The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

1 Preparación de Medios de Cultivo <ol><li> Preparar fibroblastos embrionarios de ratón (MEF) medio combinando lo siguiente: 445 ml de DMEM, 50 ml de suero bovino fetal (FBS), y 5 ml 100x penicilina / ampicilina solución madre. Mantenga el filtro de medio esterilizado a 4 ° C durante no más de 14 días. </li><li> Preparar medio de cultivo HPSC combinando lo siguiente que contiene suero: 385 ml de DMEM / F12, 100 ml de suero de reemplazo knockout (KSR), 5 ml 100x solución no esencial solución de aminoácidos de stock, 5 ml 100x penicilina / ampicilina de stock, 5 ml 100x mercaptoetanol solución madre, y 10 ng / ml de bFGF. Mantenga el filtro de medio esterilizado a 4 ° C durante no más de 10 días. </li><li> Preparar medio libre de suero mTeSR1 combinando lo siguiente: 400 ml mTeSR1 medio basal, 100 ml 5x suplemento, y 5 ml 100x penicilina / ampicilina solución stock. Mantenga medio a 4 ° C durante no más de 10 días. </li><li> Preparar 10x colagenasa solución IV existencia: pésense 0,5 g potencia colagenasa IV, ydisolver con 50 ml de DMEM / F12 y esterilizar filtro. Hacer alícuotas y almacenarlos a -20 ° C durante meses. </li><li> Prepare la solución de gelatina al 0,1%: Pesar 0,5 g de gelatina (de piel bovina, tipo B) de potencia y se disuelven con agua desionizada. Mantenga autoclave solución esterilizada a temperatura ambiente durante semanas. </li><li> Preparar medio de diferenciación KSR combinando lo siguiente: 410 ml de DMEM, 75 ml KSR, 5 ml 100x solución no esenciales de aminoácidos de stock, 5 ml 100x penicilina / ampicilina solución madre, 5 ml 100x solución madre mercaptoetanol. Mantenga el filtro de medio esterilizado a 4 ° C durante no más de 10 días. NOTA: KSR varía de un lote a otro, lo que puede afectar a la eficiencia de la diferenciación. Por tanto, es mejor probar varios lotes de KSR para encontrar el mejor para la diferenciación. </li><li> Preparar medio de diferenciación N2 mediante la combinación de lo siguiente: 98 ml de DMEM, 1 ml suplemento N2 100x y 100x 1 ml de penicilina / ampicilina solución stock. Mantenga el filtro de medio esterilizado en 4° C durante no más de 10 días. </li><li> Preparar medio de diferenciación B27 mediante la combinación de lo siguiente: medio neurobasal 480 ml, 10 ml 50x suplemento B27, 5 ml 100x solución madre glutamax, y 5 ml 100x penicilina / ampicilina solución stock. Mantenga el filtro de medio esterilizado a 4 ° C durante no más de 10 días. </li></ol> 2. Cultura de hESCs y hiPSCs sobre células alimentadoras MEF Los H9 hESCs se obtuvieron de Research Institute WiCell hiPSCs y se establecieron en el laboratorio Reijo Pera través de la transducción mediada por retrovirus de Yamanaka factores de OCT3 / 4, Sox2, Klf4 y c-MYC 18. <ol><li> Al menos un día antes de la aprobación de células, preparar algunos platos de gelatina mediante la adición de 1 ml de 0,1% de gelatina al 1 pocillo de placas de 6 pocillos, y las placas se incuban a 37 ° C durante al menos 30 min. </li><li> Después de la incubación, aspirado de gelatina a partir de las placas, y las semillas de la irradiación inactivada MEF células en las placas a una densidad de 1,6 x 10 5 células/ Pocillo en medio MEF usando un hemocitómetro para contar las células. NOTA: Opcionalmente preparar alimentadores MEF tan pronto como 3 días antes de la aprobación de la célula. </li><li> Células Passage un día después de enchapado alimentadores MEF: medianas aspirado desde hPSCs, añadir 1 mg / ml de colagenasa IV a las células en 1 ml / pocillo, y se incuban las células a 37 ° C durante 1 hora. </li><li> Durante esta incubación, lavar alimentadores MEF una vez con PBS, y luego agregar agua tibia (37 ° C) medio de cultivo que contiene suero HPSC a 1,5 ml / pocillo. </li><li> Después de 1 hora, toque en la placa de cultivo de un par de veces, así que la mayoría de las colonias indiferenciadas se desprenda de las placas, mientras que las colonias diferenciadas permanecen unidos. Recoger cuidadosamente las colonias flotantes, y transferirlos al tubo de 15 ml. </li><li> Lave suavemente la placa una vez con agua caliente (37 ° C) DMEM / F12 y transferir DMEM / F12 en el mismo tubo de 15 ml. </li><li> Centrifugar los tubos a 179 xg durante 3 min. </li><li> Aspirar medio de los tubos, teniendo cuidado de no perturbar el sedimento. Añadir guerramedio de cultivo m HPSC, pipetear las células arriba y abajo varias veces para romper en pequeños grupos y mezclar bien. </li><li> Transferir las células sobre nuevos alimentadores MEF en 1: 3-1: 6 relación. </li><li> Cambie el medio todos los días y el paso de las células cada 5-6 días. </li></ol> 3. Preparación de las células para la diferenciación <ol><li> Al menos un día antes de la preparación de las células, preparar algunos platos de Matrigel (típicamente 3 pocillos de una placa de 6 pocillos). Diluir el Matrigel con frío (4 ° C) DMEM / F12 a las 1:40 y se añade 1 ml de Matrigel por pocillo de placas de 6 pocillos. Incubar las placas Matrigel a 4 ° C durante la noche. NOTA: Se puede sellar placas Matrigel con Parafilm y almacenarlos a 4 ° C durante todo el tiempo de una semana. </li><li> Usar células (ver paso 2) 4 días después del pasaje. Retire todas las colonias diferenciadas de las placas. Aspirar medio de la placa de cultivo, añadir 1 ml accutase por pocillo, y se incuban las células a 37 ° C durante 5 min. </li><li> Durante la incubación, preparar algunos gelmérica placas mediante la adición de 1 ml de gelatina al 1 pocillo de placas de 6 pocillos. Coloque estos platos y los Matrigel placas preparadas un día antes en la incubadora. </li><li> Después de la incubación de 5 min o cuando las células se han convertido en semi-flotante, las células de la pipeta hacia arriba y abajo varias veces para hacerlos en células individuales. </li><li> Recoger las células individuales en tubos de 15 ml y centrifugar a 258 xg durante 3 min. </li><li> Resuspender las células con cantidad apropiada de suero que contiene medio de cultivo HPSC, y añadir Thiazovivin a la concentración final de 2 mM. </li><li> Células de transferencia sobre placas recubiertas con gelatina en 1: 1, e incubar las células a 37 ° C durante 30 min para eliminar los alimentadores del MEF. </li><li> Transferir las células a un tubo cónico de 15 ml, añadir el suero que contiene medio apropiado HPSC y contar las células con un hemocitómetro. </li><li> Centrifugar las células a 258 xg durante 3 min. </li><li> Durante centrífuga, añadir Thiazovivin apropiarse medio mTeSR1 para hacer una concentración de 2 mM. </li><li> Después de centrifugación, unaespirar el medio y añadir medio mTeSR1 apropiado con Thiazovivin modo que hay 1,8 x 10 5 células / ml de medio. </li><li> Saque las placas Matrigel de la incubadora, aspirar el Matrigel y añadir 2 ml de las células mixtas en 1 pocillo de las placas de 6 pocillos. NOTA: La densidad celular debe ser de 3,6 x 10 4 células / cm 2 de superficie. </li><li> Vuelva a poner las placas de nuevo a la incubadora, y dejar que las células se adhieren durante 24 horas. </li><li> 24 horas después de la siembra de las células, aspirar medio de edad y añadir 2 ml nuevo medio mTeSR1 por pozo y dejar que las células crezcan durante otras 24 horas antes de iniciar la diferenciación. </li></ol> Diferenciación Celular <ol><li> Iniciar diferenciación 48 horas después de resiembra las células en placas Matrigel. </li><li> Aspirar el medio de cultivo de la placa, lavar las células una vez con PBS, y añadir 2 ml del siguiente medio de diferenciación por pozo: medio de diferenciación KSR suplementado con 10 mM SB431542 y 100 nM LDN-193189. Marcar este día como D0 de diferenciación. </li><li> Cambie medio todos los días de D0 a D20. NOTA: Se puede preparar el medio para el uso no más de 2 días a la vez. </li><li> Utilice el siguiente medio para D1 y D2: medio de diferenciación KSR suplementado con 10 M SB431542, 100 nM LDN-193189, 0,25 M SAG, 2 M purmorphamine, y 50 ng / ml FGF8b. </li><li> Utilice el siguiente medio para D3 y D4: medio de diferenciación KSR suplementado con 10 M SB431542, 100 nM LDN-193189, 0,25 M SAG, 2 M purmorphamine, 50 ng / ml FGF8b, y 3 M CHIR99021. </li><li> Utilice el siguiente medio de D5 y D6: 75% medio de diferenciación KSR más el 25% medio de diferenciación N2 suplementado con 100 nM LDN-193189, 0,25 M SAG, purmorphamine 2 M, 50 ng / ml FGF8b, y 3 M CHIR99021. </li><li> Utilice el siguiente medio de D7 y D8: 50% medio de diferenciación KSR más el 50% medio de diferenciación N2 complementado con 100 nM LDN-193189 y 3 M CHIR99021.</li><li> Utilice el siguiente medio de D9 y D10: 25% medio de diferenciación KSR más el 75% medio de diferenciación N2 complementado con 100 nM LDN-193189 y 3 M CHIR99021. </li><li> Utilice el siguiente medio para D11 y D12: B27 diferenciación medio suplementado con 3 mM CHIR99021, 10 ng / ml BDNF, 10 ng / ml de GDNF, 1 ng / ml TGF3, ácido ascórbico 0,2 mM y 0,1 mM cAMP. </li><li> Utilice el siguiente medio para el resto de la diferenciación: medio de diferenciación B27 suplementado con 10 ng / ml BDNF, 10 ng / ml de GDNF, 1 ng / ml TGF3, ácido ascórbico 0,2 mM y 0,1 mM cAMP. </li><li> A eso de D16 de la diferenciación, preparar algunos platos / laminina / fibronectina Poly-L-ornitina. Diluir poli-L-ornitina solución madre con el frío (4 ° C) PBS a 15 mg / ml, añadir 1 ml a 1 pocillo de placas de 6 pocillos y se incuban las placas a 37 ° C durante la noche. </li><li> Aspirar la solución de poli-L-ornitina, lavar las placas 3 veces con agua estéril y luego secar al aire las placas. </li><li> Diluir la laminina de stock sollución de frío (4 ° C) PBS a 1 mg / ml, añadir 1 ml a un pocillo de placas de 6 pocillos, e incubar las placas a 37 ° C durante la noche. </li><li> Aspirar la solución de laminina, lavar las placas 3 veces con agua estéril y luego secar al aire las placas. </li><li> Diluir la solución de fibronectina de stock de frío (4 ° C) PBS a 2 mg / ml, añadir 1 ml a 1 pocillo de placas de 6 pocillos y se incuban las placas a 37 ° C durante la noche. </li><li> Placas sello con Parafilm y colocarlos a 4 ° C durante hasta dos semanas. </li><li> Después de 20 días de diferenciación, replate las células a las placas / laminina / fibronectina Poly-L-ornitina. Medio de diferenciación Aspirar, añadir 1 ml caliente (37 ° C) Accutase a 1 pocillo de placas de 6 pocillos y se incuban las células a 37 ° C durante 5 min. </li><li> Durante la incubación, colocar las placas / laminina / fibronectina Poly-L-ornitina en la incubadora. </li><li> Después de la incubación de 5 min o cuando las células se han convertido en semi-flotantes, suavemente las células de pipeta hacia arriba y abajo varias veces aconvertirlas en células individuales. </li><li> Recoger las células individuales en tubos de 15 ml cónicos, y añadir la cantidad apropiada de medio neurobasal para el recuento celular, que variará dependiendo de expansión (después de 11 días de diferenciación, las células pueden expandirse 15-20 veces). Contar las células utilizando un hemocitómetro. </li><li> Centrifugar las células a 258 xg durante 3 min. Aspirar el sobrenadante y volver a suspender las células con medio de diferenciación B27 de manera que la concentración de células es de 1 x 10 6 células / ml de medio. </li><li> Aspirar fibronectina de las placas, lavar dos veces con PBS, y añadir 2 ml de células a 1 pocillo de placas de 6 pocillos. NOTA: La densidad celular para replating debe ser de 2 x 10 5 células / cm 2 de superficie. </li><li> Cambie medio 24 horas más tarde para eliminar las células muertas sin ataduras. </li><li> Cambio de medio cada dos días hasta los puntos de tiempo deseados para un experimento dado. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Células madre pluripotentes humanos (incluyendo tanto hESCs y hiPSCs) pueden diferenciarse in vitro para generar la mayoría, si no todos, los tipos de células de nuestro cuerpo incluyendo las neuronas DA, que se ha demostrado en estudios previos 19. Aquí, basado en el conocimiento del desarrollo de neuronas dopaminérgicas embrionarias 20 y protocolos publicados de otros laboratorios 14,15, hemos optimizado las condiciones de cultivo para la generación de neuronas DA de hES...

Dopaminérgico (DA), las neuronas se pueden encontrar en varias regiones del cerebro, incluyendo el cerebro medio, el hipotálamo, la retina y bulbos olfativos. Las neuronas A9 de DA en la substantia nigra pars compacta (SNpc) controlar el comportamiento y el movimiento mediante la proyección en el cuerpo estriado en el cerebro anterior y formando el sistema motor extrapiramidal. La degeneración de las neuronas DA A9 conduce a la enfermedad de Parkinson (PD), que es el segundo trastorno neurodegenerativo más común humano y actualmente incurable. Terapia de reemplazo celular es una de las estrategias más prometedoras para el tratamiento de la EP 1, por lo tanto, ha habido un gran interés en la obtención de las neuronas DA A9 a partir de células madre pluripotentes humanas (hPSCs), incluyendo tanto las células madre embrionarias humanas (hESCs) y el células recientes de origen humano madre pluripotentes (hiPSCs). Muchas investigaciones han tratado de derivar neuronas DA A9 de hPSCs utilizando diversos métodos. Los primeros informes de todas las neuronas DA generados a través de una neuraletapa progenitor roseta. Por co-cultivo con MS5 2 o PA6 3-5 células estromales con o sin factores de crecimiento externo para 2-4 semanas, L. Studer y colegas y otros tres grupos inducidos con éxito hESCs para producir rosetas neuronales. A continuación, enriquecidos estas rosetas por la disección mecánica o digestión enzimática para su posterior diferenciación. En otros informes, los investigadores generaron rosetas neuronales a través del cuerpo embrioide (EB) la diferenciación de la cultura 6.9 flotantes. Más tarde, los investigadores establecieron un método de diferenciación monocapa basada 10,11, donde se sembraron hESCs y hiPSCs en matriz extracelular, añadieron diferentes factores de crecimiento en el cultivo para inducir la diferenciación de hPSCs a las neuronas DA, que imita el desarrollo in vivo de las neuronas DA en embrionario . Aunque todos estos estudios obtuvieron tirosina hidroxilasa (TH) células que expresaban con algunas características de las neuronas DA, todo el proceso de diferenciación es que consume tiempo y trabajo,generalmente ineficientes, y más importante, la identidad A9 de estas neuronas no se demostró en la mayoría de los estudios, excepto el uno con LMX1A expresión ectópica 12. Recientemente, una nueva placa de piso (FP) basada en protocolo se desarrolló 13-16, en el que los precursores de PF con potencial de neuronas DA se generaron por primera vez por la activación de la erizo sónico y las vías canónicas de señalización Wnt durante la etapa temprana de la diferenciación, y luego estas células de PF se especificaron aún más a las neuronas DA. Aunque este protocolo es más eficiente, todavía hay algunos problemas; por ejemplo, todo el proceso de diferenciación toma mucho tiempo (al menos 35 días) y es alimentador celular dependiente 15, o es dependiente de EB 16 o la identidad A9 no se demostró 14. Aquí, a partir del conocimiento de in vivo el desarrollo de las neuronas DA embrionario y resultados publicados de otros investigadores, hemos optimizado las condiciones de cultivo para la efciente generación de neuronas DA de ambos hESCs y hiPSCs. Se generó por primera vez las células precursoras FP por la activación de la señalización de Wnt canónica con la pequeña molécula de CHIR99021 y sonic hedgehog de señalización con moléculas pequeñas SAG y purmorphamine. Estas células expresan FP FOXA2, LMX1A, CORIN, OTX2 y Nestin. A continuación, especificamos estas células FP a las neuronas DA con factores de crecimiento incluyendo BDNF, GDNF, etc. Las neuronas DA generados son del tipo de células A9 ya que son positivos para GIRK2 mientras negativo para Calbindin 17. Este protocolo es de células de alimentación o EB independiente, altamente eficiente y reproducible. El uso de este protocolo, se puede derivar neuronas DA en menos de 4 semanas a partir de hESCs o hiPSCs de las personas normales para el estudio de trasplante de células, o de hiPSCs de los pacientes con EP para el modelado in vitro de la EP o probar agentes terapéuticos potenciales para la EP.

<table border="0" cellpadding="0" cellspacing="0" width="778">
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			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
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			<td style="width:167px;">Comments/Description</td>
		</tr>
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			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
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			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
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			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
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			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
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			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
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			<td style="width:119px;">21103-049</td>
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			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">PBS</td>
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			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
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			<td height="21" style="height:21px;width:308px;">SB431542</td>
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			<td style="width:119px;">1614</td>
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			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
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			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
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			<td height="21" style="height:21px;width:308px;">FGF8b</td>
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			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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.

Una visión general del protocolo de diferenciación se muestra en la Figura 1. La eficiencia del protocolo de diferenciación que aquí se presenta se basa en el estado de las células de partida. Por lo tanto, es crítico para asegurarse de que, primero uno elimina todas las colonias diferenciadas antes de disociarse hPSCs en células individuales para la diferenciación, y segundo, uno agota la mayoría, si no todas, las células alimentadoras MEF mediante la incubación de las células en placas rec...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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 ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.3K vistas.  Stanford University School of Medicine. 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’s disease.

Video: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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 ...

Investigación

Neurociencias

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 ...

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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 ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Replacado posterior a la diferenciación de las neuronas derivadas de células madre pluripotentes humanas para la detección de alto contenido de la neuritogénesis y la maduración de la sinapsis

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Diferenciación neuronal de las células madre embrionarias del ratón in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Derivación, expansión, criopreservación y caracterización de células endoteliales microvasculares cerebrales de células madre pluripotentes inducidas por humanos

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

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

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

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

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

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

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Cultivos organotípicos de corteza humana adulta como modelo ex vivo para el trasplante y validación de células madre humanas

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...