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).

Tutti i topi sono stati mantenuti a una Associazione americana per l&#39;accreditamento di laboratorio Animal Care (AAALAC) -accredited stabulario dell&#39;Università Johns Hopkins e alloggiati in conformità con le procedure descritte nella Guida per la cura e l&#39;uso di animali da laboratorio. La cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) ha approvato tutti i protocolli sperimentali. Preparazione 1. sperimentale <ol><li> Coat 12-pozzetti con 10% siero fetale bovino (FBS) in tampone fosfato Solution (PBS) per 60 min. </li><li> Rimuovere la soluzione di rivestimento e aggiungere 200 ml di media siero-free (SFM). Twirl piastra per assicurarsi che un film di supporto copre l&#39;intera superficie. </li></ol> 2. Procedure chirurgiche <ol><li> Euthanize topi in gravidanza che utilizzano il protocollo comitato approvato la cura degli animali dell&#39;istituzione seguita da dislocazione cervicale per garantire la completa eutanasia. </li><li> Spray e pulire la regione pancia del mouse con il 70%etanolo. Mantenere rigorose tecniche sterili per evitare la contaminazione. </li><li> Utilizzare pinze e forbici per rendere l&#39;0,5 cm 2 incisione alla regione ombelicale. </li><li> Usare le dita per pizzicare / afferrare la pelle sopra e sotto l&#39;incisione e tirare delicatamente la pelle in direzioni opposte (testa e coda). </li><li> Utilizzare pinze e forbici per fare un&#39;incisione iniziale nella membrana in corrispondenza della zona dell&#39;ombelico. Tagliare delicatamente la membrana a forma di v dall&#39;ombelico alle ovaie su ciascun lato (2 x 1,5 cm 2), rivelando così l&#39;utero. </li><li> Utilizzare pinze per pizzicare la ovidotto che collega l&#39;utero per l&#39;ovaio e forbici per pizzicare il ovidotto sul lato ovaio, liberando così l&#39;utero dall&#39;ovaio. </li><li> Tirare delicatamente l&#39;utero dal ovidotto con le pinze e tagliare l&#39;utero libera dalla regione della vescica con le forbici. </li><li> Estrarre l&#39;utero ulteriormente dalla oviduct con la pinza, e tagliare il oviduct con le forbici sull&#39;altro lato, liberando così l&#39;utero dal mouse. </li><li> Trasferire l&#39;utero di un tubo da 50 ml e aggiungere 20 ml di freddo PBS sterile con Ca 2 + e Mg 2+. PBS deve contenere Ca 2+ e Mg 2+ durante embrione dissezione. </li><li> Agitare delicatamente il tubo per 10 secondi per lavare l&#39;utero di sangue. </li><li> Trasferire l&#39;utero dal tubo con una pinza e posizionarlo in un piatto da 10 ml e aggiungere 5 ml di PBS freddo sulla parte superiore dell&#39;utero. </li><li> Porre la capsula con l&#39;utero allo stereomicroscopio. Nota: I passaggi 2,13-2,19 richiede uno stereomicroscopio. </li><li> Utilizzare due coppie di pinze per aprire delicatamente l&#39;utero e sezionare fuori cavità amniotiche con embrioni dall&#39;utero uno per uno. <ol><li> Utilizzare due coppie di pinze per aprire con attenzione il sacco amniotico che circonda l&#39;embrione, pizzicare la sacca con una pinza e delicatamente rimuoverlo dal embrione, utilizzare le altre pinze per tagliare e rilasciare il sacco amniotico dall&#39;embrione. Se un genotipo specifico necessario, tenere sacco amniotico per la genotipizzazione. </li></ol></li><li> Posizionare l&#39;embrione sul lato e utilizzare pinze per tagliare il 1 ° e 2 ° arco posteriormente al cuore tra l&#39;arco e la sacca faringea (Figura 1A &#39;&#39;). </li><li> Capovolgere delicatamente l&#39;embrione verso l&#39;altro lato e utilizzare pinze per tagliare il 1 ° e 2 ° arco posteriormente al cuore tra l&#39;arco e la sacca faringea. </li><li> Utilizzare pinze per tagliare il tratto di efflusso cardiaco che ancora collega il 1 ° e 2 ° faringei archi per l&#39;embrione. Rimuovere gli archi che sono ancora attaccati insieme nella forma di una farfalla (Figura 1B), dall&#39;embrione. </li><li> Utilizzare pinze per stringere e rilasciare il 1 ° archi faringei dal rimanente tratto di efflusso cardiaco e foregut endoderma (Figura 1B &#39;&#39; indicata con la linea tratteggiata e *). </li><li> Utilizzare pinze per pizzicare e rilasciare gli archi 2 ° faringei dal rimanente tratto di efflusso cardiacoe foregut endoderma (Figura 1B &#39;&#39; indicata con la linea tratteggiata e **). </li><li> Trasferire ogni coppia di archi separatamente utilizzando una pinza e inserire entrambe le arcate nel bel mezzo di un designato bene. Piastra 1 ° e 2 ° faringei archi in pozzetti separati. Assicurarsi che gli archi sono in contatto con la pellicola di supporto aggiunti nel passaggio 1.2. È fondamentale che gli archi non sono coperti da medium, in quanto hanno bisogno di entrare in contatto con la superficie del pozzetto per attacco durante questo periodo. </li></ol> 3. Incubazione e Imaging <ol><li> Incubare archi placcati in 12-pozzetti in 37 ° C / 5% di CO 2 per 2 ore per archi di allegare alla superficie bene. </li><li> Aggiungere delicatamente 200 ml di 37 ° C SFM lungo il lato del pozzo. Assicurarsi che gli archi rimangono attaccati e incubare O / N. </li><li> Il giorno dopo, controllare visivamente che archi faringei sono attaccati e aggiungere delicatamente 200 ml di 37 ° C SFM lungo il lato dellabene. Se archi restano distaccato e variabile, rimuovere delicatamente i media con una pipetta senza rimuovere archi fino a che solo un film di supporto è a sinistra. Utilizzare puntale per posizionare archi al centro del pozzo e ripetere il punto 3.1. </li><li> Monitorare quotidianamente; aggiungere ~ 100-200 ml di mezzi ogni 2 giorni per sostituire qualsiasi supporto evaporato. Nota: 24-48 ore dopo la migrazione delle cellule appaiono intorno l&#39;arco e si proliferare e migrare da arco per i prossimi 5-8 giorni. Dopo 36-72 ore la formazione di cardiomiociti battendo può essere osservato dal 2 faringeo arco (Figura 2B). Formazione myotube può osservare dal 1 ° arco faringeo 3-7 giorni dopo il collegamento (Figura 2A). </li></ol>

<ol>
	<li>Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+heart+field.">The second heart field.</a> Curr Top Dev Biol. 100, 33-65 (2012).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Tzahor, E., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+mesoderm+development+during+embryogenesis:+implications+for+both+heart+and+head+myogenesis.">Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis.</a> Cardiovasc Res. 91, 196-202 (2011).</li><li>Uosaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Contact+with+Endoderm-like+Cells+Efficiently+Induces+Cardiac+Progenitors+from+Mouse+and+Human+Pluripotent+Stem+Cells.">Direct Contact with Endoderm-like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells.</a> PLoS One. 7 (10), e46413 (2012).</li><li>Cheng, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+mesendodermal+cell+fate+decisions.">Fibronectin mediates mesendodermal cell fate decisions.</a> Development. 140, 2587-2596 (2013).</li><li>Kwon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+pathway+involving+Notch1/beta-catenin/Isl1+determines+cardiac+progenitor+cell+fate.">A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.</a> Nature cell biology. 11, 951-957 (2009).</li><li>Shenje, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precardiac+deletion+of+Numb+and+Numblike+reveals+renewal+of+cardiac+progenitors.">Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.</a> Elife. 3, e02164 (2014).</li><li>Grevellec, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharyngeal+pouches+and+clefts:+Development,+evolution,+structure+and+derivatives.">The pharyngeal pouches and clefts: Development, evolution, structure and derivatives.</a> Semin Cell Dev Biol. 21, 325-332 (2010).</li><li>Kwon, C., Cordes, K. R., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+acts+at+multiple+developmental+stages+to+promote+mammalian+cardiogenesis.">Wnt/beta-catenin signaling acts at multiple developmental stages to promote mammalian cardiogenesis.</a> Cell Cycle. 7, 3815-3818 (2008).</li><li>Cho, G. S., Fernandez, L., Kwon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+heart:+perspectives+on+stem-cell+therapy.">Regenerative medicine for the heart: perspectives on stem-cell therapy.</a> Antioxid Redox Signal. 21, 2018-2031 (2014).</li></ol>

The authors have nothing to disclose.

In questo video, dimostriamo come isolare e cultura prima e seconda faringei archi di 9,5 giorni antichi embrioni di topo. Archi branchiali sono transitori, rigonfiamenti che appaiono sul lato craniolateral di embrioni in via di sviluppo 9, che contengono blocchi cardiaco progenitrici delle cellule di costruzione multi-potenti per rendere il cuore durante l&#39;embriogenesi 10,11 -in secondo arco e progenitori testa muscolari prime arcate 8 segmentati . La di...

Cellule mesoderma faringei danno origine a parti del cuore e dei muscoli faringei. Durante lo sviluppo embrionale, le cellule progenitrici multipotenti cardiache dal secondo campo cuore migrano dal mesoderma faringeo e popolano il tratto di efflusso cardiaco e ventricolo destro, e il loro sviluppo anormale è strettamente associata con la malattia-cuore congenita principale causa di difetti alla nascita e nascita-difetto decessi correlati nell&#39;uomo 1-3. Recenti studi hanno dimostrato che faringea mesoderma contribuisce a testa muscoli, oltre al cuore, rendendo il mesoderma una parte critica della cardio-craniofacciale sviluppo 4. Così, i processi di sviluppo, tra cui l&#39;induzione, la proliferazione e la differenziazione di testa e del cuore progenitori del mesoderma faringea sono sotto inchiesta attivi 5-7. Fino a poco tempo fa, è rimasto sconosciuto se le cellule progenitrici cardiache subiscono espansione without differenziazione, in parte a causa della mancanza di informazioni sul loro ambiente cellulare. Il nostro recente studio suggerisce che archi branchiali fungono da microambiente per il rinnovo dei progenitori cardiaci e muscolari e possono essere coltivate ex vivo per diverse settimane 8. Questo metodo espianto offre un&#39;opportunità nuova e unica per studiare lo sviluppo dei progenitori cardio-cranio-facciale ex vivo.

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			<td>FBS</td>
			<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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		<tr>
			<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></td>
			<td></td>
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			<td colspan="2">Serum Free Media:</td>
			<td></td>
			<td></td>
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			<td>IMDM</td>
			<td>Cellgro</td>
			<td>15-016-CV</td>
			<td></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>
			<td></td>
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			<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

Il mesoderma faringeo di embrioni in via di sviluppo contribuisce ad ampie regioni della testa e la muscolatura cardiaca. Abbiamo sviluppato un nuovo metodo per studiare la testa e il cuore lo sviluppo delle cellule progenitrici con archi faringei (noto anche come archi branchiali) ex vivo. Utilizzando questo metodo, abbiamo recentemente descritto che il secondo arco faringeo contiene progenitori cardiaci auto-rinnovamento e serve come un microambiente per l&#39;espansione dei progenitori durante lo sviluppo del cuore del mouse. Le cellule progenitrici rimangono indifferenziate ed espansivo dentro l&#39;arco, ma rapidamente diventano cardiomiociti funzionali mentre migrano fuori dell&#39;arco. Abbiamo anche segnalato che il primo arco faringeo contiene progenitori muscolari che danno luogo a miotubi dopo aver lasciato l&#39;arco. Qui, dimostriamo la procedura per la dissezione e ex vivo cultura del primo e del secondo archi faringei da embrioni di topo in via di sviluppo. Il metodo permette di studiare la testa e il cuore progenitore / dev muscolareelopment, compresi cardiomiociti e myotube formazione in dettaglio ex vivo.

Durante lo sviluppo, muscolo e cuore progenitori facciali possono essere rintracciati in quanto proliferano e migrano dal 1 ° e 2 ° arco faringeo, per diventare capo e la muscolatura del cuore, rispettivamente (Figura 1A, A &#39;e A&#39; &#39;). Coltura archi faringei offre un modo unico per lo studio del cuore e sviluppo muscolare in dettaglio ex vivo. Dopo la dissezione e l&#39;attaccamento di archi faringei, cellule che migrano da archi collegati poss...

Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

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

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Ex Cultura Vivo del faringea Archi allo Studio cuore e muscoli Progenitori e la loro nicchia

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

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Research

JoVE Journal

Developmental Biology

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

7.7K 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

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.

Utilizzando<em&gt; Ex Vivo</em&gt; Upright Droplet Culture di interi organi fetali per studiare i processi evolutivi durante mouse Organogenesi

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

Ricerca

Biologia dello sviluppo

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 Cultura Vivo del Chick cerebellare fette e spazialmente mirata elettroporazione di granuli precursori delle cellule

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.

Epicardico escrescenza Cultura Assay e<em&gt; Ex Vivo</em&gt; La valutazione della migrazione delle cellule di derivazione epicardico

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.

Identificazione di progenitori eritromieloidi e loro progenie nell&#39;embrione del mouse dalla citometria del flusso

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.

Imaging in vivo del transgenico espressione genica nei singoli retinica Progenitori in embrioni di zebrafish Chimerico da Cell studio Influenze non autonomo

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.

Isolamento, la cultura e la differenziazione delle cellule stromali del midollo osseo e dei progenitori degli osteoclasti dai topi

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.

Applicazione di stimolazione cronica per lo studio del ratto indotta da attività contrattile del muscolo scheletrico fenotipica adattamenti

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 Formazione immagine del muscolo-tendine morfogenesi in Drosophila pupe

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.

Generazione del primo cuore campo-come progenitori cardiaci e ventricolare-come cardiomiociti da cellule staminali pluripotenti umane

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.

Perfusione ex vivo della placenta del roditore

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 ...