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).

Toutes les souris ont été maintenus à une association américaine pour l&#39;accréditation de laboratoire Animal Care (AAALAC) accrédité par l&#39;animalerie de l&#39;Université Johns Hopkins et logés conformément aux procédures décrites dans le Guide pour le soin et l&#39;utilisation des animaux de laboratoire. Le Comité de protection et d&#39;utilisation des animaux institutionnel (IACUC) a approuvé l&#39;ensemble des protocoles expérimentaux. 1. Préparation expérimentale <ol><li> Manteau de plaque à 12 puits avec 10% de sérum bovin fœtal (FBS) dans la solution tamponnée au phosphate (PBS) pendant 60 min. </li><li> Enlever la solution de revêtement et ajouter 200 ul de médias sans sérum (SFM). Twirl plaque pour veiller à ce que un film des médias couvre la totalité de la surface. </li></ol> 2. Interventions chirurgicales <ol><li> Euthanasier les souris enceintes utilisant le protocole approuvé par le Comité de protection des animaux de l&#39;institution suivie par dislocation cervicale pour assurer l&#39;euthanasie complète. </li><li> Pulvériser et nettoyer la région du ventre de la souris avec 70%l&#39;éthanol. Maintenir techniques stériles strictes pour éviter la contamination. </li><li> Utiliser des pinces et des ciseaux pour faire une incision de 0,5 cm 2 à la région du nombril. </li><li> Utilisez les doigts pour pincer / saisir la peau au-dessus et en dessous de l&#39;incision et tirez doucement la peau dans des directions opposées (de tête et de queue). </li><li> Utiliser des pinces et des ciseaux pour faire une incision initiale dans la membrane de la région du nombril. Couper délicatement la membrane en forme de V entre le nombril et les ovaires de chaque côté (2 x 1,5 cm 2), révélant ainsi l&#39;utérus. </li><li> Utilisez une pince pour pincer l&#39;oviducte reliant l&#39;utérus à l&#39;ovaire et des ciseaux pour pincer l&#39;oviducte sur le côté de l&#39;ovaire, libérant ainsi l&#39;utérus de l&#39;ovaire. </li><li> Tirez doucement l&#39;utérus par l&#39;oviducte avec la pince et couper l&#39;utérus libre de la région de la vessie avec des ciseaux. </li><li> Tirer l&#39;utérus en outre par l&#39;oviducte avec la pince, et couper avec des ciseaux l&#39;oviducte de l&#39;autre côté, en libérant ainsi l&#39;utérus de la mouse. </li><li> Transfert de l&#39;utérus à un tube de 50 ml et ajouter 20 ml de PBS froid stérile avec du Ca 2+ et Mg 2+. PBS doit contenir Ca 2+ et Mg 2+ au cours de la dissection embryon. </li><li> Secouer doucement le tube pendant 10 sec pour laver l&#39;utérus de sang. </li><li> Transfert de l&#39;utérus à partir du tube avec une pince et placez-le dans un plat de 10 ml et ajouter 5 ml de PBS froid sur le dessus de l&#39;utérus. </li><li> Placez le plat avec l&#39;utérus sous un stéréomicroscope. Remarque: Les étapes 2.13 à 2.19 nécessite une loupe binoculaire. </li><li> Utiliser deux paires de pinces pour ouvrir doucement l&#39;utérus et disséquer sacs amniotiques embryons avec de l&#39;utérus, un par un. <ol><li> Utilisez deux paires de pinces à ouvrir avec précaution le sac amniotique qui entoure l&#39;embryon, pincer le sac avec une pince et doucement retirer de l&#39;embryon, utiliser les autres pince à couper et à libérer le sac amniotique de l&#39;embryon. Si un génotype spécifique nécessaire, garder sac amniotique pour le génotypage. </li></ol></li><li> Placez l&#39;embryon sur le côté et utiliser une pince pour couper le 1 er et 2 ème arc postérieur au cœur entre la voûte et la poche pharyngée (figure 1A &#39;&#39;). </li><li> Retourner doucement à l&#39;embryon de l&#39;autre côté et utiliser une pince pour couper le 1er et le 2ème arc postérieur au cœur entre la voûte et la poche pharyngée. </li><li> Utilisez une pince pour couper la voie d&#39;éjection cardiaque qui est encore connecte le 1 er et 2 ème pharyngés arches à l&#39;embryon. Retirez les arches, qui sont encore attachés ensemble sous la forme d&#39;un papillon (figure 1B), à partir de l&#39;embryon. </li><li> Utilisez une pince pour pincer et libérer les 1 er pharyngés arches du reste cardiaque voies de sortie et l&#39;endoderme intestin antérieur (figure 1B &#39;&#39; indiqué par une ligne pointillée et *). </li><li> Utilisez une pince pour pincer et de libérer les arches 2 ème pharyngés de la sortie cardiaque restant voieset endoderme intestin antérieur (figure 1B &#39;&#39; indiqué par une ligne pointillée et **). </li><li> Transférer chaque paire d&#39;arcs séparément à l&#39;aide des pinces et placer les deux arches en plein milieu d&#39;un bien désigné. Planche 1 er et 2 ème pharyngés arcs dans des réservoirs séparés. Assurez-vous que les arches sont en contact avec le film de support a été ajoutée à l&#39;étape 1.2. Il est crucial que les arcs ne sont PAS couverts par le milieu, car ils ont besoin de prendre contact avec la surface du puits pour la fixation au cours de cette période. </li></ol> 3. Incubation et imagerie <ol><li> Incuber arcs métallisés dans la plaque à 12 puits à 37 ° C / 5% de CO2 pendant 2 h pour les arcs se fixer à la surface du puits. </li><li> Ajouter délicatement 200 pi de 37 ° C GDF sur le côté du puits. Assurez-vous que les arcs restent attachés et incuber O / N. </li><li> Le lendemain, vérifier visuellement que arcs pharyngés sont attachés et ajouter doucement 200 pi de 37 ° C GDF sur le côté de labien. Si arches restent seules et flottante, retirez délicatement les médias avec une pipette sans enlever arcs jusqu&#39;à ce que seul un film des médias est à gauche. Utilisez pointe de la pipette pour placer des arcs dans le milieu du bien et répétez l&#39;étape 3.1. </li><li> Daily Monitor; ajouter ~ 100-200 pi des médias tous les 2 jours pour remplacer tout support évaporé. Remarque: 24-48 heures après la migration des cellules apparaissent autour de l&#39;arc et proliférer et migrer d&#39;arc au cours des 5-8 prochains jours. Après la formation de 36-72 heures de cardiomyocytes battant peut être observée à partir de la 2 ème pharyngée arc (figure 2B). Formation Myotubes peut être observée à partir des 1 er pharyngée arc 3-7 jours après la fixation (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.

Dans cette vidéo, nous démontrons comment isoler et de la culture premier et deuxième arcs pharyngés de 9,5 jours anciens embryons de souris. Arcs pharyngés sont transitoires, renflements qui apparaissent sur ​​le côté craniolateral d&#39;embryons en développement 9, qui contiennent plusieurs puissants blocs-de construction des cellules progénitrices cardiaques à faire le cœur pendant l&#39;embryogenèse 10,11 -en deuxième arcs et des progéniteurs tête musculaires dans 8</sup...

cellules du mésoderme pharyngiens donnent lieu à des parties du coeur et les muscles pharyngés. Au cours du développement embryonnaire, multipotentes cellules progénitrices cardiaques de la deuxième champ cardiaque migrent du mésoderme pharyngé et peuplent la voie d&#39;éjection cardiaque et le ventricule droit, et de leur développement anormal est étroitement associée à la maladie du coeur congénitale principale cause de malformations congénitales et la naissance defect- décès liés à l&#39;homme 1-3. Des études récentes ont démontré que le mésoderme pharyngé contribue à la tête de muscles, en plus du coeur, ce qui rend le mésoderme un élément essentiel du développement de la cardio-craniofaciale 4. Ainsi, les processus de développement, y compris l&#39;induction, la prolifération et la différenciation des progéniteurs cardiaques tête et dans le mésoderme pharyngé sont sous enquête active 5-7. Jusqu&#39;à récemment, il est resté inconnu si les cellules progénitrices cardiaques subissent l&#39;expansion withodifférenciation ut, en partie en raison de l&#39;absence d&#39;informations sur leur environnement cellulaire. Notre étude récente suggère que les arcs pharyngiens servent un micro pour le renouvellement de progéniteurs cardiaques et musculaires et peuvent être cultivées ex vivo sur plusieurs semaines 8. Cette méthode des explants offre une occasion nouvelle et unique d&#39;étudier le développement des progéniteurs cardio-craniofaciale ex vivo.

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			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
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			<td>PBS + Ca2 + Mg2</td>
			<td>Corning</td>
			<td>21-030-CV</td>
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			<td>10cm petri plates</td>
			<td>Fishersci</td>
			<td>FB0875712</td>
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			<td>Scissor</td>
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			<td colspan="2">Dissection forceps</td>
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			<td></td>
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			<td colspan="2">Serum Free Media:</td>
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			<td>IMDM</td>
			<td>Cellgro</td>
			<td>15-016-CV</td>
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			<td>Ham&#8217;s F12</td>
			<td>Cellgro</td>
			<td>10-080-CV</td>
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			<td>N2-SUPPLEMENT</td>
			<td>Gibco</td>
			<td>17502-048</td>
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			<td>B27-retinoic acid</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
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			<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>
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			<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

Le mésoderme pharyngé d&#39;embryons en développement contribue à de larges régions de la tête et le cœur musculature. Nous avons développé une nouvelle méthode pour étudier tête et le cœur le développement des cellules progénitrices avec des arcs pharyngés (aussi connu comme arcs branchiaux) ex vivo. En utilisant cette méthode, nous avons récemment décrit que le deuxième arc pharyngien contient progéniteurs cardiaques auto-renouvellement et sert un micro pour l&#39;expansion des progéniteurs pendant le développement du cœur de souris. Les cellules progénitrices restent indifférencié et expansive intérieur de l&#39;arc, mais deviennent rapidement cardiomyocytes fonctionnels comme ils migrent hors de l&#39;arche. Nous avons également signalé que le premier arc pharyngien contient progéniteurs musculaires donnant lieu à des myotubes après avoir quitté l&#39;arc. Ici, nous démontrons la procédure pour la dissection et ex vivo culture de premier et deuxième arcs pharyngés de développement des embryons de souris. La méthode permet d&#39;étudier une tête et le cœur géniteur / dev musculaireeloppement, y compris les cardiomyocytes et myotube formation en détail ex vivo.

Au cours du développement, musculaires et cardiaques progéniteurs du visage peuvent être tracées comme ils prolifèrent et migrent de la 1 ère et 2 ème pharyngée arc, à devenir la tête et le cœur musculature, respectivement (figure 1A, A &#39;et A&#39; &#39;). Cultivant arcs pharyngés offre un moyen unique d&#39;étudier le développement du cœur et des muscles en détail ex vivo. Après dissection et la fixation des arcs pharyngés, migration ...

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.

Culture ex vivo des pharyngée Arches pour étudier cardiaques et musculaires progéniteurs et leur 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

Research

JoVE Journal

Developmental Biology

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

7.7K vues.  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.

Vidéo: 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.

Utilisation<em&gt; Ex Vivo</em&gt; Cultures Upright gouttelettes de toute fœtus organes pour étudier les processus de développement au cours de la souris Organogenèse

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

Recherche

Biologie du développement

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.

Culture ex vivo de Chick cervelet tranches et spatialement ciblées électroporation de granules précurseurs de cellules

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.

Épicardique Excroissance Culture Assay et<em&gt; Ex Vivo</em&gt; Évaluation des épicardique dérivés Migration cellulaire

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.

Identification des progéniteurs érythromyélidiens et de leur descendance dans l&#39;embryon de souris par cytométrie de flux

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.

Imagerie in vivo de Transgenic Gene Expression en Retinal individuelle progéniteurs dans chimériques Zebrafish Embryons à étudier cellulaires Influences non autonomes

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.

Isolement, Culture et différenciation des cellules stromales de la moelle osseuse et les ostéoclastes progéniteurs de souris

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.

Application d’une Stimulation chronique pour étudier les Rat d’induite par l’activité Contractile du Muscle squelettique phénotypique Adaptations

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 Imagerie de la morphogénèse du Muscle-tendon en pupes de drosophile

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.

Génération du premier cœur comme champ progéniteurs cardiaques et Cardiomyocytes ventriculaires-comme des cellules souches pluripotentes humaines

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.

Perfusion ex vivo du placenta de rongeurs

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 ...