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).

所有小鼠均维持在实验室动物护理的认可美国协会（AAALAC）-accredited在约翰·霍普金斯大学动物设施及安置按照指南中列出了实验动物的护理和使用的程序。该机构动物护理和使用委员会（IACUC）批准的所有实验方案。 1.实验准备<ol><li>外套12孔板，用10％胎牛血清（FBS）的磷酸盐缓冲溶液（PBS）中60分钟。 </li><li>除去涂布溶液，并添加200微升无血清培养基（SFM）的。捻盘，以确保媒体的膜覆盖整个表面区域。 </li></ol> 2.外科手术<ol><li>安乐死使用机构的动物护理委员会批准的协议，颈椎脱位，以确保完全安乐死孕鼠。 </li><li>喷雾和清洁鼠标的腹部区域有70％乙醇。保持严格的无菌技术，以避免污染。 </li><li>使用镊子和剪刀，使0.5 平方厘米的切口在肚脐区域。 </li><li>用手指捏住/抢皮肤上方和下方的切口，然后轻轻拉动以相反的方向（头部和尾部）的皮肤。 </li><li>使用镊子和剪刀，使在膜的初始切口在肚脐区域。轻轻地从肚脐至卵巢每侧（2×1.5 cm 2）的切成V字形的膜，从而揭示子宫。 </li><li>使用镊子夹住子宫连接卵巢和剪刀输卵管捏在输卵管卵巢端，从而释放从卵巢子宫。 </li><li>轻轻拉起子宫与镊子输卵管切摆脱用剪刀膀胱区的子宫。 </li><li>进一步向上拉子宫通过用钳子的输卵管，并用剪刀剪下输卵管上的另一侧，从而从m释放子宫乌斯河。 </li><li>子宫转移至50ml管中，并加入20毫升冷无菌PBS的与钙离子和镁离子 。 PBS必须包含在胚胎解剖钙离子和镁离子 。 </li><li>轻轻摇动管10秒以洗涤子宫血。 </li><li>从管转移子宫用镊子并将其放置在10ml的培养皿和对子宫的顶部添加冷PBS 5毫升。 </li><li>与广场立体显微镜下子宫菜。 注意:步骤2.13-2.19需要立体显微镜。 </li><li>使用两对镊子从子宫逐一轻轻打开子宫解剖用的胚胎囊羊水。 <ol><li>使用两对镊子小心地打开羊膜囊包围胚胎，掐国资委一个镊子轻轻地从胚胎中取出，用另一钳剪切和从胚胎释放羊膜囊。如果特定基因型的需要，保持羊膜囊进行基因分型。 </li></ol></li><li>在广场一侧的胚胎，并使用镊子削减1 和第 2 次向后拱到心脏的弓和咽囊（ 图1A&#39;&#39;）之间。 </li><li>轻轻翻转胚胎到另一边，并用钳子切断第一和第二拱后方的弓和咽囊之间的心脏。 </li><li>使用镊子将那仍然是连接1 和第 2 次咽弓的胚胎心脏流出道。取出拱门，目前仍在连接在一起蝴蝶（ 图1B）的形状，从胚胎。 </li><li>使用镊子夹住并从剩余的心脏流出道和肠内胚层释放1日咽弓（ 图1B&#39;标有虚线和*）。 </li><li>使用镊子夹住并从剩余的心脏流出道释放第二咽弓和前肠内胚层（ 图1B&#39;表示与虚线和**）。 </li><li>传输每对单独使用镊子拱门和放置两个拱门的中央指定的很好。板1 和第 2 次咽弓在不同的孔。确保拱在与在步骤1.2中加入介质薄膜接触。至关重要的是，拱门并不包含介质，因为它们需要在此期间，以与井用于附连的表面接触。 </li></ol> 3.孵化和影像<ol><li>孵育拱门镀在12孔板在37℃/ 5％CO 2下2小时为拱附加到孔表面面积。 </li><li>轻轻加入200微升37℃的SFM井里的一侧。确保拱门保持连接，并培育O / N。 </li><li>第二天，在视觉上检查咽弓连接，轻轻地加入200微升37℃SFM下来的边良好。如果仍然拱门未婚和漂浮，轻轻地用吸管取出介质而不删除拱门，直到只有媒体的膜离开。用枪头把拱门井和重复步骤3.1的中间。 </li><li>日常监控;添加〜100-200微升媒体每2天更换任何媒体蒸发。 注:迁移细胞后24-48小时左右出现拱，将增殖和迁移拱在未来5-8天。后36-72小时形成跳动的心肌细胞可以从第二咽弓（ 图2B）中观察到。肌管形成可以从第1次咽弓3-7天附件（ 图2A）后进行观察。 </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.

在这段视频中，我们将演示如何隔离和9.5天的小鼠胚胎文化第一和第二咽弓。咽弓是短暂的，分段出现在胚胎发育9，其中含有多能心脏祖细胞，积木胚胎10,11 -in第二拱门和头部肌肉祖细胞在第一拱8时，使心脏的craniolateral侧面凸起。 咽弓的解剖需要先进的小鼠胚胎解剖技能。一旦拱已成功解剖使用这种分步协议，并转移至预包被的孔，即拱连接到所?...

咽中胚层细胞产生的心脏的部分和咽肌肉。在胚胎发育期间，从第二心脏字段多能心脏祖细胞从咽中胚层迁移和填充心脏流出道和右心室，和它们的异常发展密切出生缺陷和出生无缺陷的先天性心脏疾病的主要原因相关相关死亡在人类1-3。最近的研究已经表明，咽中胚层有助于头部肌肉，除了心脏，使得胚层的心颅面发展4的一个关键部分。因此，在发展过程中，包括诱导，增殖，以及头部和心脏祖细胞在咽中胚层分化正在积极调查5-7。 直到最近，它仍然是未知的心脏祖细胞是否发生无覆盖扩张UT分化，部分原因是由于他们的细胞环境信息的缺乏。我们最近的研究表明，咽弓作为微环境的心脏和肌肉祖细胞的更新和可体外培养在几个星期8。该块法提供了一个新颖而独特的机会来研究心颅面祖细胞体外的发展。

<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

发育中的胚胎的中胚层咽有助于头部和心脏肌肉组织的广泛地区。我们已经开发出一种学习的头部和心脏祖细胞发育与咽弓（也称为鳃弓） 离体的新方法。使用这种方法，我们最近描述了第二咽弓包含自我更新心脏祖细胞，并作为一个微环境对祖细胞的小鼠心脏发育过程中扩张。祖细胞分化仍然膨胀和拱内，但很快就成为功能的心肌细胞，因为它们迁移出弓。我们还报道说，第一咽弓包含肌肉祖离开后拱引起肌管。在这里，我们展示了解剖，并从发展中国家小鼠胚胎的第一和第二咽弓体外培养的过程。该方法使一个研究头部和心脏祖/ dev的肌肉elopment，包括心肌细胞和肌管的详细体外形成。

在开发过程中，面部肌肉和心脏祖细胞可以作为他们增殖并从1 和第 2 次咽弓迁移，成为头部和心脏肌肉组织，分别为（ 图1A，A&#39;和A&#39;&#39;）进行跟踪。培养咽弓提供学习心脏和肌肉的发育，详细体外的独特方式。解剖和附件咽弓后，从拱门连接迁移细胞可以在24-48小时的培养中观察到。内的培养肌管形成3天可以从1次咽弓和从第二</...

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.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

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 次观看.  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.

视频: 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.

成像清除胚胎和出生后心脏在单细胞分辨率

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

科研

发育生物学

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.

心外膜细胞小鼠胚胎心脏的体外培养

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

工程学

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.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

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.

基于微注射的免疫胚胎心肌细胞体内植入系统

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.

小鼠心脏场特定心脏祖细胞的体外生成

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.

使用全载免疫作用化学和三维重建对咽弓动脉的可视化与分析

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.

产生来自多能干细胞的自组装人心脏类器官

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 ...

生物化学

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.

禽流感胚胎瓣膜祖细胞的分离和培养

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>用于研究雏鸡胚胎中人干细胞球体行为的无标记异种移植模型

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.

用于研究雏鸡胚胎中人干细胞球体行为的无标记异种移植模型

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

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

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此视频中的章节

成像清除胚胎和出生后心脏在单细胞分辨率

心外膜细胞小鼠胚胎心脏的体外培养

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

基于微注射的免疫胚胎心肌细胞体内植入系统

小鼠心脏场特定心脏祖细胞的体外生成

使用全载免疫作用化学和三维重建对咽弓动脉的可视化与分析

产生来自多能干细胞的自组装人心脏类器官

禽流感胚胎瓣膜祖细胞的分离和培养

用于研究雏鸡胚胎中人干细胞球体行为的无标记异种移植模型

用于研究雏鸡胚胎中人干细胞球体行为的无标记异种移植模型