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).

Alle Mäuse wurden an einer amerikanischen Vereinigung für die Akkreditierung von Laboratory Animal Care (AAALAC) gehalten -accredited Tierhaltung an der Johns Hopkins University und in Übereinstimmung mit den im Leitfaden für die Pflege und Verwendung von Labortieren beschriebenen Verfahren untergebracht. Das Institutional Animal Care und Verwenden Committee (IACUC) stimmten allen Versuchsprotokolle. 1. Versuchsvorbereitung <ol><li> Mantel 12-Well-Platte mit 10% fötalem Rinderserum (FBS) in Phosphat-gepufferter Lösung (PBS) für 60 min. </li><li> Entfernen Beschichtungslösung und mit 200 &amp; mgr; l serumfreien Medien (SFM). Wirbeln Platte, um sicherzustellen, dass ein Film von Medien deckt die gesamte Fläche. </li></ol> 2. Chirurgische Verfahren <ol><li> Euthanize schwangeren Mäusen mit Tierpflegeausschuss genehmigten Protokoll der Institution durch Genickbruch folgte, um eine vollständige Euthanasie zu gewährleisten. </li><li> Spray und reinigen Sie die Bauchregion des Maus mit 70%Ethanol. Strikte sterile Techniken, um Verunreinigungen zu vermeiden. </li><li> Verwenden einer Pinzette und Schere, ein 0,5 cm 2 Schnitt am Nabel Region zu machen. </li><li> Benutzen Sie die Finger zu klemmen / greifen die Haut über und unter dem Einschnitt und ziehen Sie die Haut in entgegengesetzte Richtungen (Kopf und Schwanz). </li><li> Verwenden einer Pinzette und Schere, um eine anfängliche Einschnitt in der Membran an der Nabelregion zu machen. Schneiden Sie vorsichtig die Membran in V-Form vom Nabel bis der Eierstöcke auf jeder Seite (2 x 1,5 cm 2), wodurch die Gebärmutter enthüllt. </li><li> Verwenden einer Pinzette, um die Eileiter Anschluss der Gebärmutter auf die Eierstöcke und Scheren kneifen, um die Eileiter auf dem Eierstock Seite kneifen, so befreien die Gebärmutter aus dem Eierstock. </li><li> Ziehen Sie vorsichtig die Gebärmutter durch die Eileiter mit der Pinzette und schneiden die Gebärmutter kostenlos von der Blase Region mit einer Schere. </li><li> Ziehen die Gebärmutter weiter durch den Eileiter mit der Zange, und schneiden den Eileiter mit einer Schere an der anderen Seite, wodurch der Uterus Freigabe von der mOuse. </li><li> Übertragen Sie die Gebärmutter auf eine 50-ml-Röhrchen und fügen Sie 20 ml kaltem sterilem PBS mit Ca 2+ und Mg 2+. PBS muss Ca 2+ und Mg 2+ bei Embryo Dissektion enthalten. </li><li> Schütteln Sie das Röhrchen für 10 Sekunden, um die Gebärmutter für die Blutwäsche. </li><li> Übertragen Sie die Gebärmutter aus dem Rohr mit einer Pinzette und legen Sie sie in einem 10 ml Schale und mit 5 ml kaltem PBS auf der Oberseite der Gebärmutter. </li><li> Die Schale mit der Gebärmutter unter einem Stereomikroskop. Hinweis: Die Schritte 2,13-2,19 erfordert ein Stereomikroskop. </li><li> Verwenden Sie zwei Paar Pinzette vorsichtig öffnen Sie die Gebärmutter und sezieren Fruchtblasen mit Embryonen aus der Gebärmutter einer nach dem anderen. <ol><li> Verwenden Sie zwei Paar Pinzette vorsichtig öffnen Sie die Fruchtblase, die den Embryo umgibt, kneifen den Sack mit einer Pinzette und entfernen Sie sie aus dem Embryo, verwenden Sie die andere Zange zu schneiden und lassen Sie die Fruchtblase aus dem Embryo. Wenn ein bestimmter Genotyp erforderlich, halten Fruchtblase für die Genotypisierung. </li></ol></li><li> Setzen Sie den Embryo auf der Seite und Verwenden einer Pinzette, um die 1 und 2. nach hinten wölben zum Herzen zwischen dem Bogen und dem Schlundtasche (1A &#39;&#39;) geschnitten. </li><li> Flip sanft den Embryo auf die andere Seite und verwenden Sie Zange um den 1. und 2. Bogen nach hinten, um das Herz zwischen dem Bogen und dem Schlundtasche abgeschnitten. </li><li> Verwenden einer Pinzette, um die Herzausflusstrakt, die immer noch den Anschluss wird die 1. und 2. Schlundbögen für den Embryo zu schneiden. Entfernen der Bögen, die sich noch in der Form eines Schmetterlings (1B) miteinander verbunden sind, aus dem Embryo. </li><li> Verwenden einer Pinzette zu kneifen und lassen Sie die 1. Schlundbögen von der übrigen Herzausflusstrakt und Vorderdarm Endoderm (1B &#39;&#39; mit gestrichelten Linie angedeutet und *). </li><li> Verwenden einer Pinzette zu kneifen und lassen Sie die 2. Schlundbögen von der übrigen Herzausflusstraktund Vorderdarm Endoderm (1B &#39;&#39; mit gestrichelten Linie und ** angegeben). </li><li> Übertragen jedes Paar von Bögen einzeln mit einer Pinzette und legen Sie beide Bögen in der Mitte des ein gut bezeichnet. Platte 1 und 2. Schlundbögen in getrennten Vertiefungen. Sicherzustellen, daß die Bögen in Kontakt mit dem Film von Medien in Schritt 1.2 gegeben. Entscheidend ist, dass Bögen NICHT von Medium bedeckt, wie sie benötigen, um den Kontakt mit der Oberfläche der Bohrung zur Befestigung in dieser Periode zu machen. </li></ol> 3. Inkubation und Imaging <ol><li> Bogen in der Platte mit 12 Vertiefungen plattiert Inkubieren in 37 ° C / 5% CO 2 für 2 Stunden für Bögen zum Brunnen Oberfläche zu befestigen. </li><li> Vorsichtig mit 200 &amp; mgr; l von 37 ° C SFM an der Seite des Bohrlochs. Stellen Sie sicher, dass Bögen befestigt bleiben und Inkubation O / N. </li><li> Am nächsten Tag, visuell zu überprüfen, dass Schlundbögen befestigt und sanft mit 200 &amp; mgr; l von 37 ° C SFM an der Seite dergut. Wenn Bögen bleiben ungebunden und schwimm, entfernen Medien mit einer Pipette, ohne Bögen, bis nur noch ein Film von Medien überlassen. Verwenden Pipettenspitze auf Bögen in der Mitte der gut und wiederholen Sie Schritt 3.1 zu platzieren. </li><li> Überwachen Sie täglich; fügen ~ 100-200 ul Medium jeden 2. Tag jeder verdampft Medien zu ersetzen. Hinweis: 24-48 h nach der wandernden Zellen erscheinen rund um den Bogen und sich vermehren und wandern von Bogen in den nächsten 5-8 Tage. Nach 36-72 h Bildung schlagen Kardiomyozyten können aus der 2. Schlundbogen (2B) beobachtet werden. Myotube Bildung können vom 1. Schlundbogen 3-7 Tage nach der Befestigung (2A) zu beachten. </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 diesem Video zeigen wir, wie man isolieren und Kultur ersten und zweiten Schlundbögen von 9,5 Tage alten Mausembryos. Schlundbögen sind vergänglich, segmentiert Ausbuchtungen, die auf der Seite des kraniolateral entwickelnden Embryonen 9, die multi-potenten Herzvorläuferzellen-Bausteine ​​enthalten, um das Herz während der Embryogenese 10,11 -in zweiten Bögen und Kopfmuskelvorläuferzellen in der ersten Bögen 8 erscheinen . Dissektion der Schlun...

Rachen Mesodermzellen Anlass zu Teilen des Herzens und der Rachenmuskeln. Während der embryonalen Entwicklung, multiHerzVorläuferZellen aus dem zweiten Herzfeld wandern aus dem Rachen Mesoderm und bevölkern die Herzausflusstrakt und rechten Herzkammer und ihre abnorme Entwicklung ist eng mit angeborenem Herzfehler-die häufigste Ursache von Geburtsschäden und Geburtsdefekt zugeordnet bedingten Todesfälle beim Menschen 1-3. Jüngste Studien haben gezeigt, dass die Rachen-Mesoderm trägt zur Muskulatur Kopf, zusätzlich zu Herzen, was die Mesoderm ein kritischer Teil der Herz-Kreislauf-kraniofazialen Entwicklung 4. So sind die Entwicklungsprozesse einschließlich Induktion, Proliferation und Differenzierung von Kopf und Herz-Vorläufern in der Rachen Mesoderm aktiv untersucht 5-7. Bis vor kurzem blieb es unbekannt, ob Herzvorläuferzellen Expansion witho unterziehenut Differenzierung, was teilweise auf das Fehlen von Informationen über ihre zellulären Umgebung. Unsere aktuelle Studie legt nahe, dass Schlundbögen dienen als Mikroumgebung für die Erneuerung der Herz- und Muskelvorläuferzellen und kann über mehrere Wochen 8 ex vivo kultiviert werden. Das Explantat Verfahren bietet eine neue und einzigartige Möglichkeit, die Entwicklung von Herz-Kreislauf-kraniofaziale Vorläuferzellen ex vivo zu studieren.

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			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS + Ca2 + Mg2</td>
			<td>Corning</td>
			<td>21-030-CV</td>
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		<tr>
			<td>10cm petri plates</td>
			<td>Fishersci</td>
			<td>FB0875712</td>
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			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
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			<td colspan="2">Dissection forceps</td>
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			<td></td>
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			<td></td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td colspan="2">Serum Free Media:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Cellgro</td>
			<td>15-016-CV</td>
			<td></td>
		</tr>
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			<td>Ham&#8217;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) &#224; (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>
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ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche

Die Rachen Mesoderm der sich entwickelnden Embryonen trägt zur breiten Regionen von Kopf und Herzmuskulatur. Wir haben ein neues Verfahren, um Kopf und Herz-Vorläuferzellentwicklung mit Schlundbögen (auch als Kiemenbögen bekannt) ex vivo zu studieren entwickelt. Mit dieser Methode haben wir kürzlich beschrieben, dass die zweite Schlundbogen enthält selbst erneuernden Herzvorläuferzellen und dient als Mikroumgebung für die Expansion der Vorläuferzellen der Maus während der Herzentwicklung. Die Vorläuferzellen bleiben undifferenziert und expansive innerhalb des Bogens, aber schnell funktionelle Kardiomyozyten, als sie aus dem Bogen zu migrieren. Wir haben auch berichtet, dass erste Schlundbogen enthält Muskelvorläuferzellen, die zu Myotuben nach dem Verlassen des arch. Hier zeigen wir, das Verfahren für die Präparation und ex vivo Kultur der ersten und zweiten Schlundbögen aus Entwicklungs Maus-Embryonen. Das Verfahren ermöglicht es, Kopf und Herz-Vorläufer / Muskel dev studierenelopment, einschließlich Kardiomyozyten und Myotuben Bildung im Detail ex vivo.

Während der Entwicklung können Gesichtsmuskel und Herzvorläuferzellen zurückgeführt werden, wie sie sich vermehren und wandern aus dem 1. und 2. Schlundbogen, um Kopf und Herzmuskulatur, bzw. (1A, A &#39;und A&#39; &#39;) zu werden. Kultivieren Schlundbögen bietet eine einzigartige Möglichkeit, um Herz und Muskelentwicklung im Detail ex vivo zu studieren. Nach Präparation und Befestigung der Schlundbögen können von wandernden Zellen angebracht Bögen innerh...

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-Kultur von Schlundbögen auf Herz und Muskelvorläuferzellen und ihre Nische Studieren

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

7.7K Aufrufe.  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.

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

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Verwendung<em&gt; Ex Vivo</em&gt; Upright Droplet Kulturen von Whole Fetal Orgeln zu Entwicklungsprozessen während der Maus Organogenese Studieren

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Forschung

Entwicklungsbiologie

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex-vivo-Kultur von Chick Kleinhirn-Slices und räumlich gezielte Elektroporation von Körnerzellvorstufen

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epikardiale Outgrowth Kulturtest und<em&gt; Ex Vivo</em&gt; Beurteilung von Epikardiale abgeleiteten Zellmigration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Identifizierung von Erythromyeloid Progenitoren und deren Nachkommen in der Maus Embryo Durch Durchflusszytometrie

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In - vivo - Bildgebung von Expression Transgene Gene in einzelnen Retinal Vorläufern in Chimäre Zebrabärblingembryonen Studie Zellnichtautonome Einflüsse

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolation, Kultur und Differenzierung von Stromazellen Knochenmarkzellen und Osteoklasten-Vorläufer von Mäusen

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Anwendung der chronische Stimulation kontraktilen Aktivität induzierten Ratte Skelettmuskulatur phänotypische Anpassungen zu studieren

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

In Vivo Bildgebung der Muskel-Sehnen-Morphogenese in Drosophila Puppen

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

Generation der ersten Herz Bereich-wie kardiale Stammväter und ventrikuläre wie Herzzellen aus menschlicher pluripotenter Stammzellen

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

Ex Vivo Perfusion der Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...