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

NOTA: Todos los animales procedimientos en este protocolo se llevan a cabo con la aprobación de la Universidad de Florida Institucional Cuidado de Animales y el empleo Comisión (IACUC) y están en cumplimiento de la &#39;Guía para el Cuidado y Uso de Animales de Laboratorio &quot;. 1. Experimental básico establecido antes de la administración intermitente Hipoxia <ol><li> Expose cachorros 12 a las diferentes mezclas de gas usando un conjunto de cuerpo cámaras de pletismógrafo ratón en mucho la misma manera que los roedores adultos para otros fines experimentales 5,13,14. Las cámaras tienen un diámetro de 4 pulgadas (volumen = 450 ml), que es de tamaño amplio para acomodar 3-4 crías de ratón o 1-2 crías de rata. Aquí, los protocolos de HAI se entregan a los recién nacidos el día postnatal 1-8 (P1-P8) años. </li><li> Utilice los tanques de gas de aire comprimido para entregar el gas &quot;línea de base&quot; 21%, lo que mantiene la oxigenación de cámara en un 21%. Para lograr la &quot;hipoxia relativa&quot;, utilice un tanque de gas nitrógeno 10% de oxígeno / 90%. NOE: Los tubos de plástico (3/16 &quot;de diámetro) conecta los tanques de gas para un medidor de flujo. Por lo tanto, la entrada al medidor de flujo consiste en un tubo para cada línea de base y el flujo de aire hipoxia. </li><li> Configure el tubo de gas de la siguiente manera. <ol><li> Ejecutar tubo de plástico (1/8 &quot;de diámetro) desde el medidor de flujo a un regulador de flujo de sesgo, que permite a los flujos de 1 L / min a cada cámara (es decir, 4 L / min si se utilizan cámaras 4). </li><li> Ejecutar un tubo de plástico (1/8 &quot;de diámetro) del regulador de flujo sesgo a las cámaras de pletismografía. El flujo de aire a 1 L / min a cada cámara es una velocidad de flujo predeterminada a la que se considera el intercambio de gases a ser no de inducción de estrés a los animales sobre la base de observaciones fisiológicas y de comportamiento. </li></ol></li><li> Selle todas las conexiones no utilizadas hacia y desde la unidad de flujo sesgo y todas las aberturas no utilizadas a las cámaras con tapas de manera que no hay flujo de gas se escapa a otras cámaras no están en uso, o fuera de las cámaras de pletismografía durante el protocolo. </li><li> Coloque la cámara (s) En una incubadora antes de poner los animales en la cámara para permitir el equilibrio a 37 ° C, o temperatura corporal. </li></ol> 2. Calibración de los tiempos de ciclo de hipoxia intermitente <ol><li> Inspeccione todos los tubos, entre los tanques de aire y medidor de flujo, el flujo unidad de metro y la unidad de flujo de sesgo, y el flujo de sesgo y la cámara (s) pletismografía para las conexiones adecuadas. Las conexiones se configuran como se describe en el Paso 1. Verifique la conexión de un control gradual de todas las líneas y los cierres descritos: por ejemplo, la comprobación de líneas no utilizadas lejos de la unidad de flujo de sesgo y las aberturas de la cámara no esté en uso. </li><li> Conecte un sensor de ambiente O2 con un O 2 metros de mano y luego conecte el sensor a la tapa de la cámara pletismografía. </li><li> Abra las dos tanques de gas y conecte un par de pinzas quirúrgicas, de mango largo aislados con tubo de plástico para sujetar el flujo fuera de ciclo de gas. De esta manera, sólo un tanque de gas a la vez es la entrega de la 1 L / min de flujo de gas por cámara. Sujete la mezcla de gas &quot;línea de base&quot;, mientras que la mezcla de gas &quot;hipóxica&quot; se entrega a la cámara, y viceversa. </li><li> Registre el tiempo requerido para la cámara de O 2 para llegar a los niveles objetivo de 10% y volver a 21%. Utilizar los tiempos registrados para la entrega de protocolo subsiguiente. </li></ol> 3. Administración de aguda intermitente Hipoxia <ol><li> Inspeccionar todos los tubos y las conexiones como se describe en el Paso 2.1. </li><li> Coloca los recién nacidos en la cámara (s) pletismografía y albergar a las tapas de la cámara pletismografía en la base de la cámara. Confirmar un sello propio de la junta tórica es así como el cierre de las conexiones de cámara no utilizadas. Estos pasos son fundamentales para garantizar la prestación adecuada de mezclas de gases. </li><li> Coloque las cámaras de la celebración de los cachorros para ser tratados en la incubadora a 37 ° y cierran la puerta. Permitir que la cámara se equilibre a 37 ° C antes de iniciar el protocolo. Mantener los animales de control a 37 ° C en el incubato en la sala de la oxigenación del aire. </li><li> Abra las dos tanques de gas y el uso de un par de pinzas quirúrgicas, de mango largo aislados con tubo de plástico para sujetar el flujo fuera de ciclo de gas. De esta manera, sólo un tanque de gas a la vez es la entrega de la 1 L / min de flujo de gas por cámara. </li><li> Use un cronómetro de mano para, precisamente, ciclos de tiempo y para indicar el momento de cambiar las pinzas hemostáticas entre tubos, lo que resulta en la alternancia de flujo de gas a las cámaras. </li><li> Registre la terminación de cada ciclo usando un registro de tratamiento tal como el proporcionado en la Figura 1 para marcar todos los pasos del 2-paso, protocolo de ciclo de 20. </li><li> Monitorear la temperatura de la incubadora en cada cambio de ciclo para asegurar que los animales se mantienen en el rango señalado (33-35 ° C, que es el ajuste en la incubadora que se traduce en 37 ° C). </li><li> Seguir de cerca la actividad de las crías durante todo el protocolo para asegurar que los animales toleran ciclos sin angustia visible como vocalizaciones, alteración del nivel deactividad de la integridad física o la rodadura. </li></ol> 4. Aislamiento y cultivo de células madre / progenitoras de células de la zona subventricular NOTA: El cambio en la formación neuroesfera siguiente AIH comparación con los controles es un ejemplo de un punto final que demuestra la eficacia de este protocolo para provocar cambios en tejidos y nivel celular. <ol><li> Para aislar neuroesferas forman células, retire ratón o rata crías de la cámara inmediatamente después de HAI se ha completado. Sacrificio de los animales de acuerdo a los protocolos del IACUC dentro de los 30 min de terminación de protocolo. </li><li> Recoger el tejido SVZ, coloque en la cultura neuroesfera y llevar a cabo el paso y la expansión de neuroesferas derivados estándar, según lo publicado previamente en métodos capítulos 15,16 y un protocolo de video por separado 8. </li></ol>

<ol>
	<li>Studer, L., 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=Enhanced+proliferation,+survival,+and+dopaminergic+differentiation+of+CNS+precursors+in+lowered+oxygen.">Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.</a> J Neurosci. 20 (19), 7377-7383 (2000).</li><li>Chen, H. L., 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=Oxygen+tension+regulates+survival+and+fate+of+mouse+central+nervous+system+precursors+at+multiple+levels.">Oxygen tension regulates survival and fate of mouse central nervous system precursors at multiple levels.</a> Stem Cells. 25 (9), 2291-2301 (2007).</li><li>Ling, L., 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=Chronic+intermittent+hypoxia+elicits+serotonin-dependent+plasticity+in+the+central+neural+control+of+breathing.">Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing.</a> J Neurosci. 21 (14), 5381-5388 (2001).</li><li>Mitchell, G. S., 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=Invited+review:+Intermittent+hypoxia+and+respiratory+plasticity.">Invited review: Intermittent hypoxia and respiratory plasticity.</a> J Appl Physiol. 90 (6), 2466-2475 (2001).</li><li>Fuller, D. D., Zabka, A. G., Baker, T. L., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phrenic+long-term+facilitation+requires+5-HT+receptor+activation+during+but+not+following+episodic+hypoxia.">Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.</a> J Appl Physiol. 90 (5), 2001-2006 (2001).</li><li>Fuller, D. D., Johnson, S. M., Olson, E. B., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+pathways+to+phrenic+motoneurons+are+enhanced+by+chronic+intermittent+hypoxia+after+cervical+spinal+cord+injury.">Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord injury.</a> J Neurosci. 23 (7), 2993-3000 (2003).</li><li>Baker-Herman, T. L., 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=BDNF+is+necessary+and+sufficient+for+spinal+respiratory+plasticity+following+intermittent+hypoxia.">BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia.</a> Nat Neurosci. 7 (1), 48-55 (2004).</li><li>Zhu, L. L., 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=Neurogenesis+in+the+adult+rat+brain+after+intermittent+hypoxia.">Neurogenesis in the adult rat brain after intermittent hypoxia.</a> Brain Res. 1055, 1-6 (2005).</li><li>Zhang, J. X., Chen, X. Q., Du, J. Z., Chen, Q. M., Zhu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+intermittent+hypoxia+enhances+mice+performance+in+water+maze+and+8-arm+radial+maze+tasks.">Neonatal exposure to intermittent hypoxia enhances mice performance in water maze and 8-arm radial maze tasks.</a> Journal of neurobiology. 65 (1), 72-84 (2005).</li><li>Zhu, L. L., Wu, L. Y., Yew, D. T., Fan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypoxia+on+the+proliferation+and+differentiation+of+NSCs.">Effects of hypoxia on the proliferation and differentiation of NSCs.</a> Mol Neurobiol. 31 (1-3), 231-242 (2005).</li><li>Ross, H. 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=In+vivo+intermittent+hypoxia+elicits+enhanced+expansion+and+neuronal+differentiation+in+cultured+neural+progenitors.">In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors.</a> Exp Neurol. 235 (1), 238-245 (2012).</li><li>Fuller, D. D., 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=Induced+recovery+of+hypoxic+phrenic+responses+in+adult+rats+exposed+to+hyperoxia+for+the+first+month+of+life.">Induced recovery of hypoxic phrenic responses in adult rats exposed to hyperoxia for the first month of life.</a> J Physiol. 536 (Pt 3), 917-926 (2001).</li><li>Fuller, D. D., Fregosi, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatiguing+contractions+of+tongue+protrudor+and+retractor+muscles:+influence+of+systemic+hypoxia.">Fatiguing contractions of tongue protrudor and retractor muscles: influence of systemic hypoxia.</a> J Appl Physiol. 88 (6), 2123-2130 (2000).</li><li>Baker, T. L., Fuller, D. D., Zabka, A. G., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+plasticity:+differential+actions+of+continuous+and+episodic+hypoxia+and+hypercapnia.">Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.</a> Respir Physiol. 129 (1-2), 25-35 (2001).</li><li>Marshall, G. 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=Production+of+neurospheres+from+CNS+tissue.">Production of neurospheres from CNS tissue.</a> Methods Mol Biol. 438, 135-150 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), (2010).</li></ol>

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...

El objetivo de este método es para entregar de forma reproducible y eficaz episodios intermitentes de oxígeno ambiental sistémica rebajado a los roedores neonatales. La justificación del uso de la hipoxia intermitente (IH) para manipular la biología de células madre se origina a partir de experimentos de cultivo celular in vitro en el que se altera O 2 contenido del medio de crecimiento. En concreto, si se compara con las condiciones &quot;estándar&quot; de 20% de O 2, cultivo prolongado de células madre / progenitoras células poblaciones celulares en 3% O 2 resultados en un aumento de la proliferación, la disminución de la apoptosis y el aumento de 1,2 rendimiento neuronal. Este grupo tiene una gran experiencia con la administración de IH sistémica, y ha realizado extensos estudios sobre el papel de IH en la plasticidad respiratoria 3-7. Este trabajo, y el hallazgo reciente que IH crónica aumenta la neurogénesis en el roedor CNS 8-10, constituye la base para la exploración de aguda in vivohipoxia como estímulo acondicionamiento previo (es decir., antes de la cosecha de tejidos) en el posterior cultivo de células madre / progenitoras neuronales (CPN) 11. Sorprendentemente, cuando crías de ratón fueron expuestos a un breve período (&lt;1 hr) de la hipoxia intermitente aguda (AIH), las células que fueron cosechadas de la zona subventricular (SVZ) habían aumentado significativamente la capacidad para la expansión como neuroesferas o monocapa de células adherentes. El protocolo AIH también se asoció con un aumento de la expresión de un factor de &quot;destino neuronal&quot; transcripción (Pax6). En consecuencia, en vivo protocolos AIH pueden proporcionar un medio para &quot;prime&quot; NPCs antes del cultivo. Por ejemplo, las solicitudes de este enfoque podrían incluir la expansión de poblaciones de células antes del trasplante en el sistema nervioso central lesionado, o el simple aumento de la diferenciación neuronal de células cultivadas antes de los experimentos in vitro. Además, dado que se trata de una administración sistémica, cualquierórgano, tejido o célula es un candidato para el estudio similar. Por lo tanto, el protocolo como escrito es potencialmente aplicable a una amplia gama de estudios sobre la manipulación de oxígeno intermitente en pequeños mamíferos. Hay ciertas ventajas de este enfoque. En otro trabajo publicado, los neonatos fueron tratados como una camada con la presa en cámaras hipobárica, que permite la dosificación crónica, menos manipulación antes del tratamiento, y se mantienen contactos materna durante el tratamiento 9. El enfoque actual no pasa por tratamientos repetidos a una hembra reproductora, o el uso de una presa diferente para cada experimento. Este protocolo también permite el estudio de los recién nacidos de basura de concordancia y de la misma edad precisas. Los datos representativos demuestran otra fortaleza clave de este protocolo, es decir, la rapidez con la que AIH, como entregado, provoca una respuesta biológica potente y consistente en la biología de las células madre neurales. Esto establece un precedente para este protocolo para obtener biologi tejidos y nivel celularCal cambios que alteran la biología celular. Este informe se esbozarán los procedimientos detallados utilizados para la exposición de las crías de roedores a AIH, así como el análisis de la población de las células SVZ crecido como neuroesferas.

<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.

Los experimentos iniciales, basados ​​en datos históricos, se realizaron utilizando longitudes de ciclo 1 min. Sobre la base de las calibraciones posteriores realizadas en el Paso 2 anterior, se determinó que el nivel de O 2 en la cámara era 13% en 1 min de lavado post-hipoxia y, que tomó un tiempo similar para volver a la línea de base 21%. Sin embargo, un ciclo de 2 min era suficiente para lograr tanto 10% de oxígeno y un retorno a 21% durante el ciclo de &quot;línea base&quot;. Posteriormente, s...

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

En este artículo se describe la metodología para la administración de cortos períodos de hipoxia intermitente para postnatales días 1-8 de ratón o rata crías. Este enfoque suscita efectivamente un nivel de tejido robusto &quot;efecto priming&quot; en células progenitoras neurales cultivadas que se cosechan en los 30 minutos de exposición a la hipoxia.

Entrega de<em&gt; En Vivo</em&gt; Aguda intermitente hipoxia neonatal en roedores a derivados de la Zona Prime subventricular progenitoras neurales Cultivos Celulares

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

delivery-vivo-acute-intermittent-hypoxia-neonatal-rodents-to-prime

Research

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 vistas.  University of Florida.  En este artículo se describe la metodología para la administración de cortos períodos de hipoxia intermitente para postnatales días 1-8 de ratón o rata crías. Este enfoque suscita efectivamente un nivel de tejido robusto "efecto priming" en células progenitoras neurales cultivadas que se cosechan en los 30 minutos de exposición a la hipoxia. 

Video: 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.

La inducción directa de las células madre neurales humanas a partir de células de sangre periférica progenitoras hematopoyéticas

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

Investigación

Biología del Desarrollo

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.

Celular-como Stem<em&gt; Xenopus</em&gt; Embrionarias explantes para Estudiar Características Temprana del Desarrollo Neural<em&gt; In Vitro</em&gt; Y<em&gt; En 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 Diferenciación en inmortalizadas células pluripotentes 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 de microinyección a Target a los países en desarrollo<em&gt; Xenopus</em&gt; riñón

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.

El uso de cultivos de neuroesferas primarias estudian primaria cilios

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.

Inducción de la hipoxia en los sapos vivos y los embriones de pez cebra

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.

Una nueva técnica de trasplante de grasa mamaria para visualizar la generación de vasos de células madre endoteliales 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.

Análisis de la inmunofluorescencia de Whole-mount cuantitativa de las poblaciones progenitoras cardiacas en embriones de ratón

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.

Citometría de flujo en tiempo real de células vivas para investigar la afluencia del calcio, formación de poro y fagocitosis por los receptores P2X7 en células progenitoras neurales adultas

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 de lesión cerebral de ratón con trasplante terapéutico de células neuronales derivadas de células madre pluripotentes inducidas por el hombre

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