We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

NOT: Tüm deneyler Chapel Hill&#39;deki North Carolina Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Tüketim Aort Kapağı <ol><li> Ilgi embriyonik aşaması için onaylanmış bir ötenazi tekniği kullanılarak, arzu edilen gelişme embriyonik gün (E) yavrular ile hamile bir fareyi euthanize. </li><li> Hamile kadın karnı sterilize etmek için% 70 etanol kullanın. Uzak iç organlardan alt karın yukarı ve cilt ve kas kaldırın ve uterin boynuz erişime izin vermek için açık dişinin alt karın boşluğuna teşrih. </li><li> Uterin boynuzu almak için, forseps forseps ile serviks ve dikkatlice kesilmiş kaudal tutun. Yerinde boynuz tutan herhangi bir bağ dokusu kesme, uterin boynuz kaldırın ve fallop ile uterin horn kavşak keserek kaldırma işlemini tamamlamak. </li><li> Bir Petri kabı içeren col disseke uterin boynuz yerleştirmekd 0.1 M Tris tamponu (pH 7.6) ve gerektiği gibi yıkayın. Sonra, açık uzunluğu-bilge embriyonik keselerinin maruz uterin boynuzu kesti. </li><li> Embriyo göstermek için, plasenta ve embriyo kavşağında bir embriyonik kese kesip ve embriyo serbest göbek damarları kesilmiş. Soğuk 0.1 M Tris tampon içeren ikinci bir Petri kabındaki kesilen embriyo yerleştirin. Sonraki embriyo için hazır olana kadar buz kalan undissected embriyolar ile ilk Petri dönün. </li><li> Embriyonun içinde göğüs duvarı erişimi geliştirmek için, embriyoyu başını kesmek. Genotipleme gerekli ise, kaldırma ve buz üzerine yerleştirilir, bir Eppendorf tüpü içinde ilave bir doku parçası kaydedin. </li><li> Sırtında embriyo yerleştirin ve ön ayakları yakınında, göğüs kafesinin yan boyunca dikey göğüs duvarı kesti ve yatay diyafram üstü kalp görselleştirmek için. Ardından, kalp maruz göğüs duvarı açın. </li><li> Büyük damarların boyunca yüksek tutmak için forseps kullanarak, forseps kullanarak kalp kaldırın ve aşağıdaki damarları kesilmiş.n kalbi boşaltmak için forseps yukarıdaki kesti. Akciğer damarlarında kesilmemiş kalırsa, akciğerler kalp ile kaldırılmış olabilir ve devam etmeden önce çıkarılmalıdır. </li><li> Kesme ve valfın seviyesinden pulmoner arter çıkarmak; Burada tarif edilen embriyonik aşamada, pulmoner arter kapak bölgesinden, ayrımda yardım eden, hafif opaktır. Sadece pulmoner kapak altına indi trabeküler ventriküler miyokard kaçınarak ve bu bölge de ilgi ise adımda 1.10 açıklandığı gibi toplamak. Burada ve bir sonraki aşamada önce, liziz tamponu içinde toplama uzak ilgilenilen bölgesinde aşırı Tris tamponu çizmek için kılcal hareketi vardır. </li><li> Dikkatle vananın seviyesinden, aort kapak aort distalinden kaldırmak; pulmoner arter gibi, aort, uzaklaştırılması yardım, bu embriyonik aşamada biraz opak kalır. Tekrar trabeküle ventriküler miyokard kaçınarak, sadece aort kapak altında kesilmiş ve aktarmakBuz üzerinde proteaz ve fosfataz inhibitörlerini ihtiva eden (Malzeme tabloda tarif edilmiştir) 2 ul liziz tamponu ile bir Eppendorf tüpüne, forseps kullanılarak valf. </li><li> Vanalar ayrı Eppendorf tüp her bir valfi toplayarak, tüm embriyolardan kesilip edilene kadar tekrarlayın 1,6-1,10 adımları. Tüm embriyolar aynı genotipi varsa, tüm supap tek bir Eppendorf tüpü içinde toplanabilir. </li><li> Genotipleme birlikte havuz valflarına belirlemek için gerçekleştirilecekse, hemen bir sonraki kullanıma kadar -80 ° C&#39;de lisis tamponu içinde örnekleri yer. Numuneler daha sonra (adım 2.1.1) genotipleme sonra toplanmış olabilir. </li></ol> 2. Protein Ekstraksiyonu <ol><li> 1 dakika tüpün altındaki liziz tamponunda dokuları toplamak için 13.100 x g buz ve santrifüj üzerine toplanır dokuları çözülme. <ol><li> Embriyolar genotiplendirilmiştir, hafifçe toplamak ve havuz lizis tamponu benzer genotipler ile embriyolar valf bölgeleri içeren bir pipet kullanın. Güçlü bir b sağlamakörnek önerilir başına, E 13.5 embriyolardan 3-4 kapak bölgeleri birleştirilmelidir. </li><li> Tüm embriyolar aynı genotipi ve tüm valf bölgeler Aşama 1,10 araya getirilerek durumunda Adım 2.2 &#39;de tarif edildiği gibi, örneğin tamamına lize. </li></ol></li><li> Elde edilen birleştirilmiş numune miktarını kontrol edin ve 40 ul bir toplam hacim liziz tampon eklemek; Daha sonra, bozabilir ve Eppendorf tüpleri 5 mm paslanmaz çelik boncuklar kullanarak örnekleri homojenize. Üreticinin talimatlarına uygun olarak, 50 Hz&#39;de 2-4 dakika için lize edici çalıştırın. Kısaca Spin tüpler, çözünmeyen enkaz geride bırakarak yeni tüplere (~ 13.100 1 dakika xg), ve transfer örnekleri. </li><li> Hazırlama ve bu Eslami ve ark 9 gibi bir protokol izlenerek, SDS-PAGE ve Western blot analizi daha sonra aktarım için ayrı örnekler. Bir yükleme kontrol proteini için Kurutma protein miktar tayini için önemlidir. </li></ol>

<ol>
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The authors have nothing to disclose.

Kalp kapak bölgeleri erken embriyonik fare protein seviyelerini ölçmek ve civciv yeteneği vana gelişimi için kritik hücresel sinyal olayları anlamak için ek bir araç sağlar. Burada tarif Bizim protokol standart protein izolasyon prosedürlerinin büyük ölçüde farklı değildir. Ancak, bazı önemli adımları değiştirerek, biz başarıyla derece küçük örneklem büyüklükleri fosforile proteinleri almış. Bu sonucu elde etmek için, aşağıdaki aşamalar, özel bir önem taşımaktadır. Bu kalite...

Protein ekspresyon seviyelerini belirlemek ve ölçmek için güçlü olmak bir hayvan ve hücre tabanlı deneyler için standart bir tekniktir. Ancak rağmen uzun süreli bir anda civciv ve fare hem de 1,2 immünohistokimya ile sınırlıdır gelişimi sırasında, bu özel doku içindeki protein ifadesi değerlendirilmesi, erken dönemde embriyo gelişimi kalp kapağı ilgi. En model organizmaların (örneğin, civciv ve fare) gelişmekte vanaları protein ifadesini nicel zorluk parçası elde edilebilir protein miktarını sınırlayan vanalar, küçük boyutu. Böylece, kantitatif analiz için, araştırmacılar genellikle müteakip kantitatif PCR veya mikroarray analizi 2-5 RNA ekstraksiyon ve amplifikasyon güveniyor. Bununla birlikte, RNA ve protein ekspresyon seviyeleri 6 tamamen bağıntılı değildir, bu nedenle RNA ekspresyonu odaklanan herhangi bir sinyal meydana gelen pek çok değişiklik sıkı bir hesap sağlayamazgelişmeler sırasında çeşitli zamanlarda yol. Mevcut metodolojisinde bu sınırı dayanarak, bu prosedürün amacı güvenilir önemli olan çeşitli sinyal yollarında meydana gelen değişikliklere kantitatif analiz için gelişmekte embriyonik fare kalp kapak bölgelerden proteinin yeterli miktarda elde etmek için bir protokol geliştirmek oldu Bu doku olgunlaşması. Embriyonik vanalar zaten yaygın RNA izolasyonu ve sonraki gen ekspresyon analizi 2-5 için fareden disseke edilir. Ancak bu çalışmalar, akış aşağı sinyali etkileyebilir translasyon sonrası protein modifikasyonları yer tespit algılanmasını izin vermez yolu aktivasyonunun, bir sinyalleme okuma olarak gen ekspresyonu ile sınırlı kalmıştır. Bir başlangıç ​​noktası olarak RNA izolasyon teknikleri kullanarak, ilgi konusu bölgelerin kesme ile başladı. Bizim faiz fosforillenmiş proteinleri tespit Çünkü sinyalizasyon pat göstergesidir olduğunuaort kapak geliştirme (E13.5-14.5) belirli bir süre boyunca Hway aktivitesi, bu fosfat içermeyen Tris tamponu içinde bütün tahlil yapıldı ve fosfataz ve proteaz inhibitörleri ile bir Tris-bazlı liziz tamponunda valfleri toplanır. Bizim özel durumda, sadece aort kapak bölgeleri toplandı, ancak pulmoner kapak bölgesi kolaylıkla aynı anda elde edilebilir. Valf bölgeleri, homojenize edilir ve şu anda yetişkin kalp valf 7 protein ifadesini incelemek için kullanılan bir numune tampon maddesi ile birleştirildi. Küçük örnek hacimleri (örneğin, 2 ul) kullanılarak ve aynı genotipi ile embriyolardan valf bölgeleri bir araya getirerek, biz embriyonik günde 13.5 8 de fosforlu ve fosforillenmemiş proteinleri tespit başardık. Valf bölgeler dondurulmuş ve lizis tamponunda saklanabilir için araya önce gerekirse, embriyolar genotiplenmiştir edilebilir. Bu teknik, hücre signalin değerlendirmek için kullanılabilir araçları kümesi genişletmektedirg gelişimi sırasında yollar ve özellikle gelişmekte olan kalp kapakçıkları için, immünohistokimyaya nicel bir iltifat sağlar. Bu teknik gelişim kardiyoloji değil, aynı zamanda sınırlı bir doku ihtiva eden bölümleri ile ilgili erken evre embriyoları ile çalışan tüm gelişim biyolojistlere sadece yararlı olacaktır.

<table><tbody><tr><td>Timed-pregnant mouse</td><td></td><td></td><td>To be dissected at the embryonic stage of interest</td></tr><tr><td>Stereoscopic microscope</td><td>Nikon</td><td>SMZ645</td><td></td></tr><tr><td>0.1 M Tris, pH 7.6</td><td></td><td></td><td></td></tr><tr><td>Microscissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Fine forceps, #5</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Dissecting needle holders</td><td>Ted Pella Inc.</td><td>13560</td><td></td></tr><tr><td>Dissecting needles</td><td>Ted Pella Inc.</td><td>13561-10</td><td></td></tr><tr><td>Micropipette, 20 &amp;mu;l, with tips</td><td></td><td></td><td></td></tr><tr><td>Lysis buffer</td><td></td><td></td><td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td>Add 1 tablet to 10 ml lysis buffer</td></tr><tr><td>TissueLyser LT</td><td>Qiagen</td><td>85600</td><td></td></tr><tr><td>Stainless steel beads</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr></tbody></table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

Bu hazırlık tekniği kullanarak, E13.5 embriyolar tek aort kapak bölgelerinde fosforile Smad 1,5,8 (pSmad) tespit başardık. Şekil 1A&#39;da gösterildiği gibi, hatta bir tek valf bölgesinden izole edilmiş protein bir hafif pSmad grubu tespit etmek için yeterlidir. Sinyal yoğunluğu bir araya getirilmiş, kapak bölgelerinin sayısı artar. Önemli olarak, pSmad / β-aktin oran, farklı örnek boyutları (Şekil 1C) boyunca hemen hemen sabit kalır. Nedeniyle mevcut...

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Gelişmekte Embriyonik Fare Kalp Vana Bölgesi&#39;nde Protein İzolasyonu

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.3K Görüntüleme.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Video: Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

İndüksiyon düşük Serum koşullar altında kalp Progenitor hücreler endotel farklılaşma

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Gelişim Biyolojisi

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Biyoloji

Tek Hücre Çözünürlüğünde Fare Kardiyak Progenitör Hücrelerinden Çekirdek İzolasyonu

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Yazar Spotlight: Tek Hücre Çözünürlüğünde Epigenom ve Gen İfadesi Profili Oluşturma için Fare Kardiyak Progenitör Hücrelerinden Çekirdek İzolasyonu  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

Epigenom ve Tek Hücre Çözünürlüğünde Gen Ekspresyon Profillemesi için Fare Kardiyak Progenitör Hücrelerinden Çekirdek İzolasyonu

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

İzolasyon, Kültür ve Yetişkin Fare Cardiomyoctyes Fonksiyonel Karakterizasyonu

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Embriyonik Ventriküler Endotel Hücre İzolasyonu

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Koroner Anjiyogenezin In Vitro Modeli

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

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Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Murin Vana Endotel Hücreleri İzolasyon

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Gelişmekte Embriyonik Fare Kalp Vana Bölgesi'nde Protein İzolasyonu

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Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

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Embriyonik Ventriküler Endotel Hücre İzolasyonu

Koroner Anjiyogenezin In Vitro Modeli

Murin Vana Endotel Hücreleri İzolasyon