Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC Diferenciación de células progenitoras neurales <ol><li> Crecer y expandirse hiPSCs en células madre embrionarias humanas (HES) medios de comunicación (Tabla 1) co-cultivadas en un fibroblastos embrionarios de ratón (MEF) capa alimentadora hasta grandes (pero subconfluentes) colonias están listos para la diferenciación neural a través de un cuerpo embrioide (EB) intermedia (Figura 2). Condiciones de rutina cultura hiPSC están bien descritas en otra parte 16,17; Brevemente, hiPSCs crecer en medios de HES en una capa de alimentación MEF hasta la confluencia, el paso entonces enzimáticamente con colagenasa (1 mg / ml en DMEM) y ampliar aproximadamente 1: 3 cada 7 días. </li><li> Para preparar placas recubiertas de poli-L-ornitina / laminina, placas de capa con 10 g / ml de poli-L-ornitina en agua estéril para superficies de plástico (50 g / ml para las superficies de vidrio) y se incuba a temperatura ambiente durante 3-24 horas. Lavar al menos una vez con agua estéril y luego cubra con 5 g / ml laminina en PBS estéril a temperatura ambiente durante 3-24 horas. NOTA: Si envuelto en plástico, placas pueden seralmacenada durante hasta seis meses a -20 ºC. </li><li> En el Día 1, enzimáticamente levantar hiPSCs subconfluentes (generalmente cultivadas en MEFs, o hiPSCs cultivadas en medios definidos, tales como TeSR 18 en Matrigel) de la placa como grandes colonias utilizando 1 mg / ml de colagenasa IV en DMEM / F12. NOTA: Después de 1-2 h de incubación a 37 ºC, las colonias se flotando en el plato. </li><li> Lave suavemente colonias hiPSC (por decantación, la centrifugación no) 1-2x con DMEM / F12, volver a suspender en 2 ml / pocillo de N2 / media B27 (Tabla 1) y la transferencia a los platos no adherente de 6 pocillos (combinar 3 pozos en 1 -bueno) 19. NOTA: Durante la noche, las colonias se formarán grupos esféricas flotantes denomina &quot;cuerpos embrionarios&quot; (EBS). Esperar la muerte celular sustancial. </li><li> En el día 2, las placas de inclinación y permita EBS se asiente. Retire con cuidado los medios de comunicación y lavar una vez con DMEM / F12 para eliminar los residuos. Alimentar con N2 / B27 medio suplementado con inhibidores de Smad duales (0,1 M LDN193189 y 10M SB431542) 12 </sup&gt;. </li><li> En los días 3-7, neuralization se producirá en el contexto de la inhibición SMAD dual; comprobar que los OC son redondos pero no quística. Alimente EBS cada segundo día con los medios de comunicación N2 / B27 suplementado con 0,1 M LDN193189 y 10M SB431542. </li><li> En los días 7-14, después de chapado de EBS a la poli-L-ornitina / laminina placas recubiertas, comprobar usando un microscopio de campo claro que rosetas neurales comienzan a aparecer dentro de unos pocos días (caracterizado como racimos redondos de células neuroepiteliales con la polaridad-apico basal; la Figura 1). Continuar para alimentar EBS adherentes cada segundo día con los medios de N2 / B27 suplementado con 0,1 M LDN193189, 10M SB431542 y 1 g / ml de laminina. </li></ol> 2. Cosecha de Neural Rosetas NOTA: Se recomienda que rosetas neuronales pueden cosechar enzimáticamente usando Neural Rosette Selección Reactivo 20 o reactivo de selección similar. Aunque rosetas neuronales pueden ser recogidos manualmente en 6 pocillos de poli-L-ornitina / laminina placa recubierta, thimetodología s lleva una amplia formación de dominar, y, dependiendo de la habilidad del usuario, puede requerir una segunda ronda de recoger en el día 20 para enriquecer aún más para NPCs y agotar tipos de células no neuronales. <ol><li> Para la selección enzimática de rosetas neuronales en el día 14, los medios de aspirado de EBS adheridas y añadir 1 ml de reactivo neuronal selección roseta por pozo de una placa de 6 pocillos. Se incuba a 37 ºC durante 1 hora. </li><li> Con un Pipetman P1000, retire con cuidado la enzima de cada pocillo. Añadir 1 ml de DMEM / F12 por pocillo de lavar. </li><li> Con un P1000, recoger los 1 ml de DMEM / F12 y rápidamente expulsar al DMEM / F12 de nuevo en el pozo, separando así los rosetones de la placa. Recoge las rosetas en un tubo Falcon. </li><li> Pipetear otro 1 ml de DMEM / F12 y expulsar rápidamente en el mismo pozo para separar restantes rosetas. (No se tritura. Trate de no romper los agregados de roseta). Recoge las rosetas neuronales en mismo tubo Falcon. </li><li> Repita el paso 2.4, según sea necesario. Si rosetas no se desprenden fácilmente, añadir more enzima y repetir el proceso (pasos 2.1 a 2.4) </li><li> Girar las células a 300 xg durante 3 min. Aspirar el lavado, y volver a suspender rosetas en 2 ml de medio de NPC (Tabla 1). La transferencia de células (1: 1) en una poli-L-ornitina / laminina recubiertas 6 placa de pocillos. </li><li> De 15 a 21 días, las rosetas neuronales ampliarán; rosetas de alimentación cada segundo día con los medios de comunicación de la APN. </li><li> Evaluar la calidad de las rosetas neuronales y asegurarse de que las células de tipo fibroblasto planos no están expandiendo dentro de la cultura. NOTA: Si no rosetas persisten, la cultura de la APN a veces puede ser salvado por picking manual, seleccionando sólo los mejores de los rosetones recogido en Accutase. Disociar 15 min a 37 ºC. Pellet, lavar y placa en los medios de comunicación de la APN en 24 pocillos placa recubierta de poli-L-ornitina / laminina. </li></ol> 3. Ampliación de células progenitoras neurales NOTA: hiPSC PNJ se puede cultivar en cualquier Matrigel- o poli-L-ornitina / laminina placas recubiertas. Utilizamos típicamente Matrigel-placas, ya que se pueden preparar con mayor rapidez y menor coste. <ol><li> Para preparar placas Matrigel recubiertos, descongelar y volver a suspender rápidamente una alícuota de 1 mg de Matrigel congelado en 24 ml de frío DMEM / F12 y distribuir inmediatamente 2 ml a cada pocillo de dos placas de seis pocillos. Incubar durante al menos 1 hora a 37 ºC. NOTA: Matrigel sólo necesita ser aspirado, no se lava, inmediatamente antes del uso. </li><li> Alimente NPCs cada dos días y mantener a muy alta densidad o que de forma espontánea se diferenciarán a las neuronas. NOTA: NPCs Aunque más establecidos suelen dividieron 1: 4 cada semana, NPCs muy bajos de paso se pueden dividir 1: 2 con tan poca frecuencia como una vez cada dos semanas. </li><li> Para dividir, primeros medios de aspirado y añadir 1 ml cálido Accutase (1X) por pocillo de placa de 6 pocillos. Se incuba a 37 ºC durante 10-15 minutos. </li><li> Transferir suavemente las células desprendidas en un tubo de 15 ml que contenía DMEM / F12 con la menor tensión mecánica posible. No valorar las células, mientras que en la enzima. Células de pellets por spinning a 1000 xg durante 5 min. </li><li> Aspirar el lavado, y volver a suspender NPCs en ~ 1 ml NPC medios por pozo original de 6 pocillos. NOTA: Esperar que un pozo confluente de una placa de 6 pocillos tiene 5-10 millones de células; esto puede ser confirmado contando una alícuota de 10 l de células utilizando un hematocitómetro antes de volver a chapado. </li><li> Después de la resuspensión, NPCs re-placa como NPCs, neuronas o neuroesferas. Para mantener NPCs, placa aproximadamente 1-2 millones de células por pocillo de una placa de 6 pocillos. La eficacia de la formación neurosphere puede variar entre los experimentos y las líneas celulares, pero en general se produce mejor si entre 200.000 y 1.000.000 células se sembraron por pocillo de una placa de 6 pocillos no adherente; si se produce aglutinación de las neuroesferas, reducir el número de células sembradas. Para diferenciar a neuronas, placa de aproximadamente 200.000 células por pocillo de una placa de 6 pocillos, en sustitución de los medios de comunicación de la APN con los medios de comunicación neurona. </li><li> Inmunohistoquímicamente validar cada línea NPC establecido. Una vez que el material celular suficiente ha sido expanded, NPCs etiquetas con anticuerpos para Nestin y Sox2. Líneas Validado NPC también deben diferenciarse en neuronas durante 4-6 semanas, y se marcaron con anticuerpos para III-tubulina y MAP2AB. <ol><li> Fijar las células en paraformaldehído al 4% en PBS a 4ºC durante 10 min. Permeabilizar NPCs a TA durante 15 min en 1,0% de Triton en PBS, y luego bloquear en 5% de suero de burro con 0,1% Triton a TA durante 30 min. </li><li> Incubar con el anticuerpo primario en 5% de suero de burro con 0,1% de Triton, durante la noche a 4ºC. NOTA: Se recomienda los siguientes anticuerpos: cabra anti-Sox2, 1: 200; Nestin de ratón anti-humano, 1: 200; de conejo anti-βIII-tubulina, 1: 500; ratón anti-βIII-tubulina, 1: 500; ratón anti-MAP2AB, 1: 200. (Figura 2). </li><li> Después de un lavado con PBS, se incuban las células anticuerpos secundarios con el anticuerpo secundario conjugado apropiado cabra, ratón o conejo a 1: 300, en en 5% de suero de burro con 0,1% Triton durante 1-2 horas a TA. Para visualizar los núcleos, células de tinción con 0,5 g / ml DAPI (4 &#39;, Montaje de 6-diamidino-2-fenilindol) y luego con Vectashield. </li></ol></li></ol> 4. NPC Transducción <ol><li> Utilice spinfection (centrifugación de placas de cultivo celular en presencia de virus a 1.000 xg durante 1 hr) para aumentar el porcentaje de células transfectadas 21. Aspirar los medios de comunicación y reemplazarla por la sobreexpresión relevante (o control) lentivirus (o retrovirus), tituladas a la multiplicidad deseado de infección (MOI) (típicamente 1-10), diluido en los medios de comunicación de la APN. Use 1,5 ml de volumen por pocillo de una placa de 6 pocillos (o un volumen escala apropiada para otros tipos de placas de cultivo tisular). Giran a 1000 xg y 37 ºC durante 1 hora en una centrífuga de placas. NOTA: Idealmente, la transducción de NPCs con lentiviral o vectores retrovirales dentro de 1-2 días de separación. Si el uso de virus comerciales o preparado en el laboratorio, puede ser útil para titular apropiadamente lotes de virus de antemano por dilución en serie. (Figura 3). </li><li> Para reducir celmuerte lular, sustitución de los medios dentro de 8 horas de spinfection. NOTA: la integración viral y la expresión transgénica deberían ser detectables dentro de las 24 horas por fluorescencia gen reportero. (Si el virus carece de un indicador fluorescente, esto es más difícil de evaluar; transfección exitosa mejor puede ser validada por inmunotransferencia, inmunohistoquímica o qPCR para los productos de sobreexpresión.) Expresión completa puede tardar de 3-7 días; spinfections repetidos con virus adicional puede ser necesaria para aumentar el porcentaje de células transfectadas. Spinfections recurrentes pueden ocurrir a diario, pero no tienen que ser tan frecuentes. Idealmente, si se utiliza un indicador fluorescente,&gt; 80% de las células deben ser etiquetados. </li></ol> 5. Neurosphere ensayo de migración NOTA: neuroesferas forma espontáneamente, tras la disociación enzimática de NPC (por una manera idéntica a la utilizada en la expansión NPC - los pasos 3.3 a 3.6), si las células se cultivan en suspensión en medios NPC. <ol><li> Brevemente, se disocian crecerNPCs d en los medios de comunicación de la APN para 48 horas en placas adherentes a fin de generar neuroesferas. (Figura 4). </li><li> Para la migración neurosphere, preparar placas Matrigel frescas usando los medios de comunicación de la APN frío, en lugar de DMEM, en una placa de 96 pocillos, 1-2 horas antes de la neurosphere picking. No aspirar Matrigel de los pozos. </li><li> Como se publicó originalmente para neuroesferas derivadas de células madre embrionarias de 22, recoger manualmente neuroesferas derivadas de NPC con un microscopio, después de lavar una vez en los medios de comunicación de la APN para eliminar restos celulares. Transferir una neuroesfera a cada pocillo de una placa de 96 pocillos recubierta con Matrigel. </li><li> Añadir un 0,5 mg adicionales Matrigel, diluido en medios NPC frías, a las neuroesferas en cada placa de 96 pocillos. </li><li> Centrar manualmente cada neurosphere individuo en el centro del pozo utilizando una punta de pipeta. NOTA: Es importante escoger neurosferas de tamaño similar, con el fin de reducir la variabilidad en los resultados. </li><li> Permita que la migración se produzca durante 48 horas y luego fijar las células wITH paraformaldehído al 4% y tinción con marcadores inmunohistoquímicos deseados. </li><li> Si la migración radial se determinará, neurosferas fotografía en su totalidad utilizando un objetivo 4x microscopio. Excluir las neuroesferas en contacto con el borde del pozo o una segunda neurosphere del análisis. NOTA: Si la migración se produce durante más de 48 horas, la migración radial resultante probablemente será demasiado grande para la imagen con un objetivo 4x. </li><li> Medir la migración promedio de cada neurosphere utilizando el software NIH ImageJ (Figura 5). Esto se puede hacer utilizando cualquiera de los métodos de análisis descritos a continuación: <ol><li> Migración radial: <ol><li> Trazar manualmente el borde de las células más lejanos migrar utilizando la herramienta de selección a mano alzada a continuación, utilizar la función (Ctrl + M) Medida para calcular el área de la forma resultante. Antes de medir el área, asegúrese de que la zona está marcada en la ventana Set Medidas encontrado en la ficha Analizar. Utilice lavalor de la medición de área y la ecuación para el área de un círculo (A = πr 2) para determinar el radio (exterior). </li><li> Trazar el borde de la neurosphere original, medir el área y calcular el radio (interior) de la misma manera. Calcular la migración radial total como la diferencia entre los radios exterior e interior. </li></ol></li><li> Greatest migración celular: <ol><li> Medir la distancia recorrida de las cinco células más alejadas del borde de la neuroesfera interior con la función de selección de línea recta, seguido por la función (Ctrl + M) Medida. </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

Hemos descrito los métodos por los que pueda diferenciar hiPSCs en NPCs, un tipo de célula neuronal en la que se conserva una parte importante de la firma genética de neuronas derivadas de hiPSC y que pueden servir como un sustituto de las vías de desarrollo que posiblemente contribuyen a la patogénesis de la enfermedad 8, 11. Además, como hemos detallado, NPCs son una población neuronal robusta replicativa y fácilmente transducidas, que creemos que puede ser adecuado para los estudios moleculares y b...

Estudios de expresión génica de las neuronas diferenciadas in vitro a partir de células madre pluripotentes inducidas humanas (hiPSCs) por nosotros 1 y otros 2,3 indican que las neuronas hiPSC asemejan fetal en lugar de adultos tejido cerebral. En la actualidad, los modelos basados ​​en hiPSC pueden ser más apropiados para el estudio de la predisposición a, en lugar de características tardía de la enfermedad neurológica. Hemos informado anteriormente de que una fracción significativa de la firma genética de las neuronas hiPSC derivado de la esquizofrenia se conserva en las células esquizofrenia hiPSC derivados neurales progenitoras (NPC), indicando que NPC puede ser un tipo de célula útil para estudiar las vías moleculares que contribuyen a la esquizofrenia 1 . Nosotros y otros han informado de la migración aberrante, aumento del estrés oxidativo y las especies reactivas de oxígeno, la sensibilidad a las tensiones ambientales sub-umbral y la función mitocondrial alterada en la esquizofrenia hiPSC NPC 1,4-6, así como una disminución c neuronalonectividad y la función sináptica en las neuronas hiPSC esquizofrenia 5,7-10. Si los factores moleculares que contribuyen a la migración aberrante y / o el estrés oxidativo en la esquizofrenia hiPSC NPCs también subyacen a la conectividad neuronal reducida en las neuronas hiPSC derivado de la esquizofrenia, NPC podrían ser una población neuronal robusta y altamente replicativa con el que estudiar los mecanismos responsables de la enfermedad. Además, porque se puede generar rápidamente un gran número de células y no es necesario esperar semanas o meses para la maduración neuronal, ensayos basados ​​en NPC son adecuados para el estudio de grandes cohortes de pacientes y son más susceptibles de cribado de alto rendimiento. Creemos que hiPSC NPCs puede servir como sustituto de las vías de desarrollo que posiblemente contribuyen a la patogénesis de la enfermedad, como ya se ha demostrado en los trastornos tan diversos como la esquizofrenia 1 y la enfermedad de Huntington 11. Para diferenciar los NPCs de hiPSCs, neural inicial enla producción se lleva a cabo por doble SMAD inhibición (0,1 mM y 10 mM LDN193189 SB431542) 12. Por antagonizar BMP y TGF señalización con estas pequeñas moléculas, endodermo y mesodermo especificación está bloqueada, la aceleración de la diferenciación neuronal y que conduce a la formación de rosetas neuronales visibles dentro de una semana de la siembra. Patrón Neural se produce temprano en este proceso, presumiblemente durante el período de formación de rosetas neural e inmediatamente después. A falta de otros indicios, estas células nerviosas primitivas suponen un-cerebro anterior como el destino anterior 13. Inmediatamente después de la formación de rosetas neural, y continua a lo largo de la expansión de la APN, NPCs del cerebro anterior se cultivan con FGF2 8,14. Tienen potencial linaje dual y se pueden diferenciar de las poblaciones neuronales del 70-80% neuronas III-tubulina-positivas y 20-30% de proteína ácida glial fibrilar (GFAP) -positivos astrocitos (Figura 1). La mayoría de las neuronas del cerebro anterior hiPSC son VGLUT1-positivo, Y también lo son presumiblemente glutamatérgica, aunque aproximadamente el 30% de las neuronas son GAD67-positivo (GABAérgicas) 8. NPC se pasan rutinariamente más de diez veces in vitro, mientras que el mantenimiento de los perfiles de diferenciación consistentes, y sin acumular anomalías del cariotipo. Los grupos han reportado NPCs pases más de 40 veces 15, sin embargo, nos encontramos con que más allá de diez pasajes, NPCs muestran una mayor propensión a la diferenciación de los astrocitos. NPCs bien tolera múltiples congelaciones deshielos y puede ser la transición a crecer como neuroesferas simplemente cultivando en placas no adherentes. NPCs son transducidas eficientemente por vectores virales, lo que permite una rápida evaluación de las consecuencias moleculares y celulares de la perturbación genética, y fácilmente ampliable para producir suficiente material para estudios bioquímicos. Además, porque los vectores virales permiten robusta sobre-expresión y / o desmontables de genes relevantes de la enfermedad, ya sea en el control o paciente derivada neurcélulas de Al, se puede utilizar esta plataforma para probar el efecto de los antecedentes genéticos de estas manipulaciones. Aunque no es adecuado para sináptica o ensayos basados ​​en actividades que requieren las neuronas maduras, NPCs puede ser una alternativa práctica para muchos análisis moleculares o bioquímicos directas de las células neuronales derivadas del paciente.

<table border="0" cellpadding="0" cellspacing="0" width="1174">
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			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
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			<td style="width:216px;">#11330</td>
			<td>for HES media</td>
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			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
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			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">Glutamax</td>
			<td style="width:171px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#21985-023</td>
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			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">FGF2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
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			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
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			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">#450-02</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td style="width:216px;">&#160;#05832</td>
			<td style="width:500px;"></td>
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			<td height="42" style="height:42px;width:288px;">Accutase</td>
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			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
			<td style="width:171px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
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			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Rosetas neuronales pueden ser identificadas morfológicamente, utilizando un microscopio de campo claro, por su aspecto característico como racimos redondos de células neuroepiteliales con la polaridad-apico basal (Figura 1). Aunque NPCs son típicamente cultivaron a muy alta densidad celular, inmediatamente después de los pases, ligeramente soma de forma piramidal y la estructura de neuritas bipolar es visible (Figura 1D). NPCs validados expresan nestina y Sox2 en la mayoría de las...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Una guía para la generación y uso de hiPSC NPCs derivados para el Estudio de las Enfermedades Neurológicas

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

a-guide-to-generating-using-hipsc-derived-npcs-for-study-neurological

Research

JoVE Journal

Medicine

18.1K vistas.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Video: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para el desarrollo de nuevos fármacos contra el cáncer utilizando explantes tumorales de las piezas quirúrgicas


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Investigación

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizando estimulación magnética transcraneal para Estudiar el Sistema Neuromuscular Humanos

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

El aislamiento y la inmortalización de líneas celulares derivados de los pacientes a partir de una biopsia muscular para Modelado de Enfermedades

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry con estimulación cerebral profunda para estudiar la función cerebral normal y patológica

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Un régimen del fenotipo de ratones genéticamente modificados empleadas para el estudio genes implicados en enfermedades humanas de envejecimiento

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Configuración de un equipo de carrera Algoritmo y que imparta una formación basada en la simulación en el Departamento de Emergencia - Una Guía Práctica

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Síntesis radioquímica automatizada de [<sup&gt; 18</sup&gt; F] 3F4AP: un nuevo trazador de PET para imágenes de enfermedades desmielinizantes

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Usando proyección de imagen retiniana para el estudio de la demencia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...