Nos gustaría agradecer a Marc Wozniak para la asistencia de vídeo. También queremos agradecer a Sean Morrison de UT Southwestern para el protocolo original para el cultivo de células de rata NC. Este trabajo fue apoyado Vanderbilt University Medical Centro de Soporte del Programa Académico y fue por las subvenciones del NIH (HD36720 y HD036720 11S109-) y la AHA 11GRNT7690040 a PAL, las becas predoctorales de la AHA (0615209B) y el NIH (NS065604) a Nam, y el ERP con el apoyo de una formación T32HD007502 NIH subvención.

1. Preparación de las placas <ol><li> Utilizar una técnica estéril en todo momento. </li><li> Preparar fibronectina (FN) por dilución de 100 l de plasma humano de stock FN en un volumen final de 3,3 ml en PBS de Dulbecco (DPBS). La concentración final es de 30 mcg / ml y esto se puede almacenar a 4 ° C durante 1 semana. </li><li> Cubrir el fondo de cada pocillo de una cultura estéril del tejido cuatro pocillos con solución de FN y deje reposar durante 15 minutos. Asegúrese de que toda la superficie está cubierta. Hacer medios durante este tiempo (pasos 2 y 3). </li><li> Quitar FN solución y permita que las placas que se seque. Enjuague suavemente los pocillos con 500 L DMEM, DMEM quitar y añadir 500 l de auto-renovación (SR) medio (ver más abajo). Se incuba a 37 ° C en una incubadora humidificada que contiene 5 por ciento de CO 2. Complete esta hora aproximadamente un antes de la disección de modo que las placas se seca antes de su uso. </li></ol> 2. Preparación de Medio SR <ol><li> Para la cultura de vago y tronco Carolina del Nortede diez embriones (aproximadamente una camada, en función de los antecedentes genéticos de la línea de ratón), preparar 25 ml de medio de SR. Combinar 12,5 ml de DMEM bajo de glucosa, 7,5 ml de medio Neurobasal, 25 l de ácido retinoico (117 mM concentración final), y 25 l de 2-mercaptoetanol (50 mM concentración final). Mezclar bien. </li><li> Añadir 3,75 ml de extracto de embrión del polluelo, 250 l de N 2 suplemento de sal, 500 l suplemento de B27 y 250 l de penicilina-estreptomicina (1% concentración final). Filtrar el medio a través de un filtro de 0,22 micras. </li><li> Añadir 10 l estéril IGF1 (20 ug / ml de concentración final) y 20 l bFGF estéril (20 mg / ml de concentración final). Mezclar por inversión. Almacenar a 4 ° C. </li></ol> 3. Preparación de medio de lavado <ol><li> Durante aproximadamente 10 embriones preparar 50 ml de medio de lavado. Combinar 50 mg de BSA con 35 ml de glucosa bajo DMEM, 15 ml de medio Neurobasal, y 500 l de penicilina-estreptomicina (1% de concentración final). Filtro estéril con un 0,22filtro micras. </li></ol> 4. Preparación de colagenasa / dispasa <ol><li> Añadir 50 l de 100 mg / ml de colagenasa / dispasa a 5 DPBS mL. Mezclar bien. </li><li> Jeringa filtro con un 0,2 micras filtrar y añadir 1,5 ml en cada uno de los tres pocillos de una placa de doce también. Pipeta aproximadamente 1 ml de medio de lavado en los pocillos restantes. Guarde toda la placa de hielo hasta que esté listo para digerir los tejidos disecados. </li><li> Cortar la punta de una punta de pipeta P20 y P1000 filtro con una hoja de afeitar estéril. Cortar justo debajo del borde biselado de la punta. El corte p1000 se utiliza para transferir todo el embrión mientras que el p20 se utiliza para transferir piezas de tejido aislado. </li></ol> 5. Aislar vagal y el tubo neural del tronco de 9,5 embriones DPC <ol><li> Para los embarazos programados, las presas con un tapón vaginal se consideran 0,5 dpc al mediodía por la mañana el tapón que se observa. El sacrificio y extirpar el útero en el 9,5 dpc. </li><li> Retire la decidua del útero y suavemente reMove el embrión de la decidua. Una vez experimentado con este protocolo, aislar 3-4 9,5 embriones DPC en un momento en DPBS estériles. Utilice una técnica estéril y esterilizados los instrumentos de disección. Los instrumentos pueden ser esterilizados mediante esterilización por calor autoclave, o por incubación en etanol. </li><li> Para vagal anterior, Carolina del Norte: el uso de agujas de insulina, cortar el tubo neural en la placoda ótica medio. Corta de nuevo en el borde posterior de la somite 4 º. Recorte el tejido ventral a la del tubo neural para quitar los arcos faríngeos y el corazón. </li><li> Para NC tronco: el uso de agujas de insulina, retire la parte del tubo neural entre 16-22 somitas (o la última somite si los embriones son de desarrollo anterior a la somite etapa 22). </li><li> Mantenga el saco vitelino y cualquier resto de tejido embrionario para la genotipificación. </li></ol> 6. La eliminación de los nervios no ectodermo y mesodermo <ol><li> Coloque el tubo neural que contiene segmentos en colagenasa / dispasa a temperatura ambiente durante 10 minutoss. Lavar inmediatamente en el medio de lavado. </li><li> Volver a tejido DPBS estériles. El uso de agujas estériles de insulina, retire suavemente el ectodermo no neural de los tejidos y separar los somitas lejos del tubo neural. Las últimas partes restantes del mesodermo a partir de tejido somito se puede quitar por trituración suavemente usando un corte hacia abajo p20 punta. Tenga cuidado de no dañar el tubo neural. Observe esto muy de cerca durante la trituración. </li><li> Se coloca el tubo neural aislado a través de una segunda y tercera de lavado 30 segundos de medio de lavado. </li><li> Lavar una vez en un medio de SR, y colocar el tubo neural aislado en el centro de un pozo FN-revestido que se preparó previamente (Paso 1,3). Humedezca el ambiente de la cámara de hipoxia con un plato de agua estéril. Poner la cápsula en la cámara de hipoxia y vaciar la cámara con gas mixto a 3% de O 2 (usar un tanque que contiene una mezcla de 1% de O 2, 6% de CO 2, 93% de N 2). </li><li> Siempre maneje la cámara con sumo cuidado para asegurarse de Thaexplantes son t tranquila y permanecen en el centro de los pocillos. </li><li> Se incuba a 37 ° C. Resumen de los pasos 5-6 se muestra en la Figura 1. </li></ol> 7. La eliminación del tubo neural <ol><li> Después de 24 horas de incubación, retirar el tubo neural suavemente burlas el borde del tubo neural lejos de las células que migran utilizando una aguja de insulina estéril. Retirar y desechar el tubo neural del medio utilizando un corte de p20 estéril como en el paso 6,2 y reemplazar el medio con medio fresco SR (Figura 2). Esto es más fácil hacerlo mediante un microscopio invertido. </li></ol> 8. Los resultados representativos Tras 24 horas de incubación a 37 ° C en condiciones hipóxicas, las células NC han migrado lejos del tubo neural en una población casi puro (Figura 3a). A veces, menos que los cultivos ideales no va a ceder crecimientos robustos. Por ejemplo, es posible que después de 24 horas tque el tubo neural se han enroscado sobre sí mismo y el CN no se migran fuera del tubo neural (Figura 3b). De vez en cuando el tubo neural no se adhieren a las placas recubiertas de fibronectina. En nuestra experiencia, sub-óptimo de migración Carolina del Norte o problemas con la conexión del tubo neural pueden ser afectados adversamente por las condiciones de normoxia o de concentración de la fibronectina, respectivamente. La actividad enzimática de la colagenasa / dispasa varía ligeramente por lote y el tiempo de digestión se debe ajustar apropiadamente, sin embargo, no digieren el tejido más de quince minutos. Overdigestion del tubo neural que contiene tejido en colagenasa / dispasa también dará lugar a crecimientos deficientes. Si el tejido somite no se elimina fácilmente del tubo neural después de la incubación en la colagenasa / dispasa, el tubo neural puede ser incubado durante más de diez minutos. A veces el tubo neural no se adherirá al sustrato. Si este es el caso, compruebe la fibronecestaño concentración y las condiciones de hipoxia. Aunque las condiciones normoxic se puede utilizar para cultivo de tipo salvaje NC, las condiciones hipóxicas más estrechamente imitar el entorno in vivo 29,30. En nuestra experiencia, las condiciones de hipoxia se tornó crítica cuando se cultiva mutante Carolina del Norte. Por ejemplo, cuando Foxd3 mutante NC se cultivaron en condiciones normoxic, NC tronco tenía una excrecencia celular reduce considerablemente en comparación con los controles. Esta disparidad en tamaño consecuencia se eliminó cuando los explantes se cultivaron en condiciones hipóxicas (Figura 4). Además, cuando los explantes de tipo salvaje del tubo neural se cultivaron en normoxia, el número de células positivas caspasa fue mayor que a continuación de explantes similares cultivadas en hipoxia (datos no presentados). Al mantener toda la cultura Carolina del Norte en la hipoxia, las comparaciones pueden ser más fácilmente entre la dinámica de control y culturas mutantes. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="Figura 1" /> <strong&gt; Figura 1. Esquemática general de aislamiento CN. A) Disección de las regiones de interés del embrión. B) Recopilación del tubo neural en la colagenasa / dispasa durante diez minutos (no sobrepasar los quince minutos). C) Lave en medio de lavado. D) diseccionar el ectodermo no neural y mesodermo. EF) Lavar dos veces en medio de lavado. G) Placa en el medio de auto-renovación. Se incuba a 37 ° C en el 3% de O 2 condiciones de hipoxia. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="Figura 2" /> Figura 2. La eliminación gradual del tubo neural del explante. El tubo neural deben ser removidos después de 24-48 horas para evitar la contaminación con los no-NC células. A) una notificación de la frontera entre el tubo neural y la consecuencia Carolina del Norte (línea continua). B) Cortar a lo largo del borde del tubo neural con una aguja de insulina. C) Se desecha el tubo neural y reemplazar con un nuevo medio a medio auto-renovación. La línea de puntos indica el alcance de la consecuencia. Abreviatura: NT, del tubo neural. Figura 3. Ejemplos de resultados representativos. A) fruto típico explante después de 24 horas de incubación en un medio de auto-renovación en condiciones de hipoxia (línea punteada indica el alcance de la consecuencia). B) Vista ampliada de la consecuencia Carolina del Norte después de 48 horas en la cultura. C) Menos de la cultura ideal, con rendimiento de fruto baja. A y C se cultivaron en las mismas condiciones. Imágenes demuestran la solidez en el área de distribución natural la cultura. Esto puede verse afectada por la eficiencia de aislamiento del tubo neural, la concentración de FN, las condiciones hipóxicas, y el tiempo en digestiones colagenasa / dispasa. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="Figura 4" /> Figura 4. En los análisis in vitro de cultivos de explantes NC en normoxia frente a la hipoxia. De control (tipo salvaje) NC células migraron a partir de explantes de tubo neural después de 48 horas en condiciones de cultivo normoxic. En contraste, Foxd3 mutanteCarolina del Norte se había reducido en gran medida crecimientos celulares en normoxia (de color rojo marca los contornos de los bordes de las derivaciones NC). Cuando los explantes comparables fueron cultivadas bajo condiciones de hipoxia, Foxd3 mutantes explantes de Carolina del Norte creció de forma comparable a los controles, lo que permite su posterior análisis. Tenga en cuenta que este comportamiento una buena correlación con el comportamiento de Foxd3 mutante Carolina del Norte en vivo.

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No tenemos nada que revelar.

Debe prestarse cuidadosa atención a la etapa de desarrollo del embrión para asegurar el éxito de este enfoque. Contando somitos de embriones tempranos de ratón es fundamental tanto para la etapa correspondiente embriones dentro de una camada y la determinación de las regiones correctas del tubo neural para el aislamiento. Una variación de uno o dos somitas entre embriones está dentro de una gama razonable de tiempo de desarrollo, dependiendo de la resolución del experimento llevado a cabo. Un embrión entre el 9...

<table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DMEM (glucosa baja) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Medio Neurobasal </td><td> Gibco </td><td> 21103 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BD Biosciences </td><td> 354037 </td><td> Almacenar en 50 mg / ml alícuotas a -20 ° C. </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> Tienda en 25 g / ml alícuotas a -20 ° C. </td></tr><tr><td> La fibronectina </td><td> Gibco </td><td> 33016-015 </td><td> Almacenado en 1mg/mL aliquots a -20 ° C. </td></tr><tr><td> El ácido retinoico </td><td> Sigma </td><td> R2625 </td><td> Tienda en 35 microgramos / ml alícuotas después de la reconstitución de etanol a -20 º C. </td></tr><tr><td> 2-mercaptoetanol </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N 2 suplemento </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> Suplemento B27 </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Filtros de 0,22 micras Steriflip </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> La penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0,20 micras filtros </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Jeringas (por filtración) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Cuatro placas </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Colagenasa / dispasa </td><td> Roche </td><td> 269 ​​638 </td><td> Actividad varía por lotes. Almacenar en 100 mg / ml alícuotas a -20 ° C. </td></tr><tr><td> Agujas de insulina (29 ½ medidor) </td><td> Becton Dickinson </td><td> 309306 </td><td></td></tr><tr><td> Cámara de Hipoxia </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Analizador de oxígeno </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Pinzas # 5 </td><td> Herramientas de Bellas Ciencia </td><td></td><td> Para quitar el útero y la decidua. </td></tr><tr><td> Tripsina-EDTA (0,25%) </td><td> Gibco </td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

El embrión de la cresta neural (NC) es una población de progenitores multipotentes que se origina en la cara dorsal del tubo neural, se somete a un epiteliales de transición mesénquima (TEM) y migra a través del embrión, dando lugar a diversos tipos de células de 1-3. CN también tiene la capacidad única para influir en la diferenciación y maduración de los órganos diana 4-6. Cuando explantado in vitro, los progenitores NC someterse a la auto-renovación, migrar y diferenciarse en una variedad de tipos de tejidos incluyendo las neuronas y células gliales, células del músculo liso, cartílago y hueso. Multipotencia Carolina del Norte fue descrita por primera vez a partir de explantes del tubo neural aviar 7-9. En el aislamiento in vitro de células NC facilita el estudio de la dinámica de Carolina del Norte, incluyendo la proliferación, migración y multipotencia. Seguir trabajando en los sistemas de aves y ratas demostraron que las células explantados NC conservar su potencial de Carolina del Norte cuando se trasplantan de nuevo en el embrión de 10-13. Debido a estas propiedades inherentes celulares se conservan en explantados progenitores NC, el tubo de ensayo explante neuronal proporciona una opción atractiva para el estudio de la NC in vitro. Para lograr una mejor comprensión de los mamíferos NC, muchos métodos han sido empleados para aislar poblaciones NC. NC-derivados de los progenitores puede ser cultivado a partir de post-migratorias lugares, tanto en el embrión y el adulto para estudiar la dinámica de la post-migratorias progenitores NC 11,14-20, sin embargo el aislamiento de progenitores de Carolina del Norte, ya que emigran del tubo neural proporciona una conservación óptima de NC potencial de la pila y propiedades migratorias 13,21,22. Algunos protocolos emplear fluorescencia de células activadas clasificación (FACS) para aislar una población NC enriquecido para progenitores particulares 11,13,14,17. Sin embargo, cuando se inicia con embriones en fase inicial, el número de células adecuadas para el análisis son difíciles de obtener con FACS, lo que complica el aislamiento de principios populatio NCns a partir de embriones individuales. A continuación, describimos un método que no depende de la FACS y los resultados en una población de aproximadamente el 96% pura Carolina del Norte sobre la base de un reportero Wnt1-Cre activado linaje 23. El método que aquí se presenta es una adaptación de los protocolos optimizados para el cultivo de ratas NC 11,13. Las ventajas de este protocolo en comparación con los métodos anteriores son que: 1) las células no se cultivan en una capa alimentadora, 2) FACS no se requiere para obtener una población NC relativamente puro, 3) premigratorias células aisladas se NC y 4) resultados son fácilmente cuantificados. Además, este protocolo se puede utilizar para el aislamiento de NC de cualquier modelo de ratón mutante, lo que facilita el estudio de las características de NC con diferentes manipulaciones genéticas. La limitación de este enfoque es que el CN se retira del contexto del embrión, que se sabe que influyen en la supervivencia, la migración y la diferenciación del CN 2,24-28.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Aislamiento de cresta neural embrionario del tubo neural facilita el uso de<em&gt; In vitro</em&gt; Métodos para el estudio de la migración, la auto-renovación, y multipotencia de la cresta neural.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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Research

JoVE Journal

Neuroscience

17.1K vistas.  Vanderbilt University Medical Center. Aislamiento de cresta neural embrionario del tubo neural facilita el uso de In vitro Métodos para el estudio de la migración, la auto-renovación, y multipotencia de la cresta neural.

Video: Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

La producción de extracto de embrión de pollo para el Cultivo de murino crestas de células neurales madre

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Caracterización fisiológicas, morfológicas y neuroquímicas de las neuronas modulada por el Movimiento

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Cierre del tubo neural en el cultivo de embriones de ratón entero

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Aislamiento y cultivo de células troncales de la cresta neural de los folículos del cabello humano

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Análisis de la migración de la cresta neural y la diferenciación de las especies cruzadas Trasplante

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Aislamiento y cultivo de células neuronales de ratas embrionarias: un protocolo rápido

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...