Name: ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche
Uploaded: 2015-07-20T15:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

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).

All mice were maintained at an American Association for the Accreditation of Laboratory Animal Care (AAALAC)&#8211;accredited animal facility at the Johns Hopkins University and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) approved all experimental protocols.
1. Experimental Preparation
<ol>
	<li>Coat 12-well plate with 10% Fetal Bovine Serum (FBS) in Phosphate Buffered Solution (PBS) for 60 min.</li>
	<li>Remove coating solution and add 200 &#181;l of Serum-free Media (SFM). Twirl plate to make sure that a film of media covers the entire surface area.</li>
</ol>
2. Surgical Procedures
<ol>
	<li>Euthanize pregnant mice using the institution&#39;s animal care committee-approved protocol followed by cervical dislocation to ensure complete euthanasia.</li>
	<li>Spray and clean the belly region of the mouse with 70% ethanol. Maintain strict sterile techniques to avoid contamination.</li>
	<li>Use forceps and scissors to make a 0.5 cm2 incision at the navel region.</li>
	<li>Use fingers to pinch/grab the skin above and below the incision and gently pull the skin in opposite directions (head and tail).</li>
	<li>Use forceps and scissors to make an initial incision in the membrane at the navel region. Gently cut the membrane in V-shape from the navel to the ovaries on each side (2 x 1.5 cm2), thereby revealing the uterus.</li>
	<li>Use forceps to pinch the oviduct connecting the uterus to the ovary and scissors to pinch the oviduct on the ovary side, thus freeing the uterus from the ovary.</li>
	<li>Gently pull up the uterus by the oviduct with the forceps and cut the uterus free from the bladder region with scissors.</li>
	<li>Pull the uterus further up by the oviduct with the forceps, and cut the oviduct with scissors on the other side, thereby releasing the uterus from the mouse.</li>
	<li>Transfer the uterus to a 50 ml tube and add 20 ml of cold sterile PBS with Ca2+ and Mg2+. PBS must contain Ca2+ and Mg2+ during embryo dissection.</li>
	<li>Gently shake the tube for 10 sec to wash the uterus for blood.</li>
	<li>Transfer the uterus from the tube with forceps and place it in a 10 ml&#160;dish and add 5 ml&#160;of cold PBS on top of the uterus.</li>
	<li>Place the dish with the uterus under a stereomicroscope. 
	Note: Steps 2.13-2.19 requires a stereomicroscope.</li>
	<li>Use two pairs of forceps to gently open the uterus and dissect out amniotic sacs with embryos from the uterus one by one.
	<ol>
		<li>Use two pairs of forceps to carefully open the amniotic sac that surrounds the embryo, pinch the sac with one forceps and gently remove it from the embryo, use the other forceps to cut and release the amniotic sac from the embryo. If a specific genotype needed, keep amniotic sac for genotyping.</li>
	</ol>
	</li>
	<li>Place the embryo on the side and use forceps to cut the 1st and 2nd arch posteriorly to the heart between the arch and the pharyngeal pouch (Figure 1A&#8217;&#8217;).</li>
	<li>Gently flip the embryo to the other side and use forceps to cut the 1st and 2nd arch posteriorly to the heart between the arch and the pharyngeal pouch.</li>
	<li>Use forceps to cut the cardiac outflow tract that is still connecting the 1st and 2nd pharyngeal arches to the embryo. Remove the arches, which are still attached together in the shape of a butterfly (Figure 1B), from the embryo.</li>
	<li>Use forceps to pinch and release the 1st pharyngeal arches from the remaining cardiac outflow tract and foregut endoderm (Figure 1B&#8217;&#8217; indicated with dashed line and *).</li>
	<li>Use forceps to pinch and release the 2nd pharyngeal arches from the remaining cardiac outflow tract and foregut endoderm (Figure 1B&#8217;&#8217; indicated with dashed line and **).</li>
	<li>Transfer each pair of arches separately using forceps and place both arches in the middle of a designated well. Plate 1st and 2nd pharyngeal arches in separate wells. Ensure that the arches are in contact with the film of media added in step 1.2. It is crucial that arches are NOT covered by medium, as they need to make contact with the surface of the well for attachment during this period.</li>
</ol>
3. Incubation and Imaging
<ol>
	<li>Incubate arches plated in the 12-well plate in 37 &#176;C/5% CO2 for 2 hr for arches to attach to the well surface area.</li>
	<li>Gently add 200 &#181;l of 37 &#176;C SFM down the side of the well. Make sure that arches remain attached and incubate O/N.</li>
	<li>Next day, visually check that pharyngeal arches are attached and gently add 200 &#181;l of 37 &#176;C SFM down the side of the well. If arches remain unattached and floating, gently remove media with a pipette without removing arches until only a film of media is left. Use pipette tip to place arches in the middle of the well and repeat step 3.1.</li>
	<li>Monitor daily; add ~100-200 &#181;l of media every 2nd day to replace any evaporated media. 
	Note: 24-48 hr after migrating cells appear around the arch and will proliferate and migrate from arch over the next 5-8 days. After 36-72 hr formation of beating cardiomyocytes can be observed from the 2nd pharyngeal arch (Figure 2B). Myotube formation can be observed from the 1st pharyngeal arch 3-7 days after attachment (Figure 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.

In this video, we demonstrate how to isolate and culture first and second pharyngeal arches of 9.5 days old mouse embryos. Pharyngeal arches are transient, segmented bulges that appear on the craniolateral side of developing embryos 9, which contain multi-potent cardiac progenitor cells&#8212;building blocks to make the heart during embryogenesis 10,11&#8212;in second arches and head muscle progenitors in first arches 8.
Dissection of pharyngeal arches requires advanced mouse embryo dissection skills. Once arches have been successfully dissected using this stepwise protocol and transferred to pre-coated wells, it is critical that arches attach to the bottom area. In this protocol, we coat wells with 10% FBS followed by culture in SFM resulting in attachment and growth of &#62;75% of arches, however other coating materials can be used (matrigel, gelatin, fibronectin etc.) as well as serum containing media.
The pharyngeal arches serves as primordia for a multitude of structures during development. After initial formation of the pharyngeal arches (8 days post fertilization in mice), CPCs expand in the PA2 and migrate into the cardiac outflow tract where they differentiate into cardiac cells, thereby serving as a renewable source of CPCs that supplies cells needed to sustain heart growth. As the pharyngeal arches are temporal developmental structures (E8-11.5) and migration of CPCs into the outflow tract occurs from approximately E8-E10) this brings certain limitations to the technique. For CPC studies, we recommend using pharyngeal arches dissected between E9-10, at this stage each PA2 contain approximately 5-800 RFP+ cells (Mesp1 linage trace).
With fluorescent labeling, this ex vivo culture allows us to monitor developing progenitors and their differentiation into beating cardiomyocytes or myotubes in real-time. In addition, this ex vivo culture system can be used to study roles of cell-autonomous and microenvironmental factors affecting cardiac/muscle progenitor cell proliferation, migration, and differentiation using existing Cre/loxP lines. As such, this system is expected to facilitate cardiac/muscle progenitor&#8211;niche studies, which may lead to a better understanding of heart/muscle formation and disease.

Pharyngeal mesoderm cells give rise to parts of the heart and the pharyngeal muscles. During embryonic development, multipotent cardiac progenitor cells from the second heart field migrate from the pharyngeal mesoderm and populate the cardiac outflow tract and right ventricle, and their abnormal development is closely associated with congenital heart disease&#8212;the leading cause of birth defects and birth defect-related deaths in humans 1-3. Recent studies have demonstrated that pharyngeal mesoderm contributes to head muscles, in addition to the heart, making the mesoderm a critical part of the cardio-craniofacial development 4. Thus, the developmental processes including induction, proliferation, and differentiation of head and heart progenitors in the pharyngeal mesoderm are under active investigation 5-7.
Until recently, it remained unknown whether cardiac progenitor cells undergo expansion without differentiation, partly due to the lack of information on their cellular environment. Our recent study suggests that pharyngeal arches serve as a microenvironment for the renewal of cardiac and muscle progenitors and can be cultured ex vivo over several weeks 8. This explant method offers a novel and unique opportunity to study the development of cardio-craniofacial progenitors 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

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

During development, facial muscle and heart progenitors can be traced as they proliferate and migrate from the 1st and 2nd pharyngeal arch, to become head and heart musculature, respectively (Figure 1A, A&#8217; and A&#8217;&#8217;). Culturing pharyngeal arches offers a unique way to study heart and muscle development in detail ex vivo. After dissection and attachment of pharyngeal arches, migrating cells from attached arches can be observed within 24-48 hr of culture. Within 3 days of culture myotube formation can be observed from 1st pharyngeal arches and cardiomyocyte formation from the 2nd pharyngeal arches (Figure 2A and 2B). Cardiomyocyte formation can be visually confirmed by spontaneously contracting clusters of migrating cells and/or analysis of specific cardiomyocyte markers (e.g., Cardiac Troponin T). Myotube/facial muscle formation can be visually observed by long elongated (up to 500 &#181;m in length) spontaneously twitching cells and/or analysis of specific muscle markers (e.g., Myogenin). By using the cre-lox system in mice, mesoderm progeny can be traced by fluorescent reporters upon cre-expression during ex vivo culture (Figure 2A&#8217; and 2B&#8217;). Likewise, with this method it is possible to study the fate of progeny wherein a specific gene of interest is conditionally knocked out or overexpressed, which otherwise would result in early embryonic lethality as we previously described 8.
<img alt="Figure 1" src="/files/ftp_upload/52876/52876fig1.jpg" /> 
Figure 1: Mouse embryo (Mesp1cre, Ai9) 9.5 days post fertilization (E.9.5). (A) Left side of embryo. (PA: Pharyngeal Arch, OT: Outflow tract, LV: Left ventricle. (A&#8217;) Mesp1 linage-trace; RFP marks Mesp1+ cells and their progeny. Arrow indicates RFP+ Mesp1-progeny in PA2 that is continuous with the OT. (A&#8217;&#8217;) Dashed line indicate where to cut PA1 and PA2 posterior to the heart. (B) Butterfly-like right and left PA1 and PA2 after dissection from the embryo. FE: Foregut endoderm. (B&#39;) RFP marks Mesp1 progeny in PA1 and PA2. (B&#8217;&#8217;) * and ** along with the dashed lines indicates where to cut and release PA1 and PA2 from the OT and FE. Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52876/52876fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52876/52876fig2.jpg" /> 
Figure 2: Cultured pharyngeal arches. (A) Cultured PA1 (3 days of culture). (A&#8217;) RFP marks Mesp1 progeny in PA1. Arrows and * indicate early myotube formation. (A&#8217;&#8217;) Overlay of RFP expression in PA1. (B) Cultured PA2 (8 days of culture). (B&#8217;) RFP marks Mesp1 progeny in PA2. Arrows and ** indicate beating area of cardiomyocytes (modified from Shenje et al. 8). (B&#8217;&#8217;) Overlay of RFP expression in PA2. Scale bar: 250 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52876/52876fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.8K Views.  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.

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

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.

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

Engineering

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.

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.

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.

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.

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.

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 ...

Biochemistry

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.

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

Biology

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.

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.

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

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

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

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