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.

Burada özetlenen protokol yenidoğan fareler kullanır. Hayvan bakım ve deneyleri Kuzey Carolina Üniversitesi&#39;nde Bölümü Laboratuvarı Hayvan Tıp (DLAM), Chapel Hill tarafından kurulan kurallarına uygun olarak yapılmaktadır. Tamponlar ve Medya 1. Hazırlık <ol><li> Fibroblast (FB) orta hazırlayın: 100 ml cenin sığır serumu (FBS) ve 6 ml penisilin / streptomisin, 500 ml IMDM ortamı Supplement. </li><li> 100 ml M199 medya ve 50 ml FBS ile 400 ml DMEM ortamı Supplement: ICM ortamı hazırlayın. </li><li> B27 ortamı hazırlayın: 10 ml B27 medya ile 490 ml RPMI1640 ortam Supplement. </li><li> Plat-E kültür ortamı hazırlayın: 50 ml FBS, 5 ml penisilin / streptomisin ve 5 ml, esansiyel olmayan amino asitler ile birlikte 500 ml DMEM ortamı Supplement. </li><li> Plat-E transfeksiyon ortamı hazırlayın: 50 ml FBS ve 5 ml, esansiyel olmayan amino asitler ile birlikte 500 ml DMEM ortamı Supplement. </li><li> Prepare Jelatin 24 plaka kaplanmış 24 çukurlu bir levhanın bir oyuğuna 0.5 ml% 0.1 jelatin çözeltisi ekleme, kullanımdan önce en az 10 dakika karıştırıldı ve aspirat boyunca 37 ° C&#39;de inkübe edin. </li><li> FACS markalama tamponu hazırlayın: 2 mM nihai konsantrasyon elde etmek üzere, 10 ml FBS ve 2 mi EDTA (0.5 M) ile birlikte 500 mi DPBS Supplement. 4 ° C&#39;de 0.22 mikron filtre ve mağaza üzerinden geçirin. </li><li> Tampon sıralama MACS hazırlayın: 2 mM nihai konsantrasyon elde etmek üzere 2,5 g BSA ve 2 ml EDTA (0.5 M) ile birlikte 500 mi DPBS Supplement. 4 ° C&#39;de 0.22 mikron filtre ve mağaza üzerinden geçirin. </li><li> Transfeksiyon için 2x HBS tamponu hazırlayın: 280 mM NaCI (8,1816 g), 10 mM KCI (0,37275 g), 1.5 mM Na 2 HPO 4 (0,10647 g), 12 mM glukoz ile 500 mL HEPES tamponu (50 mM) ek (1,08096 g ). 7.05-7.12 pH ayarlamak için yaklaşık 650 ul 10 N NaOH ekleyin. 4 ° C&#39;da bir 0.22 mikron gözenek filtre ve mağaza üzerinden filtre. </li><li> Transfeksiyon için 2.5 M CaCI2 çözeltisi hazırlayın: 18,376 g Ca ekleCl 2 50 ml GKD 2 O. 4 ° C&#39;da bir 0.22 mikron gözenek filtre ve mağaza üzerinden filtre. </li><li> ICC (immünositokimya) sabitleme tamponu hazırlayın:% 4 bir nihai konsantrasyon elde etmek 35 mi DPBS 5 mi paraformaldehid (PFA,% 32) ekleyin. </li><li> ICC permeabilizasyon tamponu hazırlayın:% 0.1 bir son konsantrasyona ulaşmak için 100 ml DPBS 0.1 ml Triton ekleyin. </li><li> ICC bloke tamponu hazırlayın:% 5 oranında bir nihai konsantrasyon elde etmek 100 mi DPBS 5 g sığır serum albümini (BSA) ilave edin. </li><li> ICC boyama tamponu hazırlayın:% 1 bir son konsantrasyona ulaşmak için 100 ml DPBS 1 g BSA ekleyin. </li></ol> Yenidoğan Fare Kardiyak Fibroblastları 2. Nesil <ol style="margin-left: 40px;"><li> Eksplant kültürü yöntemi kardiyak fibroblastlar oluşturun <ol><li> Temiz neonatal αMHC-GFP transgenik fare% 75 etanol ile (P2 P1). Steril makas ile kafasını kesti. Sonra diğer bir koltuk altından yatay bir kesi yapmak. Steril kullanınkünt ve bükülmüş forseps kalbini incelemek ve buz gibi soğuk PBS tampon içeren 24 plaka bir kuyuya yerleştirmek. <ol><li> Alternatif olarak, yatay kesi sonrası kalp dışarı sıkmak. </li></ol></li><li> Floresan mikroskop ile göbeğinde GFP ifade edin. GFP tanımı, bir kalp-spesifik promotör olmaktadır αMHC promotörü tarafından tahrik edilir yana negatif kalp herhangi bir floresan loş iken transgenik fare kalp yeşil 15 olmalıdır. </li><li> 60 mm merkez kuyu kültür çanak içine 3-4 GFP pozitif kalpleri alın ve steril makas ve forseps ile küçük parçalar boyutu 1 mm&#39;den az 3 içine kıyma. </li><li> 2 ml FB medya ile bir adet 10 cm&#39;lik çanak içine 3-4 kıyılmış kalpleri yerleştirin. Dokular 3 saat aşağı dinlendiriniz. </li><li> Yavaşça kalp dokuları içeren yemekleri 8 ml önceden ısıtılmış FB medya ekleyebilirsiniz. Üç gün boyunca dokuların rahatsız etmeyin. </li><li> Her üç günde medyayı değiştirin. </li><li> Gün 7, aspir Onkültür ortamı yedik ve DPBS hücreleri yıkayın. Her plaka için 3 ml% 0.05 tripsin ilave edilir ve 5 dakika boyunca 37 ° C &#39;de özet. </li><li> Tripsin gidermek için 5 ml FB Medya ekle. Yavaşça hücre kazıyıcı ile hücreleri ayırmak. Yukarı ve ileri mekanik doku ayırmak aşağı medyayı pipetle. </li><li> Hücreleri toplamak ve kalp doku parçalarının kirlenmesini önlemek için 40 mikron hücre süzgeçler süzülmeye ve daha sonra 5 dakika boyunca 200 xg&#39;de iplik pelet hücreleri. </li><li> Yıkama hücreleri bir kez MACS tamponu ve hücreler sıralamak için hazırız. </li></ol></li><li> Enzimi sindirimi yöntemi kardiyak fibroblastlar oluşturmak <ol><li> Hasat GFP 2.1.1 olumlu kalpler. 10 ml buz gibi soğuk DPBS ile 10 cm&#39;lik bir tabak içine bütün kalpleri aktarın. </li><li> Kan kaldırmak ve buz gibi soğuk DPBS ile bir kez yıkayın steril forseps ile ventriküller sıkıştırın. </li><li> Diğer doku ve yağ özgür olmak kalpleri Trim. </li><li> Hala gevşek bağlanmış kabaca 4 parçaya her kalbi kesin. </li><li> En az 20 kupa, 8 ml ılık% 0.05 tripsin-EDTA ile 15 ml konik bir tüp içine kalpleri aktarın ve 15 dakika boyunca 37 ° C&#39;de inkübe edin. <ol><li> 20-30 kalpler varsa, 10 ml ılık% 0.05 tripsin-EDTA ile 50 ml konik tüp içine kalpleri aktarın ve 15 dakika boyunca 37 ° C&#39;de inkübe edin. </li></ol></li><li> Tripsin atın ve HBSS ya da (20-30 kalpleri için) 10 ml ılık tip II kollajenin (0,5 mg / ml) (az 20 kalpleri için) 5 ml ilave edin. </li><li> Vortex 4 ila 6 civarında bir hız seviyesinde 1 dakika boyunca vortexer tüp (sıvı kapağına kadar almazsa, o zaman hız gayet). </li><li> 3-5 dakika boyunca, 37 ° C su banyosu içinde tüp inkübe edin. </li><li> 1 dakika boyunca tüp vorteksleyin. </li><li> Doku parçaları sakinleşene kadar yerçekimi ile 1 dakika boyunca sediment, 5 ml soğuk FB ortamı içeren yeni bir tüp içine sıvı toplamak. </li><li> Tekrarlayın 2.2.6-2.2.10 3-5 kez yineleyin. </li><li> 40 mikron hücre süzgecinden filtreleme tüm koleksiyonları birleştirintek bir hücre süspansiyonu yapmak. </li><li> 4 ° C&#39;de 5 dakika boyunca 200 x g&#39;de santrifüjleyin. </li><li> Süspanse 10 ml MACS tamponu hücrelerinde ve 4 ° C&#39;de 5 dakika boyunca 200 x g&#39;de santrifüje tabi tutulur. </li><li> İsteğe bağlı: 1 ml RBC liziz tamponu (150 mM NH4CI, 10 mM KHCO 3, ve 0.1 mM EDTA) ile kan hücreleri, tekrar süspansiyon hücrelerinin bir yeri vardır, daha sonra 10 ml MACS ile seyreltilir, 1 dakika boyunca buz üzerinde tutulur tampon ve 5 dakika boyunca 200 x g&#39;de santrifüje tabi tutulur. MACS tamponu ile bir kez daha yıkanır. </li></ol></li><li> MACS tarafından Thy1.2 + fibroblast İzolasyonu (manyetik aktive hücre sıralama) <ol><li> Tripan mavisi ile canlı hücre sayısını belirler. <ol><li> Adım 2.2.14 10 ml&#39;lik hücre süspansiyonu 10 ul hücreleri dışarı atın. 10 ul% 0.4 tripan mavisi çözeltisi ile karıştırılır. </li><li> Bir hemasitometre karışımı ekleyin. 3-5 dakika boyunca ayakta ve daha sonra mikroskop altında hemen incelemeye izin verin. Mavi ve canlı hücrelerde lekeli ölü hücreler boyanmamış bulunmaktadır. </li><li&gt; hemasitometre dört 1 mm köşe meydanlarda tüm canlı hücreleri saymak. Canlı hücre sayısını belirlemek: kare x 2 (seyreltme faktörü) başına ortalama sayısı 4 x 10 10 (hücre süspansiyonu toplam hacmi) x. </li></ol></li><li> Az 1 x 10 7 hücreleri için, 90 ul 10 ul Thy1.2 mikro ile tekrar süspansiyon hücreleri MACS tamponu soğutulmuş. 1&#39;den fazla x 10 7 hücre vardır orantılı ise daha çok boncuk ekleyin. İyice karıştırın ve 30 dakika boyunca bir buzdolabı (2-8 ° C) &#39;de inkübe edilir. </li><li> 10 ml MACS tamponu ve sıkma örnekler 5 dakika boyunca 200 xg&#39;de ekleyin; Tekrar 10 ml MACS tamponu ve santrifüj ile bir kez yıkanır. </li><li> MACS tamponu içinde 2 ml hacim getirin. </li><li> Bir MACS ayırıcı kaputu ayarlayın. Ayırıcı bir LS sütun ekleyin. MACS sütun tampon 3 ml bunu dengelenmeye uygulayın. </li><li> Dengelenmiş LS sütunu içinden hücre süspansiyonu geçirin. </li><li> Her tampon, 2 ml MACS ile 3 kez yıkanır. </li><li> T off LS sütun alınO Ayırıcı ve MACS yeni bir 50 ml tüp içine tampon 2 ml ile 3 kez Zehir. </li><li> 5 dakika boyunca 200 xg&#39;de Spin. </li><li> 10 ml FB medyada yeniden süspanse hücreleri, hücre sayımı ve uygun yoğunlukta plakalara tohum. Eksplant kültürü yöntemiyle üretilen fibroblastlar için, tohumlama hücreler yoğunluğu 2-3 x 10 4 hücre / kuyu 24 plaka olabilir. Enzimi sindirimi yöntemi elde fibroblastlar için, hücreler yoğunluğu yaklaşık 4-5 x 10 4 hücre / kuyu 24 oyuklu plaka olabilir. </li></ol></li></ol> ICM yeniden programlanması için retrovirüsün 3. Nesil Not: steril koşullar altında BSL2 Biyolojik Güvenlik kabinesinde aşağıdaki adımları uygulayın. Transfekte hücreler, pipet uçları ve tüplerin doğru atılması, çevre ve sağlık tehlikeleri riskini önlemek için tavsiye edilir. <ol style="margin-left: 40px;"><li> 1 ug / ml puromisin ve 10 ug / ml ile takviye edilmiş b Plat-E medya Plat-E hücreleri korumak% 5 CO2 ile 37 ° C &#39;de lasticidin. </li><li> 2 gün, puromisin ve blastisidin eksik Plat-E kültür ortamı ile orta yerine. </li><li> 0. günde, 10 cm&#39;lik kaplar yaklaşık Transfeksiyondan bir önceki gün plaka başına 4-5 x 10 6 hücre içine Plat-E hücreleri bölme. Hücreler ~ transfeksiyon günü% 80-90 ortak akışlı olmalıdır. </li><li> 1. gün önce transfeksiyondan 1 saat içinde hücreler üzerinde transfeksiyon medya değişiklik kültür ortamı. Şöyle transfeksiyon prosedürü şöyledir: <ol><li> Ticari kitler kullanılarak transfeksiyonu <ol><li> 20 ul transfeksiyon reaktifi (örneğin, Lipofectamine) ve 500 ul karıştırın düşük serum ortamı (örneğin, Opti-MEM). Serum medya azaltılmış bir 500 ul retroviral plazmid 10 mikrogram ekleyin. 5 dakika boyunca oda sıcaklığında (RT) her karışımın inkübe edin. </li><li> Yavaş yavaş transfeksiyon karışımı plazmid karışımı ekleyin. Bu adım 15-30 sn sürebilir. (Çözelti bulanık görünebilir) oda sıcaklığında 20 dakika süreyle inkübe karışımı. </li&gt; <li> Hücrelere bilge karışım damla ekleyin. </li><li> Bir 37 ° C inkübatör içinde gece boyunca inkübe edin. </li></ol></li><li> Kalsiyum Fosfat ile transfeksiyonu. <ol><li> 40 ul 2.5 M CaCl2 ve 440 uL GKD 2 O, daha sonra retroviral plazmid 20 ug (toplam hacim 500 ul böylece GKD 2 O miktarını ayarlamak) ekleyin karıştırın. </li><li> 15 ml konik tüp HBS 2x 500 ul ekleyin. </li><li> HBS vorteks ve bu arada HBS Kalsiyum-DNA karışımı damla damla ekleyin. Bu adım, her numune için 30 saniye sürer. Yani maksimum eşzamanlı 3-4 örnekleri yok. </li><li> Daha uzun bir zaman bir çökelti alacak daha geniş ve daha az çökelti hücrelere yol açar 3 dakika süre ile, oda sıcaklığında inkübasyon için inkübe edilir. </li><li> Karışım hücrelere damla damla ve 20X mikroskop altında gözlemlemek ekleyin. İnce çökeltiler (küçük olanlar bir sürü iyi ama birkaç büyük olanları iyi değildir) görülmelidir. </li><li> 37 ° C inkübatör gecede inkübe. </li&gt; </ol></li></ol></li><li> 2 gün, kültür ortamı önceden ısıtılmış 8 ml hücreler üzerinde ortamı değiştirmek. 24 saat süreyle inkübe edin. </li><li> Gün 3 ve 4. günde, 0.45 mikron gözenek boyutlu selüloz asetat filtre boyunca, 10 ml steril bir şırınga kullanılarak ve 50 ml&#39;lik bir tüp içine aktarılarak yemekler kültür süpernatan toplamak. Her 8 ml virüs içeren süpernatan için retrovirüs çökeltme çözeltisinin 2 ml ilave edilir. Yavaşça karıştırın ve 4 ° C&#39;de gece boyunca kuluçkaya yatmaktadır. </li><li> 5. günde, 30 dakika boyunca 4 ° C&#39;de 1500 x g&#39;de viral karışımı dönerler. Viral parçacıklar tüpün alt kısmında beyaz pelet görünmelidir. </li><li> Süpernatantı atın. 5 dakika boyunca 1500 x g&#39;de santrifüjleme ile bir artık çözeltinin dönerler. Pelet Çöken retroviral parçacıklar rahatsız etmemek için büyük bir özen, aspirasyon yoluyla sıvının tüm izlerini silmek. </li><li> Her 8 ml viral süpernatan için 100 ml DPBS tamponu ile retroviral pelet yeniden süspanse edin. TümbölenBuz üzerinde virüsü. Hemen kullanın veya -80 ° C&#39;de saklayın. </li></ol> Kardiyak Fibroblastları 4. yeniden programlanması <ol><li> 10 ml ICM ortama 10 mg / ml polybrene solüsyonu 4 ul ekleyin ve yukarı ve aşağı pipetleme hafifçe karıştırın. Polibren nihai konsantrasyonu 4 ug / ml&#39;dir. </li><li> Fibroblastlar ile tohumlanmış 24 çukurlu bir levhanın her çukuruna 500 ul ICM ortam ihtiva eden polybrene ekleyin. </li><li> Eksplant kültürü veya enzimi sindirimi yöntemi 4-5 x 10 4 hücrelerinden içeren her birini 2-3 x 10 4 hücrelere 10 ul retrovirüs ekleyin. Virüs miktarı orantılı olarak, farklı kültür plakaları ya da bulaşıklar için artmış bir hücre sayısı ile yükseltilmelidir. Viral enfeksiyon izin vermek için 24-48 saat için 37 ° C inkübatör plaka koyun. Kalp yeniden programlama için zaman çizgisi Şekil 1&#39;de gösterilmiştir. </li><li> Viral iletimi sonrasında 500 ul düzenli ICM medya ile virüs içeren medya değiştirin. </li><li> 2-3 günde ortamı değiştirmek. Viral transduse hücreleri, pozitif seleksiyon için, hedef hücrelere 2 ug / ml&#39;de puromisin ile desteklenmiş ICM ortamı eklenir. Üç gün boyunca tutun. Bundan sonra, 1 ug / ml konsantrasyonunda da ICM ortam içinde puromisin korumak. </li><li> Üç gün viral enfeksiyondan sonra, inkübatör dışarı plaka almak ve GFP ifade gözlemlemek için bir ters floresan mikroskop (20X) altına yerleştirin. Not: On gün viral enfeksiyondan sonra, hücreler, FACS analizi ve yeniden programlama etkinliğini belirlemek için ICC analizi için hasat edilebilir (adım 5 ve 6). </li><li> Ondört gün viral enfeksiyondan sonra, B27 medya ile ICM ortamı değiştirin. Her üç günde ortamı değiştirmek. Spontan dayak hücre loci üç (enzim sindirim yöntemi hücrelerin) veya viral transdüksiyon sonra dört (eksplant kültürü yöntemiyle elde edilen hücreler) hafta görünebilir. </li></ol> Yeniden programlanması Verimliliğinin 5. İmmünositokimyasal Analizi <ol><li> Durulayın hücrelerüç kez PBS soğuk buz ile; Fazla çözüm kaldırmak. </li><li> 24 oyuklu plakalar, her çukuruna 0.5 ml ICC sabitleme tamponu ekleyin. 15-20 dakika boyunca oda sıcaklığında hücreleri saptamak. </li><li> PBS ile üç kez hücreleri durulayın. </li><li> 24 oyuklu plakalar, her çukuruna 0.5 ml ICC permeabilizasyon tamponu ekleyin. 15 dakika boyunca oda sıcaklığında hücreleri geçirgenliği. </li><li> PBS ile üç kez hücreleri durulayın. </li><li> 24 çukurlu bir levhanın her çukuruna bloklama tamponu, 0.5 ml ICC ekleme, 1 saat süre ile oda sıcaklığında inkübe edilir. </li><li> ICC lekeleme tampon maddesi ile seyreltilmiş birincil antikorlar. De 200 ul her bir antikor çözeltileri ilave edin ve gece boyunca 4 ° C enkübe edilir. Antikor seyreltme faktörleri Malzeme listelenmiştir. </li><li> Bir sonraki gün, PBS ile üç kez yıkama hücreleri. </li><li> Hücrelere 200 ul ikincil antikor çözüm ekleyin ve karanlıkta oda sıcaklığında 1 saat süreyle inkübe edin. </li><li> Üç kez PBS ile hücrelerin yıkayın. </li><li> Solüsyonu her bir çukura DAPI içeren montaj 150 ul ekle. 1 dakika boyunca çekirdekleri leke izin verin. Daha sonrayuvarlak cam kapak slip ile her iyi kapak ve yavaşça hava kabarcıklarını çıkarmak için bir forseps ile taban yüzeyine karşı kayma basın. </li><li> Fazla solüsyonun aspire ve örnekler görüntüleme için hazırdır. D14 de ifade yeniden programlanan hücreleri kalp işaretleyici cTnT ve αActinin gösteren temsili resim Şekil 2B&#39;de gösterilmektedir. </li></ol> Yeniden programlanması Verimliliği 6. FACS Analizi <ol><li> İyice DPBS ile bir kez yıkama hücreleri. Her oyuğa 0.3 ml tripsin (% 0.05) ilave edin ve 5 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Yavaşça hücreleri kaldırma kolaylaştırmak için plaka dokunun. Mikroskop altında hücre ayrışması edin. En hücreleri kesilerek zaman, her bir kuyucuğa 1 mi FACS tampon ekleyin. Pipet yukarı ve ileri mekanik hücreleri ayırmak için aşağı. </li><li> Kuyuya bir 96 derin oyuklu plakaya 24 oyuklu bir plaka içerisinde hücrelerin aktarın. 4 ° C&#39;de 5 dakika boyunca 200 x g&#39;de Spin hücreleri toplamak için. </li><li> Bir kez yıkayın hücreleriFACS tamponu. </li><li> 4 ° C&#39;de 20 dakika boyunca hücreler tekrar süspansiyon 100 ul sabitleme / permeabilizasyon çözümler ekleyin. </li><li> 1x yıkama solüsyonu, pelet ile iki kez hücre yıkayın ve süpernatant kaldırmak. </li><li> 1x yıkama tamponu GFP ve cTnT karşı birincil antikor çözüm hazırlayın. İyice 50 ul antikor çözeltisi ile her bir örnek tekrar süspansiyon ve 30 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> 1x yıkama solüsyonu, pelet kez hücreleri yıkayın ve süpernatant kaldırmak. </li><li> Her bir örnek için Alexa Fluor 488 konjüge edilmiş eşek anti-tavşan IgG ve Alexa Fluor 647 konjüge edilmiş eşek anti-fare IgG içeren 50 ul ikincil antikor solüsyonu ilave. Karanlıkta 30 dakika boyunca 4 ° C&#39;de inkübe edilir. </li><li> 1x yıkama solüsyonu, pelet kez hücreleri yıkayın ve süpernatant kaldırmak. </li><li> 400 ul sabitleme tampon maddesi (% 1 PFA ile DPBS) içinde süspanse edin hücreleri. Hücre süzgecinden kapaklı FACS tüpü hücreyi aktarın. Numuneler, FACS tespiti için hazırdır. Yeniden programlama efficienc temsili FACS analiziy pMxs-puromycin MGT Şekil 2A gösterilmiştir veya puromisin seçimi olmadan inşa kullanarak. </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.

Bu protokolü kullanarak başarılı ICM nesil için, genel verimliliği üzerinde bir etkiye sahip birkaç önemli faktör vardır. Özellikle başlangıç ​​fibroblast koşulları ve retrovirüs kodlama MGT kalitesini büyük ölçüde yeniden programlama verimliliğini etkileyebilir. Mümkün olduğunca taze ve sağlıklı fibroblastlar oluşturmak için önemlidir. Eksplantlar yedi gün sonra tabaklara önce eksplant kültürü yöntemi için, fibroblastlar kullanılabilir. Enzimi si...

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

Kaynaklı kardiyomiyositlere kardiyak fibroblastlar (CFS) Doğrudan dönüşüm (ICMS) kalp hastalıklarının tedavisi için alternatif stratejiler sunarak rejeneratif tıp için büyük bir potansiyele sahiptir. Bu dönüşüm Gata4 (G), Mef2c (M) ve Tbx5 (T) gibi tanımlanmıştır faktörler zorla ifade ile elde edilmiştir. Geleneksel olarak, bu ICMS bireysel faktörlerin ifade virüslerin bir kokteyl ile üretilmiştir. Bununla birlikte, verim yeniden programlama nispeten düşük olan ve in vitro G en büyük kısmı, E, fibroblastlar zor yeniden programlama mekanizmaları incelemek için yapım tamamen yeniden programlanması olmazlar T-transdüksiyonlu. Biz son zamanlarda G, M stokiyometrisi, T verimli ICM yeniden programlama için çok önemli olduğunu göstermiştir. Bir M ve polisistronik MGT vektörü (bundan sonra MGT olarak anılacaktır) önemli ölçüde artmıştır yeniden programlama etkinliğini kullanılarak elde G ve T düşük seviyelerde göreli yüksek düzeyde G, M, T optimal stoikiometri ve in vitro olarak geliştirilmiş ICM kalitesi. Burada kalp fibroblastlardan MGT yapısı ile ICMS oluşturmak için kullanılan yöntemlerin ayrıntılı bir açıklama sağlar. Kalp fibroblastlar, yeniden programlama ve yeniden programlama sürecinin değerlendirilmesi için virüsün nesil izolasyonu da ICMS verimli ve tekrarlanabilir nesil için bir platform sağlamak için dahil edilmiştir.

Yeniden programlama adımlar. Şekil 1&#39;de şematik özetlenmiştir MGT transdüksiyon sonra, hücreleri yeniden programlama GFP ifade gibi erken transduced hücrelerin 3. Puromycin seçimi 3 gün başlar ve ilk iki haftasında korunur gün olarak tespit olabilir eğer PMX-puro -MGT yapı kullanılır. Gün 14 10. günde, cTnT ve αActinin gibi kardiyak markerlerinin ifadesi başlangıç ​​fibroblastlar, bir kalp hücre doğru yeniden programlamayı geçiren belirten iki ICC (Şekil</stro...

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.

Gata4, Mef2c ve Tbx5 Optimal Oranı ifade bir polisistronik Construct kullanma Kaynaklı kardiyomiyositlerde Geliştirilmiş Nesil

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 Görüntüleme.  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.

Küçük Molekül-modüle Farklılaşma ve sonraki Glikoz şiddetli açlık kullanma İnsan uyarılmış pluripotent kök hücrelerin yüksek oranda saf kardiyomiyositlerde türetilmesi

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

Gelişim Biyolojisi

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.

İzolasyon ve Neonatal Rat kardiyomiyositlerde Kryoprezervasyon

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.

Bütünleşmeyen episomal plasmidler kullanılarak dondurulmuş Buffy tüylerdeki uyarılmış pluripotent kök hücrelerin üretilmesi

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.

Saç türetilmiş keratinositlerden Kullanarak Entegrasyon serbest İnsan uyarılmış pluripotent kök hücrelerin üretilmesi

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.

Besleyici içermeyen şartlar Sendai virüs kullanılarak insan periferik T hücrelerinden uyarılmış pluripotent kök hücrelerin üretilmesi

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.

Romatoid Artrit Hastalarının Mafsallar İzole fibroblast benzeri sinoviyosit kullanma Kaynaklı-pluripotent Kök Hücre Üretimi

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.

Sendai Virüs ve santrifüj kullanılarak Kan Hücreleri indüklenmiş pluripotent kök hücre Üretimi

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.

Epizomal vektörlerin kullanılması insan periferik kan mononükleer hücrelerinden entegrasyon içermeyen uyarılmış pluripotent kök hücrelerin üretilmesi

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.

Elektriksel Uyarı Biowires İnsan Kök Hücre kaynaklı kardiyomiyositlerde Olgunlaşma

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.

İskele-free, üç boyutlu insülin ifade Pancreatoids fare pankreas ataları Vitro gelen nesil

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