene fino al coperchio, allora la velocità è bene). </li><li> Incubare la provetta in 37 ° C bagnomaria per 3-5 minuti. </li><li> Vortex la provetta per 1 min. </li><li> Sedimenti per 1 min per gravità fino a che i pezzi di tessuto stabilirsi, raccogliere il liquido in un nuovo tubo contenente 5 ml di medie FB freddo. </li><li> Ripetere i punti 2.2.6-2.2.10 3-5 volte. </li><li> Unire tutte le collezioni filtrando attraverso 40 micron colino cellaper rendere sospensione singola cella. </li><li> Centrifugare a 200 xg per 5 minuti a 4 ° C. </li><li> Risospendere le cellule in 10 ml MACS buffer e centrifugare a 200 xg per 5 minuti a 4 ° C. </li><li> Opzionale: se ci sono un sacco di cellule del sangue, risospendere le cellule con 1 ml di RBC tampone di lisi (150 mm NH 4 Cl, 10 KHCO mm 3, e 0.1 mM EDTA), tenere in ghiaccio per 1 minuto, quindi diluire con 10 ml MACS buffer e centrifugare a 200 xg per 5 min. Lavare una volta con il tampone MACS. </li></ol></li><li> Isolamento di Thy1.2 + fibroblasti da MACS (magnetico attivato separazione delle cellule) <ol><li> Determinare il numero di cellule vitali attraverso tripano colorazione blu. <ol><li> Prendere 10 celle microlitri dalla sospensione cellulare 10 ml al punto 2.2.14. Mescolare con 10 microlitri 0,4% trypan soluzione blu. </li><li> Aggiungere il composto di un emocitometro. Lasciare riposare per 3-5 minuti e poi esaminare immediatamente al microscopio. Le cellule morte vengono colorate in cellule blu e vitali sono senza macchia. </li><li&gt; contare tutte le cellule vitali in quattro piazze 1 millimetro d&#39;angolo del emocitometro. Determinare il numero di cellule vitali: il numero medio per quadrato x 2 (fattore di diluizione) x 10 4 x 10 (volume totale della sospensione cellulare). </li></ol></li><li> Per meno di 1 x 10 7 cellule, risospendere le cellule con 10 microlitri microsfere Thy1.2 in 90 microlitri di buffer MACS refrigerati. Aggiungere più perline proporzionalmente se ci sono più di 1 x 10 7 cellule. Mescolare bene e incubare in frigorifero (2-8 ° C) per 30 min. </li><li> Aggiungere 10 ml MACS tampone e campioni di spin a 200 xg per 5 minuti; lavare una volta con tampone 10 ml MACS e centrifugare di nuovo. </li><li> Portare il volume a 2 ml in tampone MACS. </li><li> Impostare un separatore MACS in cappuccio. Inserire una colonna LS al separatore. Applicare 3 ml MACS Buffer alla colonna di equilibrare esso. </li><li> Passare la sospensione cellulare attraverso la colonna LS equilibrata. </li><li> Lavare 3 volte con 2 ml di buffer MACS ogni volta. </li><li> Prendere colonna LS fuori tlui separatore ed eluire 3 volte con 2 ml MACS tampone in un nuovo tubo da 50 ml. </li><li> Spin a 200 xg per 5 min. </li><li> Risospendere le cellule in 10 ml di FB media, contano le cellule e le sementi in piastre a giusta densità. Per fibroblasti generati dal metodo di coltura espianto, la semina di cellule densità potrebbe essere circa 2-3 x 10 4 cellule / pozzetto di 24 pozzetti. Per fibroblasti generati dal metodo di digestione enzimatica, la densità di cellule potrebbe essere di circa 4-5 x 10 4 cellule / pozzetto di 24 pozzetti. </li></ol></li></ol> 3. Generazione di Retrovirus per ICM Riprogrammazione Nota: Effettuare le seguenti operazioni in un BSL2 Cappa di sicurezza biologica in condizioni sterili. Il corretto smaltimento delle cellule transfettate, puntali e tubi si consiglia di evitare il rischio di rischi ambientali e per la salute. <ol style="margin-left: 40px;"><li> Mantenere le cellule Plat-E a Plat-E multimediale integrato con 1 mg / ml puromicina e 10 ug / ml blasticidin a 37 ° C con 5% di CO 2. </li><li> Il giorno 2, sostituire il mezzo con Plat-E terreno di coltura privo di puromicina e blasticidin. </li><li> Il giorno 0, dividere le celle Plat-E in 10 cm piatti del giorno prima trasfezione a circa 4-5 x 10 6 cellule per piastra. Le cellule devono essere ~ 80-90% confluenti il ​​giorno della trasfezione. </li><li> Il giorno 1, il cambiamento culturale dei media ai media di trasfezione su cellule entro 1 ora prima della trasfezione. Trasfezione procedimento è il seguente: <ol><li> Trasfezione utilizzando kit commerciali <ol><li> Mescolare 20 ml di reagente di trasfezione (ad esempio Lipofectamine) e 500 microlitri mezzi siero ridotto (ad esempio, Opti-MEM). Aggiungere 10 ug del plasmide retrovirale per altri 500 microlitri ridotte supporti siero. Incubare ogni miscela a temperatura ambiente (RT) per 5 min. </li><li> Aggiungere lentamente il composto plasmide di miscela di trasfezione. Questo passaggio potrebbe richiedere 15-30 secondi. Incubare la miscela per 20 minuti a temperatura ambiente (soluzione può apparire torbida). </li&gt; <li> Aggiungete il composto goccia a goccia per le cellule. </li><li> Incubare per una notte in un incubatore a 37 °. </li></ol></li><li> Trasfezione con fosfato di calcio. <ol><li> Mescolare 40 ml 2.5 M CaCl 2 e 440 ml DDH 2 O, quindi aggiungere 20 mg del plasmide retrovirale (regolare la quantità di DDH 2 O in modo che il volume totale è di 500 microlitri). </li><li> Aggiungere 500 microlitri 2x HBS in una provetta da 15 ml. </li><li> Vortex la HBS e nel frattempo aggiungere la miscela Calcio-DNA alla HBS cadere saggio. Questa fase dura circa 30 secondi per ogni campione. Quindi al massimo fare 3-4 campioni contemporaneamente. </li><li> Incubare a temperatura ambiente per 3 minuti di incubazione per più tempo porta a grandi precipitato e meno cellule assumerà il precipitato. </li><li> Aggiungere il composto cadere saggio alle cellule e osservare sotto 20X microscopio. Precipitati fini (un sacco di piccoli sono buone, ma pochi grandi non sono buone) dovrebbe essere visto. </li><li> Incubare una notte a 37 ° C incubatore. </li&gt; </ol></li></ol></li><li> Il giorno 2, cambiare media su cellule con 8 ml preriscaldata coltura. Incubare per 24 ore. </li><li> Il giorno 3 e il giorno 4, raccogliere coltura supernatante dai piatti utilizzando una siringa monouso 10 ml sterile, filtrandola attraverso un filtro di acetato di cellulosa dimensioni 0,45 micron pori, e il trasferimento in un tubo da 50 ml. Aggiungere 2 ml di soluzione retrovirus precipitazioni per ogni 8 ml contenente il virus surnatante. Mescolare delicatamente e incubare una notte a 4 ° C. </li><li> Il giorno 5, girare la miscela virale a 1.500 xg a 4 ° C per 30 min. Le particelle virali dovrebbero apparire in pellet bianco sul fondo della provetta. </li><li> Scartare il surnatante. Spin down soluzione residua mediante centrifugazione a 1500 xg per 5 min. Eliminare tutte le tracce di liquido per aspirazione, facendo molta attenzione a non disturbare le particelle retrovirali precipitati in pellet. </li><li> Risospendere pellet retrovirali con 100 microlitri di buffer DPBS per ogni 8 ml surnatante virale. Aliquotail virus su ghiaccio. Utilizzare immediatamente o conservare a -80 ° C. </li></ol> 4. La riprogrammazione dei fibroblasti cardiaci <ol><li> Aggiungere 4 ml di 10 mg / ml soluzione polibrene nel Icm medio di 10 ml, e mescolare delicatamente pipettando su e giù. La concentrazione finale di polibrene è 4 mg / ml. </li><li> Aggiungere 500 microlitri mezzi ICM contenente polibrene a ciascun pozzetto di 24 pozzetti seminato con fibroblasti. </li><li> Aggiungere 10 microlitri retrovirus a ciascun pozzetto contenente sia 2-3 x 10 4 cellule della cultura espianto o 4-5 x 10 4 cellule da metodo digestione enzimatica. La quantità di virus deve essere aumentato proporzionalmente con l&#39;aumento del numero di cellule in diverse piastre di coltura o piatti. Mettere la piastra a 37 ° C per 24-48 hr incubatore per consentire l&#39;infezione virale. La linea di tempo per la riprogrammazione cardiaca è mostrato in Figura 1. </li><li> Dopo trasduzione virale, sostituire il virus contenente media con 500 microlitri mezzi regolare ICM. </li><li> Modificare i media ogni 2-3 giorni. Per la selezione positiva delle cellule trasdotte virali, aggiungere media ICM integrato con puromicina a 2 mg / ml alle cellule bersaglio. Keep it per tre giorni. Dopo di che, mantenere puromicina nel mezzo ICM alla concentrazione di 1 mg / ml. </li><li> Tre giorni dopo l&#39;infezione virale, prendere la targa fuori dalla incubatrice e posizionarlo sotto un microscopio a fluorescenza invertito (20X) per osservare l&#39;espressione della GFP. Nota: Dieci giorni dopo l&#39;infezione virale, le cellule possono essere raccolte per l&#39;analisi FACS e analisi ICC per determinare l&#39;efficienza riprogrammazione (nel passaggio 5 e 6). </li><li> Quattordici giorni dopo l&#39;infezione virale, sostituire ICM media con i media B27. Modificare i media ogni tre giorni. Spontaneo pestaggio loci cella può apparire da tre (cellule da metodo di digestione enzimatica) o quattro (cellule da espianto metodo cultura) settimane dopo la trasduzione virale. </li></ol> 5. Analisi immunocitochimica di efficienza Riprogrammazione <ol><li> Cellule risciacquocon ghiaccio PBS freddo per tre volte; rimuovere la soluzione in eccesso. </li><li> Aggiungere 0,5 ml di tampone di fissaggio ICC a ciascun pozzetto di piastre da 24 pozzetti. Fissare le cellule a temperatura ambiente per 15-20 minuti. </li><li> Risciacquare le cellule con PBS per tre volte. </li><li> Aggiungere 0,5 ml di buffer permeabilizzazione ICC a ciascun pozzetto di piastre da 24 pozzetti. Permeabilize le cellule a temperatura ambiente per 15 min. </li><li> Risciacquare le cellule con PBS per tre volte. </li><li> Aggiungere 0,5 ml ICC tampone di bloccaggio in ciascun pozzetto di 24 pozzetti, incubare a temperatura ambiente per 1 ora. </li><li> Anticorpi primari diluiti con tampone colorazione ICC. Aggiungere 200 ml di soluzioni di anticorpi a ogni pozzetto e incubare una notte a 4 ° C. I fattori di diluizione anticorpo sono elencati in Materiali. </li><li> Il giorno dopo, lavare le cellule con PBS per tre volte. </li><li> Aggiungere 200 microlitri di soluzioni di anticorpi secondari alle cellule e incubare per 1 ora a RT nel buio. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Aggiungere 150 ml soluzione contenente DAPI in ogni pozzetto di montaggio. Lasciare macchia nuclei per 1 min. Poicoprire ogni pozzetto con copertura in vetro rotondo slittamento e premere delicatamente la polizza contro la superficie inferiore con una pinza per rimuovere le bolle d&#39;aria. </li><li> Aspirare la soluzione in eccesso ed i campioni sono pronti per l&#39;imaging. Le immagini rappresentative che mostrano le cellule riprogrammate che esprimono marcatore cardiaco cTnT e αActinin a d14 sono mostrati in figura 2B. </li></ol> 6. Analisi FACS di Riprogrammazione Efficienza <ol><li> Lavare accuratamente le cellule una volta con DPBS. Aggiungere 0,3 ml di tripsina (0,05%) a ciascun pozzetto e incubare a 37 ° C per 5 min. </li><li> Battere delicatamente la piastra per facilitare il sollevamento delle cellule. Controllare il distacco delle cellule sotto il microscopio. Aggiungere 1 ml di FACS tampone di quando la maggior parte delle cellule sono dissociate. Pipetta su e giù per dissociare ulteriormente meccanicamente le cellule. </li><li> Trasferire le cellule nella piastra 24 pozzetti ad una piastra ben pozzo profondo 96 pozzo a pozzo. Spin a 200 xg per 5 minuti a 4 ° C per raccogliere le cellule. </li><li> Lavare le cellule una volta conBuffer di FACS. </li><li> Aggiungere 100 ml di soluzioni di fissaggio / permeabilizzazione per risospendere le cellule per 20 minuti a 4 ° C. </li><li> Lavare le cellule due volte con soluzione di lavaggio 1x, pellet, e rimuovere il surnatante. </li><li> Preparare la soluzione anticorpo primario contro GFP e cTnT in tampone di lavaggio 1x. Risospendere accuratamente ciascun campione con soluzione di anticorpi 50 microlitri e incubare a 4 ° C per 30 min. </li><li> Lavare le cellule una volta nella soluzione di lavaggio 1x, pellet, e rimuovere il surnatante. </li><li> Aggiungere 50 ml soluzione di anticorpo secondario contenente Alexa Fluor IgG anti-coniglio 488 asino coniugato e Alexa Fluor 647 asino coniugato anti-topo IgG per ogni campione. Incubare a 4 ° C per 30 minuti in scuro. </li><li> Lavare le cellule una volta nella soluzione di lavaggio 1x, pellet, e rimuovere il surnatante. </li><li> Risospendere le cellule in 400 microlitri di buffer di fissazione (DPBS con 1% PFA). Trasferimento delle cellule in provetta FACS con tappo colino cella. I campioni sono pronti per il rilevamento di FACS. Il rappresentante analisi FACS di riprogrammazione efficiency utilizzando pMxs-puromicina-MGT costrutto con o senza selezione puromicina è mostrato in Figura 2A. </li></ol>

<ol>
	<li>Mozaffarian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2015+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2015 Update: A Report From the American Heart Association.</a> Circulation. , (2014).</li><li>Whelan, R. S., Kaplinskiy, V., Kitsis, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+the+pathogenesis+of+heart+disease:+mechanisms+and+significance.">Cell death in the pathogenesis of heart disease: mechanisms and significance.</a> Annu Rev Physiol. 72, (2010).</li><li>Senyo, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+heart+renewal+by+pre-existing+cardiomyocytes.">Mammalian heart renewal by pre-existing cardiomyocytes.</a> Nature. 493, 433-436 (2013).</li><li>Soonpaa, M. H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am J Physiol. 272, 220-226 (1997).</li><li>Hosoda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonality+of+mouse+and+human+cardiomyogenesis+in+vivo.">Clonality of mouse and human cardiomyogenesis in vivo.</a> Proc Natl Acad Sci U S A. 106, 17169-17174 (2009).</li><li>Choi, W. Y., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration.">Cardiac regeneration.</a> Curr Top Dev Biol. 100, 319-344 (2012).</li><li>Souders, C. A., Bowers, S. L., Baudino, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast:+the+renaissance+cell.">Cardiac fibroblast: the renaissance cell.</a> Circ Res. 105, 1164-1176 (2009).</li><li>Ieda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblasts+regulate+myocardial+proliferation+through+beta1+integrin+signaling.">Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling.</a> Dev Cell. 16, 233-244 (2009).</li><li>Moore-Morris, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+fibroblast+lineages+mediate+pressure+overload-induced+cardiac+fibrosis.">Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis.</a> J Clin Invest. 124, 2921-2934 (2014).</li><li>Addis, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+direct+fibroblast+reprogramming+to+cardiomyocytes+using+calcium+activity+as+a+functional+measure+of+success.">Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.</a> J Mol Cell Cardiol. 60, 97-106 (2013).</li><li>Chen, J. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+reprogramming+of+fibroblasts+into+cardiomyocytes+using+Gata4,+Mef2c,+and+Tbx5.">Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5.</a> Circ Res. 111, 50-55 (2012).</li><li>Christoforou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+MYOCD,+SRF,+Mesp1+and+SMARCD3+enhance+the+cardio-inducing+effect+of+GATA4,+TBX5,+and+MEF2C+during+direct+cellular+reprogramming.">Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.</a> PLoS One. 8, e63577 (2013).</li><li>Fu, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+human+fibroblasts+toward+a+cardiomyocyte-like+state.">Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.</a> Stem Cell Reports. 1, 235-247 (2013).</li><li>Hirai, H., Katoku-Kikyo, N., Keirstead, S. A., Kikyo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+direct+reprogramming+of+fibroblasts+into+cardiomyocyte-like+cells+with+the+MyoD+transactivation+domain.">Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain.</a> Cardiovasc Res. 100, 105-113 (2013).</li><li>Ieda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142, 375-386 (2010).</li><li>Inagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cardiomyocyte-like+cells+in+infarct+hearts+by+gene+transfer+of+Gata4,+Mef2c,+and+Tbx5.">Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.</a> Circ Res. 111, 1147-1156 (2012).</li><li>Islas, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+ETS2+and+MESP1+transdifferentiate+human+dermal+fibroblasts+into+cardiac+progenitors.">Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.</a> Proc Natl Acad Sci U.S.A. 109, 13016-13021 (2012).</li><li>Jayawardena, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-mediated+in+vitro+and+in+vivo+direct+reprogramming+of+cardiac+fibroblasts+to+cardiomyocytes.">MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes.</a> Circ Res. 110, 1465-1473 (2012).</li><li>Muraoka, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-133+promotes+cardiac+reprogramming+by+directly+repressing+Snai1+and+silencing+fibroblast+signatures.">MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.</a> Embo J. 33, 1565-1581 (2014).</li><li>Nam, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141, 4267-4278 (2014).</li><li>Nam, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+fibroblasts+toward+a+cardiac+fate.">Reprogramming of human fibroblasts toward a cardiac fate.</a> Proc Natl Acad Sci USA. 110, 5588-5593 (2013).</li><li>Protze, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+transcription+factor+screening+for+reprogramming+of+fibroblasts+to+cardiomyocyte-like+cells.">A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells.</a> J Mol Cell Cardiol. 53, 323-332 (2012).</li><li>Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+mouse+fibroblasts+into+cardiomyocyte-like+cells+in+vitro.">Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.</a> Nat Protoc. 8, 1204-1215 (2013).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Molecules+Enable+Cardiac+Reprogramming+of+Mouse+Fibroblasts+with+a+Single+Factor,+Oct4.">Small Molecules Enable Cardiac Reprogramming of Mouse Fibroblasts with a Single Factor, Oct4.</a> Cell Rep. , (2014).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+Gata4,+Mef2c,+and+Tbx5+Influences+the+Efficiency+and+Quality+of+Induced+Cardiac+Myocyte+Reprogramming.">Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming.</a> Circ Res. , (2014).</li><li>Song, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485, 599-604 (2012).</li><li>Qian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes.">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485, 593-598 (2012).</li><li>Mathison, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cardiac+cellular+reprogramming+efficacy+is+enhanced+by+angiogenic+preconditioning+of+the+infarcted+myocardium+with+vascular+endothelial+growth+factor.">In vivo cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the infarcted myocardium with vascular endothelial growth factor.</a> J Am Heart Assoc. 1, e005652 (2012).</li><li>Srivastava, D., Ieda, M., Fu, J., Qian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+repair+with+thymosin+beta4+and+cardiac+reprogramming+factors.">Cardiac repair with thymosin beta4 and cardiac reprogramming factors.</a> Ann NY Acad Sci. 1270, 66-72 (2012).</li><li>Muraoka, N., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+transcription+factors+is+critical+for+cardiac+reprogramming.">Stoichiometry of transcription factors is critical for cardiac reprogramming.</a> Circ Res. , 116-216 (2015).</li><li>Srivastava, D., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+factors+for+cardiac+reprogramming.">Critical factors for cardiac reprogramming.</a> Circ Res. 111, 5-8 (2012).</li></ol>

The authors have nothing to disclose.

Per il successo generazione ICM quando si utilizza questo protocollo, ci sono diversi fattori importanti che hanno un impatto sull&#39;efficienza complessiva. In particolare le condizioni di fibroblasti di partenza e la qualità di codifica retrovirus MGT possono influenzare notevolmente l&#39;efficienza riprogrammazione. È importante generare fibroblasti fresco e sano possibile. Per espianto metodo di coltura, i fibroblasti possono essere utilizzati prima di sette giorni dopo gli espianti ...

Cardiovascular disease remains the leading cause of death worldwide, accounting for 17.3 million deaths per year1. Loss of cardiomyocytes resulting from myocardial infarction (MI) or progressive heart failure is a major cause of morbidity and mortality2. Due to limited regenerative capacity, adult mammalian hearts usually suffer from impaired pump function and heart failure following injury3-6. As such, efficient (re)generation of cardiomyocytes in vivo and in vitro for treatment of heart disease and for disease modeling is a critical issue needing to be addressed.
Recent development of direct reprogramming, which directly reprograms cells from one differentiated phenotype to another without transitioning through the pluripotent state, offers a promising alternative approach for regenerative medicine. The mammalian heart contains abundant cardiac fibroblasts (CFs), which account for approximately half of the cells in heart and massively proliferate upon injury7-9. Thus, the vast pool of CFs could serve as an endogenous source of new CMs for regenerative therapy if they could be directly reprogrammed into functional CMs. It has been shown that a combination of transcription factors, such as Gata4 (G), Mef2c (M) and Tbx5 (T), with or without microRNAs or small molecules can reprogram fibroblasts into iCMs10-26. Importantly, this conversion can also be induced in vivo, and results in an improvement in cardiac function and a reduction in scar size in an infarcted heart16,27-29. These studies indicate that direct cardiac reprogramming may be a potential avenue to heal an injured heart. However, the low efficiency of iCM reprogramming has become a major hurdle for further mechanistic studies. In addition, the reproducibility of cardiac reprogramming is another controversial issue of this technology11,30,31. 
Very recently, we generated a complete set of polycistronic constructs encoding G,M,T in all possible splicing orders with identical 2A sequences in a single mRNA. These polycistronic constructs yielded varied G, M and T protein expression levels, which led to significantly different reprogramming efficiency25. The most efficient construct, named MGT, which showed a relatively high Mef2c and low Gata4 and Tbx5 expression, significantly improved reprogramming efficiency and produced large amounts of iCMs with CM markers expression, robust calcium oscillation and spontaneous beating25. Moreover, by using MGT polycistronic construct, our study avoided the use of multiple vectors and generated cells with homogenous expression ratio of G,M,T, thus providing an improved platform for cardiac reprogramming research. To increase experimental reproducibility, here we describe in detail how to isolate fibroblasts, produce retrovirus carrying MGT cassette, generate iCMs and evaluate the reprogramming efficiency.

<table border="0" cellpadding="0" cellspacing="0" width="1088">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">anti-cardiac troponin T</td>
			<td style="width:236px;">Thermo Scientific</td>
			<td style="width:200px;">MS-295-PO</td>
			<td style="width:191px;">1:200 for FACS and 1:400 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-GFP</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11122</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti- aActinin</td>
			<td>Sigma-Aldrich</td>
			<td>A7811</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Connexin43</td>
			<td>Sigma-Aldrich</td>
			<td>C6219</td>
			<td>1:500 for&#160; ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anit-Mef2c</td>
			<td>Abcam</td>
			<td style="width:200px;">ab64644</td>
			<td>1:1000 for ICC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Gata4</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:200px;">sc-1237</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Tbx5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17866</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 488&#8211;conjugated donkey anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>711-545-152</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 647&#8211;conjugated donkey anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>715-605-150</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Cytofix/Cytoperm kit for intracellular staining</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">554722</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rhod-3 Calcium Imaging Kit</td>
			<td>Life Technologies</td>
			<td style="width:200px;">R10145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thy1.2 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-049-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectashield solution with DAPI</td>
			<td>Vector labs</td>
			<td style="width:200px;">H-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td style="width:200px;">F-2442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">25-052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PRMI1640 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Life Technologies</td>
			<td style="width:200px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td style="width:200px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">30-002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11130-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11668500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">blasticidin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11139-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Millipore</td>
			<td style="width:200px;">TR-1003-G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td style="width:236px;">Fisher</td>
			<td style="width:200px;">BP151-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>PX1405</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:200px;">15714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Retrovirus Precipitation Solution</td>
			<td>ALSTEM</td>
			<td style="width:200px;">VC-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4%Trypan blue solution</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">G1393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS, 1x)</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS (Hanks Balanced Salt Solution)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">21022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LS column</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;m cellulose acetate filter&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">190-2545</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well plates</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm Tissue culture dishes&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">172958</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mm center well culture dish&#160;&#160;</td>
			<td>Corning</td>
			<td style="width:200px;">3260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Clear V-Bottom 2mL Polypropylene Deep Well Plate</td>
			<td>Denville Scientific</td>
			<td style="width:200px;">P9639</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Polystyrene round-bottom tubes with cell-strainer cap&#160;</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:200px;">5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortexer MINI</td>
			<td style="width:236px;">VWR</td>
			<td style="width:200px;">58816-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EVOS&#174; FL Auto Cell Imaging System</td>
			<td>Life Technologies</td>
			<td style="width:200px;">AMAFD1000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MidiMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Round glass cover slip</td>
			<td style="width:236px;">Electron Microscopy Sciences</td>
			<td style="width:200px;">72195-15</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved generation of induced cardiomyocytes using a polycistronic construct expressing optimal ratio of gata4, mef2c and tbx5

La conversione diretta dei fibroblasti cardiaci (CFS) in cardiomiociti indotte (ICMS) ha un grande potenziale per la medicina rigenerativa, offrendo strategie alternative per il trattamento di malattie cardiache. Questa conversione è stato ottenuto mediante espressione forzata di fattori definiti come Gata4 (G), MEF2C (M) e Tbx5 (T). Tradizionalmente, ICMS sono generati da un cocktail di virus che esprimono questi singoli fattori. Tuttavia, riprogrammazione efficienza è relativamente basso e la maggior parte in vitro G, M, T-trasdotte fibroblasti non diventano completamente riprogrammati, rendendo difficile lo studio dei meccanismi di riprogrammazione. Abbiamo recentemente dimostrato che la stechiometria di G, M, T è fondamentale per un&#39;efficiente riprogrammazione ICM. Un stechiometria ottimale di G, M, T ad alto livello relativo di M e bassi livelli di G e T ottenuti utilizzando il nostro vettore MGT policistronico (di seguito denominato MGT) significativamente maggiore efficienza riprogrammazione e una migliore qualità Icm in vitro. Qui forniamo una descrizione dettagliata della metodologia utilizzata per generare ICMS con MGT costrutto da fibroblasti cardiaci. Isolamento di fibroblasti cardiaci, la generazione di virus per la riprogrammazione e la valutazione del processo di riprogrammazione sono inclusi anche di fornire una piattaforma per la generazione efficiente e riproducibile di ICMS.

Le fasi di riprogrammazione sono riassunti da schema in figura 1. Dopo MGT trasduzione, l&#39;espressione della GFP in riprogrammazione delle cellule potrebbe essere rilevato fin dal giorno 3. puromicina selezione di cellule trasdotte parte da giorno 3 e viene mantenuta durante le prime due settimane se PMX-puro è usato costrutto -MGT. Di giorno 10 al giorno 14, espressione di marcatori cardiaci come cTnT e αActinin potrebbe essere rilevato da entrambi ICC (Figura 2B, punto 5) e FACS ...

Watch this Scientific Journal Video about Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5 at JoVE.com

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Migliorata la generazione di cardiomiociti indotte usando un costrutto policistronico Esprimendo rapporto ottimale di Gata4, MEF2C e Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

improved-generation-induced-cardiomyocytes-using-polycistronic

Research

JoVE Journal

Developmental Biology

8.6K Views.  University of North Carolina, Chapel Hill. We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming. 

Video: Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Derivazione di Cardiomyocytes altamente purificato da cellule staminali umane pluripotenti indotte utilizzando piccoli Differenziazione Molecola-modulato e successiva glucosio Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Ricerca

Biologia dello sviluppo

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Isolamento e Crioconservazione di neonatali Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generazione di pluripotenti indotte cellule staminali da congelata Buffy cappotti con non-integrazione episomiale Plasmidi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Generazione di pluripotenti indotte cellule staminali da umani periferici T Cellule con Sendai virus in condizioni di libera-Feeder

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Generazione di cellule staminali pluripotenti indotte-Uso sinoviociti fibroblasti-come isolato da Giunti dell&#39;artrite reumatoide pazienti

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Pluripotenti indotte generazione di cellule staminali da cellule del sangue Utilizzando Sendai Virus e centrifugazione

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generazione di integrazione-liberi cellule staminali pluripotenti indotte dalle cellule mononucleari di sangue periferico umano Utilizzando episomiale Vettori

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Maturazione di staminali umane cardiomiociti derivati ​​dalle cellule in Biowires Utilizzando Stimolazione Elettrica

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Generazione di insulina senza impalcatura, tridimensionale esprimendo Pancreatoids dal Mouse progenitori pancreatico In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...