Esta investigación fue apoyada por el Programa de Investigación Intramural del Instituto Nacional de Salud, NINDS. Los autores desean agradecer a todos los miembros del Laboratorio de Medicina Molecular y Neurociencias, Rick Dreyfuss por ayudar con microscopía y tamizado Pamela C. por su ayuda en la edición.

Nota: Para el cultivo de rutina de las células progenitoras neurales y oligodendrocíticos linaje, la incubación se realiza a 37 ° C en un humidificado 5% de CO 2 atmósfera. Cada 2 días, el medio se sustituye con 50 a 100% de medio fresco si la cultura es 40-70% de confluencia. En el momento de confluencia cerca, los cultivos se pasaron a 2-2.5e6/T75 matraz por lo general con un programa semanal. 1. Preparación del frasco recubierto <ol><li> Para preparar matraces recubiertos diluir 5 mg de poli-D-lisina (PDL) en 100 ml de agua desionizada (DI-agua), luego capa matraces T75 con 50 ug / ml de PDL a temperatura ambiente (TA) en la oscuridad. Después de 1 ½ hr, aspirar el PDL y enjuague los frascos una vez con DI-agua. Que los matraces secos antes de la siembra de las células (Tabla 1). </li></ol> 2. A partir diferenciación de células progenitoras neurales progenitoras en oligodendrocitos derivados (DOP) El protocolo esolato humanos células progenitoras del SNC se describe en una publicación anterior y no es parte de este protocolo 1. Humanos de las células progenitoras del SNC se aislaron a partir del telencéfalo de un cerebro 8-semanas de gestación fetal, obtenido de acuerdo con las directrices del NIH. <ol><li> Grow población multipotencial de las células progenitoras neurales en PDL matraces recubiertos en medio neurobasal suplementado con 25 ng / ml de bFGF y 20 ng / ml de factor de crecimiento epidérmico (EGF) (Figura 1A). No los use si es superior a paso 11 (p11). </li><li> Iniciar para diferenciar las células progenitoras neurales cuando el cultivo es de aproximadamente 70% de confluencia. </li><li> Hacer medio de diferenciación de oligodendrocitos completa utilizando suero libre de DMEM / Hams F12 01:01 suplementado con albúmina de suero bovino, L-glutamina, gentamicina, componentes N2, T3, Shh, NT-3, bFGF y PDGF-AA (Tabla 1) . Este medio se define como medio oligo con factores de crecimiento (oligo medio GF +). </li><li> Retire Progenmedios Itor de los matraces, enjuagar las células una vez con solución salina tamponada con fosfato (PBS), a continuación se añade medio de oligo + GF (detalles descritos en 2,3) para iniciar la diferenciación de oligodendrocitos. Sólo las células comprometidas con el linaje oligodendrocíticos crecerá (Figura 1B). Cultivar las células en este medio durante 3 semanas. </li><li> Cada semana, cuando las células están en 90-95% de confluencia, paso utilizando tripsina (0,05%)-EDTA (0,1%) (4 ml para un matraz T75). Pasar el medio completo de las células a ser pasados ​​en un tubo cónico de 50 ml; este medio acondicionado se utiliza para apagar la tripsina. Asegúrese de que no se sequen las células. A continuación, añadir tripsina. </li><li> Se incuban las células en tripsina durante 5 min a TA, golpeando suavemente el matraz varias veces para ayudar desprendimiento celular. </li><li> Se inactiva la tripsina mediante la adición de 4-5 ml de medio condicionado al matraz con las células separadas. </li><li> Pasar el medio y las células con el mismo tubo de 50 ml cónico. Se centrifuga el medio y las células a 1.200 rpm (~300 xg) durante 5 min a TA. </li><li> Aspirar el sobrenadante, resuspender el sedimento celular, inclinando suavemente el tubo y, a continuación añadir medio fresco oligo + GF. Suavemente medio de la pipeta y las células arriba y abajo para realizar una suspensión celular uniforme. </li><li> Contar las células y 2.5e6 de transferencia en cada nueva PDL recubierto matraz T75. </li></ol> 3. Etapa Final en la diferenciación de oligodendrocitos <ol><li> Después de 3 semanas de cultivo en medio oligo + GF, cuando las células están en 70-80% de confluencia, inicie el proceso final de la diferenciación (Figura 1C). </li><li> Preparar el medio de la misma manera como el medio de oligo + GF pero sin añadir el GF cuatro: Shh, NT-3, bFGF y PDGF-AA. Este medio sin el GF cuatro se define como medio oligo - GF. </li><li> Aspirar el medio oligo + GF del matraz, se lava una vez con PBS y luego añadir un volumen total de 16 ml de medio, de los cuales ¾ (12 ml) es oligo medio - GF y ¼ (4 ml) es medio acondicionado PDN, por 6-10 Días). El uso de medio acondicionado de PDN no es crítica para el proceso de diferenciación o la supervivencia de las células. Hemos observado que, de hecho, sólo el contacto directo de las células con DOP con NEM en experimentos de co-cultivo tuvo un efecto sobre la duración de la supervivencia PDO. El tiempo de supervivencia medio en oligo - GF podría prolongarse a dos o tres semanas si las células PDO se co-cultivadas con células NEM. El protocolo para diferenciar las células progenitoras neurales en PDN se describe en una publicación anterior y no es parte de este protocolo 1. Crecimiento factor de retirada de los resultados del medio de cultivo en cultivo progresivamente diferenciadas de células con múltiples procesos (Figura 1D). </li></ol> 4. Ensayo de citometría de flujo El ensayo de citometría de flujo se compara la adquisición de marcadores de oligodendrocitos durante el proceso de diferenciación en relación con los de la población parental. <ol><li> Use pro neuralgenitores como control. Semillas DOP células en 2.5x10 6 células en dos matraces T75 recubiertos con poli-D-lisina en medio de oligo + GF. </li><li> Una semana más tarde, sustituir medio con medio fresco oligo + GF en matraces como un cultivo de control para la viabilidad celular, mientras que otros frascos tenía medio oligo - GF. </li><li> Para disociar células PDO en ambos medios utilizar 20 U / ml de papaína y tripsina no. Esto es debido a la papaína tiene un efecto suave con las células durante la disociación preservar la morfología celular. </li><li> Añadir 5 ml de solución salina equilibrada de Earle (EBSS) a un vial de papaína. Colocar el vial a 37 º C durante 10 min o hasta que la papaína está completamente disuelta y la solución aparece clara. Esta es la solución de papaína utilizado para disociar las células. </li><li> Añadir 0,5 ml de EBSS a una desoxirribonucleasa I (ADNasa) vial (Tabla 1). Mezclar suavemente. Añadir 0,25 ml de esta solución en el vial de la papaína disuelto. Esta preparación contiene una concentración final de aproximadamente 20 U / ml de papaína y 00.005% DNasa. </li><li> Place papaína solución (como se describió anteriormente) en las células y se incuba a 37 ° C durante 15-30 min; controlar el desprendimiento de las células utilizando un microscopio. Detener la reacción con 5 ml de medio con o sin GF y centrifugar a 1.200 rpm durante 7 min. </li><li> Transferir los sedimentos celulares de ambos matraces en dos diferentes tubos de 5 ml de polipropileno y lavar con medio fisiológico normal (NPM: 145 mM NaCl, 5 mM KCl, 1,8 mM CaCl 2, 10 mM Hepes y 10 mM de glucosa) suplementado con 1 mg / ml albúmina de suero bovino (BSA + NPM). </li><li> Realizar superficie inmunotinción a 4 º C durante 20 min utilizando anticuerpos primarios específicos y se titula apropiadamente para A2B5, O4, GalC (Tabla 2). </li><li> Lavar las células con NPM + BSA a 4 ° C durante 5 min y se incuban con anticuerpos secundarios específicos a 4 º C durante 20 min (Tabla 2). </li><li> Para la tinción de antígenos intracelulares incluyendo nestina, proteína ácida glial fibrilar (GFAP), clase III y serta;-tubulina y la proteína básica de la mielina (MBP) (Tabla 2), fijar las células en 2% de paraformaldehído (PFA) durante 20 min a TA y se permeabilizan con etanol frío al 70% durante 15 min a -20 ° C. </li><li> Lavar las células con PBS 1x y se incuban con anticuerpos secundarios específicos (Tabla 2). </li><li> Para discriminar entre las células intactas y los desechos subcelular durante el análisis de citometría de flujo, tinción de células con 4,6-diamidino-2-phenyllindole, dihidrocloruro (DAPI) (Life Technologies, Grand Island, NY). </li><li> En la optimización del protocolo anterior, hemos realizado todos los experimentos de control adecuadas para confirmar la especificidad de cada inmunorreactivo. En resumen, para los anticuerpos marcados directamente, el control negativo apropiado era un anticuerpo conjugado directamente de la clase de inmunoglobulina mismo isotipo (o subclase) y conjugado con fluorocromo como el anticuerpo primario (es decir, el control de isotipo). Para inmunotinción indirecta de anticuerpos primarios, el adecuado control negativo wcomo el anticuerpo secundario conjugado con el fluorocromo de interés que específicamente la clase de inmunoglobulina (o subclase) de la máquina en la que se generó el anticuerpo primario. Cuando los anticuerpos primarios desde múltiples hosts (ratón, conejo, pollo) se utilizan conjuntamente, se utilizó el anticuerpo secundario dirigido el anticuerpo primario de cada host y cada anticuerpo secundario utilizado fue típicamente cruzada adsorbida contra las inmunoglobulinas de los otros anfitriones para minimizar cruzada acoger inmunorreactividad. Los controles positivos incluidos inmunotinción directa o indirecta de preparaciones de células se sabe que expresan los antígenos de interés usando ya sea inmunorreacciones directa o indirecta con los anticuerpos primarios específicos para estos antígenos. Los métodos de optimización más arriba se han llevado a cabo una vez por cada tipo de inmunotinción experimentos. Inmunorreacciones controles no reveló cruzada significativa epítopo inmunoreactividad entre los anticuerpos primarios y secundarios. Citometría de flujo se realizó en uniformementesuspendidas las células marcadas con fluorescencia utilizando un clasificador de células FACVantage SE (BD Biosciences, San Jose, CA) equipado con tres rayos láser, que proporcionan longitudes de onda de excitación sintonizados a 488 nm, 647 nm, y un amplio UV (351-364 nm) (Figura 2) . </li></ol> 5. Ensayo de inmunofluorescencia Nota: Para los experimentos de inmunofluorescencia, PDO se sembraron en medio oligo + GF o medio oligo - GF en 2.5e5 en placas de 6 pocillos recubiertas con PDL. Las células se fijan en 2% de PFA en diversos puntos temporales después de la retirada de factores de crecimiento. El anticuerpo mancha se realiza en placas de 6 pocillos de plástico en lugar de cubreobjetos de vidrio. <ol><li> Retire el medio y añadir el 2% PFA. Incubar durante 10 min a TA. Deseche PFA. Lavar las células 3 veces con PBS, 5 minutos cada vez. Tenga cuidado de no secar las células. </li><li> Permeabilizar las células para marcadores intracelulares utilizando 0,25% de solución de Triton en PBS durante 10 min a TA. </li><li> Para evitar que las células no específicas vinculantes, bloque fo 10 min con HHG (1 mM de tampón HEPES, 2% de suero de caballo, y 10% de suero de cabra en solución salina equilibrada de Hanks). Diluir específicos anticuerpos primarios (Tabla 2) para una concentración predeterminada en HHG. Coloque anticuerpos diluidos sobre las células. Incubar 1 hora a temperatura ambiente con agitación mecánica o a 4 º C durante la noche. </li><li> Lavar las células 3 veces con PBS, durante 5 min cada vez. Diluir anticuerpos secundarios apropiados (Tabla 2) junto con un fluorocromo a una concentración predeterminada en HHG. Coloque la mezcla diluida de anticuerpos secundarios en las células. Incubar 1 hora a temperatura ambiente en un agitador en la oscuridad. </li><li> Lavar las células 3 veces con PBS, durante 5 min cada vez. </li><li> Para evitar photobleaching añadir 10 l de prolongar oro con DAPI a las células y colocar un cubreobjetos de vidrio en la parte superior de ellos. Visualizar las células marcadas utilizando un microscopio de fluorescencia Axiovert 200M (Zeiss, Thornwood, NY) (Figura 3). </li></ol>

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Este protocolo describe la forma de obtener los oligodendrocitos fetales a partir de células humanas primarias progenitoras neurales y caracterizar su fenotipo utilizando tanto la citometría de flujo y tinción de inmunofluorescencia. La expansión y el crecimiento de células progenitoras neurales fetales desde CNS ha sido muy bien descrita 1-4. Sin embargo, la obtención de suficientes oligodendrocitos humanos para la experimentación in vitro sigue siendo difícil, a pesar de que es pos...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Concentración final </td></tr><tr><td> DME / JAMONES F12 01:01 </td><td> Omega Científico </td><td> DM-251 </td><td> 1X </td></tr><tr><td> Albúmina bovina </td><td> Sigma </td><td> A9418 </td><td> 1% </td></tr><tr><td> Gentamicina </td><td> Calidad de Productos Biológicos </td><td> 120-098-031 </td><td> 50 ug / ml </td></tr><tr><td> L-Glutamina </td><td> Calidad de Productos Biológicos </td><td> 118-084-061 </td><td> 2 mM </td></tr><tr><td> T3 </td><td> Sigma </td><td> T2877 </td><td> 3 nM </td></tr><tr><td> N2 Componentes </td><td> Gibco BRL </td><td> 17502 </td><td> 1:100 </td></tr><tr><td> NT-3 </td><td> PeproTech Inc </td><td> 450-03 </td><td> 2 ng / ml </td></tr><tr><td> Shh </td><td> R &amp; D System </td><td> 1314-SH/CF </td><td> 2 ng / ml </td></tr><tr><td> bFGF </td><td> PeproTech Inc </td><td> 100-18B </td><td> 20 ng / ml </td></tr><tr><td> PDGF-AA </td><td> PeproTech Inc </td><td> 100-13A </td><td> 10 ng / ml </td></tr><tr><td> PDL </td><td> Sigma </td><td> P6407 </td><td> 50 g / m </td></tr><tr><td> PFA </td><td> Microscopía Electrónica de Ciencias </td><td> 15712 </td><td> 2% </td></tr><tr><td> Tripsina </td><td> Calidad de Productos Biológicos </td><td> 118-087-721 </td><td></td></tr><tr><td> Papaína </td><td> Worthington </td><td> LK003178 </td><td> 20 U / ml </td></tr><tr><td> DNasa viales </td><td> Worthington </td><td> LK003172 </td><td> 0,005% </td></tr><tr><td> EBSS </td><td> Worthington </td><td> LK003188 </td><td></td></tr><tr><td> Prolongar Oro conDAPI </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 1. Reactivos. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Primaria Abs (todos a 1 mg / ml) </td><td></td><td> Especies Isotipo </td><td> Fuente </td><td> Secundaria Abs </td></tr><tr><td colspan="5"> Citometría de Flujo </td></tr><tr><td> A2B5-Biotina </td><td> Ratón </td><td> IgM </td><td> Regalo J. Nielson </td><td> Estreptavidina PETR, Invitrogen, CA </td></tr><tr><td> O4FITC </td><td> Ratón </td><td> IgM </td><td> Regalo de J. Nielson </td><td></td></tr><tr><td> A2B5 </td><td> Ratón </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> O4 </td><td> Ratón </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> GalC </td><td> Ratón </td><td> IgG 3 </td><td> Millipore, MA </td><td> gαm IgG3-PE, Southern Biotech, AL </td></tr><tr><td> MBP </td><td> Pollo </td><td> IgY </td><td> Millipore, MA </td><td> dαck IgY-AMCA, Jackson Immu., PA </td></tr><tr><td> Nestin </td><td> Ratón </td><td> IgG 1 </td><td> Messam et al. 2000 28 </td><td> gαm IgG 1-PECy5, Invitrogen, CA </td></tr><tr><td> GFAP </td><td> Conejo </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-PE, Jackson Immu, PA </td></tr><tr><td> βIII tubulina </td><td> Ratón </td><td> IgG 2 </td><td> Covance, CA </td><td> gαm IgG-2 a PETR, Invitrogen, CA </td></tr><tr><td colspan="6"> La inmunocitoquímica </td></tr><tr><td> βIII tubulina (1:1,500) </td><td> Ratón </td><td> IgG2a </td><td> Covance, CA </td><td> gαm IgG2a-FITC, Invitrogen, CA (1:1,000) </td></tr><tr><td> GFAP (1:1,000) </td><td> Conejo </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-FITC, Jackson Immu, PA (1:500) </td></tr><tr><td> MBP (1:50) </td><td> Pollo </td><td> IgY </td><td> Millipore, MA </td><td> dαck IgY-FITC, Jackson Immu, PA (1:100) </td></tr><tr><td> O4 (1:100) </td><td> Ratón </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-AF546, Invitrogen, CA (1: 100) </td></tr><tr><td> GalC (1:10) </td><td> Conejo </td><td> IgG </td><td> Millipore, MA </td><td> gαm IgM-AF750, Invitrogen, CA (1:100) </td></tr></tbody></table> . Tabla 2 anticuerpos (Abs) utilizados para la citometría de flujo y ensayos de inmunocitoquímica conjugados de anticuerpos: PE, ficoeritrina; PETR, ficoeritrina Texas Red; AMCA, amino-metil-cumarina-acetato; Cy, cianina; FITC, isotiocianato de fluoresceína. Ig: inmunoglobulina. . AF: Alexa Fluor; gαm: de cabra anti-ratón; gαrb: de cabra anti-conejo; dαck: burro anti-pollo. </td></tr></tbody></table>

progenitor-derived oligodendrocyte culture system from human fetal brain

La diferenciación de los progenitores neurales humanas en tipos de células neuronales y gliales ofrece un modelo para estudiar y comparar la regulación molecular del desarrollo neural linaje celular. Expansión in vitro de células progenitoras neurales a partir de tejido del SNC fetal ha sido bien caracterizado. A pesar de la identificación y el aislamiento de células progenitoras gliales de adulto humano sub-cortical materia blanca y el desarrollo de diversas condiciones de cultivo para la diferenciación directa de progenitores neurales fetales en oligodendrocitos de mielina producir, adquirir suficientes oligodendrocitos humanos para la experimentación in vitro sigue siendo difícil. Diferenciación de galactocerebrósido + (GalC) y O4 + precursoras de oligodendrocitos o células progenitoras (OPC) a partir de células precursoras neurales ha informado mediante cerebro fetal segundo trimestre. Sin embargo, estas células no proliferan en ausencia de células de apoyo incluyendo los astrocitos y las neuronas, y se pierden rápidamente con el tiempo en cultivo. Persiste la necesidad de un sistema de cultivo para producir las células del linaje de oligodendrocitos adecuado para la experimentación in vitro. Cultura de oligodendrocitos humanos primarios podría, por ejemplo, ser un modelo útil para estudiar la patogénesis de la neurotrópicos agentes infecciosos como el virus de polioma humano, JCV, que in vivo infecta las células. Estas células cultivadas también podrían proporcionar modelos de otras enfermedades desmielinizantes del sistema nervioso central (SNC). Primario, humano fetal derivado del cerebro, las células progenitoras neurales multipotenciales proliferan in vitro, mientras que el mantenimiento de la capacidad de diferenciarse en neuronas (neuronas derivadas progenitor-, PDN) y astrocitos (derivado progenitor-astrocitos, PDA) Este estudio muestra que los progenitores neurales pueden ser inducidas para diferenciar a través de muchas de las etapas del desarrollo de linaje oligodendrocíticos (derivado progenitor-oligodendrocitos, PDO). Nosotros, las células progenitoras neurales en cultivo DMEM-F12 sin suero supcomplementado con factor de crecimiento de fibroblastos básico (bFGF), factor de crecimiento derivado de plaquetas (PDGF-AA), Sonic hedgehog (Shh), factor neurotrófico 3 (NT-3), N-2 y triyodotironina (T3). Las células cultivadas se someten a pasadas en células 2.5e6 por matraces de 75 cm aproximadamente cada siete días. Usando estas condiciones, la mayoría de las células en cultivo mantener una morfología caracterizada por pocos procesos y expresan marcadores de oligodendrocitos pre-células, tales como A2B5 y O 4-. Cuando eliminar los factores de crecimiento de cuatro (GF) (bFGF, PDGF-AA, Shh, NT-3) y añadir medio acondicionado de PDN, las células comienzan a adquirir más procesos y expresan marcadores específicos de diferenciación de oligodendrocitos, tales como GalC y de la mielina proteína básica (MBP). Se realizó la caracterización fenotípica mediante citometría de flujo multicolor para identificar marcadores únicos de oligodendrocitos.

Es muy importante para iniciar el proceso de diferenciación de un 70% -80% confluentes cultura neural de células progenitoras (Figura 1A). Muchas células morirán después de cambiar el medio de cultivo a partir de progenitor a medio oligo ya que incluye factores de crecimiento específicos. Esto indica que el crecimiento de las células progenitoras neurales no comprometidos a un fenotipo oligodendrocíticos no será soportado por el nuevo medio (Figura 1B). La incubación en medio ...

Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Primario, fetales humanos derivado del cerebro, células progenitoras multipotenciales proliferan<em&gt; In vitro</em&gt; Mientras se mantiene la capacidad de diferenciarse en neuronas y astrocitos. Este trabajo muestra que los progenitores neurales pueden ser inducidas a diferenciarse a través de las etapas del linaje oligodendrocíticos por aire con factores de crecimiento seleccionados.

Progenitor derivado de sistema de cultivo de oligodendrocitos de cerebro fetal humano

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

progenitor-derived-oligodendrocyte-culture-system-from-human-fetal

Research

JoVE Journal

Neuroscience

14.8K vistas.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Primario, fetales humanos derivado del cerebro, células progenitoras multipotenciales proliferan In vitro Mientras se mantiene la capacidad de diferenciarse en neuronas y astrocitos. Este trabajo muestra que los progenitores neurales pueden ser inducidas a diferenciarse a través de las etapas del linaje oligodendrocíticos por aire con factores de crecimiento seleccionados.

Video: Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Investigación

Neurociencias

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivación de las Culturas enriquecido oligodendrocitos y neuronas oligodendrocitos / myelinating Co-culturas de Post-natal tejidos murinos

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Un método de expansión de GMPc aplicable para agregados de Neural humano madre y células progenitoras derivadas de células madre pluripotentes o tejido cerebral fetal

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Proyección de imagen de las células de la corteza Cerebral primaria usando un sistema 2D de la cultura en vivo

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Sencilla generación de una cultura de alto rendimiento de las neuronas inducidas de fibroblastos de piel humana adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

Generación de oligodendrocitos y medio condicionado por oligodendrocitos para experimentos de cocultura

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Disección y aislamiento de la glía murina de múltiples regiones del sistema nervioso central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Obtención de Microglia Humana del Tejido Cerebral Humano Adulto

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Generación de neuronas humanas y oligodendrocitos a partir de células madre pluripotentes para modelar las interacciones neuronal-oligodendrocitos

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...