Agradecemos a Jonatan Lucas por su asistencia técnica en cultivo de células de alimentador, Servei Estabulari de la UAB para el cuidado de los animales y Domènec Martín para grabar el video. Este trabajo ha sido apoyado por el Ministerio de Economia y Competitividad AGL2014-52408-R y la Generalitat de Catalunya 2014 SGR-524. MVC es beneficiario de una beca de PIF-UAB.

el uso de los animales descritos en las secciones a continuación ha sido aprobado por el Comité de ética Animal y de la investigación humana de la Universitat Autònoma de Barcelona y por el Departamento d &#39; Agricultura, Ramaderia, Pesca Alimentació de la Generalitat de Catalunya (protocolo #8741).
 1. inactivación de células de alimentador y almacenamiento Nota: fibroblastos de prepucio humano (HFF-1) fueron congelados previamente en 1 mL de la solución congelada que consiste en 90% suero bovino Fetal (FBS) y 10% dimetil sulfóxido ( DMSO) y almacenadas en nitrógeno líquido hasta uso.
<ol><li>Descongelar un vial criogénico de HFF-1 (1 mL) sumergiendo el frasco en un baño de agua de 37 ° C.</li>
	<li>Agregar el contenido descongelado del frasco criogénico a precalentado Dulbecco &#39; s Modified Eagle &#39; s medio (DMEM) suplementado con 10% FBS para hacer un 1:10 dilución.</li>
	<li>Las células diluidas (10 mL) de semillas en un frasco de T75 y cultivo a 37 ° C y 5% CO 2.</li>
	<li>Al día siguiente, cambiar el medio para eliminar los restos de DMSO. Quite el medio y añadir 10 mL de precalentado DMEM suplementado con 10% FBS.</li>
	<li>Cultura las HFFs y cambiar el medio cada 48 h hasta que la cultura llegue a la etapa confluente. Esto puede tardar aproximadamente 7 a 10 días.</li>
	<li>Para preparar la solución de inactivación, diluir la mitomicina C en DMEM suplementado con 10% FBS a una concentración final de 10 μg/mL. Incubar las células con la solución de inactivación por 3 h a 37 ° C y 5% CO 2.</li>
	<li>Eliminar la inactivación de la solución y enjuague las células tres veces con 2 mL de Hanks balanceada sal solución (HBSS) para eliminar restos de mitomicina C.</li>
	<li>Para separar las células inactivadas, añadir 1 mL de solución de tripsina-EDTA precalentado e incubar 5 min a 37 ° C y 5% CO 2.</li>
	<li>Observar las células bajo un microscopio invertido para asegurar que están separadas (objetivo de 10 x 0.3 apertura numérica o NA). Si no, los incuban durante un par de minutos más a 37 ° C y 5% CO 2. Compruebe las células otra vez bajo el microscopio invertido.</li>
	<li>Pipetear la solución con una pipeta Pasteur para disociar las matas celular.</li>
	<li>Neutralizar la solución enzimática añadiendo 4 mL de DMEM suplementado con 10% FBS y resuspender.</li>
	<li>Toma una alícuota de la suspensión de células y hace una dilución 1: 100 en HBSS. Para determinar el número de células, cargar 10 μl de la dilución en un Neubauer &#39; cámara de s. Contar las células situadas en una de las grandes plazas de la cámara en un microscopio invertido (objetivo 20 x con un NA 0.45).
	<ol><li>Repetir el recuento en un total de cuatro cuadrados grandes. Calcular el número total de células (N) n = número promedio de células contadas x volumen total x dilución x 10 4.</li>
	</ol></li> <li>Centrifugar el resto de la muestra durante 5 min a 200 x g. descartar el sobrenadante y resuspender el precipitado con congelación de la solución en una concentración de 10 6 células / mL.</li>
	<li>Alícuota la suspensión en frascos criogénicos con 10 6 celdas (1 mL) cada uno. Coloque los frascos criogénicos en un recipiente de congelación y congelación a -80 ° C.</li>
	<li>Al día siguiente, guarde los viales criogénicos de nitrógeno líquido.</li>
</ol> 2. Cultivo de células de alimentador <ol><li>uno a cuatro días antes de iniciar el proceso de derivación, inactivado de descongelar un vial criogénico que contiene 1 x 10 6 células de HFF-1 en un baño de agua a 37 ° C.</li>
	<li>Hacer una dilución de 1:6 del HFF-1 descongelado en pre calentado DMEM suplementado con 10% FBS y mantenga la temperatura a 37 ° C.</li>
	<li>Llene cada pocillo de una placa de 4 pozos con 500 μl de precalentado 0.2% de gelatina de piel porcina y mantenga la placa a 37 ° C durante 10 minutos</li>
	<li>Quitar el 500 μl de la gelatina de los pozos y agregar 500 μl de la dilución de HFFs. Muévalo suavemente la placa para asegurar una distribución uniforme de las HFFs en la parte inferior de los pozos. Evitar movimientos circulares, de lo contrario las células se concentrarán en el centro del pozo.</li>
	<li>Incubar la placa por lo menos 24 h a 37 ° C y 5% CO 2 para obtener una monocapa de HFFs inactivados en cada pozo.</li>
	<li>Al día siguiente, observar las células bajo un microscopio invertido (objetivo de 10 X con NA 0.3) y compruebe que están conectados y distribuidos homogéneamente. Cambiar el medio para eliminar los restos de DMSO. Quite el medio y agregar 500 μl de precalentado DMEM suplementado con 10% FBS.</li>
</ol> 3. Colección de embriones y de la cultura <ol><li>cuatro días antes de la recogida de embriones, administrar 5 UI de yegua preñada &#39; gonadotropina del suero de s por inyección intraperitoneal a ratones hembra 6-12 semanas de edad. 
	Nota: Hemos utilizado las hembras de los 129S2 o cepas de B6CFAF1, aunque las hembras de otras cepas de ratón pueden ser utilizadas. Sin embargo, cuando trabajo con hembras de cepas no-permisivas (por ejemplo, CBA), medio debería complementarse con 2i como se menciona en el paso 4.2.</li>
	<li>Después de 48 h, administrar 5 UI de gonadotropina coriónica humana por inyección intraperitoneal y aparearse las hembras con los machos, por lo menos 8 semanas de edad, en una proporción 1:1. Separar las hembras de los machos al día siguiente.</li>
	<li>El día antes de embrión, preparar un cultivo de células de 35 mm placa de Petri con varias gotas de μl 40 KSOMaag suplementado con 4 mg/mL de albúmina sérica bovina (BSA) y llenar el plato con aceite mineral para cubrir completamente las gotas. Incubar la placa a 37 ° C y 5% CO 2 para equilibrar el medio.</li>
	<li>Para preparar pipetas de la manipulación del embrión, calienta la parte más delgada de un cristal con punta larga pipeta Pasteur utilizando un mechero Bunsen. Una vez suave, retirar del fuego y separe los dos extremos. Romper a la longitud deseada. El diámetro interior en la punta debe ser 100-150 μm.</li>
	<li>Eutanasia los ratones hembra por dislocación cervical 45-48 h post-coitum (p.c.).</li>
	<li>Disecar ratones para exponer la cavidad abdominal mediante la reducción de la piel con unas tijeras afiladas lisas curvadas y peritoneo con tijeras recta agudos. Agarrar un útero con pinzas, corte el oviducto utilizando unas tijeras afiladas rectas y colóquelo en CZB tamponado con Hepes medio (HCZB) 13 a 37 ° C. Repita el proceso para eliminar el otro oviducto.</li>
	<li>Descarga los oviductos con una jeringa de 1 mL cargados con HCZB sostenido por pinzas de relojero para recoger los embriones de 2 células. 
	Nota: Para preparar la aguja lavado, tomar una aguja de hipodérmica 30 G, corte el extremo y una punta Roma en una piedra abrasiva de tierra.</li>
	<li>Usando el aparato de la pipeta de boca, recoger los embriones, lavar en HCZB y les cultura en la placa de cultivo de embriones previamente preparado a 37 ° C y 5% de CO 2 hasta la fase de blastocisto. Hasta 50 embriones pueden ser cultivados en cada gota de 40 μl. Supervisar el desarrollo del embrión cada 24 h bajo un estereomicroscopio. 
	Nota: Como alternativa, los embriones pueden recogerse en la etapa de blastocisto para acortar en vitro el cultivo de embriones. En este caso, las hembras de eutanasia en día 3.5-4.5 p.c., diseccionar el útero. y enjuague con HCZB.</li>
</ol> 4. Derivación de células madre <ol><li>preparación de las placas de derivación <ol><li>en el día de la siembra de embriones, preparar el medio de derivación complementando el medio DMEM con 100 μm 2-β-mercaptoetanol, 1 x de los aminoácidos no esenciales , el 20% de reemplazo de suero y 10 3 U/mL LIF.</li>
		<li>Si lo desea, también añadir 1 μm del inhibidor MEK PD0325901 y 3 μm del inhibidor GSK3 CHIR99021 para obtener un medio de 2i.</li>
		<li>Tomar la placa de 4 pozos con la monocapa de HFFs inactivadas (paso 2.6) de la incubadora. Quite el medio y añadir 800 μl de medio con derivación a cada pocillo. Devolver la placa a la incubadora de.</li>
	</ol></li> <li>Retiro de la Zona pelúcida y embrión siembra <ol><li>preparar un cultivo celular de 35 mm placa de Petri con gotas tres 30 μl de ácido Tyrode &#39; s solución 14 y gotas tres 30 μl de medio de derivación. Cubrir las gotas con aceite mineral y mantener el plato a 37 ° C.</li>
		<li>Tomar la placa de cultivo de embrión de la incubadora. Uno por uno, transfiera los blastocistos de las gotas de cultura a un ácido Tyrode &#39; gota de la solución de s con una pipeta de manipulación de embriones. Si es posible, seleccione sólo los blastocistos expandidos para iniciar el proceso de derivación.</li>
		<li>Monitor la zona pelúcida digestión bajo el estereomicroscopio. Tan pronto como desaparece la zona pelúcida, transferir el blastocisto a una gota media de derivación para lavar el ácido Tyrode &#39; solución s.</li>
		<li>Tomar la placa de 4 pozos de derivación de la incubadora y semilla de una zona libre blastocisto en el alimentador de células en cada pozo.</li>
		<li>Volver la placa de 4 pozos de derivación a la incubadora y la cultura en 37 ° C y 5% CO 2.</li>
	</ol></li> <li>Crecimiento de la célula de vástago de monitoreo y medio cambio <ol><li>vigilar los cultivos cada 24 h debajo de un microscopio invertido (objetivo 20 X con NA 0.45). Es importante observar si hay signos de diferenciación de células madre, como células refractivas inflamadas que rodea el fruto o incluso empujar el alimentador células aparte.</li>
		<li>Día, cambiar el medio de la cultura. Tomar la placa de 4 pozos de la incubadora, retire el medio de cada bien y añadir 800 μl de medio con derivación suplementado con LIF y 2i, si se utiliza. Mantener la cultura hasta crecimientos se observan (días 6-8).</li>
	</ol></li></ol> 5. Mantenimiento y cultivo de células de vástago asas de Kolle-como <ol><li>preparar y pipetas de manipulación de células madre. Siga el paso 3.4 preparar pipetas de manipulación de la célula de vástago con diámetro interno en la punta de 250-300 μm. utilizar la otra parte de la pipeta de Pasteur tirada para preparar el mango de Kolle-como. Calor y la forma hasta obtener un gancho de.</li>
	<li>Tomar la placa de cultivo de 4 pozos fuera de la incubadora y localizar la consecuencia bajo un estereomicroscopio. Use la manija para alejar las células diferenciadas y los alimentadores que rodean el fruto. 
	Nota: Para evitar eventual diferenciación de la consecuencia, es importante eliminar todas las células diferenciadas del borde de la extensión.</li>
	<li>Aspire el fruto con una pipeta de la manipulación de células madre y coloque en un plato nuevo en una gota de tripsina-EDTA 50 μl.</li>
	<li>Utilizar un bisturí para cortar el fruto en varias partes para desagregación mecánica y enzimáticamente.</li>
	<li>, Transferencia de los fragmentos del fruto en una dulce gota de medio de derivación para neutralizar la acción de la tripsina.</li>
	<li>Transferencia de los fragmentos del fruto a una nueva placa de 4 pozos con una monocapa de alimentadores. Transferencia de fragmentos de hasta 10 por pozo y de la cultura con un medio de derivación a 37 ° C y 5% CO 2.</li>
	<li>Mantener las culturas de célula de vástago-como por lo menos seis semanas antes de considerar la línea mESC establecida. Cambiar el medio cada otro día y subcultura ellas una vez por semana.</li>
</ol>

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Los autores no tienen nada que revelar.

Aunque mESC línea derivación es un procedimiento bien conocido en muchos laboratorios utilizado habitualmente, su eficiencia no siempre es tan alta como se esperaba debido a los múltiples factores que pueden perturbar el mantenimiento de la pluripotencia. En el presente artículo mostramos, paso a paso, cómo establecer líneas mESC con alta eficiencia utilizando un protocolo sencillo y fiable. Estas eficiencias son similares a los reportados en la literatura.
Para asegurar la máxima efici...

Las células madre embrionarias (ESC) son células pluripotentes derivadas de embriones preimplantación, que conservan la capacidad de autorrenovación o diferenciar bajo condiciones específicas1. Partiendo de estas propiedades, ESC se han convertido en una herramienta útil para la medicina regenerativa, modelado de la enfermedad y tejido ingeniería estudios2.
ESC se derivan por primera vez embriones de preimplantación ratón, origina líneas ESC (mESC) de ratón con éxito bajo3,4. Durante años, la eficacia de la derivación seguía siendo baja debido a la dificultad de mantenimiento de la pluripotencia in vitro. Además de la presencia de alimentador células5, que mejorar la eficiencia de la derivación, promover la fijación de la Colonia y mejorar estabilidad cariotípica6, varias modificaciones del protocolo conducen a una mejora en el mantenimiento de la pluripotencia. El más importantes eran la adición de Factor inhibitorio de leucemia (LIF) para mESC medio de cultivo7,8, el uso de embriones de la 129S2 fondo permisiva9 y define la cultura de la mESC con suplementado con suero, libre de potenciales factores de diferenciación10. Más recientemente, convincente evidencia ha demostrado que la adición de un cóctel de dos moduladores de molécula pequeña de vías de señalización al medio de cultivo mESC (conocido como 2i) es mejor mantener la pluripotencialidad. El 2i cóctel consiste en una combinación de inhibidor de la MEK PD0325901, que reduce las señales de diferenciación mediante la inhibición de la vía de la quinasa (MAPK) de proteína mitogen-activados, y el inhibidor de la GSK3β CHIR99021, que mejora la supervivencia de la célula a baja densidad mediante la activación de la vía Wnt del11.
En el presente artículo, se describe un protocolo simple para derivar eficazmente mESC líneas de blastocistos de ratón. Seguimos procedimientos estándar, cultivo de embriones de cepas permisivas en las células de alimentador en medio de la derivación con LIF y un recambio de suero12. Siguiendo este protocolo, mESC líneas puede derivarse con rendimientos equivalentes a las obtenidas en medio 2i. A pesar de esto, pueden agregarse 2i para evitar posibles problemas con el mantenimiento de la pluripotencia o cuando se trabaja con cepas no permisiva.

<table><tbody><tr><td>1 mL sryringe</td><td>BD&amp;nbsp;</td><td>309628</td><td></td></tr><tr><td>2-&amp;beta;-mercaptoethanol</td><td>Life Technologies</td><td>31350-010</td><td></td></tr><tr><td>4-well plate</td><td>Thermo Scientific Nunc</td><td>176740</td><td></td></tr><tr><td>Acidic Tyrode&#039;s solution&amp;nbsp;</td><td>Home-made</td><td></td><td>See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4&amp;ordm;C. Alternatively, for long-term storage keep it at -20&amp;ordm;C. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M.&amp;nbsp;</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>Chicken anti-Mouse IgG, Alexa Fluor 488</td><td>Molecular Probes - Invitrogen</td><td>A-21200</td><td>1:500 dilution. Secondary antibody for Oct4, Tuj1, &amp;alpha;SMA and AFP mouse antibodies.&amp;nbsp;</td></tr><tr><td>CHIR990212</td><td>Axon Medchem&amp;nbsp;</td><td>1386</td><td>Reconstitute with 2.15 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&amp;ordm;C. Avoid freeze and thaw cycles.&amp;nbsp;</td></tr><tr><td>CryoTube</td><td>Thermo Scientific Nunc</td><td>3754518</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>41640</td><td></td></tr><tr><td>DMEM</td><td>BioWest</td><td>L0107</td><td></td></tr><tr><td>FBS</td><td>BioWest</td><td>S1810</td><td>Inactivate it prior to use by heating for 30 mins at 56&amp;ordm;C. Aliquot and store at -20&amp;ordm;C</td></tr><tr><td>Flushing needle</td><td>BD&amp;nbsp;</td><td>304000</td><td>Cut the end of the needle and ground to a blunt tip on an abrasive stone</td></tr><tr><td>Gelatin from porcine skin</td><td>Fluka</td><td>48724</td><td>Dilute at 0,2% in destilled water and autoclave it. Each dilution can be used for a month.</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 594</td><td>Molecular Probes - Invitrogen</td><td>A-11037</td><td>1:500 dilution. Secondary antibody for Sox2 rabbit antibody.&amp;nbsp;</td></tr><tr><td>HBSS</td><td>BioWest</td><td>L0611</td><td></td></tr><tr><td>Hepes-buffered CZB&amp;nbsp;</td><td>Home-made</td><td></td><td>See composition in Chatot et al, 1989</td></tr><tr><td>Human chorionic gonadotropin</td><td>Divasa-Farmavic</td><td>Veterin-Corion 3000 UI</td><td>Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&amp;ordm;C for up to two months. Once thawed do not freeze again.&amp;nbsp;</td></tr><tr><td>Human foreskin fibroblasts</td><td>ATCC</td><td>ATCCSCRC-1041</td><td>For long term storage it is recommended to store them in liquid nitrogen.</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>&amp;nbsp;10828-028</td><td>Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20&amp;ordm;C (check the expiration date). Avoid freeze and thaw cycles.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>KSOMaag Evolve</td><td>Zenith Biotech</td><td>ZEKS-050</td><td>Supplement with 4 mg/ml BSA. Equilibrate at 37&amp;ordm;C and 5% CO2 a day prior to use.</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Merk Millipore</td><td>ESG1106</td><td></td></tr><tr><td>Mice&amp;nbsp;</td><td>Charles River/Harlan</td><td></td><td>It is recommended to use mice from a permissive strain in terms of mESC derivation (such as 129S2 or C57BL). However, inbreed strains are less efficient producing embryos. Therefore, it is recomended to use a hybrid strain, such as 129S2 x C57BL or B6CBAF1 to take advantage of the hybrid vigor.&amp;nbsp;</td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M8410</td><td>Embryo tested. Photosensitive.&amp;nbsp;</td></tr><tr><td>Mitomycin C</td><td>Serva</td><td>2980501</td><td>Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4&amp;ordm;C protected from light. Attention, it is harmful and suspected of causing cancer.&amp;nbsp;</td></tr><tr><td>Mouse anti-tubulin &amp;beta; III (Tuj1)</td><td>Biolegend</td><td>MMS-435P</td><td>1:500 dilution</td></tr><tr><td>Mouse monoclonal anti-&amp;alpha;SMA</td><td>Sigma</td><td>A5228</td><td>1:200 dilution</td></tr><tr><td>Mouse monoclonal anti-AFP</td><td>R&amp;amp;D Systems</td><td>MAB1368</td><td>1:50 dilution</td></tr><tr><td>Mouse monoclonal anti Oct3/4&amp;nbsp;</td><td>Santa cruz</td><td>Sc-5279</td><td>1:50 dilution</td></tr><tr><td>Non-Essential Amino Acids</td><td>Life Technologies</td><td>11140-035</td><td></td></tr><tr><td>PD0325901</td><td>Axon Medchem&amp;nbsp;</td><td>1408</td><td>Reconstitute with 2.08 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&amp;ordm;C. Avoid freeze and thaw cycles.</td></tr><tr><td>Petri dish (35mm)</td><td>Thermo Scientific Nunc</td><td>153066</td><td></td></tr><tr><td>Petri dish (60mm)</td><td>Thermo Scientific Nunc</td><td>150288</td><td></td></tr><tr><td>Pregnant mare&#039;s serum gonadotropin</td><td>Foligon</td><td>Foligon-1000 UI</td><td>Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&amp;ordm;C for up to two months. Once thawed do not freeze again.&amp;nbsp;</td></tr><tr><td>Rabbit policlonal anti-Sox2</td><td>Merck Millipore</td><td>AB5603</td><td>1:200 dilution</td></tr><tr><td>T75 falcon</td><td>Thermo Scientific</td><td>130190</td><td></td></tr><tr><td>Trypsin-EDTA 10x</td><td>BioWest</td><td>X0930</td><td>Dilute 1:10 in HBSS to obtain a 1x solution</td></tr></tbody></table>

derivation of stem cell lines from mouse preimplantation embryos

Derivación de células madre embrionarias (mESC) de ratón es el proceso por que pluripotentes se establecen líneas celulares de embriones de preimplantación. Estas líneas conservan la capacidad de autorrenovación o diferenciar bajo condiciones específicas. Debido a estas propiedades, mESC son una herramienta útil en la medicina regenerativa, modelado de la enfermedad y estudios de ingeniería de tejidos. Este artículo describe un protocolo simple para obtener líneas mESC con eficiencias de derivación alta (60-80%) por cultivo de blastocistos de cepas permisivas ratón células de alimentador en definido suplementado con factor inhibidor de leucemia. El protocolo se puede aplicar también para derivar eficazmente mESC líneas de cepas de ratón no permisiva, por la simple adición de una combinación de dos inhibidores de molécula pequeña al derivación medio (2i). Se proporcionan procedimientos detallados sobre la preparación y cultivo de células de alimentador, recolección y cultivo de embriones de ratón y derivación y cultura de mESC líneas. Este protocolo no requiere equipo especializado y puede realizarse en cualquier laboratorio con experiencia en cultivo células de mamífero básico.

Siguiendo este protocolo, debe lograrse más 95% de la formación del fruto después de la primera semana de la cultura (figura 1A). Establecimiento de líneas de la mESC luego de seis pasos es variable entre los distintos recipientes experimentales, con un éxito promedio de 60-80%. La adición de 2i no mejora significativamente los resultados cuando se trabaja con cepas de ratón permisiva, como aquellas con antecedentes de 129S2, pero es necesario cuando s...

Watch this Scientific Journal Video about Derivation of Stem Cell Lines from Mouse Preimplantation Embryos at JoVE.com

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Derivación de líneas de células madre de embriones de preimplantación de ratón

Este artículo describe un protocolo y eficiente derivar líneas de células madre pluripotentes de embriones de ratón de la cultura en la etapa de blastocisto.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

derivation-of-stem-cell-lines-from-mouse-preimplantation-embryos

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.3K vistas.  Universitat Autònoma de Barcelona. Este artículo describe un protocolo y eficiente derivar líneas de células madre pluripotentes de embriones de ratón de la cultura en la etapa de blastocisto.

Video: Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Investigación

Biología

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Análisis cuantitativo de la expresión de la proteína para el Estudio de la especificación del linaje de ratón de preimplantación de embriones

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Biología del Desarrollo

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Deficitaria y en el enfoque de ganancia de función de investigar la célula destino temprano Determinantes de preimplantación embriones de ratón

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivación de células madre de ratón a partir de blastocistos Trofoblasto

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

Aplicación de células madre embrionarias haploides partegenéticas del ratón como sustituto de los espermatozoides

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

La diferenciación de ratón recién nacido piel derivados de las células madre en células germinales-como<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Etiquetado de la célula y de inyección en Desarrollo de embriones de ratón Corazones

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...