The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).

NOTA: Todos os procedimentos com animais neste protocolo são realizadas com a aprovação da University of Florida Animal Care Institucional e Comitê de Uso (IACUC) e estão em conformidade com o &quot;Guia para o Cuidado e Uso de Animais de Laboratório &#39;. 1. Básica Experimental Set up antes da administração intermitente hipóxia <ol><li> Expor filhotes 12 a diferentes misturas de gás usando uma de corpo inteiro câmaras rato pletismógrafo em muito da mesma forma que roedores adultos para outros fins experimentais 5,13,14. As câmaras têm um diâmetro de 4 polegadas (volume = 450 ml), que é de tamanho suficiente para acomodar 3-4 filhotes de rato ou 1-2 crias de rato. Aqui, os protocolos de AIH são entregues aos recém-nascidos com idade pós-natal 1-8 dia (P1-P8). </li><li> Use tanques de gás de ar comprimido para entregar a 21% do gás &quot;linha de base&quot;, que mantém a oxigenação câmara em 21%. Para alcançar o &quot;hipóxia relativa&quot;, use um / 90% tanque de gás nitrogênio de 10% de oxigênio. NÃOE: tubos de plástico (3/16 &quot;de diâmetro) conecta os tanques de gás de um medidor de caudal. Assim, a entrada para o medidor de fluxo é composto por um tubo para cada linha de base do fluxo de ar e hipóxia. </li><li> Configurar o tubo de gás como se segue. <ol><li> Executar tubo de plástico (diâmetro de 1/8 &quot;) a partir do medidor de fluxo para um regulador de fluxo de polarização, o que permite que os fluxos de 1 L / min para cada câmara (ou seja, 4 litros / min, se são usadas câmaras 4). </li><li> Executar tubo de plástico (diâmetro de 1/8 &quot;) a partir do regulador de fluxo de polarização para as câmaras de pletismografia. O fluxo de ar a 1 L / min, para cada uma das câmaras é um caudal predeterminado em que a troca gasosa é considerada não-indução de stress para os animais, com base em observações comportamentais e fisiológicas. </li></ol></li><li> Vede todas as ligações não utilizadas de e para a unidade de fluxo de polarização e de todas as aberturas não utilizadas para as câmaras com tampas de modo que nenhum fluxo de gás escapa a outras câmaras não em uso, ou para fora das câmaras da pletismografia durante o protocolo. </li><li> Coloque a câmara (s) Em uma incubadora antes de colocar os animais na câmara para permitir o equilíbrio a 37 ° C, ou a temperatura do corpo. </li></ol> 2. Calibração de Ciclo Times por hipóxia intermitente <ol><li> Inspecione toda a tubulação, entre tanques de ar e medidor de fluxo, medidor de fluxo e unidade de fluxo de polarização, e fluxo de viés unidade e câmara (s) pletismografia para as conexões adequadas. As conexões são configurados como descrito no Passo 1. Verifique a conexão por uma verificação passo a passo de todas as linhas descritas e fechamentos: por exemplo, verificando linhas não utilizadas fora da unidade de fluxo de polarização e as aberturas de câmara não estiver em uso. </li><li> Ligue um ambiente sensor de O2 com um O 2 metros de mão e, em seguida, conecte o sensor à tampa da câmara pletismografia. </li><li> Abra os dois tanques de gás e anexar um par de hemostats cirúrgicos, de cabo longo isolados com tubos de plástico para prender o fluxo fora de ciclo de gás. Desta forma, apenas um tanque de gás num momento está a fornecer o / min de fluxo de gás de 1 L por câmara. Prenda a mistura de gases de &quot;linha de base&quot;, enquanto a mistura de gás &quot;hipóxica&quot; é entregue à câmara, e vice-versa. </li><li> Grave o tempo requerido para a câmara de O 2 para alcançar os níveis pretendidos de 10% e regresso a 21%. Utilizar os tempos registrados para entrega protocolo subsequente. </li></ol> 3. Administração de hipóxia intermitente aguda <ol><li> Inspecione toda a tubulação e conexões conforme descrito no Passo 2.1. </li><li> Coloque recém-nascidos na câmara (s) pletismografia e abrigar as tampas de câmara pletismografia na base da câmara. Confirmar uma vedação apropriada para o anel de vedação é assim como o fechamento da câmara de quaisquer ligações não utilizadas. Estes passos são críticos para assegurar o fornecimento adequado de misturas de gás. </li><li> Coloque as câmaras que prendem os filhotes a ser tratada no 37 ° C incubadora e feche a porta. Permitir câmara para equilibrar a 37 ° C antes de se iniciar o protocolo. Manter os animais de controlo a 37 ° C no incubatou na sala de ar oxigenação. </li><li> Abra os dois tanques de gás e usar um par de hemostats cirúrgicos, de cabo longo isolados com tubos de plástico para prender o fluxo fora de ciclo de gás. Desta forma, apenas um tanque de gás num momento está a fornecer o / min de fluxo de gás de 1 L por câmara. </li><li> Use um cronômetro de mão para precisamente ciclos de tempo e para indicar a hora de mudar os hemostats entre tubos, resultando, assim, na alternância do fluxo de gás para as câmaras. </li><li> Grave a conclusão de cada ciclo de tratamento usando um log, como o fornecido na Figura 1 para marcar todas as etapas do 2-step, 20 protocolo de ciclo. </li><li> Monitorar a temperatura da incubadora em cada mudança de ciclo para assegurar animais são mantidos no intervalo designado (33-35 ° C, que é a configuração na incubadora que resulta em 37 ° C). </li><li> Acompanhar de perto a atividade filhote ao longo do protocolo para garantir que os animais tolerar ciclos sem desgaste visível, como vocalizações, alteração do nível deatividade membro ou de rolamento. </li></ol> 4. Isolamento e Cultura de Células tronco / progenitoras da zona subventricular NOTA: A alteração na formação neurosphere seguinte AIH comparados aos controles é um exemplo de um desfecho que demonstra a eficácia deste protocolo para provocar mudanças Tecido e de nível celular. <ol><li> Para isolar neurospheres formando células, remova ratinho ou um rato filhotes da câmara imediatamente após AIH está concluída. Sacrificar os animais de acordo com protocolos IACUC dentro de 30 min de cessação do protocolo. </li><li> Colher o tecido SVZ, coloque na cultura neurosphere e conduzir passagem padrão e expansão da neurospheres derivativos, conforme publicado anteriormente em métodos capítulos 15,16 e um protocolo de vídeo separada 8. </li></ol>

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The authors have nothing to disclose.

This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emph...

O objetivo deste método é entregar reprodutível e eficaz ataques intermitentes de oxigênio sistêmico ambiente reduzido para roedores neonatais. A justificativa para o uso de hipóxia intermitente (IH) para manipular a biologia de células-tronco origina de em experimentos de cultura de células in vitro em que O 2 conteúdo do meio de crescimento é alterada. Especificamente, quando comparadas com as condições &quot;standard&quot; de 20% de O2, cultura prolongada de células estaminais / progenitoras de células em populações de células 3% de O2 resulta num aumento da proliferação, diminuição e aumento da apoptose neuronal 1,2 rendimento. Este grupo tem uma experiência significativa com a administração de IH sistêmica, e já realizou extensos estudos sobre o papel do IH na plasticidade respiratória 3-7. Este trabalho, ea recente descoberta de que IH crônica aumentou a neurogênese no roedor CNS 10/08, constitui a base para a exploração do aguda in vivohipóxia como um estímulo pré-condicionamento (ie., antes da colheita de tecidos) sobre a cultura subsequente de células estaminais / progenitoras neurais (NPCs) 11. Notavelmente, quando as crias de rato foram expostos a uma breve (&lt;1 hora) período de hipoxia aguda intermitente (AIH), as células que foram colhidas a partir da zona subventricular (SVZ) tinha aumentado significativamente a capacidade de expansão como neuroesferas ou células em monocamadas aderentes. O protocolo AIH também foi associado com o aumento da expressão de um fator de transcrição &quot;destino neuronal&quot; (Pax6). Deste modo, in vivo protocolos AIH pode proporcionar um meio para &quot;Prime&quot; NPCs antes da cultura. Por exemplo, os pedidos de esta abordagem poderia incluir a expansão das populações de células antes do transplante para o sistema nervoso central lesionado, ou simplesmente aumentando a diferenciação neuronal de células cultivadas antes de experiências in vitro. Além disso, porque esta é uma entrega sistémica, qualquerórgão, tecido ou célula é um candidato para o estudo semelhante. Portanto, o protocolo é como escrito potencialmente aplicáveis ​​a uma ampla gama de estudos sobre a manipulação de oxigénio intermitente em pequenos mamíferos. Existem algumas vantagens para esta abordagem. Em outro trabalho publicado, recém-nascidos foram tratados como uma ninhada com a barragem em câmaras hipobáricas, que permite a administração crónica, menos manuseio antes do tratamento, e manteve contato materno durante o tratamento 9. A abordagem actual ultrapassa tratamentos repetidos para uma fêmea de reprodução, ou o uso de uma barragem diferente para cada experimento. Este protocolo também permite o estudo de recém-nascidos pareados por ninhada e da mesma idade precisos. Os dados representativos demonstrar outra força fundamental deste protocolo, ou seja, a rapidez com que AIH, como entregue, provoca uma resposta biológica poderosa e consistente em biologia de células-tronco neurais. Isto estabelece um precedente para esse protocolo para provocar biologi Tecido e-nível celularcal mudanças que alteram a biologia celular. Este relatório irá delinear os procedimentos detalhados utilizados para expor os filhotes de roedores a AIH, bem como a análise da população de células SVZ cultivadas como neurospheres.

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:243px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Mouse plethysmography chambers</td>
			<td style="width:147px;">Buxco</td>
			<td style="width:136px;">PLY4211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Flow meter&#160;</td>
			<td style="width:147px;">Porter</td>
			<td style="width:136px;">F150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bias flow unit</td>
			<td style="width:147px;">AFPS</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Baseline Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">AIZ300</td>
			<td>Compressed Air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hypoxic Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">X03NI72C2000189</td>
			<td>10% Oxygen, balance nitrogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Oxygen Meter</td>
			<td style="width:147px;">Teledyne</td>
			<td style="width:136px;">AX-300</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Os experimentos iniciais, com base em dados históricos, foram conduzidos utilizando comprimentos de ciclo 1 min. Com base nos calibrações subsequentes realizados no Passo 2 acima, determinou-se que o nível de O2 na câmara era de 13% em 1 min de lavagem pós-hipoxia e, que teve um tempo semelhante para voltar à linha de base de 21%. No entanto, um ciclo de 2 minutos foi suficiente tanto para alcançar 10% de oxigénio e um retorno a 21% durante o ciclo de &quot;linha de base&quot;. Subsequentemente, os c...

Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Este artigo descreve a metodologia para a administração de curtos períodos de hipóxia intermitente para pós-natal 1-8 dia ratinho ou um rato filhotes. Esta abordagem suscita efectivamente um nível do tecido robusto &quot;efeito priming&quot; sobre as células progenitoras neurais em cultura que são colhidas no prazo de 30 min de exposição hipóxia.

Entrega de<em&gt; In Vivo</em&gt; Aguda intermitente hipóxia em Neonatal Roedores ao primeiro-subventricular Zona derivados de culturas de células progenitoras neurais

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

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JoVE Journal

Developmental Biology

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

7.2K visualizações.  University of Florida.  Este artigo descreve a metodologia para a administração de curtos períodos de hipóxia intermitente para pós-natal 1-8 dia ratinho ou um rato filhotes. Esta abordagem suscita efectivamente um nível do tecido robusto "efeito priming" sobre as células progenitoras neurais em cultura que são colhidas no prazo de 30 min de exposição hipóxia. 

Vídeo: Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Indução direta de Neurais Human células-tronco de sangue periférico células progenitoras hematopoéticas

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Biologia do Desenvolvimento

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stem Cell-like<em&gt; Xenopus</em&gt; Embrionárias explantes Estudar características Neural desenvolvimento precoce<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Iniciando A diferenciação em células multipotentes imortalizadas Otic progenitoras

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Técnica ao Target microinjeção para o desenvolvimento<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

Usando culturas neurosphere primárias para o Estudo Primário Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Indução de hipóxia em sapos vivos e embriões de peixe-zebra

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Uma nova técnica de transplante de almofada de gordura mamária para visualizar a geração de vasos de células vasculares endoteliais vasculares

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Análise quantitativa da imunofluorescência toda a montagem das populações do Progenitor cardíaca em embriões de rato

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Citometria de fluxo de viver-pilha em tempo real para investigar o influxo de cálcio, formação de poros e fagocitose pelos receptores P2X7 em células progenitoras neurais adulto

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Modelo de impacto cortical controlado da lesão cerebral do rato com transplantação terapêutica de células neurais pluripotentes induzidas humanas da pilha de haste

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...