Este trabajo fue apoyado por una beca para investigadores avanzados de la Fundación Nacional de Ciencia de Suiza y por medio de donaciones de NYSTEM (C026446; C026447) y la iniciativa de células madre Tri-institucional (Fundación Starr).

1. Preparación de medios de cultivo, placas revestidas y Mantenimiento de hPSCs Preparación 1.1 Medios Nota: todos los medios de filtro para la esterilización y se almacenan a 4 ° C en la oscuridad durante un máximo de 2 semanas. Números de los nombres de Reactivos para la empresa y catálogo se listan en la Tabla de Materiales. <ol><li> DMEM/10% de SFB: Combinar 885 ml DMEM, FBS 100 ml, 10 ml penicilina / estreptomicina y 5 ml de L-glutamina. </li><li> HES-medio: Combinar 800 ml de DMEM/F12, KSR 200 ml, 5 ml de L-glutamina, 5 ml de penicilina / estreptomicina, 10 ml de MEM mínimo de solución aminoácidos esenciales, 1 ml β-mercaptoetanol. Añadir 10 ng / ml de FGF-2 después de filtrar el medio. PRECAUCIÓN: β-mercaptoetanol es tóxico, evitar la inhalación, ingestión y contacto con la piel. </li><li> Medio KSR-diferenciación: Combinar 820 ml Knockout DMEM, 150 ml KSR, 10 ml de L-glutamina, 10 ml penicilina / estreptomicina, 10 ml MEM mínima solución aminoácidos esenciales y 1 ml β-mercaptoetanol. </li><li> N2-differentiatien un medio: Disolver 12 g de polvo en 980 ml ​​de DMEM/F12 dH 2 O, añadir 1,55 g de glucosa, 2 g Bicarbonato sódico y 100 mg APO transferrina humana. Mezclar 2 ml de dH2O con 25 mg de insulina humana y 40 l de NaOH 1 N, añadir la solución disuelta para el medio. Añadir 100 l de dihidrocloruro de putrescina, 60 l de selenito, 100 l progesterona y llevar el volumen hasta 1 litro con dH 2 O. </li></ol> 1.2 Recubrimiento de placas de cultivo <ol><li> Revestimiento Matrigel: Descongelar 1 ml congelados alícuota matrigel al vaso 19 ml DMEM/F12 sobre la alícuota hasta que se haya disuelto. Eliminar grumos haciéndola pasar a través de un filtro de células de 40 micras y la placa 8 ml/10 cm plato. Incubar los platos durante 1 hora a TA. Aspirar el matrigel inmediatamente antes placas las células. Nota: Trabaje rápidamente con matrigel porque puede agruparse a temperaturas superiores a 4 ° C. </li><li> Recubrimiento PO / Lam / FN: Añadir 10 ml de 1x PBS que contenía 15 mg / ml bromhidrato de poli-L ornitina a una placa de 10 cm. Incubar la placa durante la noche a 37 ° C. Lavar las placas con PBS 1x vez y añadir 10 ml/10 cm plato de 1x PBS que contenía 2 mg / ml ratón laminina-I y 2 mg / ml de fibronectina. Incubar los platos durante la noche a 37 ° C. Antes de placas las células, eliminar completamente la solución y deje que los platos se sequen completamente a temperatura ambiente durante 15 a 20 minutos en pie para arriba en la pared campana de cultivo de tejidos sin la tapa puesta. Nota: Los platos son completamente seca y lista para la siembra celular cuando uno puede ver las estructuras de cristal en la superficie (visible a simple vista). Las placas se pueden mantener a temperatura ambiente durante unas pocas horas en este estado seco. Platos PO / Lam / FN tienen que estar preparados 2 días antes de tiempo. En casos de emergencia Lam / FN puede incubarse por tan solo 2-4 horas. Sin embargo, esto puede poner en riesgo los resultados de la diferenciación / supervivencia subóptimos. </li></ol> 1.3 Mantenimiento de hPSCs Nota: hPSCs se mantienen en el 0,1% de gelatina y el ratón mitóticamente inactivados fibroblastos embrionarios (MEFS) en HES-medio suplementado con 10 ng / ml de FGF-2 como se describe previamente 10,12. Las células deben dividirse cada 6-8 días. <ol><li> Escudo un plato de 10 cm con 8 ml de 0,1% de gelatina (en 1x PBS sin magnesio o calcio) a TA durante 5 min. </li><li> Descongele MEFs congelados rápidamente en un baño de agua a 37 ° C. Añadir 1 millón de MEFs a 10 ml de DMEM/10% de FBS. </li><li> Aspirar la gelatina y la placa de las células. Incubar a 37 ° C durante al menos 6 horas. </li><li> Aspirar el FBS DMEM/10% de la placa de MEF preparado, lavar la placa con 1x PBS una vez y añadir 10 ml de HES-medio suplementado con 10 mM de Y-27632 dihidrocloruro. Dejar que el medio se caliente a 37 ° C durante 20 min. Nota: Para la división manual hPSCs se utiliza una campana de flujo laminar con un microscopio incorporado. Sin embargo, las células pueden ser pasadas utilizando métodos alternativos apropiados. </li><li> Bajo una campana de flujo laminar con un microscopio incorporado, separar colonias separadas de utilizar una grúa móvil y permiten flotar. </li><li> Utilice un1 ml jeringa para aspirar las colonias flotantes y enviarlos a la placa MEF fresco, cálido. Transferir aproximadamente un cuarto de las células para el nuevo plato. Se incuba a 37 ° C. </li><li> Alimente células diariamente con medio HES fresco. </li></ol> 2. Plating de hPSCs de Diferenciación Nota: hPSCs deben dividir o chapada de diferenciación cuando las colonias son grandes, pero todavía tienen bordes afilados con tan poco como posibles células que se diferencian en sus fronteras (véase la Figura 1B). Cuando las células se mantienen utilizando pases manual de las colonias deben ser lo suficientemente grandes para ser fácilmente visto por el ojo. Para conseguir la sensación adecuada para este punto en el tiempo se puede mantener un plato HPSC separada por dos semanas sin pases y ver las células alcanzan y pasar el punto de tiempo ideal para pases / diferenciarlas. <ol><li> Preparar platos de 10 cm con matrigel 1 hora antes de iniciar la diferenciación. Revestimiento de matrigel acertada puede serverificado en la magnificación de 4x. </li><li> Cuando los hPSCs están listos para ser dividido, aspirar el medio y añadir 4 ml de 0,05% de tripsina-EDTA a las células. </li><li> Agite el plato horizontalmente durante 2 min vigorosamente hasta que uno puede ver los MEFs como células individuales de elevación de la placa bajo el microscopio. Las colonias HPSC permanecen unidos como colonias. </li><li> Inmediatamente ya fondo, aspirar la tripsina y dejar reposar la placa a temperatura ambiente durante 2-4 min. </li><li> Añadir 2 ml de HES-medio a la placa y el uso de una pipeta P1000, separar las células. Nota: Si las células no se pueden levantar fácilmente la superficie, la placa se puede incubar vacío a TA durante otros 2-3 min. </li><li> Transferir las células a 8 ml de HES-medio suplementado con 10 mM de Y-27632 dihidrocloruro. </li><li> Aspirar el Matrigel a partir de un preparado previamente placa de 10 cm. Placa las células a una proporción de 1:1 o 1:2 (por ejemplo: Las células de una placa de 10 cm se dividen en dos placas de 10 cm) en la placa de Matrigel. Incubar a 37 °C durante la noche. Nota: Este chapado debe dar lugar a aproximadamente 100.000 células / cm 2. </li></ol> 3. La inducción de la diferenciación neuronal Nota: La diferenciación puede ser iniciado (día 0) cuando las células son confluentes al 90-100% (véase la Figura 1C), por lo general el día siguiente. Si la confluencia exacta no se alcanza, sin embargo, las células pueden ser alimentados diariamente con HES-medio hasta que estén listos para la diferenciación. Alternativamente, el número inicial de células sembradas se puede aumentar. <ol><li> En el día 0 a 3, alimentar a las células diariamente con 10 ml/10 cm plato medio KSR-diferenciación que contiene 0,1 LDN193189 mu M y 10 M SB431542. </li><li> En el día 4 y 5 de alimentación de las células con 75% de medio KSR-diferenciación y 25% medio N2-diferenciación tanto que contiene LDN193189 y SB431542. </li><li> En el día 6 y 7 de alimentación de las células con 50% de medio KSR-diferenciación y 50% medio N2-diferenciación tanto que contiene LDN193189 y SB431542. </li><li> En el día 8 y 9 de alimentación de las células con 25% de medio KSR-diferenciación y 75% medio N2-diferenciación tanto que contiene LDN193189 y SB431542. </li><li> El día 9 y 10 se preparan platos PO / Lam / FN, como se indica en el punto 1.2.2 para replating de las células en el día 11. </li><li> El día 10 las células de alimentación con medio N2-diferenciación que contiene LDN193189 y SB431542. </li></ol> 4. Replating en Gotitas por NC Especificaciones <ol><li> El día 11 de aspirado Lam / FN de las placas preparadas con anterioridad y deje que se sequen por completo a temperatura ambiente durante 20 a 30 min, como se explica en el punto 1.2.2. </li><li> Retire medio de las células diferenciadoras, se lavan las células una vez con PBS 1x y añadir 8 ml accutase/10 cm plato. Incubar durante 20 minutos a 37 ° C. </li><li> Utilizando un elevador de celular, separar las células y resuspender en accutase con una pipeta de 5 ml. Transferir la suspensión a un tubo de 15 ml. </li><li> Añadir 5 ml de 1x PBS y girar las células durante 5 min a 114 x g. </li><li> Resuspender las células en 10 ml de 1x PBS. Para garantizar una única suspensión de células, filtrado a través de un filtro de células de 40 micras y contar las células usando un hemocitómetro o una técnica equivalente. </li><li> Girar las células hacia abajo y volver a suspender en medio N2-diferenciación que contiene 200 mM de ácido ascórbico (AA), 20 ng / ml de BDNF, 100 ng / ml de FGF8, 20 ng / ml de SHH, 10 mM de Y-27632 de dihidrocloruro a la concentración de 100 000 -150.000 células/10 l. </li><li> Usando una pipeta de repetición-, placa 10 l gotitas cerca uno del otro sin que toquen a la PO seca / Lam / FN 15 cm de platos. Nota: Una placa de 10 cm de células diferenciadas que normalmente se traduce en aproximadamente 2-3 veces 15 cm de platos o de aproximadamente 100 x 10 cm l droplets/15 plato. </li><li> Deje que las gotitas de pie a temperatura ambiente durante 20 a 30 min, luego con mucho cuidado (no molestar a las gotas) añadir 30 ml de N2/AA/BDNF/FGF8/SHH/Y y se incuba a 37 ° C. </li><li> El día 12, se alimentan con cuidado las células con 20 ml/15 cm N2/AA/BDNF/FGF8/SHH plato. </li><li> De day 14-17 alimentar las células cada dos o tres días con N2/AA/BDNF/FGF8. </li><li> El día 16 y 17 de la preparación / placas Lam / FN PO a replate NC células después de la separación FACS. </li></ol> 5. Fluorescencia de células activadas clasificación (FACS) de células NC Nota: La preparación de las células para FACS requiere aproximadamente 2 horas. <ol><li> El día 18 a medio quitar, lavar las células una vez con PBS 1x en el plato y añadir 12 ml accutase. Se incuban las células durante 20 min a 37 ° C. </li><li> Utilizando un elevador de celular, separar las células de la superficie y permiten flotar. Pipetear las células en suspensión utilizando una pipeta de 5 ml, transferir a un tubo de 50 ml y añadir 30 ml de PBS 1x. Haga girar las células durante 5 minutos a 114 x g. </li><li> Usando una pipeta P1000, resuspender las células en 1 ml de 2% de FBS / HBSS (el HBSS contiene 15 mM de HEPES). Añadir 19 ml de 2% de FBS / HBSS y filtrar las células a través de un filtro de células de 40 micras para asegurar una suspensión de células individuales. Se incuban las células en hielo durante 15 men para el bloqueo de anticuerpos y luego contarlos utilizando un hemocitómetro. </li><li> Girar las células hacia abajo y resuspender a la concentración de 10 millones de células / ml en FBS al 2% / HBSS. </li><li> Ponga a un lado 1 millón de células cada uno por la mancha, de un solo manchado (HNK-1 sólo y únicamente p75) y anticuerpo secundario manchado solamente (APC solamente y 488 solamente) FACS controla. </li><li> Añadir 5 l de HNK-1 y 5 l de p75 anticuerpo primario por 10 millones de células en 1 ml de suspensión a las muestras adecuadas y se incuba durante 20 min en hielo. </li><li> Lavar las células en 10 ml de PBS 1x (centrífuga 5 min a 114 xg) y resuspender las células en 1 ml de 2% de FBS / HBSS. Añadir 2 l de APC y 1 l de anticuerpo secundario 488 por 10 millones de células en 1 ml de suspensión a las muestras adecuadas y se incuba durante 20 min en hielo en la oscuridad. Nota: APC y 488 son los anticuerpos secundarios utilizados aquí para la tinción HNK-1 y p75, respectivamente. </li><li> Se lavan las células en 10 ml de 1x PBS dos veces, volver a suspender 10 millones de células en 500 l de2% de FBS / HBSS que contenía DAPI (a 0,5 ng / l) o tinción de células alternativa apropiada en vivo para excluir las células muertas del análisis FACS. </li><li> Transferir las muestras de apropiarse tubos de FACS. </li><li> Preparar tubos de FACS con 0,5 ml de FBS al 2% / HBSS para la recolección de células. Nota: Las células y los tubos de recogida se mantienen en hielo en la oscuridad durante el tiempo de clasificación. </li><li> Usando una máquina de clasificación de células con rayos láser que pueden detectar DAPI, APC y 488 especie DAPI-/HNK-1 + / P75 + células dobles positivas. Nota: El tiempo de preparación y la manipulación de las células conduce a un pequeño porcentaje de muerte celular, DAPI manchas células muertas sólo por lo que permite a estas células para ser excluidos de la población ordenada. Cualquier mancha en vivo / muerto alternativa funciona igual de bien para este propósito. DAPI en sí no causa citotoxicidad en la población de células vivas. </li></ol> 6. Replating de células clasificadas, Mantenimiento NC y Expansión Nota: las células FACS ordenados deben ser parteconducido con especial cuidado para asegurar la supervivencia óptima. Manténgalos en hielo hasta resiembra ellos. No agite ni pipetear con dureza. Las células se pueden resuspendieron accionando el tubo. <ol><li> Después de la separación FACS, contar las células NC, a continuación, girar hacia abajo y resuspender en medio N2-diferenciación suplementado con 10 ng / ml de FGF2, 20 ng / ml de EGF y 10 mM de dihidrocloruro de Y-27632 en el volumen apropiado para replate ellos en 30.000 células / 10 gotas mu l. </li><li> Seque bien previamente preparado / placas Lam / FN PO y placa aproximadamente 50 a 70 gotas por placa de 10 cm. Deje que los platos se destacan a temperatura ambiente durante 20 a 30 min. </li><li> Añadir mucho cuidado 20 ml/10 cm plato de N2/FGF2/EGF/Y-27632 sin molestar a las gotas. Se incuban las células a 37 ° C. </li><li> Alimentar a las células cada 2-3 días con N2/FGF2/EGF. Nota: NC células se expanden o se pasaron aproximadamente cada 4-5 días o cuando empiezan a acumular dentro de las gotitas. </li><li> Para el paso de las células NC, eliminar el medio, wash las células una vez con PBS 1x y añadir 8 ml accutase/10 cm plato. Incubar durante 20 minutos a 37 ° C. </li><li> Pipetear las células de la placa usando una pipeta de 5 ml y transferir a un tubo de 15 ml. Añadir 6 ml 1x PBS. </li><li> Haga girar las células durante 5 minutos a 114 x g. </li><li> Resuspender las células en medio N2-diferenciación suplementado con 10 ng / ml de FGF2, 20 ng / ml de EGF y 10 mM de dihidrocloruro de Y-27632 con el fin de tener 20.000 células/10 l gotita. </li><li> Replate las células en 10 gotas mu l en placas de PO / Lam / FN secas. Deje que los platos se destacan a temperatura ambiente durante 20 a 30 min. </li><li> Añadir con cuidado 20 ml/10 cm plato de N2/FGF2/EGF/Y-27632. Se incuba a 37 ° C. Nota: NC células pueden mantenerse o ampliarse hasta por 2 semanas como progenitoras población relativamente homogénea. Cultura ya puede llevarlos a diferenciarse en diversos tipos de células progenie. </li></ol>

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Los autores no tienen conflictos de intereses a revelar.

Para la diferenciación de las células exitosa R-NC de células madre / hiPSCs deben hacerse las siguientes consideraciones. Es fundamental trabajar en condiciones de cultivo estériles en todo momento. En particular, es importante para poner a prueba las culturas HPSC para contaminación por micoplasma regularmente, ya que esto impedirá la contaminación diferenciación exitosa, pero no puede fácilmente ser detectado visualmente en cultivos de HPSC. La diferenciación R-NC debe iniciarse con la densidad celular 90-1...

Las células madre embrionarias (células madre) y las células madre pluripotentes inducidas humanas (hiPSC) han demostrado un enorme potencial, en particular para la investigación y el tratamiento futuro de enfermedades humanas para las que están disponibles ni los buenos modelos animales ni los tejidos primarios. Ejemplos de aplicación de la tecnología de células madre / hiPSC son los siguientes: Las células de especial interés pueden ser generados a partir de células madre / hiPSCs para la medicina regenerativa en cantidad ilimitada 1. Las células pueden producirse a partir de pacientes portadores de una enfermedad específica y se utiliza para establecer en modelos de enfermedad in vitro 2,3. Tales modelos de enfermedad pueden ser empleados para la detección de drogas a gran escala en la búsqueda de nuevos compuestos farmacológicos 4, así como las pruebas de los medicamentos existentes para la eficacia y la toxicidad 5. Modelos in vitro de enfermedades pueden conducir a la identificación de los mecanismos de las enfermedades nuevas. Para todas las aplicaciones de la tecnología de células madre / IPSC es importante trabajar con específica, bien definirtipos d células afectadas en la enfermedad de interés. Por lo tanto, la disponibilidad de protocolos de diferenciación in vitro sólidas y reproducibles en es crucial para todas las aplicaciones de la tecnología de células madre / hiPSC. Los protocolos son deseables que muestran una variabilidad mínima, gasto de tiempo, esfuerzo, dificultad y costo, así como la reproducibilidad máxima entre las líneas de células madre / hiPSC y diferentes investigadores. Las células de la cresta neural (NC) emergen durante neurulation vertebrados entre la epidermis y el epitelio neural. Ellos proliferan y migran ampliamente en todo el embrión en desarrollo y dan lugar a una impresionante diversidad de tipos de células progenie, incluyendo el hueso / cartílago, el esqueleto craneofacial, los nervios sensoriales, las células de Schwann, melanocitos, células de músculo liso, las neuronas entéricas, las neuronas autónomas, células cromafines , células cardiacas tabique, los dientes y las células glandulares suprarrenal / tiroideas 6. Por lo tanto, las células NC son un tipo de célula atractivo para el campo de células madre e importante para lamodelización de una variedad de enfermedades, como la enfermedad de Hirschsprung 7, disautonomía familiar 8, así como cánceres tales como el neuroblastoma 9. Además, ofrecen la posibilidad de estudiar los aspectos del desarrollo embrionario humano in vitro. El actualmente disponibles y ampliamente aplicado en protocolo de diferenciación in vitro para la derivación de células NC de hESCs 10,11 requiere hasta 35 días de diferenciación y que implica la inducción neural en células alimentadoras tales como células del estroma MS5 y por lo tanto se lleva a cabo bajo condiciones de mal definidos. Si bien puede ser en mayor escala para generar grandes cantidades de células NC, por ejemplo se requiere para la detección de drogas de alto rendimiento 4, se trata de mano de obra y costos de obra. Además, se trata de pases manual de las rosetas de los nervios, que pueden ser difíciles de reproducir y por lo tanto está sujeta a la variabilidad general, en particular cuando se aplica a una gran variedad de células madre o hiPSClíneas. Aquí, se muestra la derivación gradual de células NC en un protocolo de 18 días que está libre de células alimentadoras. Este método es más corto y más definido que el protocolo utilizado actualmente. Además, es muy robusto en la generación de células NC entre diferentes líneas hiPSC. Es importante destacar que se ha demostrado que las células NC producidas por ambos protocolos surgen en la frontera de los rosetones de los nervios (en lo sucesivo denominado roseta-NC o R-NC). Las células derivadas utilizando cualquiera de los dos protocolos parecen morfológicamente idénticas, expresan los mismos marcadores NC y se agrupan en el análisis de microarrays. NC células derivadas usando el nuevo protocolo (R-NC) son funcionales, similar a las células derivadas NC usando el protocolo antiguo (MS5-R-NC) tal que pueden migrar y diferenciarse en neuronas más. Por lo tanto, las células se pueden utilizar simultáneamente con las células MS5-R-NC. El protocolo de células R-NC para la derivación de células NC de células madre / IPSC será útil para todas las aplicaciones de la tecnología de células madre / IPSC que implica el linaje NC. &lt;/ P&gt;

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			<td height="21" style="height:21px;width:353px;">Regents</td>
			<td style="width:219px;">company</td>
			<td style="width:160px;">cataloge number</td>
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			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>11965-092</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
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			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Gibco-Life Technologies</td>
			<td>11330-032</td>
			<td></td>
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			<td height="20" style="height:20px;">Knockout Serum Replacement</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td>Lot should be tested</td>
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			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
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			<td height="20" style="height:20px;">Penicinlin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td></td>
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			<td height="20" style="height:20px;">MEM minimum essential amino acids solution&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-050</td>
			<td></td>
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			<td height="20" style="height:20px;">&#946;-Mercaptoethanol&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>toxic</td>
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			<td height="20" style="height:20px;">Recombinant human FGF basic (FGF2)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td></td>
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			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
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			<td height="20" style="height:20px;">DMEM/F12 powder&#160;</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
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			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
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			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
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			<td height="20" style="height:20px;">APO human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
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			<td height="20" style="height:20px;">Human insulin&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
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			<td height="20" style="height:20px;">Putriscine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
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			<td height="20" style="height:20px;">Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
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			<td height="20" style="height:20px;">Poly-L Ornithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td></td>
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			<td height="20" style="height:20px;">Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td></td>
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			<td height="20" style="height:20px;">Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dispase in Hank&#39;s Balanced Salt Solution 5U/ml</td>
			<td>Stem Cell Technologies</td>
			<td>7013</td>
			<td></td>
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			<td height="20" style="height:20px;">Trypsin-EDTA&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
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			<td height="20" style="height:20px;">Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
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			<td height="20" style="height:20px;">LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
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			<td height="20" style="height:20px;">Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
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			<td height="20" style="height:20px;">Ascorbic Acid&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
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			<td height="20" style="height:20px;">BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td></td>
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			<td height="20" style="height:20px;">FGF8</td>
			<td>R&#38;D Systems</td>
			<td>423-F8</td>
			<td></td>
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			<td height="20" style="height:20px;">Mouse recombinant sonic hedgehog (SHH)</td>
			<td>R&#38;D Systems</td>
			<td>464SH</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
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			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco-Life Technologies</td>
			<td>14170-112</td>
			<td></td>
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			<td height="20" style="height:20px;">HEPES</td>
			<td>Gibco-Life Technologies</td>
			<td>1563-080</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant EGF</td>
			<td>R&#38;D Systems</td>
			<td>236EG</td>
			<td></td>
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			<td height="20" style="height:20px;">gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Anti HNK-1/N-Cam (CD57) mIgM</td>
			<td>Sigma</td>
			<td>C6680-100TST</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Anti-p75 mIgG1 (Nerve Growth factor receptor)</td>
			<td>Advanced Targeting Systems</td>
			<td>AB-N07</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;">APC rat anti-mIgM</td>
			<td>BD Parmingen</td>
			<td>550676</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 goat anti-mouse IgG1&#160;</td>
			<td>Invitrogen</td>
			<td>A21121</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Anti Oct4 mIgG2b (used at 1:200 dilution)</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Material/Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-6105M</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Cell culture dishes: 10cm and 15 cm plates, centrifuge tubes, FACS tubes,&#160; pipetts, pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass hematocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture incubator (CO2, humidity and temperature controlled)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture laminar flow hood with embedded microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture biosafety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell sorting machine, i.e. MoFlo</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml TB syringe 27Gx1/2</td>
			<td>BD Biosciences</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell lifter Polyethylene</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td></td>
		</tr>
	</tbody>
</table>

feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells

Células madre pluripotentes humanas (hPSCs) tienen un gran potencial para el estudio de desarrollo embrionario humano, para el modelado de enfermedades humanas en el plato y como una fuente de células trasplantables para aplicaciones de regeneración después de la enfermedad o accidentes. Células de la cresta neural (NC) son los precursores para una gran variedad de células somáticas adultas, tales como células del sistema nervioso periférico y glía, melanocitos y células mesenquimales. Ellos son una valiosa fuente de células para estudiar aspectos del desarrollo embrionario humano, incluida la especificación del destino celular y la migración. Además diferenciación de las células progenitoras NC en tipos de células diferenciadas terminalmente ofrece la posibilidad de modelar enfermedades humanas in vitro, investigar mecanismos de la enfermedad y generar células para la medicina regenerativa. En este artículo se presenta la adaptación de un protocolo vitro disponibles actualmente en la diferenciación para la derivación de células NC desde hPSCs. Este nuevo protocolo requiere 18 días de diferenciación, es-alimentador libre, fácilmente escalable y altamente reproducible entre células madre embrionarias humanas (hESC) líneas, así como células madre pluripotentes inducidas humanas (hiPSC) líneas. Ambos viejos y nuevos protocolos producen células NC de igual identidad.

Las dos mejoras más importantes del protocolo R-NC a través del protocolo MS5-R-NC 11 son las condiciones de diferenciación, definidos libres de alimentador y el acortamiento general del requisito de tiempo. Células alimentadoras MS5 13 son de médula ósea células del estroma derivadas murinas que han sido mostrados para apoyar la diferenciación neural de hESCs 14. Células madre embrionarias humanas cultivadas sobre células alimentadoras MS5 en estructuras de baja densidad de fo...

Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Las células de la cresta neural (NC) derivadas de células madre pluripotentes humanas (HPSC) tienen un gran potencial para modelar el desarrollo humano y las enfermedades y para las terapias de reemplazo celular. Aquí, una adaptación libre del alimentador ampliamente utilizado en la actualidad<em&gt; In vitro</em&gt; Protocolo de diferenciación para la derivación de células NC de hPSCs se presenta.

Derivación-Feeder libres de Células Progenitoras de la cresta neural Pluripotentes Células Madre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

feeder-free-derivation-neural-crest-progenitor-cells-from-human

Research

JoVE Journal

Neuroscience

14.1K vistas.  Sloan-Kettering Institute for Cancer Research. Las células de la cresta neural (NC) derivadas de células madre pluripotentes humanas (HPSC) tienen un gran potencial para modelar el desarrollo humano y las enfermedades y para las terapias de reemplazo celular. Aquí, una adaptación libre del alimentador ampliamente utilizado en la actualidad In vitro Protocolo de diferenciación para la derivación de células NC de hPSCs se presenta.

Video: Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural formadoras de colonias ensayo de células: un ensayo para discriminar Bona Fide células troncales nerviosas de células progenitoras neurales

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Investigación

Neurociencias

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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

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

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

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

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

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivación eficiente de los Derechos Humanos progenitores neuronales y neuronas de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

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

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

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

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

La especificación de telencefálicas neuronas glutamatérgicas a partir de células madre pluripotentes

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dirigida dopaminérgica Neurona La diferenciación de las células madre pluripotentes humanas

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Derivación de adultos fibroblastos humanos y de su conversión directa en células progenitoras neurales expandible

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificación de marcadores de superficie celular de células madre neuronales primarias y células progenitoras por etiquetado metabólico de sialoglicano

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...