A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

Tutte le procedure sperimentali che coinvolgono le pratiche degli animali sono stati approvati dal Comitato Istituzionale Animal cura e l&#39;uso a UT Southwestern Medical Center. 1. Isolamento di HCN4-GFP E12.5 embrionali di topo fibroblasti (MEF) <ol><li> Impostare accoppiamenti cronometrati tra omozigoti maschi HCN4-GFP e CD-1 femmine. </li><li> Sacrifica femmina incinta a E12.5 dal biossido di carbonio l&#39;eutanasia e la successiva dislocazione cervicale. <ol><li> Rimuovere corna uterine con dissettore come descritto in precedenza 22, 23, e metterli in una capsula di Petri sul ghiaccio con soluzione salina 1x tampone fosfato (PBS) senza Ca 2+ e Mg 2+. </li></ol></li><li> Eseguire tutti i passaggi successivi nella cappa di coltura utilizzando la tecnica sterile. <ol><li> Rimuovere gli embrioni dall&#39;utero e amniotico sac con le forbici e pinze dissezione. Mantenere la placenta allegata per una migliore maneggevolezza. pregnant CD-1 le femmine in genere partoriscono tra 10 e 14 cuccioli. </li><li> Utilizzando dissezione pinze, prendere gli embrioni isolate e rapidamente sciacquare due volte in 70% (v / v) EtOH. NOTA: I lavaggi devono essere veloci per ridurre al minimo la morte delle cellule. </li><li> Togliere la testa, gli arti, la coda, e gli organi interni, compreso il cuore dagli embrioni isolate. </li><li> Posizionare il tessuto rimanente in un 10 cm Piatto con 1 ml di PBS 1x e tritare finemente usando una lama di rasoio sterile a circa 1 mm 3 dimensioni. </li><li> Trasferire il tessuto tritato in una provetta da 50 ml con PBS. </li><li> Spin a 300 xg per 3 min. Con attenzione aspirare l&#39;eccesso di PBS. </li><li> Aggiungere 1 ml di sterile 0,25% tripsina-EDTA per ogni embrione. Incubare le cellule in un bagno d&#39;acqua a 37 ° C per 15 min. Mescolare delicatamente il tubo ogni 4 min. Over-digestione del tessuto abbasserà notevolmente la resa. </li><li> miscela di cellule Vortex a velocità massima (3.200 rpm) per 4 s. </li><li> Aggiungere 2 ml di media fibroblasti per embrioni e mescolare. Filtro tttraverso un colino cella di 100 micron con una pipetta per aiutare le cellule attraverso il filtro. Fare riferimento alla Tabella 1 per la formulazione di tutti i mezzi successive. </li><li> Spin a 300 xg per 4 minuti. Con attenzione aspirare il surnatante. </li><li> Aggiungere 10 ml di mezzi di fibroblasti fresca per 3 embrioni e triturare 6-10 volte. </li><li> Piastra le cellule in 1 15 cm piatto di coltura di tessuti per ogni 3 embrioni preparati. Cultura overnight a 37 ° C, 5% CO 2 incubatore. </li><li> Dopo l&#39;incubazione durante la notte, sostituire il supporto con freschi 30 ml di supporti per piastra. Mettere le cellule di nuovo nel incubatore durante la notte. NOTA: Verificare la presenza di contaminazione delle cellule HCN4-GFP + sotto un microscopio a fluorescenza. La cultura dovrebbe essere GFP - e diventare solo GFP + sulla riprogrammazione. </li><li> Il giorno dopo, le cellule raccolto con 3 ml di fresca pre-riscaldato 0,25% tripsina-EDTA. Contare e congelare le cellule. Tipicamente, congelare cellule a 3 x 10 6 cellule per ml. Il Yie previstold dovrebbe essere di 3 x 10 6 cellule per dell&#39;embrione. </li></ol></li></ol> 2. Retrovirus Produzione e riprogrammazione Attenzione: il seguente protocollo richiede la produzione e la gestione dei retrovirus infettive. Effettuare le seguenti operazioni in un armadio biosicurezza di livello 2 sotto BSL-2 linee guida e tecnica sterile. Utilizzare il 10% di candeggina per smaltire tutti i materiali esposti a retrovirus. <ol><li> La produzione Retrovirus e preparazione MEF NOTA: Il seguente protocollo è per la produzione di retrovirale in 6 pozzetti e infezioni MEF in piastre da 24 pozzetti. Per gli altri formati, fare riferimento alla Tabella 2. MEF sono placcati in occasione della Giornata -1, così dovrà essere coordinata in modo appropriato per ogni esperimento (fare riferimento alla sezione 2.3 e Figura 2) la tempistica. <ol><li> Mantenere le cellule secondo le raccomandazioni del produttore Plat-E (PE). In breve, le cellule cultura PE in DMEM supplementato con 10% FBS, 1 mg / ml puromycin, 10 mg / ml blasticidin, la penicillina, e streptomicina. cellule Passage 1: 4 ogni due giorni in cui la cultura raggiunge il 70-90% di confluenza. </li><li> Giorno -2: Il giorno prima trasfezione, le cellule seme Plat-E a cellule 1 x 10 6 / bene su un 6-pozzetti nei media trasfezione. Le cellule devono essere 70-80% confluenti al momento della transfezione. </li></ol></li><li> Trasfezione utilizzando un agente trasfezione commerciale. NOTA: I reagenti commerciali devono essere a temperatura ambiente (RT) prima di trasfezione. Per la trasfezione del DNA, aggiungere ogni DNA plasmide retrovirale singolarmente (G, H, M e T) per formare un cocktail GHMT 9. <ol><li> Giorno -1: In una provetta di polistirene da 15 ml conica, mescolare 60 ml di mezzi siero ridotti con 6 ml di trasfezione reagente per la reazione per un formato 6-pozzetti. Incubare la miscela per 5 minuti a temperatura ambiente. NOTA: Dal momento che il reagente di trasfezione usato qui si lega alla plastica, undd direttamente ai media siero ridotta per evitare qualsiasi diminuzione di efficienza di trasfezione. </li><li> Aggiungere un totale di 2 mg di GHMT cocktail a reazione e delicatamente toccare per mescolare. Non farlo vortice. Incubare la reazione per 15 minuti a RT. </li><li> Aggiungere il composto dal punto 2.2.3 alle cellule PE in maniera saggia goccia. </li><li> Incubare le cellule trasfettate Plat-E durante la notte in un 37 ° C, 5% CO 2 incubatore. Registrare il tempo di trasfezione. </li></ol></li><li> Semina di HCN4-GFP fibroblasti embrionali di topo. <ol><li> 1 h prima della placcatura MEF, preparare un 24-pozzetti per immunocitochimica. <ol><li> Aggiungere un fibronectina coprioggetto 12 millimetri per pozzetto. </li><li> Pozzi cappotto con 300 ml di soluzione di collagene bovino (ad esempio, SureCoat) e incubare in un incubatore a 37 ° per 1 ora. </li><li> soluzione di rivestimento Aspirare immediatamente prima placcatura MEF. </li></ol></li><li> Scongelare una fiala congelata di HCN4-GFP MEF e lavare x1 con fibrobl pre-riscaldatosupporti AST a 500 xg per 5 min. </li><li> Determinare la vitalità delle cellule utilizzando trypan esclusione blu o coloranti simili. Calcolare il numero di cellule vitali per ml di coltura utilizzando la seguente formula: % cellule vitali = [1.00 - (Numero di celle blu / numero di cellule totali)] x 100 <ol><li> Calcolare il numero totale di cellule vitali utilizzando la seguente formula: Le cellule vitali =% cellule vitali x fattore di diluizione x 10.000 x volume totale della sospensione cellulare </li></ol></li><li> Seed 3 x 10 4 cellule per pozzetto su una piastra da 24 pozzetti con collagene bovino coprioggetto soluzione fibronectina precedentemente preparato. </li></ol></li><li> La trasduzione e riprogrammazione di MEF NOTA: Secondo le note del produttore, corretta manutenzione cellule Plat-E producono un titolo medio di 1 x 10 7 unità di infezione / mL. Anche se il titolo non viene misurato direttamente per ogni esperimento, un controllo GFP è sempre inclusa come un surrogato per l&#39;efficienza infezione.Espressione alta GFP e l&#39;intensità (GFP +&gt; 95%) correla generalmente con successo generazione GHMT-mediata di iCLMs. <ol><li> Giorno 0: 24 ore dopo la trasfezione, filtrare il medium retrovirale PE attraverso una dimensione del filtro di acetato 0,45 micron-pori senza tensioattivi di cellulosa e trasferimento in un tubo da 15 ml. Aggiungere polibrene ad una concentrazione finale di 8 mg / mL. ricostituire con attenzione le cellule con 2 ml di terreno fresco trasfezione. NOTA: Le cellule facilmente staccano dalla piastra se il supporto è cambiato troppo rapidamente. </li><li> Aspirare il mezzo di coltura delle MEF e aggiungere il supporto retrovirale appena raccolti; esso dovrebbe produrre 1,7 ml di mezzi per pozzetto di un 6-pozzetti. Aggiungere ~ 800 ml per pozzetto di una piastra da 24 pozzetti. Rientro piatto MEF per l&#39;incubatore e incubare una notte. </li><li> Giorno 1: ripetere le fasi 2.4.1 e 2.4.2. Eliminare le celle dopo la collezione di virus 2 °. Rientro MEF infetti per l&#39;incubatore e lasciarli riposare durante la notte. </li><li> 2 ° giorno: 48 ore dopo l&#39;induzione, aspirare il cellulare condizionata media e lavare x1 con PBS 1x. Aggiungere i media iCLM 500 ml di pre-riscaldato per pozzetto di una piastra ben 24. </li><li> Sostituire i media iCLM ogni 2 - 3 giorni. Placca di 14 giorni dopo l&#39;induzione virale per immunocitochimica (ICC) analisi della riprogrammazione cardiaco. </li></ol></li></ol> 3. Immunocolorazione di MEF riprogrammato <ol><li> 14 giorni post-induzione, aspirare accuratamente la media. </li><li> Lavare ogni pozzetto con 300 ml di 1x PBS ghiacciato. Aspirare la soluzione in eccesso. </li><li> Fissare le cellule con 250 ml di paraformaldeide soluzione al 4% (PFA) per pozzetto di una piastra ben 24. Incubare 15 minuti a temperatura ambiente. NOTA: fisso cellule possono essere conservate in PBS a 4 ° C per 1 - 2 settimane prima colorazione. </li><li> cellule permeabilize da Wells lavaggio x3 con 300 microlitri di 0.1% PBS-TritonX 100 (PBST). Incubare 5 minuti a temperatura ambiente tra i lavaggi. Aspirare la soluzione in eccesso dopo l&#39;ultimo lavaggio. </li><li> Blocco per 10minuti a temperatura ambiente con 1x universale blocchetto a 300 ml / pozzetto. </li><li> Preparare buffer di colorazione (ICC): Aggiungere 1: 1 di PBS 1x e 1x universale tampone di bloccaggio. Diluire gli anticorpi primari nel buffer di colorazione ICC e incubare anticorpi notte a 4 ° C. Fare riferimento alla sezione materiale per diluizioni raccomandate. <ol><li> Macchia un paio di diapositive con il mouse α-actinina, αGFP pollo e coniglio NPPA per iPM e l&#39;identificazione iAM. </li><li> Macchia un paio di diapositive con il mouse α-actinina, αGFP pollo e di coniglio per MYL2 iPM e IVM identificazione. </li></ol></li><li> Il giorno seguente, lavare i pozzetti x3 con 300 microlitri di 0.1% PBST. Incubare 5 minuti a temperatura ambiente tra i lavaggi. Aspirare la soluzione in eccesso dopo l&#39;ultimo lavaggio. </li><li> Preparare le diluizioni degli anticorpi secondari nel buffer ICC colorazione. Fare riferimento alla sezione materiale per diluizioni raccomandate. Incubare anticorpi secondari 1 ora a temperatura ambiente, al riparo dalla luce. <ol><li> Macchia tutte le diapositive con il seguente antibodie secondarias: il mouse Alexa-555, pollo Alexa-488, e coniglio Alexa-647. </li></ol></li><li> Lavare pozzi x3 con 300 microlitri di 0.1% PBST. Incubare 5 minuti a temperatura ambiente tra i lavaggi. Proteggere dalla luce. </li><li> Aggiungere 2,4 ml di mezzo di montaggio contenenti 1,5 mg / mL di 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) su un vetrino per microscopio. Rimuovere con attenzione il vetrino dal pozzo della piastra 24 pozzetti, rimuovere la soluzione in eccesso, e trasferire al vetrino con mezzi di montaggio. Premere delicatamente il vetrino per rimuovere l&#39;eccesso di volume e l&#39;aria. </li><li> diapositive Seal con smalto preferito o sigillante di plastica. Conservare diapositive montate a 4 ° C al riparo dalla luce. </li></ol> 4. Identificazione di cardiache sottotipi Utilizzando Microscopia confocale NOTA: Per l&#39;imaging, un microscopio confocale dotato di almeno 2 rivelatori fluorescenti capaci di rilevazione spettrale a 405, 488, 555 e 639 nm lunghezza d&#39;onda necessaria per identificare IPM, Iams, e IVMS. imagcellule e utilizzando un piano-Apochromat 20X / 0.75 obiettivo o migliore. Utilizzando il software di analisi di immagine del produttore, la digitalizzazione delle immagini dello zoom può ottenere immagini di qualità ad immersione 40X-petrolio. <ol><li> libreria Immagine: prendere immagini a 8 bit con DAPI, Alexa-488, Alexa-555, e Alexa-647 canali (cap.). Pixel tempo di sosta di 6 s, 1024 dimensione del frame, passo la linea a 2, e la media dei 2 è sufficiente per immagini ad alta risoluzione. </li><li> Per ogni diapositiva, iniziare da un bordo e iniziare la scansione su e giù nel canale rosso fluorescente (ch.) Per le cellule sarcomere + α-actinina + (Fare riferimento alla Figura 3 e Figura 5A per gli esempi). striature sarcomere sono più facili da identificare visivamente nella lunghezza d&#39;onda 555 nm. <ol><li> Una volta che un α-actinina + cellule sarcomere + è stato identificato, passare alla ch verde. e di tenere nota se è positivo (IPM). Passare al computer per valutare il far-rosso 647 ch. (IAM o IVM). Nota: le celle che sonoα-actinina + / sarcomero + / Hnc4-GFP + / NPPA - / MYL2 - sono designati come IPMS. Espressione GFP sarà visto in tutta la cella (Figura 4A). </li><li> diapositive macchia con α-actinina (mouse Alexa555), HCN4-GFP (pollo-Alexa488), NPPA (coniglio-Alexa647) e DAPI. Le cellule che sono α-actinina + / sarcomero + / Hnc4-GFP - / NPPA + sono iAM. NPPA colorazione apparirà perinucleare e puntiformi (Figura 4B). </li><li> diapositive macchia con α-actinina (mouse Alexa555), HCN4-GFP (pollo-Alexa488), MYL2 (coniglio-Alexa647). Cellule positive per la α-actinina + / sarcomero + / Hnc4-GFP - / + MYL2 sono IVMS. MYL2 colorazione esporrà una forma striato lungo il filamento sarcomero. A causa di variazioni nella qualità della colorazione e Z-plane, striature non sono sempre facilmente visibili (Figura 4C). </li></ol></li></ol> NOTA: Per valutare il numero effettivo di MEF potenzialmente riprogrammati, 2 pozzetti di una piastra da 24 pozzetti vengono seminate in parallelo ai pozzetti sperimentali sono raccolte un giorno dopo la placcatura. Il numero totale di cellule piastrate è quindi determinata dalla media dei due pozzetti. Questo diventa le cellule totali effettivi cromato (atotal). <ol><li> sarcomere + <ol><li> Visivamente ogni cella su un vetrino per la corretta α-actinina + / sarcomero + (pannelli di destra Figura 3) e registrare (Figura 5B-i). </li><li> Tabulare il numero totale di α-actinina + / + sarcomere su ogni vetrino e dividere per le cellule totali effettivi cromato (atotal) (Figura 5B-III). Ad esempio, se atotal = 12.500 cellule, e 100 cellule sono state alfa-actinina + / + sarcomere poi, 0,8% dei MEF placcati stati riprogrammato. Una mediareprograming esperimento produrrà cellule 1% alfa-actinina + / sarcomero + (Figura 5C). </li></ol></li><li> sottotipo + NOTA: Per le seguenti operazioni, fare riferimento alla Figura 5B / C per un flusso di lavoro rappresentante iCLM quantificazione. In breve, per ogni sarcomero + cellulare, tabulare se è unica sia per il sottotipo (Figura 5B-i). Calcolare% sottotipo (Figura 5B-iii) dividendo il numero di cellule sottotipo + sul sarcomere media + cellule x 100 (Figura 5B-i). Per calcolare l&#39;assoluta efficienza% sottotipo (Figura 5B-IV), dividere il numero sottotipo + cellulare da figura 5B-i per il numero totale di cellule seminate x 100 (Figura 5B-ii). <ol><li> Per le celle ciascuno di α-actinina + / + sarcomere, valutare se sono GFP +, NPPA +, o MYL2 <sup&gt; +. </li><li> Tabulare numero totale di α-actinina + / sarcomero + / Hnc4-GFP + / NPPA - / MYL2 -, actinina + / sarcomero + / Hnc4-GFP - / NPPA +, e α-actinina + / sarcomero + / Hnc4-GFP - + cellule / MYL2 (Figura 5B-i). </li><li> Per calcolare la percentuale di ciascun sottotipo, dividere il numero di cellule Sottotip + per il numero totale di α-actinina + / + cellule sarcomere in che pure e moltiplicare per 100. GHMT genera IPMS, Iams, e IVMS più o meno uguali rapporti (Figura 5B-III). </li><li> Per calcolare il numero assoluto di sottotipo + cellule, dividere il numero totale di sottotipo + cellule per la condizione sperimentale atotal e moltiplicare per 100 (Figura 5B-ii). Su IPMS media rappresentano lo 0,3% della popolazione cellulare infettata totale, Iams 0,3%, eIVMS 0,25% (Figura 5B-iv). </li></ol></li></ol>

<ol>
	<li>Sissman, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+landmarks+in+cardiac+morphogenesis:+comparative+chronology.">Developmental landmarks in cardiac morphogenesis: comparative chronology.</a> Am J Cardiol. 25 (2), 141-148 (1970).</li><li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6 (11), 826-835 (2005).</li><li>Ali, S. R., 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=Existing+cardiomyocytes+generate+cardiomyocytes+at+a+low+rate+after+birth+in+mice.">Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.</a> Proc Natl Acad Sci U S A. 111 (24), 8850-8855 (2014).</li><li>Bergmann, O., 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=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</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 (7432), 433-436 (2013).</li><li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6 (239), 239rv231 (2014).</li><li>Writing Group, 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=Heart+Disease+and+Stroke+Statistics-2016+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), e38-e360 (2016).</li><li>Takahashi, K., Yamanaka, S. <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+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</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 (7400), 599-604 (2012).</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 (3), 375-386 (2010).</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 (7400), 593-598 (2012).</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 (3), 235-247 (2013).</li><li>Zhou, H., Dickson, M. E., Kim, M. S., Bassel-Duby, R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/protein+kinase+B+enhances+transcriptional+reprogramming+of+fibroblasts+to+functional+cardiomyocytes.">Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes.</a> Proc Natl Acad Sci U S A. 112 (38), 11864-11869 (2015).</li><li>Zhou, Y., 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=Bmi1+Is+a+Key+Epigenetic+Barrier+to+Direct+Cardiac+Reprogramming.">Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming.</a> Cell Stem Cell. 18 (3), 382-395 (2016).</li><li>Zhao, Y., 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=High-efficiency+reprogramming+of+fibroblasts+into+cardiomyocytes+requires+suppression+of+pro-fibrotic+signalling.">High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.</a> Nat Commun. 6, 8243 (2015).</li><li>Miki, K., Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+steady+progress+on+direct+cardiac+reprogramming+toward+clinical+application.">Making steady progress on direct cardiac reprogramming toward clinical application.</a> Circ Res. 113 (1), 13-15 (2013).</li><li>Atkinson, A., 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=Anatomical+and+molecular+mapping+of+the+left+and+right+ventricular+His-Purkinje+conduction+networks.">Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks.</a> J Mol Cell Cardiol. 51 (5), 689-701 (2011).</li><li>Bootman, M. D., Smyrnias, I., Thul, R., Coombes, S., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+cardiomyocyte+calcium+signalling.">Atrial cardiomyocyte calcium signalling.</a> Biochim Biophys Acta. 1813 (5), 922-934 (2011).</li><li>Miquerol, L., Beyer, S., Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+mouse+ventricular+conduction+system.">Establishment of the mouse ventricular conduction system.</a> Cardiovasc Res. 91 (2), 232-242 (2011).</li><li>Sp&#228;ter, D., Hansson, E. M., Zangi, L., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+cardiomyocyte.">How to make a cardiomyocyte.</a> Development. 141 (23), 4418-4431 (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 (22), 4267-4278 (2014).</li><li>Conner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (2001).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. (64), (2012).</li><li>Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+G+glycoprotein+pseudotyped+retroviral+vectors:+concentration+to+very+high+titer+and+efficient+gene+transfer+into+mammalian+and+nonmammalian+cells.">Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.</a> Proc Natl Acad Sci U S A. 90 (17), 8033-8037 (1993).</li><li>Ichim, C. V., Wells, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+high-titer+viral+preparations+by+concentration+using+successive+rounds+of+ultracentrifugation.">Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation.</a> J Transl Med. 9, 137 (2011).</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 (6), 1204-1215 (2013).</li><li>Yang, R., 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+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors.">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nat Commun. 5, 5807 (2014).</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=Direct+reprogramming+of+fibroblasts+into+myocytes+to+reverse+fibrosis.">Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.</a> Annu Rev Physiol. 76, 21-37 (2014).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E., Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Bass, G. 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=Automated+image+analysis+identifies+signaling+pathways+regulating+distinct+signatures+of+cardiac+myocyte+hypertrophy.">Automated image analysis identifies signaling pathways regulating distinct signatures of cardiac myocyte hypertrophy.</a> J Mol Cell Cardiol. 52 (5), 923-930 (2012).</li></ol>

The authors have nothing to disclose.

Il presente studio fornisce una strategia a riprogrammazione diretta per la conversione di MEF in un insieme diversificato di sottotipi cardiaci tramite espressione retrovirus-mediata della trascrizione cardiaco fattori Gata4, MEF2C, Tbx5, e Hand2 (GHMT). Utilizzando un approccio immunocolorazione multiplex in combinazione con un PM-specifica del mouse giornalista, siamo in grado di identificare iAM, IVMS, e IPMS ad una risoluzione di singola cellula. Tale test permette una sperimentazione in vitro sistema in g...

Il cuore è il primo organo funzionale a sviluppare nell&#39;embrione 1, 2. In combinazione con il sistema circolatorio, fornisce l&#39;ossigeno, nutrienti, e un meccanismo di smaltimento durante lo sviluppo. Tre settimane dopo la fecondazione, il cuore umano batte per la prima volta e la sua regolamentazione adeguata è mantenuta da cardiomiociti (CMS). La perdita irreversibile di queste cellule specializzate è quindi la questione fondamentale sottostante insufficienza cardiaca progressiva. Mentre alcuni organismi come zebrafish e Xenopus hanno il potenziale per la rigenerazione cardiaca, cuore adulto dei mammiferi è più limitata 3, 5, 6. Così, data la funzione critica del cuore, non è sorprendente che la malattia di cuore è la principale causa di morte nel mondo, che rappresentano 600.000 morti nel solo 7 negli Stati Uniti. Ilto, è terapie a base di cellule per riparare o sostituire il miocardio danneggiato in modo efficiente sono di grande interesse clinico. Lo studio fondamentale di Yamanaka e colleghi hanno mostrato che 8 espressione forzata di quattro fattori di trascrizione è sufficiente per convertire i fibroblasti completamente differenziate di cellule staminali pluripotenti. Tuttavia, la capacità tumorigenico di tutte le strategie di cellule staminali pluripotenti è stata una preoccupazione fondamentale per il loro uso per scopi terapeutici. Questo ha motivato il campo scientifico per la ricerca di metodi alternativi per transdifferenziare cellule, evitando uno stadio di pluripotenza. Recentemente, diversi gruppi hanno dimostrato la fattibilità di questa strategia per la visualizzazione di conversione diretta dei fibroblasti di topo a cellule cardiomiociti come indotte (iCLMs) con l&#39;espressione ectopica di fattori di trascrizione Gata4, MEF2C, Tbx5, e più tardi, Hand2 (GMT e GHMT rispettivamente) 9, 10. Furthermore, la stessa strategia può essere eseguita in vivo e in tessuti umani di derivazione 9, 11, 12. Recenti studi hanno evidenziato fattori complementari o vie di segnalazione che possono essere modulati per migliorare ulteriormente l&#39;efficienza riprogrammazione cardiaco 13, 14, 15. Presi insieme, questi studi dimostrano il potenziale di transdifferenziazione diretta per terapie rigenerative. Tuttavia, la scarsa efficienza della riprogrammazione CM, i meccanismi molecolari sconosciuti, riproducibilità incoerente a causa delle differenze metodologiche 16, e la natura eterogenea del iCLMs rimangono senza indirizzo. Al fine di valutare direttamente iCLM eterogeneità, abbiamo progettato un saggio unicellulare discreto e robusto per l&#39;individuazione di sviluppo sarcomere e cardiaco specificatio lignaggion-due necessarie caratteristiche di cardiomiociti funzionali. Ci sono almeno tre tipi principali di CM nel cuore come definite dalla loro posizione e uniche proprietà elettriche: atriale (AM), ventricolare (VM) e pacemaker (PM) 17, 18, 19, 20. In una combinazione orchestrato, permettono il corretto pompaggio di sangue. Durante la ferita del cuore, uno o tutti i sottotipi potrebbero essere interessati, e il tipo di terapia cellulare dovrebbero essere affrontate caso per caso. Attualmente, la maggior parte delle strategie si concentrano sulla generazione complessiva di cardiomiociti, mentre poco lavoro è stato fatto per studiare i meccanismi molecolari che regola specifica sottotipo. Il seguente studio dettagli come quantificare correttamente sarcomeri ben organizzati e identificare un insieme diversificato di sottotipi di cardiomiociti. Utilizzando un pacemaker (PM) topo giornalista SPECIFICI, siamo in grado di applicare un iapproccio mmunocytochemical distinguere miociti atriali indotte-like (IAM), miociti ventricolari come indotta (IVM), e indotti miociti PM-like (IPMS) 21. Sulla base delle nostre osservazioni, solo le cellule che presentano l&#39;organizzazione sarcomere sono in grado di battere spontanea. Questa piattaforma unica riprogrammazione permette di valutare il ruolo di alcuni parametri nell&#39;organizzazione sarcomere, specifica sottotipo, e l&#39;efficienza della riprogrammazione CM ad una risoluzione di singola cellula.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">DMEM</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D5796</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Medium 199</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Fetal bovine serrum (FBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">F2442</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Insulin-Transferrin-Selenium G</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">41400-045</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM vitamin solution</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11120-052</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1601149</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Non-Essential amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Antibiotic-Antimycotics</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15240062</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">B-27 supplement</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17504044</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Heat-Inactivated Horse Serum</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">26050-088</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">NaPyruvate</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11360-70</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Penicillin/Streptomycin</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1514022</td>
			<td style="width:347px;">Component of Plat-E media and fibroblast media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Puromycin&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A11139-03</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Blasticidin&#160;&#160;</td>
			<td style="width:91px;">Gemini Bio-Products</td>
			<td>400-128P</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Glutamax</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td>Component of Fibroblast media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Confocal laser scanning LSM700</td>
			<td style="width:91px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>For confocal analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">FuGENE 6 transfection Reagent</td>
			<td style="width:91px;">Promega</td>
			<td style="width:119px;">E2692</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Opti-MEM Reduced Serum Medium</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">31985-070</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Polybrene</td>
			<td style="width:91px;">Millipore</td>
			<td style="width:119px;">TR-1003-G</td>
			<td>Induction reagent. Use at a final concentration of 8um/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td>
			<td style="width:91px;">CellBiolabs</td>
			<td style="width:119px;">RV-101</td>
			<td>Retroviral pacaking cell line</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Trypsin 0.25% EDTA</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For MEFs and Plat-E dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti &#945;-Actinin (Clone EA-53)</td>
			<td style="width:91px;">Sigma</td>
			<td>A7811</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chicken anti-GFP IgY</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A10262</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti-NPPA&#160;</td>
			<td style="width:91px;">Abgent</td>
			<td>AP8534A</td>
			<td>Antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti Myl2 IgG&#160;</td>
			<td style="width:91px;">ProteinTech</td>
			<td>10906-1-AP</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Vectashield solution with DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:91px;">Vector Labs</td>
			<td style="width:119px;">H-1500</td>
			<td>Dye for confocal analysis</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Superfrost Plus Microscope slides</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">BioCoat Fibronectin 12mm coverslips</td>
			<td style="width:91px;">NeuVitro Corp</td>
			<td>GG-12-1.5</td>
			<td>Coverslips for confocal analysis</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">100um cell strainer</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">0.45um Syringes filters SFCA 25MM</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-740-106</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6ml Syringes</td>
			<td style="width:91px;">Covidien</td>
			<td>8881516937</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Chicken IgY (H&#38;L) A488</td>
			<td style="width:91px;">Abcam</td>
			<td>AB150169</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Donkey anti-rabbit A647 IgG(H+L)&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A31573</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Goat anti-mouse IgG(H+L) A555</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A21422</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Triton X-100</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">93443-100ml</td>
			<td>For cell permeabilization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Power Block 10X Universal Blocking reagent</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NC9495720</td>
			<td>Dilute to 1X in H20</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:319px;">16% Paraformaldehyde aqueous solution (PFA)</td>
			<td style="width:91px;">Electro Microscopy Sciences&#160;</td>
			<td style="width:119px;">15710</td>
			<td>Use at 4% diluted in dH20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6 cm&#160; plates</td>
			<td style="width:91px;">Olympus</td>
			<td style="width:119px;">25-260</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">6-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-105</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">24-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">10 cm Tissue culture dishes</td>
			<td style="width:91px;">Corning</td>
			<td>4239</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">15 cm&#160; Tissue culture dishes</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>5442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">15 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">50 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4249</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">0.4% Trypan blue solution</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td>For viability</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Ethyl Alcohol 200 proof</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">7005</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Bleach</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">6009</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

Approfittando della PM-specifica del mouse giornalista, abbiamo sviluppato una strategia immunocolorazione multiplex per identificare i diversi miociti endogeni come illustrato nella figura 1. Seguendo la procedura di riprogrammazione mostrati nella Figura 2, l&#39;induzione di CM specifici del sottotipo può essere rilevato fin dal giorno 4 21, anche se ad un basso tasso. Entro il giorno 14, l&#39;esperimento può essere fermato ...

Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Valutare cardiomiociti sottotipi A seguito di riprogrammazione Fattore di trascrizione-mediata di fibroblasti embrionali di topo

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

assessing-cardiomyocyte-subtypes-following-transcription-factor

Research

JoVE Journal

Developmental Biology

7.4K Views.  UT Southwestern Medical Center. This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5. 

Video: Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Ricerca

Biologia dello sviluppo

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

Analisi di Cardiomyocyte Sviluppo usando immunofluorescenza in cuore embrionali di topo

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Stabile ed efficiente Modificazione genetica di cellule nel topo adulto V-SVZ per l&#39;analisi di cellule staminali neurali autonoma ed effetti non autonomi

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Trascrittoma Profiling di<em&gt; In-Vivo</em&gt; Embrioni bovini pre-impianto prodotto con piattaforma microarray a due colori

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

La valutazione simultanea di Cardiomyocyte DNA Sintesi e Ploidia: Un metodo per assistere Quantificazione del Cardiomyocyte Rigenerazione e fatturato

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Protocollo per la conversione diretta dei fibroblasti murini embrionali in trofoblasto cellule staminali

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

Riprogrammazione fibroblasti embrionali di topo con fattori di trascrizione per indurre un programma Hemogenic

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissezione e cultura del rene embrionale del mouse

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Soppressione della segnalazione Pro-fibrotici rafforza fattore-mediata riprogrammazione di fibroblasti embrionali di topo in cardiomiociti indotti

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Diretto lignaggio riprogrammazione di fibroblasti di topo adulto ai progenitori eritroidi

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Valutare cardiomiociti sottotipi A seguito di riprogrammazione Fattore di trascrizione-mediata di fibroblasti embrionali di topo

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