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 los ratones fueron mantenidos a una Asociación Americana para la Acreditación de Cuidado de Animales de Laboratorio (AAALAC) acreditado por animalario de la Universidad Johns Hopkins y alojados de conformidad con los procedimientos establecidos en la Guía para el Cuidado y Uso de Animales de Laboratorio. El Cuidado y Uso de Animales Comité Institucional (IACUC) aprobó todos los protocolos experimentales. 1. Preparación Experimental <ol><li> Escudo placa de 12 pocillos con 10% de suero bovino fetal (FBS) en solución tamponada de fosfato (PBS) durante 60 min. </li><li> Retire solución de revestimiento y añadir 200 l de los medios de comunicación libre de suero (SFM). Girar la placa para asegurarse de que una película de los medios cubre toda la superficie. </li></ol> 2. Procedimientos Quirúrgicos <ol><li> La eutanasia a los ratones embarazadas que utilizan cuidado de los animales protocolo comité aprobados de la institución seguido por dislocación cervical para asegurar la eutanasia completa. </li><li> Rocíe y limpie la región del vientre del ratón con el 70%etanol. Mantener estrictas técnicas estériles para evitar la contaminación. </li><li> Use pinzas y tijeras para hacer un 0,5 cm2 incisión en la región del ombligo. </li><li> Utilice los dedos para pellizcar / agarrar la piel por encima y por debajo de la incisión y tire suavemente la piel en direcciones opuestas (cabeza y cola). </li><li> Utilice pinzas y tijeras para hacer una incisión inicial en la membrana en la región del ombligo. Cortar con cuidado la membrana en forma de V desde el ombligo hasta los ovarios en cada lado (2 x 1,5 cm 2), revelando así el útero. </li><li> El uso de fórceps para pellizcar el oviducto que conecta el útero con el ovario y tijeras para pellizcar el oviducto en el lado del ovario, liberando así el útero desde el ovario. </li><li> Tire suavemente hasta el útero por el oviducto con las pinzas y cortar el útero libre de la región de la vejiga con unas tijeras. </li><li> Tire del útero más arriba por el oviducto con las pinzas, y cortar el oviducto con unas tijeras en el otro lado, liberando de este modo el útero de la mOuse. </li><li> Transferir el útero a un tubo de 50 ml y añadir 20 ml de PBS frío estéril con Ca2 + y Mg2 +. PBS debe contener Ca 2+ y Mg 2+ durante la disección de embriones. </li><li> Agite suavemente el tubo durante 10 segundos para lavar el útero para la sangre. </li><li> Transferir el útero desde el tubo con pinzas y colocarlo en una placa de 10 ml y añadir 5 ml de PBS frío en la parte superior del útero. </li><li> Coloque el plato con el útero bajo un microscopio estereoscópico. Nota: Los pasos 02/13 a 02/19 requiere un microscopio estereoscópico. </li><li> Use dos pares de pinzas para abrir suavemente el útero y diseccionar sacos amnióticos con embriones desde el útero uno por uno. <ol><li> Use dos pares de pinzas para abrir cuidadosamente el saco amniótico que rodea al embrión, pellizcar el saco con uno fórceps y suavemente sacarlo de la embrión, utilizar las otras pinzas para cortar y liberar el saco amniótico a partir del embrión. Si un genotipo específico necesario, mantenga saco amniótico para el genotipado. </li></ol></li><li> Coloque el embrión en el lado y utilizar pinzas para cortar el 1 ° y 2 ° arco posterior al corazón entre el arco y la bolsa faríngea (Figura 1 A &#39;&#39;). </li><li> Voltear suavemente el embrión para el otro lado y utilizar pinzas para cortar la primera y segunda arco posterior al corazón entre el arco y la bolsa faríngea. </li><li> Utilice unas pinzas para cortar el tracto de salida cardíaco que todavía se está conectando el 1 y 2 de la faringe arcos al embrión. Retire los arcos, que todavía están unidos juntos en la forma de una mariposa (Figura 1B), a partir del embrión. </li><li> Utilice pinzas para pellizcar y suelte los arcos 1 st faríngeas del restante del tracto de salida cardíaco y endodermo del intestino anterior (Figura 1B &#39;&#39; se indica con línea discontinua y *). </li><li> El uso de fórceps para pellizcar y suelte los arcos faríngeos 2º del resto del tracto de salida cardíacoy endodermo del intestino anterior (Figura 1B &#39;&#39; se indica con línea discontinua y **). </li><li> Transferir cada par de arcos por separado utilizando fórceps y colocar ambas arcadas en el medio de un así designado. Plate 1 y 2 de la faringe arcos en pocillos separados. Asegúrese de que los arcos están en contacto con la película de los medios agregados en el paso 1.2. Es crucial que los arcos no están cubiertos por medio, ya que necesitan para hacer contacto con la superficie del pozo para la fijación durante este período. </li></ol> 3. Incubación e Imagen <ol><li> Incubar arcos plateados en la placa de 12 pocillos en 37 ° C / 5% de CO 2 durante 2 horas para arcos para fijar a la superficie también. </li><li> Añadir suavemente 200 l de 37 ° C SFM por el lado del pozo. Asegúrese de que los arcos permanecen unidos e incubar O / N. </li><li> Al día siguiente, comprobar visualmente que arcos faríngeos se unen y agregan suavidad 200 l de 37 ° C SFM por el lado de labien. Si arcos permanecen sin ataduras y flotante, retire con cuidado los medios de comunicación con una pipeta sin remover arcos hasta que sólo queda una película de los medios de comunicación. Utilice punta de la pipeta para colocar arcos en el medio de la etapa de bien y de repetición 3.1. </li><li> Monitorear diariamente; añadir ~ 100-200 l de los medios de comunicación cada segundo día para reemplazar cualquier medio evaporó. Nota: 24-48 h después de la migración de las células aparecen alrededor del arco y proliferar y migrar de arco en los próximos 5-8 días. Después de 36 a 72 horas de formación de los cardiomiocitos que baten se puede observar desde el 2º arco faríngeo (Figura 2B). Formación myotube se puede observar a partir de los 1 st faríngea arco 3-7 días después de la unión (Figura 2). </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.

En este vídeo, se demuestra cómo aislar y cultura primero y segundo arcos faríngeos de 9,5 días de edad embriones de ratón. Arcos faríngeos se transitoria, segmentados abultamientos que aparecen en el lado craneolateral de embriones en desarrollo 9, que contienen bloques cardiaca de creación de células progenitoras múltiples potentes para hacer que el corazón durante la embriogénesis 10,11 -en segundo arcos y progenitores musculares cabeza en primera arcos 8 . <p class="jo...

Las células del mesodermo faríngeos dan lugar a partes del corazón y los músculos faríngeos. Durante el desarrollo embrionario, las células progenitoras multipotentes cardiacos desde el segundo campo corazón migran desde el mesodermo faríngea y pueblan el tracto de salida del ventrículo derecho y cardiaco, y su desarrollo anormal está estrechamente asociada con la enfermedad-el corazón congénita causa de defectos de nacimiento y-defecto de nacimiento muertes relacionadas en seres humanos 1-3. Estudios recientes han demostrado que mesodermo faríngea contribuye a la cabeza músculos, además del corazón, haciendo que el mesodermo una parte crítica del desarrollo cardio-4 craneofacial. Por lo tanto, los procesos de desarrollo incluyendo la inducción, proliferación, y diferenciación de progenitores de la cabeza y del corazón en el mesodermo faríngea están bajo investigación activa 5-7. Hasta hace poco, seguía siendo desconocida si las células progenitoras cardíacas experimentan witho expansióndiferenciación ut, debido en parte a la falta de información sobre su entorno celular. Nuestro estudio reciente sugiere que los arcos faríngeos sirven como un microambiente para la renovación de los progenitores cardíacos y musculares y pueden ser cultivadas ex vivo durante varias semanas 8. Este método de explante ofrece una novedosa y única oportunidad de estudiar el desarrollo de progenitores cardio-craneofacial 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

El mesodermo faríngea de los embriones en desarrollo contribuye a amplias regiones de la cabeza y la musculatura del corazón. Hemos desarrollado un nuevo método para estudiar la cabeza y progenitor corazón desarrollo de las células con arcos faríngeos (también conocido como arcos branquiales) ex vivo. Usando este método, hemos descrito recientemente que el segundo arco faríngeo contiene progenitores corazón auto-renovación y sirve como un microambiente para la expansión de los progenitores durante el desarrollo del corazón de ratón. Las células progenitoras permanecen indiferenciadas y expansivo en el interior del arco, pero rápidamente se convierten en cardiomiocitos funcionales a medida que migran fuera del arco. También informó que el primer arco faríngeo contiene progenitores musculares que dan lugar a miotubos después de dejar el arco. Aquí, se demuestra el procedimiento para la disección y la cultura ex vivo de primer y segundo arcos faríngeos de desarrollo de embriones de ratón. El método permite a uno para estudiar la cabeza y el corazón progenitor / dev muscularelopment, incluyendo cardiomiocitos y myotube formación en detalle ex vivo.

Durante el desarrollo, los progenitores del músculo y del corazón faciales se pueden remontar a medida que proliferan y migran desde el 1 ° y 2 ° arco faríngeo, para convertirse en la cabeza y la musculatura del corazón, respectivamente (Figura 1 A, A &#39;y A&#39; &#39;). El cultivo de arcos faríngeos ofrece una forma única para estudiar el corazón y el músculo de desarrollo en detalle ex vivo. Después de la disección y la unión de los arcos faríngeos,...

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 Vivo Cultura de faríngea Arcos para el Estudio de corazón y músculo Progenitores y su 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 ...

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 vistas.  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.

Video: 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.

Borrado de imágenes embrionario y postnatal Corazones en la Resolución de celda única

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

Investigación

Biología del Desarrollo

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.

Cultivo in vitro de células a partir de epicárdicos del corazón de embriones de ratón

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

Ingeniería

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.

Análisis de la inmunofluorescencia de Whole-mount cuantitativa de las poblaciones progenitoras cardiacas en embriones de ratón

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 basado en la microinyección en Vivo implantación de cardiomiocitos embrionarios en el embrión aviar

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 Generación de Células Progenitoras Cardíacas específicas de Mouse Field

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.

Visualización y análisis de arterias de arco faríngeo utilizando inmunohistoquímica de montaje completo y reconstrucción 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.

Generación de organoides del corazón humano autoensamblables derivados de células madre 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.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

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

Biología

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>Un modelo de xenoinjerto sin etiquetas para investigar el comportamiento de los esferoides de células madre humanas en embriones de pollo

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.

Un modelo de xenoinjerto sin etiquetas para investigar el comportamiento de los esferoides de células madre humanas en embriones de pollo

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

Ex Vivo Cultura de faríngea Arcos para el Estudio de corazón y músculo Progenitores y su nicho

Resumen

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Un modelo de xenoinjerto sin etiquetas para investigar el comportamiento de los esferoides de células madre humanas en embriones de pollo

Un modelo de xenoinjerto sin etiquetas para investigar el comportamiento de los esferoides de células madre humanas en embriones de pollo