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.

注：全ての実験は、ノースカロライナ大学チャペルヒル校で施設内動物管理使用委員会によって承認された。 1消費税大動脈弁<ol><li>興味のある胚の段階のために承認された安楽死の技術を使用して、開発の目的の胚日（E）で子犬を妊娠マウスを安楽死させる。 </li><li>妊娠中の女性の腹部を滅菌する70％エタノールを使用してください。下腹部の皮膚と筋肉を持ち上げ、離れて内臓から、および子宮角へのアクセスを許可するように、女性の下腹部キャビティを開い解剖。 </li><li>子宮角を取得するには、鉗子の鉗子で子宮頸部、慎重にカット尾を開催しています。所定の位置にホーンを保持し、任意の結合組織を切断、子宮角を持ち上げ、卵管と子宮角の接合部を切断して削除を完了。 </li><li>コルを含むペトリ皿に解剖子宮角を置きdは、0.1Mトリス緩衝液（pH7.6）で、必要に応じてすすぎます。その後、胚嚢を露出するために子宮角開放長さ方向をカット。 </li><li>胚を公開するには、胎盤や胚の接合部での胚嚢を開いてカットし、胚を解放するために臍帯血管を切った。冷たい0.1Mトリス緩衝液で二ペトリ皿に解剖した胚を置きます。次の胚の準備ができるまで氷に残りundissected胚を持つ最初のペトリ皿を返します。 </li><li>胚内の胸壁へのアクセスを改善するために、胚を首を切る。遺伝子型決定が必要な場合は、削除し、氷上に置いエッペンドルフチュー​​ブに余分な組織片を保存する。 </li><li>その背中に胚を置き、前肢の近くで、胸郭の側面に沿って垂直に胸壁をカットし、水平方向に振動板の上に心を視覚化する。その後、心臓を露出させ、胸壁を開きます。 </li><li>大血管に沿って高く保持するために鉗子を使用して、鉗子を用いて心臓を持ち上げ、下の血管を切る。ザ·nは、心を解放するために鉗子の上にカット。肺血管が切断されていない残っている場合、肺は心で除去してもよいし、続行する前に削除する必要があります。 </li><li>カットし、バルブのレベル以上の肺動脈を取り除く。明細書に記載の胚の段階で、肺動脈弁領域からその区別に役立つ、これわずかに不透明である。肉柱心室筋を避け、単に肺動脈弁の下にカットし、ステップ1.10で説明したように、この領域に関心がある場合にも収集しています。ここで、次のステップで、離れて溶解緩衝液中に収集する前に、関心領域からの過剰のトリス緩衝液を描画するために毛管作用を使用。 </li><li>慎重にちょうどバルブのレベルを超えて、大動脈弁に大動脈遠位を削除。肺動脈のように、大動脈はその除去を助ける、これらの胚の段階でわずかに不透明のまま。再び肉柱心室筋を避け、ちょうど大動脈弁の下にカットして、転送する氷上でプロテアーゼおよびホスファターゼ阻害剤を含む（材料表に記載）を2μlの溶解緩衝液をエッペンドルフチュー​​ブに鉗子を使用してバルブ。 </li><li>バルブは別個のエッペンドルフチュー​​ブに各バルブを集め、すべての胚から切除されるまで繰り返します1.6から1.10を繰り返します。すべての胚が同じ遺伝子型を持っている場合は、すべてのバルブは、単一のエッペンドルフチュー​​ブに収集することができる。 </li><li>遺伝子型決定は、一緒にプールするバルブかを決定するために実行される場合には、直ちにさらなる使用まで-80℃で溶解緩衝液中に試料を置く。次に、試料を（ステップ2.1.1）を遺伝子型決定した後にプールすることができる。 </li></ol> 2タンパク質抽出<ol><li>チューブの底に溶解緩衝液中で組織を収集するために1分間13,100×gで遠心し、氷上に採取した組織を解凍する。 <ol><li>胚を遺伝子型決定した場合は、ゆっくりと類似の遺伝子型を持つ胚からバルブ領域を含有する溶解緩衝液を収集し、プールするために、ピペットを使用しています。強いBを確実にするために、そして、サンプルあたりE13.5胚から3-​​4バルブ領域をプールすることをお勧めします。 </li><li>すべての胚が同じ遺伝子型のものであり、すべてのバルブ領域がステップ1.10で一緒に収集された場合は、ステップ2.2で説明したように、サンプル全体を溶解。 </li></ol></li><li>結果としてプールされた試料の体積をチェックして、40μlの全量を溶解バッファーを追加します。次に、エッペンドルフチュー​​ブ5mmのステンレススチールビーズを用いて試料を破壊し、均質化する。メーカーの指示に従って、50 Hzで2〜4分間Lyser装置を操作します。不溶性の残骸を残す新しいチューブに短時間スピンチューブ、（1分間〜13,100×g）で、転写サンプル。 </li><li>そのようなEslami ら 9などのプロトコルに従って、その後のウエスタンブロット分析のために準備し、SDS-PAGEおよび転送のための独立したサンプル。ローディング対照タンパク質をブロッティングするタンパク質定量のために必須である。 </li></ol>

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The authors have nothing to disclose.

初期胚のマウスとニワトリの心臓弁領域中のタンパク質レベルを定量化する能力は、バルブの開発のための重要な細胞シグナル伝達事象を理解するための追加のツールを提供しています。明細書に記載の当社のプロトコルは、標準的なタンパク質単離手順から大きく異なるものではない。しかし、いくつかの重要な手順を変更することによって、私たちは非常に小さなサンプルサイズからリ?...

タンパク質発現レベルを同定および定量化することができることは、動物 - 、および細胞ベースの実験のための標準的な技術である。しかし、開発中にこの特定の組織におけるタンパク質発現を評価し、初期胚心臓弁の開発における長年の関心にもかかわらず、現在、ヒヨコとマウス1,2の両方で免疫組織に限定されている。ほとんどのモデル生物（ 例えば 、ニワトリおよびマウス）の現像バルブにおけるタンパク質発現を定量化することの難しさの一部を得ることができるタンパク質の量を制限するバルブのサイズが小さいことである。このように、定量分析のために、研究者は、一般的に、その後の定量的PCRやマイクロアレイ解析2-5 RNA抽出と増幅に依存しています。しかし、RNAおよびタンパク質発現レベルが6完全相関ではないため、RNA発現に焦点を当てたことは、任意のシグナル伝達に発生する多数の変更を厳密にアカウントを提供することはできません開発中のさまざまな時間での経路。現在利用可能な方法でこの制限に基づいて、この手順の目的は、確実に重要であるさまざまなシグナル伝達経路に発生する変化の定量分析のための現像マウス胎児心臓弁領域からのタンパク質の十分な量を得るためのプロトコルを開発することであったこの組織の成熟。 胚弁はすでに一般的にRNAの単離およびその後の遺伝子発現解析2-5マウスから解剖されています。しかし、これらの研究は、下流のシグナル伝達に影響を与える可能性が翻訳後タンパク質修飾を検出する検出を許可していない経路の活性化をシグナル伝達の読み出しなどの遺伝子発現を使用することに限定されている。出発点として、RNA単離技術を用いて、関心領域を解剖始まった。私たちの関心は、パットのシグナルを示していたリン酸化タンパク質を検出したため大動脈弁の開発（E13.5-14.5）の特定の期間にhway活性は、私たちはリン酸塩を含まないトリス緩衝液内のすべての解剖を実施し、ホスファターゼおよびプロテアーゼ阻害剤を用いたトリスベースの溶解緩衝液中でバルブを集めた。私たちの特定の場合において、唯一の大動脈弁領域は、収集されたが、肺動脈弁領域を容易に同時に得ることができた。弁領域は、その後、現在成人の心臓弁7におけるタンパク質発現を研究するために使用されるサンプル緩衝液でホモジナイズした後、混合した。少量の試料（ 例えば 、2μl）を使用し、同じ遺伝子型を持つ胚からバルブ領域をプールすることで、私たちは胎生13.5 8でリン酸化および非リン酸化タンパク質を検出することができた。弁領域が凍結し、溶解緩衝液中で保存することができるので、プールする前に、必要に応じて、胚を遺伝子型を決定することができる。 この技術は、細胞signalinを評価するための利用可能なツールのセットを広げるグラム開発中の経路とは、具体的には発展途上心臓弁のために、免疫組織化学への定量的な賛辞を提供しています。この技術は発達心臓専門医にも限定された組織が含まれていた領域に興味を持っている初期段階の胚を扱うすべての発達生物学者のみならず有益である必要があります。

<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.

この調製技術を用いて、E13.5胚からの単一の大動脈弁の領域にリン酸化されたSmad 1,5,8（pSmad）を検出することができた。 図1Aに示すように、単一の弁領域から単離されたタンパク質は、かすかなpSmadバンドを検出するのに十分である。信号強度は、プールされ、弁領域の数に比例して増加する。重要なことは、pSmad /βアクチン比率が異なるサンプルサイズ（ 図1C）</s...

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.

途上胚性マウス心臓弁地域からのタンパク質の単離

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

Research

JoVE Journal

Biology

9.3K ビュー.  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.

ビデオ: 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.

低血清条件下で心臓前駆細胞の内皮への分化の誘導

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

発生生物学

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.

鳥類胚弁膜前駆細胞の単離と培養

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

生物学

マウス心臓前駆細胞からの単一細胞分解能での核単離

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.

著者スポットライト:マウス心臓前駆細胞からの核単離によるエピゲノムおよび遺伝子発現プロファイリング(シングルセル分解能でのエピゲノムおよび遺伝子発現プロファイリング)  

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.

シングルセル分解能でのエピゲノムおよび遺伝子発現プロファイリングのためのマウス心臓前駆細胞からの核分離(英語)

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;

マウスの心臓から初代メラノサイト様細胞を単離する

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.

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

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.

胚心室内皮細胞の単離

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.

冠動脈血管新生のインビトロモデル

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

医学

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.

ネズミ弁内皮細胞の単離

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

途上胚性マウス心臓弁地域からのタンパク質の単離

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低血清条件下で心臓前駆細胞の内皮への分化の誘導

鳥類胚弁膜前駆細胞の単離と培養

シングルセル分解能でのエピゲノムおよび遺伝子発現プロファイリングのためのマウス心臓前駆細胞からの核分離(英語)

シングルセル分解能でのエピゲノムおよび遺伝子発現プロファイリングのためのマウス心臓前駆細胞からの核分離(英語)

シングルセル分解能でのエピゲノムおよび遺伝子発現プロファイリングのためのマウス心臓前駆細胞からの核分離(英語)

マウスの心臓から初代メラノサイト様細胞を単離する

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

胚心室内皮細胞の単離

冠動脈血管新生のインビトロモデル

ネズミ弁内皮細胞の単離