Este trabajo fue apoyado por la Fundación Nacional de Ciencias Naturales de China (Nº 31501099) y el Departamento de Educación Media y Joven de la Provincia de Hubei, China (No. Q20191104). Además, agradecemos al profesor Wensheng Deng de la Universidad de Ciencia y Tecnología de Wuhan por proporcionar las líneas de células madre embrionarias del ratón A2lox.

1. Cultivo de células madre embrionarias del ratón
<ol><li>Prepare platos o platos de cultivo celular recubiertos de gelatina al 0,1%.<ol>
<li>Añadir 2 ml de gelatina esterilizada del 0,1% (0,1% w/v en agua) a platos de cultivo celular de 60 mm. Rockear suavemente para asegurar incluso el recubrimiento de los platos de cultivo celular.</li>
		<li>Coloque los platos en una incubadora de CO2 al 5% a 37 °C y deje el recubrimiento durante 1 h.</li>
		<li>Retire la solución de gelatina del 0,1% antes de sembrar las células. NOTA: Después de retirar la gelatina, no es necesario secar o lavar los platos recubiertos.</li>
	</ol>
</li>
	<li>Cultivo de células madre embrionarias del ratón (A2lox y 129)<ol>
<li>Incubar células mESC (A2lox y 129) en el 0,1% de gelatina recubierta de 60 mm de platos de cultivo celular en medio de crecimiento mESC a 37 °C en una incubadora de CO2 del 5%, respectivamente. El medio de crecimiento del mESC consiste en un 85% de eliminación dmem/F12, 15% reemplazo sérico knock-out (KSR), 0,1 mM β-mercaptoetanol (2ME), 2 mM GlutaMAX, 1% aminoácido no esencial (NEAA), 1% penicilina/estreptomicina (P/S), 1000 factor inhibitorio de leucemia U/mL (LIF), 10 nM CHIR-99021 (inhibidor de GSK-3) y 0,33 nM PD0325901 (inhibidor de MEK). PRECAUCIÓN: β-mercaptoetanol es inflamable y tiene toxicidad por inhalación. Manténgase alejado de las fuentes de fuego y use una máscara para evitar la inhalación cuando se utilice.</li>
		<li>Cambie el medio de crecimiento del MESC diariamente para un mejor crecimiento de A2lox y 129.</li>
		<li>Cuando las células alcancen el 80% de confluencia, retire el medio y agregue 1 ml de 0,1% de trippsina al plato. Mezcle suavemente durante 30 s para asegurar una cobertura uniforme de trypsina en todas las células.</li>
		<li>Deje las celdas durante aproximadamente 1 minuto para probar eltamaño de la triptina y luego retire la trippsina usando pipeta de 1 ml.</li>
		<li>Añadir 2 ml de medio de crecimiento mESC al plato, pipeta arriba y abajo varias veces para hacer una sola suspensión celular.</li>
		<li>Contar la densidad de las células en la suspensión con la mayor precisión posible utilizando hemociclo.</li>
		<li>Divida las células en 7 grupos e induzca la diferenciación utilizando diferentes protocolos que se muestran en la Tabla 1.</li>
	</ol>
</li>
</ol>2. Diferenciación de mESC a PNJ (Fase I)
<ol><li>Prepare placas de cultivo celular recubiertas de gelatina al 0,1% o cubre manchas.<ol>
<li>Antes de su uso, prepare 0.1% de placas de gelatina recubiertas de 6 pozos o cubrecubres como en el paso 1.1.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 1 (Tabla 1)<ol>
<li>Semillas de aproximadamente 2 x 104 mESC en 2 ml de medio de diferenciación basal I por pozo en las placas de 6 pozos recubiertas de gelatina al 0,1%. Compruebe la densidad celular bajo un microscopio.</li>
		<li>Incubar las células a 37 °C en una incubadora de CO2 al 5% durante 6 h para permitir el apego.</li>
		<li>Después del apego, saque de las células de la incubadora y lave las células dos veces con 2 ml de PBS.</li>
		<li>Agregue 2 ml de medio de diferenciación basal I(Tabla 1)a cada pozo y vuelva a colocar las células en la incubadora.</li>
		<li>Deje las células para la diferenciación durante 8 días. Reemplace el medio de diferenciación basal I cada 2 días.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 2 (Tabla 1)<ol>
<li>Añadir 1,5 x 106 mESC en un plato bacteriano nodhesivo en 10 ml de medio de diferenciación basal I para permitir la formación de cuerpos embrionados a 37 °C en una incubadora de CO2 del 5%.</li>
		<li>Después de 2 días, transfiera los agregados celulares a 15 tubos centrífugas de ml y déjelos establecer por gravedad.</li>
		<li>Retire el sobrenadante y agregue 10 ml de medio de diferenciación basal fresco I para resuspend los cuerpos embrionados. Replantarlos en un nuevo plato bacteriano nodhesivo y permitir la diferenciación durante otros 2 días.</li>
		<li>Compruebe la formación de cuerpos embrionarios bajo el microscopio (Figura 2A).</li>
		<li>Recoger cuerpos embrionarios como se describe en los pasos 2.3.2-2.3.3. Semillas alrededor de 50 cuerpos embrionarios en 2 ml de medio de diferenciación basal I por pozo en placas de 6 pozos recubiertas de gelatina del 0,1%.</li>
		<li>Prepare 1 mM de caldo ra totalmente trans (en DMSO) y guárdelo lejos de la luz en un congelador de -80 °C después del subenvasado. NOTA: La AR es inestable, y se debe prestar atención a mantenerla alejada de la luz y reducir el contacto con el aire durante la preparación del caldo de RA.</li>
		<li>Para la inducción de AR, agregue 2 μL de caldo de AR en cada pozo para hacer una concentración final de 1 μM.</li>
		<li>Coloque la placa en la incubadora de CO2 al 5% a 37 °C y diferencie durante otros 4 días.</li>
		<li>Cambie los 2 ml enteros del medio de diferenciación basal I (con 1 μM RA) cada 2 días.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 3 (Tabla 1)<ol>
<li>Plantar 1,5 x 106 mESC en un plato bacteriano nodhesivo en 10 ml de medio de diferenciación basal I. Dejar durante 2 días para la formación de cuerpos embrionados a 37 °C en una incubadora de CO2 del 5%.</li>
		<li>Compruebe la formación de cuerpos embrionarios bajo un microscopio(Figura 2B).</li>
		<li>Transfiera los agregados celulares a tubos centrífugas de 15 ml y déjelos establecer por gravedad.</li>
		<li>Retire el sobrenadante cuidadosamente y agregue 10 ml de medio de diferenciación basal fresco I para volver a usarlos.</li>
		<li>Sembrar alrededor de 50 cuerpos embrionarios en 2 ml de medio de diferenciación basal I por pozo en placas de 6 pozos recubiertas de gelatina al 0,1%.</li>
		<li>Para la inducción de AR, agregue 2 μL de caldo de AR en cada pozo para hacer una concentración final de 1 μM.</li>
		<li>Coloque la placa en la incubadora de CO2 al 5% a 37 °C y diferencie durante otros 6 días. Cambie todo el medio de diferenciación basal I (con 1 μM RA) cada 2 días.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 4 (Tabla 1)<ol>
<li>Semillas de aproximadamente 2 x 104 mESC dentro de 2 ml de medio de diferenciación basal I por pozo en las placas de 6 pozos recubiertas de gelatina al 0,1%. Coloque la placa en la incubadora de CO2 al 5% a 37 °C para permitir la fijación durante 6 h.</li>
		<li>Después de la fijación, lave las células dos veces con 2 ml de PBS. Añadir 2 ml de medio de diferenciación basal I a cada pozo y permitir la diferenciación durante 4 días en la incubadora de CO2 del 5% a 37 °C.</li>
		<li>Para la inducción por AR, añadir 2 μL de caldo de AR trans en cada pozo (la concentración de trabajo es de 1 μM) para inducir la diferenciación durante otros 4 días.</li>
		<li>En todo el proceso, reemplace todo el medio cada 2 días.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 5 (Tabla 1)<ol>
<li>Semillas de aproximadamente 2 x 104 mESC dentro de 2 ml de medio de diferenciación basal I por pozo en las placas de 6 pozos recubiertas de gelatina al 0,1%. Coloque la placa en la incubadora de CO2 al 5% a 37 °C para permitir la fijación durante 6 h.</li>
		<li>Lave las células dos veces con 2 ml de PBS. Añadir 2 ml de medio de diferenciación basal I a cada pozo y permitir la diferenciación durante 2 días en la incubadora de CO2 al 5% a 37 °C.</li>
		<li>Para la posterior inducción de AR, añadir 2 μL de caldo de AR en cada pozo para hacer una concentración final de 1 μM. Coloque la placa en la incubadora de CO2 al 5% a 37 °C para inducir la diferenciación durante otros 6 días.</li>
		<li>En todo el proceso, reemplace todo el medio cada 2 días.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 6 (Tabla 1)<ol>
<li>Plantar 1,5 x 106 mESC en un plato bacteriano nodhesivo en 10 ml de N2B27 medio II(Tabla 1)para permitir la formación de cuerpos embrionarios.</li>
		<li>Eldía 2, recoja los agregados celulares como se describe en los pasos 2.3.2-2.3.3 y resuspend los cuerpos embrionarios usando 10 ml de N2B27 medio II fresco.</li>
		<li>Replantarlos en un nuevo plato bacteriano nodhesivo y permitir la diferenciación durante otros 2 días en la incubadora de CO2 al 5% a 37 °C. Compruebe la formación de cuerpos embrionarios bajo microscopio.</li>
		<li>El día4, recoge cuerpos embrionarios. Sembrar alrededor de 50 cuerpos embrionarios por pozo en placas de 6 pozos recubiertas de gelatina del 0,1% con 2 ml de N2B27 medio II.</li>
		<li>Añadir 2 μL de caldo de AR totalmente trans en cada pozo e inducir la diferenciación durante otros 4 días. Reemplace todo el medio (N2B27 medio II por 1 μM RA) cada dos días.</li>
	</ol></li>
	<li>Diferenciación de fase I mediante el protocolo 7 (Tabla 1)<ol>
<li>Semillas de aproximadamente 2 x 104 mESC dentro de 2 ml de medio de diferenciación basal I por pozo en las placas de 6 pozos recubiertas de gelatina al 0,1%. Coloque la placa en la incubadora de CO2 al 5% a 37 °C para permitir la fijación durante 6 h.</li>
		<li>Lave las células dos veces con PBS. A continuación, añadir 2 ml de N2B27 medio II a cada pozo y permitir la diferenciación durante 8 días a 37 °C en una incubadora de CO2 del 5%.</li>
		<li>Cambie todo el medio N2B27 II cada 2 días.</li>
	</ol></li>
</ol>3. Observación morfológica celular
<ol><li>Compruebe el estado de diferenciación de los 7 grupos mencionados anteriormente diariamente bajo un microscopio de luz de contraste de fase invertido.</li>
	<li>Seleccione aleatoriamente al menos 12 campos y tome fotos para registrar los cambios morfológicos de cada grupo en D8.</li>
</ol>4. Tinción de inmunofluorescencia
<ol><li>Preparación de la muestra: Plecciones de semillas en 0.1% coverslips recubiertos de gelatina y permiten la diferenciación durante 8 días utilizando los protocolos mencionados en el paso 2.</li>
	<li>Enjuague: El día8, saque las muestras de la incubadora y retire el medio de diferenciación por aspiración. Enjuague suavemente las células una vez con 1 ml de PBS durante 5 minutos.</li>
	<li>Fijación: Agregue 1 ml de paraformaldehído al 4% a cada muestra y fije las celdas durante 20 minutos a temperatura ambiente (RT).</li>
	<li>Enjuague: Después de la fijación, enjuague suavemente las células con 1 ml de PBS 3 veces, durante 5 minutos cada una.</li>
	<li>Permeabilización: Añadir 1 ml de 0.2% TritonX-100 en PBS a cada muestra y dejar durante 8 minutos en RT.</li>
	<li>Enjuague: Después de la permeabilización, enjuague suavemente las células con 1 ml de PBS 3 veces, durante 5 minutos cada una.</li>
	<li>Bloqueo: Añadir 1 ml de suero de cabra al 10% en PBS a cada muestra e incubar en RT durante 1 h para bloquear cualquier interacción no específica.</li>
	<li>Incubación con anticuerpo primario<ol>
<li>Diluir el anticuerpo anti-Nestin a una proporción de 1:100 usando 5% suero de cabra en PBS.</li>
		<li>Aplicar 500 μL de anticuerpo diluido en diferentes muestras e incubar durante la noche a 4 °C.</li>
	</ol>
</li>
	<li>Enjuagar: Retire el anticuerpo y enjuague las muestras suavemente con 1 ml de PBS 3 veces durante 8 minutos cada una.</li>
	<li>Incubación con anticuerpo secundario<ol>
<li>Diluya el IgG anti-ratón de cabra con etiqueta Alexa Fluor 488 a una proporción de 1:500 usando un 5% de suero de cabra en PBS.</li>
		<li>Aplicar 500 μL de anticuerpo diluido en diferentes muestras e incubar en la oscuridad durante 2 h en RT. NOTA: Después de aplicar anticuerpo secundario fluorescente, realice todos los pasos posteriores en la oscuridad para evitar que se calme la fluorescencia.</li>
	</ol>
</li>
	<li>Enjuague: Retire el anticuerpo secundario y enjuague las muestras suavemente con 1 ml de PBS 3 veces durante 8 minutos cada una.</li>
	<li>Tinción y montaje nuclear<ol>
<li>Coloque una gota del medio de montaje DAPI en el microsluro limpio.</li>
		<li>Retire cuidadosamente las muestras de las placas y coloque la muestra encima del medio de montaje DAPI con la celda boca abajo. Dejar en la oscuridad durante 5 minutos en RT.</li>
		<li>Retire el exceso de medio de montaje DAPI con papel absorbente.</li>
	</ol>
</li>
	<li>Observación de microscopía de fluorescencia<ol>
<li>Coloque las muestras bajo la microscopía de fluorescencia y detecte la señal de DAPI y Alexa Fluor 488 utilizando filtros adecuados.</li>
		<li>Elija de manera uniforme y aleatoria 10-15 campos visuales diferentes para cada muestra y grabe las imágenes con una cámara CCD.</li>
	</ol>
</li>
</ol>5. Diferenciación de los PNJ a las neuronas (Fase II)
<ol><li>Preparar derivados mESC en la diferenciación de fase I utilizando el protocolo 3 (8 días, Cuadro 1)como se detalla en el paso 2.4, que tiene la mayor eficiencia de diferenciación (véase la Figura 3). NOTA: Después de la diferenciación de la fase I, el control de calidad debe llevarse a cabo utilizando la observación de morfología celular y el ensayo de inmunofluorescencia mencionado anteriormente, para garantizar un PNJ sano y de alto rendimiento.</li>
	<li>Semillas de aproximadamente 5 x 105 derivados mESC dentro de 2 ml de medio de diferenciación basal I por pozo en las placas de 6 pozos recubiertas de gelatina al 0,1%. Divida aleatoriamente los derivados del mESC en 3 grupos, como el protocolo de fase II 1, el protocolo 2 y el protocolo 3, respectivamente.</li>
	<li>Coloque la placa en la incubadora de CO2 al 5% a 37 °C para permitir la fijación durante 6 h. Lávalo dos veces con 2 ml de PBS.</li>
	<li>Añadir 2 ml de medio de diferenciación basal I, N2B27 medio I y N2B27 medio II(Tabla 2),respectivamente, a cada pozo de los grupos anteriores.</li>
	<li>Coloque las placas en la incubadora y deje diferenciar durante otros 10 días. Cambie el medio correspondiente cada 2 días.</li>
	<li>Compruebe el estado de diferenciación y registre los cambios morfológicos mencionados en el paso 3.</li>
	<li>El día 18, evaluar la generación de neuronas (β-Tubulin III positivo) y determinar la eficiencia de diferenciación de los 3 protocolos que se utilizan en el paso 4.</li>
</ol>

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E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

Los autores no tienen nada que revelar.

En el presente estudio, establecimos un método simple y eficaz para la diferenciación neuronal de los mESC, con materiales de bajo costo y fácil obtención. En este método, 2 días de formación de cuerpos embrionados seguidos de 6 días de inducción por AR pueden promover eficazmente la diferenciación de los MESC en LOSP (Fase I-protocolo 3). Para la diferenciación de fase II, N2B27 medio II (Fase II-protocolo 3) más eficazmente inducen la diferenciación de los NPC en las neuronas. Para garantizar el éxito, se...

Las células madre embrionarias (ESC) son pluripotentes y pueden diferenciarse en células precursoras neuronales (NPC) y posteriormente en neuronas bajo ciertas condiciones1. La neurogénesis basada en ESC proporciona la mejor plataforma para imitar la neurogénesis, sirviendo así como una herramienta útil para estudios de biología del desarrollo y potencialmente ayuda en medicina regenerativa2,3. En las últimas décadas, muchas estrategias se han divulgado para inducir neurogénesis embrionaria, como el método transgénico4,utilizando moléculas pequeñas5,utilizando una matriz 3D microambiente6,y la técnica de co-cultivo7. Sin embargo, la mayoría de estos protocolos son condicionantes o difíciles de operar, por lo que no son adecuados para su uso en la mayoría de los laboratorios.
Para encontrar un método fácil de operar y de bajo costo para lograr una diferenciación neuronal eficiente de los mESC, aquí se utilizó una estrategia de cribado combinatoria. Como se describe en la Figura 1,todo el proceso de neurogénesis embrionaria se dividió en 2 fases. La Fase I se refiere al proceso de diferenciación de los MESC a los PNJ, y la fase II se relaciona con la diferenciación posterior de los PNJ en las neuronas. Sobre la base de los principios de fácil operación, bajo costo, materiales fácilmente disponibles y alta eficiencia de diferenciación, se eligieron siete protocolos en la Fase I y tres protocolos en la Fase II basados en el sistema tradicional de cultivo monocapa adherente o sistema de formación de cuerpos embrionados8,9. La eficiencia de diferenciación de los protocolos en ambas fases se evaluó utilizando la observación de morfología celular y el ensayo de inmunofluorescencia. Mediante la combinación del protocolo más eficiente de cada fase, establecimos el método optimizado para la diferenciación neuronal de los mESC.

<table>
	<tbody>
		<tr>
			<td>Anti-Nestin antibody [Rat-401]</td>
			<td>Abcam</td>
			<td>Ab11306</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor human</td>
			<td>Sigma</td>
			<td>L5283</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson</td>
			<td>005-000-121</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nonadhesive bacterial dish</td>
			<td>Corning</td>
			<td>3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Stem-Cellbanker (DMSO free)</td>
			<td>ZENOAQ</td>
			<td>stem cellbanker DMSO free</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>6 - well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

neuronal differentiation from mouse embryonic stem cells in vitro

La diferenciación neuronal de las células madre embrionarias del ratón (MESC) es una herramienta potencial para dilucidar los mecanismos clave implicados en la neurogénesis y potencialmente ayudar en la medicina regenerativa. Aquí, establecimos un método eficiente y de bajo costo para la diferenciación neuronal de los MESC invitro, utilizando la estrategia de cribado combinatorio. En las condiciones definidas aquí, la formación de cuerpos embrionados de 2 días + protocolo de inducción de ácido retinoico de 6 días permite una diferenciación rápida y eficiente de los mESC en células precursoras neuronales (NPC), como lo ve la formación de A2lox bien apilado y similar a un neurite y 129 derivados que son nestin positivos. El estado saludable de los cuerpos embrionarios y el punto de tiempo en el que se aplica el ácido retinoico (AR), así como las concentraciones de AR, son críticos en el proceso. En la diferenciación posterior de los PNJ en las neuronas, N2B27 medio II (complementado por medio neurobasal) podría apoyar mejor el mantenimiento a largo plazo y la maduración de las células neuronales. El método presentado es altamente eficiente, de bajo costo y fácil de operar, y puede ser una poderosa herramienta para la neurobiología y la investigación en biología del desarrollo.

La formación de cuerpos embrionados de 2 días + la inducción por AR de 6 días funciona mejor en la dirección de la diferenciación de los MESC en PNJ (Fase I). Para determinar el protocolo óptimo que mejor promueve la diferenciación de los MESC en PNJ (Fase I), se probaron 7 protocolos tanto en A2lox como en 129 mESCs(Cuadro 1)y se monitorizó el estado de diferenciación de cada grupo utilizando microscopio ligero. Como se muestra en la Figura 3A, la mayoría de los ...

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Aquí, establecimos un método de bajo costo y fácil de operar que dirige la diferenciación rápida y eficiente de las células madre embrionarias a las neuronas. Este método es adecuado para la popularización entre laboratorios y puede ser una herramienta útil para la investigación neurológica.

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 ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Research

JoVE Journal

Neuroscience

8.7K vistas.  Wuhan Center for Disease Control and Prevention. Aquí, establecimos un método de bajo costo y fácil de operar que dirige la diferenciación rápida y eficiente de las células madre embrionarias a las neuronas. Este método es adecuado para la popularización entre laboratorios y puede ser una herramienta útil para la investigación neurológica.

Video: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Culturas organotípicos rebanada de mesencéfalo ventral embrionario: Un sistema para estudiar el desarrollo neuronal dopaminérgica In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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 ...

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Diferenciación de células madre embrionarias de ratón en precursores corticales interneurona

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Aislamiento y cultivo de las células madre neurales embrionarias de ratón

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In Vitro Wedge Slice Preparación para imitar la conectividad del circuito neuronal In Vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Modelado in vitro para enfermedades neurológicas utilizando la conversión directa de fibroblastos a células progenitoras neuronales y la diferenciación en astrocitos

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

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 ...