We thank Kwon laboratory members for helpful discussions. This work was supported by grants from NHLBI/NIH (R01HL111198) and Maryland Stem Cell Research Fund (MSCRF).

Todos os ratos foram mantidos a uma associação americana para o Credenciamento de Laboratório Animal Care (AAALAC) -accredited biotério da Universidade Johns Hopkins e alojados em conformidade com os procedimentos descritos no Guia para o Cuidado e Uso de Animais de Laboratório. O Animal Care e Use Comitê Institucional (IACUC) aprovou todos os protocolos experimentais. 1. Experimental Preparação <ol><li> Revestimento de 12 poços com 10% de Soro Fetal Bovino (FBS) em solução tamponada de fosfato (PBS) durante 60 min. </li><li> Remover solução de revestimento e adicionar 200 mL de meio de soro-free (SFM). Girar em placa para se certificar de que um filme de meios abrange toda a área de superfície. </li></ol> 2. Procedimentos Cirúrgicos <ol><li> Euthanize camundongos grávidas usando o protocolo aprovado pelo comitê de cuidados com os animais da instituição seguido por deslocamento cervical para garantir a eutanásia completa. </li><li> Spray e limpar região da barriga do mouse com 70%etanol. Manter rigorosas técnicas estéreis para evitar a contaminação. </li><li> Use uma pinça e tesouras para fazer um 0,5 centímetros 2 incisão na região do umbigo. </li><li> Use os dedos para apertar / agarrar a pele acima e abaixo da incisão e puxe a pele em sentidos opostos (cabeça e cauda). </li><li> Use fórceps e tesouras para fazer uma incisão inicial na membrana na região do umbigo. Suavemente cortar a membrana em forma de V a partir do umbigo até os ovários em ambos os lados (2 x 1,5 cm2), revelando assim o útero. </li><li> Use uma pinça para beliscar o oviduto de ligar o útero ao ovário e uma tesoura para beliscar a tuba uterina do lado ovário, libertando assim o útero do ovário. </li><li> Puxe cuidadosamente o útero pelo oviduto com a pinça e cortar o útero livre da região de bexiga com uma tesoura. </li><li> Puxar o útero mais acima pelo oviduto com as pinças, e cortar o oviduto com tesouras por outro lado, libertando assim o útero do mouse. </li><li> Transferir o útero para um tubo de 50 ml e adicionar 20 ml de PBS estéril frio com Ca 2+ e Mg 2+. PBS deve conter Ca 2+ e Mg 2+ durante a dissecação de embriões. </li><li> Agite suavemente o tubo para 10 segundos para lavar o útero de sangue. </li><li> Transferir o útero do tubo com uma pinça e colocá-lo em um prato de 10 ml e adicionam-se 5 ml de PBS frio na parte superior do útero. </li><li> Coloque o prato com o útero sob microscópio estereoscópico. Nota: Os passos 2,13-2,19 requer um microscópio estereoscópico. </li><li> Usar dois pares de pinças para abrir suavemente o útero e dissecar sacos amniótico com embriões a partir do útero, um por um. <ol><li> Usar dois pares de pinças para abrir cuidadosamente o saco amniótico que envolve o embrião, comprimir o saco com uma pinça e removê-lo suavemente a partir do embrião, utilizar as outras pinças de cortar e libertar o saco amniótico do embrião. Se um genótipo específico necessário, manter saco amniótico para genotipagem. </li></ol></li><li> Coloque o embrião do lado e utilize uma pinça para cortar o 1 º e 2 º arco posteriormente ao coração entre o arco ea bolsa faríngea (Figura 1A &#39;&#39;). </li><li> Gentilmente virar o embrião para o outro lado e utilize uma pinça para cortar o 1º e 2º arco posteriormente ao coração entre o arco ea bolsa faríngea. </li><li> Use uma pinça para cortar a via de saída cardíaca que ainda está se conectando o 1º e 2º da faringe arcos para o embrião. Remover os arcos, os quais ainda estão ligados em conjunto na forma de uma borboleta (Figura 1B), a partir do embrião. </li><li> Use uma pinça para apertar e soltar os arcos um st de faringe da endoderme via de saída cardíaca e foregut remanescente (Figura 1B &#39;&#39; indicado com linha tracejada e *). </li><li> Use uma pinça para apertar e soltar os arcos 2º da faringe do restante da via de saída cardíacae foregut endoderme (Figura 1B &#39;&#39; indicado com linha tracejada e **). </li><li> Transferir cada par de arcos separadamente utilizando uma pinça e colocar ambos os arcos no meio de um assim designado. Placa 1 º e 2 º da faringe arcos em poços separados. Certifique-se de que os arcos estão em contacto com a película de meios adicionados no passo 1.2. É crucial que os arcos não são cobertos por meio de, como eles precisam fazer contato com a superfície do poço para fixação durante este período. </li></ol> 3. Incubação e Imagem <ol><li> Incubar arcos plaqueadas na placa de 12 poços em 37 ° C / 5% de CO 2 durante 2 horas para arcos para anexar a área de superfície do poço. </li><li> Adicionar cuidadosamente 200 ul de 37 ° C SFM para baixo do lado do poço. Certifique-se de que arcos permanecer solidários e incubar O / N. </li><li> No dia seguinte, verificar visualmente que arcos faríngeos estão ligados e adicione delicadamente 200 mL de 37 ° C SFM para o lado dabem. Se arcos permanecer solto e flutuante, remover suavemente com uma pipeta de mídia sem remover arcos até que apenas um filme de mídia for deixado. Use ponta da pipeta para colocar os arcos no meio do poço e repetir o passo 3.1. </li><li> Monitorar diariamente; adicionar ~ 100-200 mL de mídia a cada segundo dia para substituir qualquer evaporado mídia. Nota: 24-48 h após a migração de células aparecem ao redor do arco e vão proliferar e migrar do arco ao longo dos próximos 5-8 dias. Depois 36-72 h formação de cardiomiócitos batendo pode ser observado a partir do 2º faríngea arco (Figura 2B). Formação miotubo pode ser observado a partir de 1º arco faríngeo 3-7 dias depois de anexo (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.

Neste vídeo, demonstramos como isolar e cultura primeira e segunda faringe arcos de 9,5 dias antigos embriões de camundongos. Arcos faríngeos são transitórios, segmentado protuberâncias que aparecem no lado craniolateral de embriões em desenvolvimento 9, que contêm multi-potentes blocos cardíaca progenitoras de células-construção para fazer o coração durante a embriogênese 10,11 -em segundo arcos e progenitores musculares cabeça na primeira arcos 8 . <p class="jove_co...

Mesoderme células da faringe dar origem a partes do coração e os músculos da faringe. Durante o desenvolvimento embrionário, as células progenitoras cardíacas multipotentes do segundo campo coração migrar a partir da mesoderme faríngea e preencher a via de saída do ventrículo direito cardíaco e, e seu desenvolvimento anormal está intimamente associada com doença cardíaca congênita-a principal causa de defeitos de nascimento e defect- nascimento mortes relacionadas nos seres humanos 1-3. Estudos recentes têm demonstrado que faríngea mesoderme contribui para dirigir músculos, para além do coração, tornando a mesoderme uma parte crítica do desenvolvimento cardio-craniofacial 4. Assim, os processos de desenvolvimento, incluindo a indução, proliferação e diferenciação de progenitores de cabeça e do coração na mesoderme da faringe estão sob investigação activa 5-7. Até recentemente, ele permaneceu desconhecido se as células progenitoras cardíacas submetidos a witho expansãout diferenciação, em parte devido à falta de informação sobre o seu ambiente celular. Nosso estudo recente sugere que arcos faríngeos servir como um microambiente para a renovação de progenitores cardíacos e musculares e podem ser cultivadas ex vivo ao longo de várias semanas 8. Este método de explante oferece uma oportunidade nova e única para estudar o desenvolvimento de células progenitoras cardio-craniofacial 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>
			<td></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 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>
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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

A mesoderme da faringe dos embriões em desenvolvimento contribui para amplas regiões da cabeça e musculatura cardíaca. Temos desenvolvido um novo método para estudar cabeça e coração progenitor desenvolvimento de células com arcos faríngeos (também conhecido como arcos branquiais) ex vivo. Usando este método, que descrevemos recentemente que o segundo arco faríngeo contém auto-renovação progenitores cardíacos e serve como um microambiente para a expansão das células progenitoras durante o desenvolvimento do coração de rato. As células progenitoras permanecem indiferenciadas e expansivo dentro do arco, mas rapidamente se tornou cardiomiócitos funcionais à medida que migram para fora do arco. Nós também informou que primeiro arco faríngeo contém progenitores musculares que dão origem a myotubes depois de deixar o arco. Aqui, nós demonstramos o procedimento de dissecção e ex vivo cultura de primeiro e segundo arcos faríngeos de desenvolvimento de embriões de camundongos. O método permite o estudo a cabeça eo coração progenitor / dev muscularolvimento, incluindo cardiomiócitos e miotubo formação em detalhe ex vivo.

Durante o desenvolvimento, músculo e coração progenitores faciais podem ser rastreados como elas proliferam e migram do 1 º e 2 º da faringe arco, para se tornar cabeça e musculatura cardíaca, respectivamente (Figura 1A, A &#39;e A&#39; &#39;). Cultura arcos faríngeos oferece uma maneira única de estudar coração e músculo desenvolvimento em detalhe ex vivo. Após a dissecção e fixação dos arcos faríngeos, migrando células de arcos anexado...

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 de Pharyngeal Arcos para estudo do coração e do músculo Progenitores e seu nicho

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

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Developmental Biology

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

7.7K visualizações.  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.

Vídeo: 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.

Usando<em&gt; Ex Vivo</em&gt; Culturas Vertical Gota de toda fetais Órgãos para estudar processos de desenvolvimento durante Rato Organogenesis

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

Biologia do Desenvolvimento

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 de Pintainho Cerebelar Slices e voltadas para o espaço Eletroporação do grânulo precursores de células

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.

Epicárdio Conseqüência Cultura Ensaio e<em&gt; Ex Vivo</em&gt; Avaliação da migração celular Epicárdica derivado

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.

Identificação de progenitores eritromoelóides e sua progênie no embrião de mouse por citometria de fluxo

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 de imagens de Gene Transgenic Expressão em Individual Retinal Progenitores em embriões Zebrafish Quimérico para estudar celular Influências não-autônomos

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, cultura e diferenciação de células do estroma da medula óssea e osteoclasto progenitores dos ratos

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.

Aplicação da estimulação crônica para estudar ratos induzida pela atividade contrátil adaptações fenotípica de músculo esquelético

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.

Na Vivo Imagem latente da morfogênese músculo-tendão em pupas de Drosophila

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.

Geração do primeiro coração campo-como progenitores cardíaca e Cardiomyocytes Ventricular, como de células-tronco pluripotentes humanas

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.

Perfusão ex vivo da placenta de roedores

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 ...