We thank Kwon laboratory members for helpful discussions. This work was supported by grants from NHLBI/NIH (R01HL111198) and Maryland Stem Cell Research Fund (MSCRF).

すべてのマウスは、ジョンズ·ホプキンス大学の動物施設を-accredited実験動物管理の認定のためのアメリカ協会（AAALAC）に維持し、実験動物の管理と使用に関するガイドに概説された手順に従って飼育しました。制度動物実験委員会（IACUC）は、すべての実験プロトコールを承認しました。 1。実験の準備<ol><li>リン酸緩衝液中の10％ウシ胎児血清（FBS）（PBS）を用いてコート12ウェルプレートで60分間。 </li><li>コー​​ティング溶液を除去し、無血清培地（SFM）の200μlを添加します。メディアのフィルムは、表面領域全体を覆っていることを確認するために、プレートを振り回します。 </li></ol> 2.外科的処置<ol><li>完全な安楽死を確実にするために、頸椎脱臼に続いて、金融機関の動物管理委員会が承認したプロトコルを使用して妊娠マウスを安楽死させます。 </li><li>スプレーし、70％のマウスの腹の領域をきれいにエタノール。汚染を避けるために、厳密な無菌技術を維持しています。 </li><li>へその領域で0.5 cm 2の切開を作るために鉗子やハサミを使用してください。 </li><li> /ピンチ切開の上および下の皮膚をつかみ、ゆっくりと反対方向（ヘッドとテール）に皮膚を引っ張って指を使用してください。 </li><li>へその領域における膜内の最初の切開を作るために鉗子やハサミを使用してください。ゆっくりそれによって子宮を明らかに、それぞれの側の卵巣にへそからV字状に（2×1.5 cm 2で）膜をカット。 </li><li>このように卵巣から子宮を解放し、卵巣側の卵管を挟ま卵巣およびはさみに子宮を接続卵管を挟ま鉗子を使用してください。 </li><li>優しく鉗子で卵管によって子宮をプルアップし、はさみで膀胱領域のない子宮を切りました。 </li><li>ピンセットで卵管によってさらにアップ子宮を引き出し、それによってMから子宮を解放する、反対側にハサミで卵管をカットウーズ。 </li><li> 50mlのチューブに子宮を転送およびCa 2+およびMg 2+との無菌冷PBS 20mlのを追加します。 PBSは、胚の解剖中のCa 2+およびMg 2+を含まれている必要があります。 </li><li>ゆっくり血を子宮を洗浄するために10秒間チューブを振ります。 </li><li>鉗子で管から子宮を移し、10mlの皿にそれを置き、子宮の上に冷PBSを5ml加えます。 </li><li>実体顕微鏡下での子宮で皿を置きます。 注：2.13から2.19には実体顕微鏡を必要とする手順。 </li><li>静かに子宮を開き、子宮一つ一つからの胚で羊膜嚢を解剖するために鉗子の二組を使用してください。 <ol><li>慎重に、胚を囲む羊膜を開く1鉗子で嚢をつまんで優しく胚から削除し、胚から羊膜を切断し、解放するために他の鉗子を使用するために鉗子の二組を使用してください。特定の遺伝子型は、必要に応じて、遺伝子型決定のための羊膜を維持します。 </li></ol></li><li>側に胚を配置し、 第 1および第 2の切断に鉗子を使用NDアーチと咽頭嚢（ 図1A &#39;&#39;）との間の心に後方アーチ。 </li><li>静かに反対側に胚を反転し、アーチや咽頭嚢の間心に後方に1番目と2番目のアーチをカットする鉗子を使用しています。 </li><li>まだ胚に1 番目と2 番目の咽頭弓を接続している心臓の流出路を切断するために鉗子を使用してください。胚から、まだ蝶（ 図1B）の形で一緒に取り付けられているアーチを、削除します。 </li><li>ピンチする鉗子を使用し、残りの心臓の流出路と前腸内胚葉から1 番目の咽頭弓を解放します（ 図1（b） &#39;&#39; *破線で示すと）。 </li><li>ピンチする鉗子を使用し、残りの心臓の流出路から2 番目の咽頭弓をリリースと前腸内胚葉（ 図1B &#39;&#39;破線で示し、**）。 </li><li>ピンセットを用いて別々のアーチの各ペアを移し、よく指定の途中で両方のアーチを配置します。別々のウェルでプレート1 番目と2 番目の咽頭弓。アーチは、ステップ1.2で追加されたメディアのフィルムに接触していることを確認してください。彼らは、この期間中に取り付けるためのウェルの表面と接触する必要があるように、それは、アーチが媒体によって覆われていないことが重要です。 </li></ol> 3.インキュベーションとイメージング<ol><li>ウェル表面領域に取り付けるためのアーチのために2時間、37℃/ 5％CO 2で12ウェルプレートにプレーティングアーチをインキュベートします。 </li><li>ゆっくりウェルの側面の下、37℃SFMの200μlを添加します。アーチが取り付けられたままとO / Nをインキュベートすることを確認します。 </li><li>翌日、視覚的に咽頭弓が接続されていることを確認し、穏やか側下37°C SFMの200μlのを追加よく。アーチが取り付けられていないとフローティング残っている場合、メディアの膜のみが残っているまで、静かにアーチを除去せずにピペットでメディアを削除します。よく繰り返しステップ3.1の中央にアーチを配置するピペットチップを使用してください。 </li><li>毎日の監視;任意の蒸発したメディアを交換するごとに2日目のメディアの〜100〜200μlを添加します。 注：細胞を移行した後24〜48時間は、アーチの周りに表示され、増殖し、次の5-8日にわたって弓から移行されます。拍動心筋細胞の36から72時間の形成は、2 番目の咽頭弓（ 図2B）から観察することができます後。筋管形成は、3-7日付着（ 図2A）した後、 第 1 の咽頭弓から観察することができます。 </li></ol>

<ol>
	<li>Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+heart+field.">The second heart field.</a> Curr Top Dev Biol. 100, 33-65 (2012).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Tzahor, E., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+mesoderm+development+during+embryogenesis:+implications+for+both+heart+and+head+myogenesis.">Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis.</a> Cardiovasc Res. 91, 196-202 (2011).</li><li>Uosaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Contact+with+Endoderm-like+Cells+Efficiently+Induces+Cardiac+Progenitors+from+Mouse+and+Human+Pluripotent+Stem+Cells.">Direct Contact with Endoderm-like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells.</a> PLoS One. 7 (10), e46413 (2012).</li><li>Cheng, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+mesendodermal+cell+fate+decisions.">Fibronectin mediates mesendodermal cell fate decisions.</a> Development. 140, 2587-2596 (2013).</li><li>Kwon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+pathway+involving+Notch1/beta-catenin/Isl1+determines+cardiac+progenitor+cell+fate.">A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.</a> Nature cell biology. 11, 951-957 (2009).</li><li>Shenje, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precardiac+deletion+of+Numb+and+Numblike+reveals+renewal+of+cardiac+progenitors.">Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.</a> Elife. 3, e02164 (2014).</li><li>Grevellec, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharyngeal+pouches+and+clefts:+Development,+evolution,+structure+and+derivatives.">The pharyngeal pouches and clefts: Development, evolution, structure and derivatives.</a> Semin Cell Dev Biol. 21, 325-332 (2010).</li><li>Kwon, C., Cordes, K. R., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+acts+at+multiple+developmental+stages+to+promote+mammalian+cardiogenesis.">Wnt/beta-catenin signaling acts at multiple developmental stages to promote mammalian cardiogenesis.</a> Cell Cycle. 7, 3815-3818 (2008).</li><li>Cho, G. S., Fernandez, L., Kwon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+heart:+perspectives+on+stem-cell+therapy.">Regenerative medicine for the heart: perspectives on stem-cell therapy.</a> Antioxid Redox Signal. 21, 2018-2031 (2014).</li></ol>

The authors have nothing to disclose.

このビデオでは、我々は9.5日の咽頭弓古いマウス胚第一および第二の単離および培養する方法を示します。咽頭のアーチは、最初のアーチ8を第二のアーチとヘッド筋前駆細胞-in胚10,11の間に心臓をするために、マルチ強力な心臓前駆細胞ビルディングブロックを含む発生中の胚9のcraniolateral側に表示されるバルジを過渡セグメント化されています。 <p class="jove_conten...

咽頭中胚葉細胞は、心臓の部品や咽頭の筋肉を生じさせます。胚発生の間に、第2の心臓フィールドから多能心臓前駆細胞は、咽頭中胚葉から移行し、心臓流出路および右心室を移入し、それらの異常な発達は密接先天性心疾患、先天性欠損症の主な原因と出生defect-に関連付けられていますヒト1-3関連死。最近の研究では、咽頭中胚葉が中胚葉心臓頭蓋顔面の開発4の重要な部分作る、心臓に加えて、筋肉を頭に寄与することを実証しました。このように、誘導、増殖、および咽頭中胚葉で頭と心前駆細胞の分化を含む発達プロセスは、活発な調査5-7中です。 最近まで、心臓前駆細胞は、拡張withoを受けるかどうかは不明のままで部分的には、それらの細胞環境に関する情報の欠如UT分化。我々の最近の研究では、咽頭のアーチは、心臓や筋肉前駆細胞の更新のための微小環境として機能し、数週間かけて8 ex vivoで培養することができることを示唆しています。この外植法は心臓頭蓋顔面前駆細胞ex vivoでの開発を研究するための新規かつユニークな機会を提供しています。

<table><tbody><tr><td>FBS</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>PBS + Ca&lt;sub&gt;2&lt;/sub&gt; + Mg&lt;sub&gt;2&lt;/sub&gt;</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>10 cm Petri plates</td><td>Fishersci</td><td>FB0875712</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Dissection forceps</td><td></td><td></td><td></td></tr><tr><td>Serum Free Media:</td><td></td><td></td><td></td></tr><tr><td>IMDM</td><td>Cellgro</td><td>15-016-CV</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>Cellgro</td><td>10-080-CV</td><td></td></tr><tr><td>N2-SUPPLEMENT</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>B27-retinoic acid</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>10% BSA (in PBS) (Invitrogen Cat#. P2489)</td><td>Life Sciences</td><td>P2489</td><td></td></tr><tr><td>100x Glutamine (Gibco Cat.# 25030-081)</td><td>Gibco</td><td>35050-61</td><td></td></tr><tr><td>100x Pen/Strep (Gibco Cat#. 15070-063)</td><td>Gibco</td><td>15140-122</td><td></td></tr></tbody></table>

ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche

発生中の胚の咽頭中胚葉は、頭と心筋組織の広範な地域に貢献しています。我々は、ex vivoで （も鰓のアーチと呼ばれる）咽頭弓で頭と心前駆細胞の発達を研究するための新規な方法を開発しました。この方法を使用して、我々は最近、第二の咽頭弓が自己複製心臓前駆細胞が含まれており、マウスの心臓の開発中に前駆細胞の拡大のための微小環境として機能することを記載しています。前駆細胞は、アーチの内側未分化と広大なままであるが、すぐに彼らはアーチの外に移動するような機能心筋細胞になります。また、最初の咽頭弓は弓を離れた後の筋管を生じさせる筋肉前駆細胞が含まれていることを報告しました。ここでは、解剖とマウス胚の開発からの第1および第2の咽頭弓のex vivo培養のための手順を示しています。この方法は、頭と心前駆/ devの筋肉を研究することを可能ex vivoで詳細に心筋細胞および筋管形成を含むelopment、。

彼らは増殖し、1 番目と2 番目の咽頭弓からの移行のように、開発時には、顔の筋肉や心臓前駆細胞は（ 図1A、A &#39;とA&#39; &#39;）は、それぞれ、頭と心の筋肉組織になるために、追跡することができます。咽頭弓を培養すると、ex vivoで詳細に心臓や筋肉の発達を研究するためのユニークな方法を提供しています。解剖と咽頭弓の付着した後、?...

Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽頭アーチのex vivo培養は、ハートと筋前駆細胞とそのニッチを研究します

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

ex-vivo-culture-pharyngeal-arches-to-study-heart-muscle-progenitors

Research

JoVE Journal

Developmental Biology

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

7.8K ビュー.  Johns Hopkins University School of Medicine. Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

ビデオ: Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

イメージングは​​、単一細胞の解像度で胚および出生後の心をクリア

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

発生生物学

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

マウス胚心臓から心外膜細胞の体外培養で

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

工学

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.

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

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

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

鳥胚胚心筋細胞の生体内注入にマイクロインジェクション ベース システム

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

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.

マウス心臓分野特異的心臓前駆細胞のインビトロ生成

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

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

全実装免疫学化学と3D再構成を用いた咽頭弓動脈の可視化と解析

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

多能性幹細胞由来の自己組織化ヒト心臓オルガノイドの生成

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

生化学

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

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

<meta charset="utf8"></head><body>ニワトリ胚におけるヒト幹細胞スフェロイドの挙動を調査するためのラベルフリー異種移植モデル

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

ニワトリ胚におけるヒト幹細胞スフェロイドの挙動を調査するためのラベルフリー異種移植モデル

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

咽頭アーチのex vivo培養は、ハートと筋前駆細胞とそのニッチを研究します

要約

さらに動画を探す

この動画の章

イメージングは、単一細胞の解像度で胚および出生後の心をクリア

マウス胚心臓から心外膜細胞の体外培養で

マウス胚における心臓前駆細胞集団の定量的ホールマウント免疫染色解析

鳥胚胚心筋細胞の生体内注入にマイクロインジェクションベースシステム

マウス心臓分野特異的心臓前駆細胞のインビトロ生成

全実装免疫学化学と3D再構成を用いた咽頭弓動脈の可視化と解析

多能性幹細胞由来の自己組織化ヒト心臓オルガノイドの生成

鳥類胚弁膜前駆細胞の単離と培養

ニワトリ胚におけるヒト幹細胞スフェロイドの挙動を調査するためのラベルフリー異種移植モデル

ニワトリ胚におけるヒト幹細胞スフェロイドの挙動を調査するためのラベルフリー異種移植モデル

咽頭アーチのex vivo培養は、ハートと筋前駆細胞とそのニッチを研究します

要約

さらに動画を探す

この動画の章

イメージングは​​、単一細胞の解像度で胚および出生後の心をクリア

マウス胚心臓から心外膜細胞の体外培養で

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

鳥胚胚心筋細胞の生体内注入にマイクロインジェクション ベース システム

マウス心臓分野特異的心臓前駆細胞のインビトロ生成

全実装免疫学化学と3D再構成を用いた咽頭弓動脈の可視化と解析

多能性幹細胞由来の自己組織化ヒト心臓オルガノイドの生成

鳥類胚弁膜前駆細胞の単離と培養

ニワトリ胚におけるヒト幹細胞スフェロイドの挙動を調査するためのラベルフリー異種移植モデル

ニワトリ胚におけるヒト幹細胞スフェロイドの挙動を調査するためのラベルフリー異種移植モデル

イメージングは、単一細胞の解像度で胚および出生後の心をクリア

マウス胚における心臓前駆細胞集団の定量的ホールマウント免疫染色解析

鳥胚胚心筋細胞の生体内注入にマイクロインジェクションベースシステム