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.

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			<td>Biochrom AG</td>
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			<td>Biochrom AG</td>
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			<td>PAN-Biotech</td>
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			<td>PAN-Biotech</td>
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			<td height="20" style="height:20px;">Leukemia inhibitory factor</td>
			<td>Phoenix Europe GmbH</td>
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			<td>TPP</td>
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			<td>92424</td>
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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

Universidad de Cartagena UDEC

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

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Kimyasal Güvenlik Tarama ve Sistem Biyolojisi Veri Üretimi Gelişim Toksisite Tahliller Tabanlı İnsan Pluripotent Kök Hücre

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Gelişim Biyolojisi

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

Fare Embriyonik Kök Hücre Agregaların Üretimi Simetri Kırma, Polarizasyon ve Acil Toplu Davranış göster o<em&gt; İn Vitro</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Güçsüz İnsan dermis kullanma Genetiği Değiştirilmiş Organotipik Cilt Kültürlerin Üretimi

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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 ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Vasküler Endotelyal Kök Hücrelerin Damar Oluşumunu Görselleştirmek İçin Yeni Bir Göğüs Göğsü Transplantasyon Tekniği

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Nicel bütün Dağı ayirt analiz fare embriyo kardiyak progenitör nüfusun

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

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 ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

Bütün Mount Epifluorescence kullanarak fare embriyogenez sırasında kalp odası gelişimin analizi

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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 ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Diferansiyel Tripsinizasyon a göre Mozaik Meme Organoidlerinin Üretimi

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