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).

Todos os ratos foram mantidos a uma associação americana para o Credenciamento de Laboratório Animal Care (AAALAC) -accredited biotério da Universidade Johns Hopkins e alojados em conformidade com os procedimentos descritos no Guia para o Cuidado e Uso de Animais de Laboratório. O Animal Care e Use Comitê Institucional (IACUC) aprovou todos os protocolos experimentais. 1. Experimental Preparação <ol><li> Revestimento de 12 poços com 10% de Soro Fetal Bovino (FBS) em solução tamponada de fosfato (PBS) durante 60 min. </li><li> Remover solução de revestimento e adicionar 200 mL de meio de soro-free (SFM). Girar em placa para se certificar de que um filme de meios abrange toda a área de superfície. </li></ol> 2. Procedimentos Cirúrgicos <ol><li> Euthanize camundongos grávidas usando o protocolo aprovado pelo comitê de cuidados com os animais da instituição seguido por deslocamento cervical para garantir a eutanásia completa. </li><li> Spray e limpar região da barriga do mouse com 70%etanol. Manter rigorosas técnicas estéreis para evitar a contaminação. </li><li> Use uma pinça e tesouras para fazer um 0,5 centímetros 2 incisão na região do umbigo. </li><li> Use os dedos para apertar / agarrar a pele acima e abaixo da incisão e puxe a pele em sentidos opostos (cabeça e cauda). </li><li> Use fórceps e tesouras para fazer uma incisão inicial na membrana na região do umbigo. Suavemente cortar a membrana em forma de V a partir do umbigo até os ovários em ambos os lados (2 x 1,5 cm2), revelando assim o útero. </li><li> Use uma pinça para beliscar o oviduto de ligar o útero ao ovário e uma tesoura para beliscar a tuba uterina do lado ovário, libertando assim o útero do ovário. </li><li> Puxe cuidadosamente o útero pelo oviduto com a pinça e cortar o útero livre da região de bexiga com uma tesoura. </li><li> Puxar o útero mais acima pelo oviduto com as pinças, e cortar o oviduto com tesouras por outro lado, libertando assim o útero do mouse. </li><li> Transferir o útero para um tubo de 50 ml e adicionar 20 ml de PBS estéril frio com Ca 2+ e Mg 2+. PBS deve conter Ca 2+ e Mg 2+ durante a dissecação de embriões. </li><li> Agite suavemente o tubo para 10 segundos para lavar o útero de sangue. </li><li> Transferir o útero do tubo com uma pinça e colocá-lo em um prato de 10 ml e adicionam-se 5 ml de PBS frio na parte superior do útero. </li><li> Coloque o prato com o útero sob microscópio estereoscópico. Nota: Os passos 2,13-2,19 requer um microscópio estereoscópico. </li><li> Usar dois pares de pinças para abrir suavemente o útero e dissecar sacos amniótico com embriões a partir do útero, um por um. <ol><li> Usar dois pares de pinças para abrir cuidadosamente o saco amniótico que envolve o embrião, comprimir o saco com uma pinça e removê-lo suavemente a partir do embrião, utilizar as outras pinças de cortar e libertar o saco amniótico do embrião. Se um genótipo específico necessário, manter saco amniótico para genotipagem. </li></ol></li><li> Coloque o embrião do lado e utilize uma pinça para cortar o 1 º e 2 º arco posteriormente ao coração entre o arco ea bolsa faríngea (Figura 1A &#39;&#39;). </li><li> Gentilmente virar o embrião para o outro lado e utilize uma pinça para cortar o 1º e 2º arco posteriormente ao coração entre o arco ea bolsa faríngea. </li><li> Use uma pinça para cortar a via de saída cardíaca que ainda está se conectando o 1º e 2º da faringe arcos para o embrião. Remover os arcos, os quais ainda estão ligados em conjunto na forma de uma borboleta (Figura 1B), a partir do embrião. </li><li> Use uma pinça para apertar e soltar os arcos um st de faringe da endoderme via de saída cardíaca e foregut remanescente (Figura 1B &#39;&#39; indicado com linha tracejada e *). </li><li> Use uma pinça para apertar e soltar os arcos 2º da faringe do restante da via de saída cardíacae foregut endoderme (Figura 1B &#39;&#39; indicado com linha tracejada e **). </li><li> Transferir cada par de arcos separadamente utilizando uma pinça e colocar ambos os arcos no meio de um assim designado. Placa 1 º e 2 º da faringe arcos em poços separados. Certifique-se de que os arcos estão em contacto com a película de meios adicionados no passo 1.2. É crucial que os arcos não são cobertos por meio de, como eles precisam fazer contato com a superfície do poço para fixação durante este período. </li></ol> 3. Incubação e Imagem <ol><li> Incubar arcos plaqueadas na placa de 12 poços em 37 ° C / 5% de CO 2 durante 2 horas para arcos para anexar a área de superfície do poço. </li><li> Adicionar cuidadosamente 200 ul de 37 ° C SFM para baixo do lado do poço. Certifique-se de que arcos permanecer solidários e incubar O / N. </li><li> No dia seguinte, verificar visualmente que arcos faríngeos estão ligados e adicione delicadamente 200 mL de 37 ° C SFM para o lado dabem. Se arcos permanecer solto e flutuante, remover suavemente com uma pipeta de mídia sem remover arcos até que apenas um filme de mídia for deixado. Use ponta da pipeta para colocar os arcos no meio do poço e repetir o passo 3.1. </li><li> Monitorar diariamente; adicionar ~ 100-200 mL de mídia a cada segundo dia para substituir qualquer evaporado mídia. Nota: 24-48 h após a migração de células aparecem ao redor do arco e vão proliferar e migrar do arco ao longo dos próximos 5-8 dias. Depois 36-72 h formação de cardiomiócitos batendo pode ser observado a partir do 2º faríngea arco (Figura 2B). Formação miotubo pode ser observado a partir de 1º arco faríngeo 3-7 dias depois de anexo (Figura 2A). </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.

Neste vídeo, demonstramos como isolar e cultura primeira e segunda faringe arcos de 9,5 dias antigos embriões de camundongos. Arcos faríngeos são transitórios, segmentado protuberâncias que aparecem no lado craniolateral de embriões em desenvolvimento 9, que contêm multi-potentes blocos cardíaca progenitoras de células-construção para fazer o coração durante a embriogênese 10,11 -em segundo arcos e progenitores musculares cabeça na primeira arcos 8 . <p class="jove_co...

Mesoderme células da faringe dar origem a partes do coração e os músculos da faringe. Durante o desenvolvimento embrionário, as células progenitoras cardíacas multipotentes do segundo campo coração migrar a partir da mesoderme faríngea e preencher a via de saída do ventrículo direito cardíaco e, e seu desenvolvimento anormal está intimamente associada com doença cardíaca congênita-a principal causa de defeitos de nascimento e defect- nascimento mortes relacionadas nos seres humanos 1-3. Estudos recentes têm demonstrado que faríngea mesoderme contribui para dirigir músculos, para além do coração, tornando a mesoderme uma parte crítica do desenvolvimento cardio-craniofacial 4. Assim, os processos de desenvolvimento, incluindo a indução, proliferação e diferenciação de progenitores de cabeça e do coração na mesoderme da faringe estão sob investigação activa 5-7. Até recentemente, ele permaneceu desconhecido se as células progenitoras cardíacas submetidos a witho expansãout diferenciação, em parte devido à falta de informação sobre o seu ambiente celular. Nosso estudo recente sugere que arcos faríngeos servir como um microambiente para a renovação de progenitores cardíacos e musculares e podem ser cultivadas ex vivo ao longo de várias semanas 8. Este método de explante oferece uma oportunidade nova e única para estudar o desenvolvimento de células progenitoras cardio-craniofacial ex vivo.

<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

A mesoderme da faringe dos embriões em desenvolvimento contribui para amplas regiões da cabeça e musculatura cardíaca. Temos desenvolvido um novo método para estudar cabeça e coração progenitor desenvolvimento de células com arcos faríngeos (também conhecido como arcos branquiais) ex vivo. Usando este método, que descrevemos recentemente que o segundo arco faríngeo contém auto-renovação progenitores cardíacos e serve como um microambiente para a expansão das células progenitoras durante o desenvolvimento do coração de rato. As células progenitoras permanecem indiferenciadas e expansivo dentro do arco, mas rapidamente se tornou cardiomiócitos funcionais à medida que migram para fora do arco. Nós também informou que primeiro arco faríngeo contém progenitores musculares que dão origem a myotubes depois de deixar o arco. Aqui, nós demonstramos o procedimento de dissecção e ex vivo cultura de primeiro e segundo arcos faríngeos de desenvolvimento de embriões de camundongos. O método permite o estudo a cabeça eo coração progenitor / dev muscularolvimento, incluindo cardiomiócitos e miotubo formação em detalhe ex vivo.

Durante o desenvolvimento, músculo e coração progenitores faciais podem ser rastreados como elas proliferam e migram do 1 º e 2 º da faringe arco, para se tornar cabeça e musculatura cardíaca, respectivamente (Figura 1A, A &#39;e A&#39; &#39;). Cultura arcos faríngeos oferece uma maneira única de estudar coração e músculo desenvolvimento em detalhe ex vivo. Após a dissecção e fixação dos arcos faríngeos, migrando células de arcos anexado...

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.

Ex Cultura Vivo de Pharyngeal Arcos para estudo do coração e do músculo Progenitores e seu nicho

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

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JoVE Journal

Developmental Biology

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

7.8K visualizações.  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.

Vídeo: 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.

Imagiologia Cleared embrionário e pós-natal Corações na resolução de célula única

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

Biologia do Desenvolvimento

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.

Cultura in vitro de células epicárdicas De Coração embrionárias de camundongos

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

Engenharia

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.

Análise quantitativa da imunofluorescência toda a montagem das populações do Progenitor cardíaca em embriões de rato

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.

Sistema baseado em microinjeção para na implantação de Vivo do Cardiomyocytes embrionárias no embrião aviária

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.

In vitro geração de células progenitoras cardíacas específicas do coração do rato

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.

Visualização e Análise de Artérias ariníngeos usando Imunohistoquímica de montagem integral e Reconstrução 3D

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.

Gerando organoides cardíacos humanos auto-montados derivados de células-tronco pluripotentes

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 ...

Bioquímica

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.

Isolamento e Cultura da Avian embrionárias Células Progenitoras Valvular

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

Biologia

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>Um modelo de xenoenxerto sem rótulo para investigar o comportamento de esferoides de células-tronco humanas em embriões de galinhas

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.

Um modelo de xenoenxerto sem rótulo para investigar o comportamento de esferoides de células-tronco humanas em embriões de galinhas

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

Ex Cultura Vivo de Pharyngeal Arcos para estudo do coração e do músculo Progenitores e seu nicho

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Isolamento e Cultura da Avian embrionárias Células Progenitoras Valvular

Um modelo de xenoenxerto sem rótulo para investigar o comportamento de esferoides de células-tronco humanas em embriões de galinhas

Um modelo de xenoenxerto sem rótulo para investigar o comportamento de esferoides de células-tronco humanas em embriões de galinhas