1. Öneriler Başlamadan Önce <ol><li> Onlar sinüs düğümü hücrelerine doğru ayırt çünkü mikoplazma ile kontamine PSC&#39;ler kullanmayın. Protokolü başlamadan önce mikoplazma kontaminasyonu için test. Mikoplazmalar hızlı, son derece duyarlı bir PCR kiti kullanılarak bu yapın ve, üreticinin protokolü izleyin. </li><li> Her bir Petri kabı (aşama 2.3.4), kaplama, bir 10 sm, 37 ° C&#39;de 1 saat boyunca soğuk su balığı deri steril 7 mi,% 0.1 jelatin ile 2 hücre kültür çanağı için. Steril bir tezgah steril koşullar altında çanak kuru jelatin çıkarın ve izin. </li><li> Bir memeli ifade vektörü kullanılarak TBX3 i) Kurucu aşırı ifadesi: Eğer farklılaşma protokolü başlamadan önce aşağıdaki özellikleri içeren bir çift transfekte kararlı fare ES hücre hattı klonu gerekir. Ii) Myh6-destekleyici 5 kontrolü altında G418 direnç geni. </li><li> farklılaşmamış PSC hücreler, bir P-yetiştirmek olmalıdırtarif edilen 1 gibi standart koşullar altında ışınlanmış besleyici hücreler d. </li></ol> 2. Farklılaşma Prosedürü <ol><li> İki Gün Farklılaşma önce - besleyici hücreleri PSC&#39;ler çıkarın. <ol><li> 10 ml fosfat tamponlu tuzlu su, PBS ile yıkama hücreleri, ortam aspire 7 mi kollajenaz IV, bir çözeltisi eklenmekte ve 37 ° C&#39;de 10 dakika boyunca kuluçkalayın. 50 ml&#39;lik bir tüp içinde bir steril 40 mikron filtre yerleştirin. </li><li> Dikkatle PSC koloniler durulama yukarı ve aşağı 5 kez kollajenaz çözüm pipetleme (besleyici hücreleri çıkarmak için kaçının). </li><li> , 40 mikron filtre hücre süspansiyonu aktarın 8 ml PBS ile filtreyi üç kez yıkayın. Filtre etrafında döndürün ve baş aşağı bir petri yerleştirin. Filtrenin tabanına 10 mi PBS pipetleme PSC hücre kolonileri çıkarın. </li><li> 300 x g, 5 dakika boyunca 15 ml&#39;lik bir tüp ve santrifüj hücre süspansiyonu aktarın. </li><li> 1 ml Accutase ve i hücreleri askıya, PBS Kaldır5 dakika boyunca 37 ° C&#39;de ncubate. </li><li> 10 ml PBS ilave 300 x g 5 dakika boyunca aşağı 5 kez ve santrifüj yukarı pipetleme ve hücre çözeltisi karıştırılır. </li><li> , PBS çıkarın, 10 ml ekimi ortamda hücrelerin askıya ve hücre sayısını belirlemek. </li><li> Tohum 15,000 hücre / 75 cm2 filtre şişesi üzerine dayanan cm2 ve 37 ° C,% 5 CO2 ve 2 gün süreyle yetiştirilir. 2 gün sonra, şişe,% 50-70 konfluent olmalıdır. </li></ol></li><li> Gün 0 - Farklılaşma Başlangıç <ol><li> Ortamı çıkarın ve 10 ml PBS ile yıkama hücreleri. </li><li> , PBS aspire 2 mi Accutase ilave et ve 5 dakika boyunca 37 ° C&#39;de kuluçkalayın. 50 ml&#39;lik bir tüp içinde bir steril 40 mikron filtre yerleştirin. </li><li> 40 um&#39;lik bir filtreden hücre süspansiyonu transferi, 10 mi PBS ekleyin 10 ml PBS ilavesiyle ve santrifüj ile filtre durulama akış yoluyla 300 x g, 5 dakika karıştırıldı. </li><li> 10 ml farklılaşma ortamda hücreleri Askıya ve hücre sayısını belirlemek. </li><li> T seyreltinO 20,000 hücre / ml&#39;lik nihai bir konsantrasyona kadar farklılaşma ortamı ile süspansiyon hücre. </li><li> Pipet 20 ml su ve HDS kurutulması çıkmasını engellemek için bir ikinci derece Petri kabındaki damla (HD) çözeltisi asılı 5 mi. NOT: Her 24 plaka içeren iSABs için 16 Petri kapları ile başlar (bölüm 2.8.6.4/2.8.7 bakınız). </li><li> Bir tepsiye 50 mi hücre süspansiyonu kadar Pipet. </li><li> Petri kapağı etrafında çevirin. 12 kanallı pipet kullanarak kapak üzerine 180 HDS her içeren 20 ul (400 hücre / HD) hücre süspansiyonu yerleştirin. </li><li> Dikkatle kapak dönüp Petri kabı üzerine yerleştirin. NOT: Kapağın etrafında dönme hızı çok önemlidir. Kapak çok yavaş ya da çok hızlı döndü ise, hücre süspansiyonu yüzey gerilimi asılı kapağındaki damlaları korumak için yeterince yüksek değil. Orta 20 ul damla kapak etrafında dönen ilk kez uygulama için HDS üretmeye çalışıyor önce. </li><li> 3 2 gün boyunca hücrelerin kültive7 ° C,% 5 CO 2 onları EBS formu izin. NOT: 5 Petri kapları bir yığının üzerine su dolu yerleştirin, bir petri. Aksi takdirde, üst petri HD kurur. </li></ol></li><li> Gün 2 <ol><li> Dikkatle kuadratik Petri kabı kapağının etrafında çevirin ve 50 ml tüp iki Petri kapları (360 EBS) türetilen EBS aktarmak. </li><li> EBS tüpün dibinde yerleşmek izin 10 dakika bekleyin (EBS santrifüj etmeyin). </li><li> Aspire mümkün olduğu kadar ortam olarak, 10 mi farklılaşma ortamda EBS askıya ve 10 cm Petri için süspansiyonun aktarın. </li></ol></li><li> Gün 2-6 Süspansiyon kültürü <ol><li> 37 ° C&#39;de 4 gün boyunca süspansiyon içinde EBS yetiştirmek,% 5 CO2, 2 gün sonra ortam değiştirin. Not: EBS genellikle yüzeye eklemek (bir hücre kültürü tabağına aksine) petri tabağı kaplı değildir bile. EBS ekli EBS, her gün ve gerektiğinde kopmakta eğer petri kapları kontrolNazik pipetleme yüzeyinden. İsteğe bağlı: süspansiyon kültürü döneminde sürekli petri kapları çalkalayın. Çalkalayıcı hızıyla dikkatli olun. EBS ne ortasında birikir ne Petri yemekleri sınırına yüzer gerekir. </li></ol></li><li> Gün 6 <ol><li> Bir jelatin kaplı 10 cm 2 hücre kültürü çanak bir Petri kabı EBS aktarın. </li></ol></li><li> Gün 7-12 <ol><li> Hücreler 8-12 gün sonra yenmek için başlamalıdır. Günlük mikroskop altında hücrelerin odakları yenerek kontrol edin. hücreler, bir tabaka oluşturmak için başlar. </li><li> Orta gazetesine değiştirin. NOT: orta değiştirdikten sonra, hücreler kurtarmak ve tekrar dayak başlamak için 3-4 saat gerektirir. </li></ol></li><li> Sinüs düğüm hücrelerin Gün 12-15 seçimi. <ol><li> Hücreler (günlük 12-15 civarında) çarpmaya başladı olan üç gün sonra, hücreler, 400 ug / ml G418 içeren taze farklılaştırma ortamının orta ve pipet 10 ml aspire. </li></ol></li><li&gt; Dört gün seçimi başlangıcından sonra hücre katmanı ayırmak. NOT: Bu hücre tabakası önceden yüzeyinden ayrılır mümkündür. <ol><li> Hücre katmanı aspire etmeden dikkatlice orta çıkarın. </li><li> PBS 10 ml ekleyin ve hücre tabakası aspire etmeden dikkatlice PBS kaldırmak. </li><li> 50 ml&#39;lik bir tüpe hücreleri transferi ve 5 dakika süreyle 37 ° C&#39;de inkübe hücreleri, kollajenaz IV, 10 ml solüsyonu ekleyin. </li><li> Şiddetle yukarı pipetleme ve 10 kat aşağı süspansiyon karıştırın ve 5 dakika boyunca 37 ° C&#39;de inkübe hücreleri. Küçük kümeler bir süspansiyon meydana gelmelidir. Hala sol katmanın büyük parçalar varsa, o adım 2.8.4 iki kez daha tekrarlayın. </li><li> 300 x g, 10 dakika boyunca 20 ml PBS ve santrifüj ekleyin. </li><li> PBS dikkatle aspire. NOT: iSABs adım 2.8.7 devam elde edilecek ise. Isab türetilmiş sinüs düğüm tek hücre oluşturulur olursak adım 2.9 ile devam. </li><li> Hücreleri Askıya12 ml farklılaşma karışımı, 400 ug / ml G418 ihtiva eden ve kaplanmış jelatin 6 kuyu 24 kuyu (/ oyuk 2 mi) üzerine tohum. </li></ol></li><li> Tek düğüm hücrenin üretilmesi. <ol><li> 5 mi Accutase adım 2.8.6 gelen süspanse edildi ve 5 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Şiddetle yukarı pipetleme ve 10 kat aşağı süspansiyon karıştırın ve 5 dakika boyunca 37 ° C&#39;de inkübe hücreleri. </li><li> 300 x g, 5 dakika boyunca 20 ml PBS ve santrifüj ekleyin. </li><li> 400 ug / ml G418 ihtiva eden 12 mi farklılaşma ortamı içinde süspanse 24 kuyu (2 ml / oyuk) kaplanmış bir jelatin 6 kuyu bunları tohum. </li></ol></li><li> Ayrışma sonrası Gün 2, orta aspire ve PBS ile hücreler üç kez yıkayın. Iyi / 1 ml farklılaşma ortamı ekleyin. </li><li> Gün 4-8 ayrışma sonra, her 2. gün Orta değiştirin. Ayrılma kullanımı iSABs veya isab edilen sinüs 10 gün sonra düğüm tek hücre, daha fazla analiz için kullanılabilir. </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

yetenek &quot;biyolojik kalp pili&quot; anlamında uygun kalp ritminin sulandırma izin verebilir hücre kaynaklı kalp pili kök hücreler üretmek için. Aynı şekilde, in vitro uyuşturucu testi onların durumu yararlanacaktır. PSC&#39;leri kalp pili hücresi özellikleri 8,9,10,11,12,13 ile kardiyomiyositlere dahil memeli vücudunun herhangi bir hücre türüne yol açabilir. Ancak, genellikle &quot;Embriyoid Bodies&quot; içinde hücre popülasyonları kaçınılmaz güvenilir bir seçim ve izolasyon s...

dönem &quot;hasta sinüs sendromu&quot; kalp pili sisteminin bozulmasına yol açan birden hastalıkları özetler. Bu patolojik, semptomatik sinüs bradikardi, sinoatrial blok, sinüs tutuklama yanı sıra taşikardi-bradikardi sendromu oluşur. Böylece, bir &quot;hasta sinüs sendromu&quot;, genellikle böyle bir iskemik kalp hastalığı, kardiyomiyopati ya da miyokardit gibi genel kalp hastalıkları eşlik ediyor. Şu anda, tedavi yaklaşımları elektrik pili implantasyonu dayanmaktadır. Ancak, bu tür enfeksiyonların ve pil arızası gibi risklerin bir dizi ile birlikte gider. Genel olarak, komplikasyonların insidansı halen hastaların yapay kalp pili implante olan çok yüksektir. Endojen kalp karşı Ayrıca, bu cihazlar, hormon stimülasyonuna yanıt vermez. Gelecekteki bir alternatif olan PSC&#39;ler hizmet verebilir &quot;biyolojik kalp pili&quot; durumu güveniyor olabilirUygun bir hücre kaynağı olan ve aynı zamanda in vitro ilaç testi için oldukça yararlı olacaktır. Ancak, büyük bir sorun embriyoid organları (EBS) içindeki sinüs düğüm hücrelerin çok nadir görünüm yatıyor - bu genellikle ~% 0.5 1 aşmaz. Daha önce, bu özel kardiyomiyosit alt tiplerine karşı &quot;ileri programlama&quot;, yani ilgili Mesoderm-özgü arka 1 (MesP1) ve NK2 transkripsiyon faktörü, lokus 5 (Nkx2.5) 2 gibi farklı erken kardiyovasküler transkripsiyon faktörlerinin aşırı ifadesi yoluyla mümkün olduğu gösterilmiştir, 3. Normal boyut ve sinoatriyal düğüm (SAN) bir fonksiyonu için, T-box transkripsiyon faktörü Tbx3 pili gen programının başlatılmasını ve SAN 4 farklılaşmasını kontrol ettiği gösterilmiştir olan, son derece önemlidir. Bu fonksiyonel kalp pili hücrelerinin görünümünü gelişmiş iken, içeriği hala tüm cardiomyocytic hücre popülasyonu içinde ~ 40% geçmedi. Bu nedenle, ek bir Myh6-organizatörü bazlı antibiyotik seçimi adım 5 bizim tarafından tanıtıldı. In vitro ekili bir kemirgen kalbin olanlar ve karşılaştırılabilir yaklaşıldığıdır ilk kez in vitro yüksek artış yenerek frekansları (&gt; 400 bpm) sergilerler; Bu sonuçta henüz farkedilmemiş kardiyomiyosit agrega (iSABs &quot;&quot; uyarılmış Çin-atriyal organları &quot;) yol açar bir murin kalp 6 izole sinüs düğüm hücreleri. İzoprenalin yönetimi altında 550 bpm bile dayak frekansları elde edilir. Özellikle, iSABs 7 analizleri geniş fizyolojik itibaren belirgin% 80&#39;in üzerinde fonksiyonel düğüm hücreleri oluşur. Son zamanlarda, çeşitli yaklaşımlar doğrudan yeniden programlama 19 kullanılarak Sinus düğüm hücreleri oluşturmak için, yüzey 16,17 tarif edildi küçük moleküller ile 14 ya da farmakolojik tedavi işaretler. Ancak, bu yöntemlerin hiçbiri kalp pili hücrelerinin böyle bir yüksek saflıkta yol açtı ve murin yakın frekansları yenerek osanat iSABs görüldüğü gibi. Dahası, spontan dayak aktivitesini kaybetmiş ekili yetişkin fare ventriküler dilimleri bir ex vivo modelde, iSABs, dilim dokusu içine entegre böylece kendiliğinden aktif kalan ve sağlam kasılmaların 7 kalp dilimleri pacing yeteneğine sahiptir. Bu iSABs üretimi için ayrıntılı bir protokol bu yazıda açıklanmıştır.

<table><tbody><tr><td>Iscove&#039;s liquid medium with stable glutamine</td><td>Biochrom AG</td><td>FG 0465</td><td></td></tr><tr><td>DMEM liquid medium without Na-pyruvate, with stable glutamine</td><td>Biochrom AG</td><td>FG 0435</td><td></td></tr><tr><td>CLS type IV, CLS IV</td><td>Biochrom AG</td><td>C4-22</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Biochrom AG</td><td>K 0293</td><td></td></tr><tr><td>FBS Superior</td><td>Biochrom AG</td><td>S 0615</td><td></td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Biochrom AG</td><td>L 0473</td><td></td></tr><tr><td>G 418-BC liquid (ready-to-use solution), sterile</td><td>Biochrom AG</td><td>A 2912</td><td></td></tr><tr><td>Reagent reservoir PP f.multichannel pip. 60ml,sterile</td><td>Brand</td><td>703409</td><td></td></tr><tr><td>Accutase</td><td>eBioscience,Inc.</td><td>00-4555-56</td><td></td></tr><tr><td>Eppendorf Xplorer/Xplorer plus, electronic pipette</td><td>Eppendorf</td><td>4861000155</td><td></td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>GE Healthcare</td><td>P11-010</td><td></td></tr><tr><td>Petri Dishes</td><td>Greiner BioOne</td><td>663102</td><td></td></tr><tr><td>DPBS without Ca and Mg</td><td>PAN-Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Fetal bovine serum</td><td>PAN-Biotech</td><td>P30-3302</td><td></td></tr><tr><td>Leukemia inhibitory factor</td><td>Phoenix Europe GmbH</td><td>LIF-250</td><td></td></tr><tr><td>Quadratic petri dishes</td><td>Roth</td><td>PX67.1</td><td></td></tr><tr><td>Gelatin from cold water fish skin</td><td>SigmaAldrich</td><td>G7765</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>SigmaAldrich</td><td>M3148&amp;nbsp;</td><td></td></tr><tr><td>1-Thioglycerol</td><td>SigmaAldrich</td><td>M6145</td><td></td></tr><tr><td>Tissue culture dishes</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>Tissue culture flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>Tissue culture test plates (24 well)</td><td>TPP</td><td>92424</td><td></td></tr></tbody></table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

&quot;Hasta sinüs sendromu&quot; Tedavi yapay kalp pili dayanmaktadır. Bunlar pil arızası ve enfeksiyonlar gibi tehlikeleri ayı. Ayrıca, hormon eksikliği yanıt ve genel prosedür maliyeti yoğundur. PSC&#39;ler oluşturulan &quot;Biyolojik kalp pilleri&quot; bir alternatif haline gelebilir, ancak Embriyoid Oluşumu kalp pili hücrelerinin tipik içeriği (EBS) son derece düşüktür. açıklanan protokol Myh6-organizatörü bazlı antibiyotik seçimi ile sinüs düğümü indükleyici TBX3 yoluyla kemirgen PSC&#39;lerin &quot;ileri programlama&quot; birleştirir. Bu&gt;% 80 fizyolojik fonksiyonel kalp pili hücrelerinin tutarlı kardiyomiyosit agrega verir. Bu &quot;uyarılmış-sinoatrial-organları&quot; (&quot;iSABs&quot;) kendiliğinden fare kalplerinden izole düğüm hücrelere karşılık gelen henüz ulaşılmamış frekanslarda (400-500 bpm) olarak sözleşme ve kemirgen miyokard ex vivo pace edebiliyoruz. Açıklanan protokol oldukça saf sinüs kullanmaNodal tek hücreler in vitro ilaç testi için kullanılabilir, ki burada, örneğin oluşturulabilir. Ayrıca, bu protokole göre üretilen iSABs kalp doku mühendisliği yolunda çok önemli bir adım olabilir.

açıklanan protokol fare kalp dayak frekansına yakın (film gösterilir) PSC&#39;ler yaklaşık 450 bpm bir dayak frekansı ile iSABs üretimi sağlar. Şekil 1 &#39;de gösterildiği gibi, iSABs ayrışmasından (aşama 2.8.8) sonra gözlenen tek hücre sinüs düğümü (mil ve örümcek hücreleri) hücrelerin, tipik şeklini gösterir. Bu hücreler bilinen yüksek hızlı protein fonksiyonu için önemli olduğu hiperpolarizasyon aktive siklik nükleotid kapılı katyon kanalı 4 (Hcn4), Connexin4...

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

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

Bu protokol, kemirgen pluripotent kök hücreler (PSC) fonksiyonel sinüs düğüm doku üretmek açıklamaktadır. T-Box3 (TBX3) ekspresyonu artı kalp Myosin ağır zincir (Myh6) promotör antibiyotik seçimi son derece saf kalp pili hücre agrega yol açar. Bu &quot;İsteyerek-sinoatrial-organları&quot; (&quot;iSABs&quot;), son derece dayak oranları arttı göstermek% 80&#39;in üzerinde kalp pili hücreleri içeren ve miyokard ex vivo pace edebiliyoruz.

Myh6-Promotörle Seçim ile kombinasyonu ES-Hücre-Programlama dayanarak Kemirgen Kardiyak Kalp Pili Hücre Agregaların Üretimi

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

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Research

JoVE Journal

Developmental Biology

9.6K Görüntüleme.  University of Rostock.  Bu protokol, kemirgen pluripotent kök hücreler (PSC) fonksiyonel sinüs düğüm doku üretmek açıklamaktadır. T-Box3 (TBX3) ekspresyonu artı kalp Myosin ağır zincir (Myh6) promotör antibiyotik seçimi son derece saf kalp pili hücre agrega yol açar. Bu "İsteyerek-sinoatrial-organları" ("iSABs"), son derece dayak oranları arttı göstermek% 80'in üzerinde kalp pili hücreleri içeren ve miyokard ex vivo pace edebiliyoruz. 

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Embriyonik Kök Hücreleri Kardiyak Progenitör Hücrelerinin türetilmesi

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Gelişim Biyolojisi

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

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

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 ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

BMP rakip GREM2 kullanma Pluripotent Kök Hücreleri Atriyal kardiyomiyositlerde Farklılaşma

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

İnsan pluripotent kök hücrelerin Süspansiyon tabanlı Kardiyak Farklılaşma için mikro Agrega boyutu Optimizasyon

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Mikrokontak baskı sayesinde Bağlantısızlar Fonksiyonel Miyokard Doku Üretimi

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Biyomühendislik

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.

Pro-fibrotik sinyal bastırma faktörü-aracılı fare embriyonik fibroblastlar indüklenen Cardiomyocytes yeniden programlama Potentiates

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

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Tanımlı İnsan Engineered Kalp Dokuların inşaatı Kardiyak Hücre Tedavisinin Mekanizmaları Eğitim için

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In vitro Generation fare kalp alanı-spesifik kardiyak progenitor hücreler

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biyoloji

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

İnsan kardiyomiyositleri Üretimi: Besleyici-ücretsiz İnsan Bağlı Pluripotent Kök Hücre A Farklılaşma Protokolü

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...