Un agradecimiento especial al Dr. Boris Greber para la creación de la activina un protocolo de ELISA que se publica en Greber et al. De 2007. Estamos especialmente agradecidos a la Sra. Mónica Shevack para preparar el resumen gráfico. Estamos muy agradecidos al Dr. Heiko Fuchs por toda la ayuda y sus valiosas sugerencias antes y durante el rodaje. Nos gustaría dar las gracias a todos los miembros del laboratorio de Adjaye, especialmente Elisabeth Socha para mantener un suministro constante de MEFs y CM. También agradecemos a nuestros colegas en las instalaciones de los animales de la MPIMG por su apoyo permanente. Este trabajo fue financiado en parte por la Sociedad Max Planck y el [BMBF, el número de concesión 0315717A], los socios de la ERASysBio + iniciativa apoyada en la UE ERA-NET Plus esquema en el 7PM.

1. El aislamiento de fibroblastos de ratón embrionarias (MEFs) Los dos pasos siguientes se llevan a cabo bajo condiciones no asépticas. <ol><li> Sacrificar un ratón embarazada (CF1, Harlan, EE.UU.) a 13 o 14 dpc (días post-coitum) por dislocación cervical. </li><li> Diseccionar los cuernos uterinos, una breve aclaración en un 70% (v / v) de etanol y colocarlo en un tubo Falcon con PBS sin Ca 2 + Mg 2 + (Gibco, Invitrogen). </li></ol> Los pasos siguientes se llevan a cabo en una campana de cultivo de tejidos en condiciones asépticas y utilizando instrumentos estériles. <ol start="3"><li> Coloque cuernos uterinos en una placa de Petri y separar cada embrión a partir de la placenta y el saco embrionario. </li><li> Diseccionar la cabeza y órganos rojos, lavar en PBS y colocar todos los embriones en una placa de Petri limpia. Finamente picada el tejido usando una cuchilla de afeitar estéril hasta que se hace posible pipeta. </li><li> Añadir 1 ml de 0,05% de tripsina / EDTA (Gibco, Invitrogen), inclusiónding Kunitz 100 unidades de DNasa I (USB), por embrión. </li><li> Transferir el tejido en un tubo Falcon de 50 ml y se incuba durante 15 min a 37 ° C. Después de cada 5 min de incubación, las células se disocian pipeteando arriba y hacia abajo a fondo. </li><li> Inactivar la tripsina mediante la adición de aproximadamente 1 volumen de medio MEF recién preparada. MEF medio de cultivo (componentes para hacer 500 ml de los medios de comunicación, mezclar todos los componentes y el filtro): 450 ml de DMEM, 50 ml de FBS (10% (v / v)), 5 ml de 200 mM de L-glutamina (1/100 (v / v)), 5 ml de penicilina-estreptomicina (1/100 (v / v)). </li><li> Centrifugar las células con baja velocidad (300 xg), a 5 minutos, retirar con cuidado el sobrenadante de células y resuspender el pellet en medio de MEF caliente. </li><li> Placa aproximadamente un número de células que es equivalente a 3-4 embriones en cada T150 (TPP) matraz recubiertas con 0,2% de gelatina (gelatina de piel bovina, Tipo B, Sigma) durante 2 h. Los fibroblastos (P0, el paso 0) son las únicas células que tienen la capacidad de unir a los frascos de gelatina recubiertos con película. </li&gt; <li> Idealmente, las células son 80-90% confluentes después de 24 horas y en esta etapa una parte importante de P0 células se congela para uso futuro. </li><li> Expanda el restante T150 matraz (s) de células P0 hasta P3 o P4, a continuación, inactivar y utilizar como alimentadores para replate hESCs o para producir medio condicionado (CM). </li></ol> 2. Desactivación y Revestimiento MEFs (Alimentador Preparación células) Todas las etapas se llevan a cabo en una campana de cultivo de tejidos en condiciones asépticas. <ol><li> Capa T150 matraces con 0,2% de gelatina y se incuba a temperatura ambiente durante al menos 2 horas. </li><li> Diluir mitomicina C en PBS (1 mg / ml) y se filtro. </li><li> Aspirar los medios de comunicación de MEFs y lavar con PBS sin Ca 2 + Mg 2 +. </li><li> , 20 ml de medio que contiene 10 mg / ml de mitomicina C en MEFs. </li><li> Incubar durante 2 horas a 37 ° C con mitomicina C que contiene medio lavar dos veces con PBS, trypsinize, se centrifuga (durante 5 minutos a 300 xg) y Resuspender las células en medio caliente. </li><li>Contar las células y la placa a una densidad de 56.000 células / cm 2 en matraces T150 y utilizar para la producción de CM para las siguientes 6 días. </li></ol> 3. Medio condicionado (CM) Preparación Todas las etapas se llevan a cabo en una campana de cultivo de tejidos en condiciones asépticas. <ol><li> El día después de placas inactivada MEFs a una densidad de 56.000 células / cm 2 reemplazar el medio con medio MEF hCME (MU, medio condicionado) (0,5 ml / cm 2) suplementado recién con 4 ng / ml de FGF2. </li><li> Recoger CM de matraces de alimentación después de 24 h de incubación y se añade medio fresco que contiene hCME 4 ng / ml de FGF2 a los alimentadores. </li><li> Repita este procedimiento para los próximos 6 días. Cada almacén de recogida CM días a -20 ° C. </li><li> Después de 6 días mezcle todas las alícuotas del medio y el filtro (Corning, 0,22 micras, PAS). Hacer alícuotas de 50 ml y se almacena a -80 ° C. </li><li> Suplemento CM con más de 4 ng / ml de FGF2 antes de añadir a hESCs cultivadas en Matrigel. &lt;/ Li&gt; </ol> 4. Medición de la activina A en los medios condicionados (ELISA) 15 <ol><li> Lleve todas las muestras y los reactivos a temperatura ambiente. </li><li> Diluir la captura de anticuerpos (Humanos \ Mouse \ Rata activina A MAB, R &amp; D Systems) en PBS con BSA al 1%, se suman a la microplaca (100 l / pocillo) y se incuba durante la noche a temperatura ambiente. </li><li> Después de 24 horas lavar los pocillos tres veces con PBST (PBS con 0,05% de Tween 20) (300 l / pocillo) entonces el bloque (1% de BSA / PBS, 300 l / pocillo) durante 1 hora a temperatura ambiente. </li><li> En este tiempo de preparación Un activina (R &amp; D Systems) curva estándar, incluyendo 7 diluciones (concentración de menos de 30 ng / ml) y la muestra en blanco. El rango lineal de trabajo de la activina A es entre 0,25 y 32 ng / ml. </li><li> Añadir duplicados de patrones y muestras a los pocillos (100 l / pocillo) y se incuba durante 2 horas a temperatura ambiente. </li><li> Lavar los pocillos tres veces (300 l de PBST). </li><li> Añadir el secundario (biotina) de anticuerpos (humano / ratón / rata Biotinylated activina A MAb, R &amp; D Systems) (0.25 ug / ml en 1% BSA / PBS) y se incuba durante 2 horas a temperatura ambiente. </li><li> Lavar los pocillos tres veces (300 l de PBST), añadir estreptavidina-HRP (diluida en 1% BSA / PBS, R &amp; D Systems) y se incuba durante 20 min a temperatura ambiente. </li><li> Lavar los pocillos tres veces (300 l de PBST), añadir 100 ml de solución de sustrato (Quantikine, R &amp; D Systems) y se incuba durante 30 min a temperatura ambiente en la oscuridad. </li><li> Añadir 100 l de solución de parada (Quantikine, R &amp; D Systems) a cada pozo y mezcle suavemente. </li><li> Conjunto Lector de microplacas (Molecular Devices Spectra Max 250, Instrumentación Médica Global, Inc., Minnesota) a 450 nm, con corrección de longitud de onda a 540 o 570 nm, para determinar la densidad óptica de cada pocillo. </li></ol> 5. Los resultados representativos El esquema general del procedimiento de aislamiento se presenta en la Figura 1. La morfología típica de hESCs y hiPSCs cultivadas bajo condiciones diferentes se presentan en la Figura 2. La morfología de<html> MEFs y células inactivadas alimentadores utilizados para preparar CM se presentan en la Figura 3. En general, las células deben ser confluente 24 horas post-aislamiento y listo para ser congelada o expandido. Sin embargo, a veces puede ser que tome 2-3 días antes de la obtención de cultivos confluentes. El CM debe ser preparado a partir de las células en el paso 4 y no más tarde. Esto es crucial porque las células primarias sólo puede ser ampliado por 4-5 pasajes antes del inicio de la senescencia. La citoquina, activina A, es considerado como el factor más crítico secretada por las células de conexión para el apoyo del crecimiento indiferenciado de las células pluripotentes 14. La medición del nivel de activina A en CM (Figura 4) es un ensayo cuantitativo muy conveniente para controlar la calidad de MEFs. <img alt="Figura 1" src="/files/ftp_upload/3854/3854fig1.jpg" /> Figura 1. Una representación esquemática del procedimiento de aislamiento MEFs. CONTENIDO &quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/3854/3854fig2.jpg" /> Figura 2. La morfología típica de hESCs indiferenciados cultivados en la presencia de células alimentadoras (A), (B) medio condicionado y el medio (C) definido. La morfología típica de hiPSCs cultivadas en presencia de células alimentadoras (D, E). <img alt="Figura 3" src="/files/ftp_upload/3854/3854fig3.jpg" /> Figura 3. La morfología típica de fibroblastos de embriones de ratón (MEFs). (A) el pasaje 0 (P0) dos días después de placas / aislamiento, (B) de la capa alimentador inactivada a una densidad de 56.000 células / cm. 2 <img alt="Figura 4" src="/files/ftp_upload/3854/3854fig4.jpg" /> Figura 4. Ligado a enzimas (ELISA)-basados ​​en las mediciones de la concentración de activina A en medio condicionado (CM) prepared con fibroblastos de embriones de ratón derivadas de la cepa de ratón CF1. CM se recogió durante 6 días y se reunieron a continuación. CM &quot;1&quot; y CM &quot;2&quot; se refieren a los diferentes lotes de medios de comunicación y mensajería unificada a los medios de comunicación no acondicionados. Como la función del proceso de acondicionamiento se activina A la secreción en el medio por MEFs, activina A es casi indetectable en la UM.

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No hay conflictos de interés declarado.

El procedimiento de aislamiento MEFs presentado aquí permite el establecimiento de una condición de cultivo estandarizado para hESCs y hiPSCs. Además, el sistema basado en ELISA utilizado para evaluar la producción de citocinas por células alimentadoras es un indicador útil de la calidad de los medios condicionados MEF-derivados. El mantenimiento de rutina de la cepa de ratón (CF1) proporcionar fibroblastos de apoyo es necesario para evitar lote a lote variación de medios de cultivo. Además, se recomienda para aislar embriones de varios ratones simultáneamente para obtener una calidad uniforme de las células. Ciertamente, recién aisladas MEFs se puede mantener congelada a P0 y P1. También inactiva MEFs se puede mantener congelado en alícuotas de las cantidades apropiadas dependiendo de los requerimientos para el cultivo celular. Por lo general, alrededor de 250.000 células deben ser cultivadas en un solo pozo de una placa de 6 pocillos de hESCs cultura y las células iPS. El método establecido y optimizados pueden ser usados ​​rutinariamente para minimizar la variabilidad entre los difealquiler de experimentos.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DMEM (glucosa alta) </td><td> Gibco, Invitrogen </td><td> 41966-052 </td><td></td></tr><tr><td> FBS </td><td> Biochrom </td><td> S0115 </td><td></td></tr><tr><td> L-glutamina </td><td> Gibco, Invitrogen </td><td> 25030-024 </td><td></td></tr><tr><td> La penicilina-estreptomicina </td><td> Gibco, Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Vacío filtro sistema </td><td> Corning </td><td> 431097 </td><td> 500 ml, 0,22 m, PAS </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650 </td><td></td></tr><tr><td> Knockout DMEM </td><td> Gibco, Invitrogen </td><td> 10829-018 </td><td></td></tr><tr><td> Ser golpe de graciaum reemplazo </td><td> Gibco, Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> No aminoácidos esenciales </td><td> Gibco, Invitrogen </td><td> 11140-035 </td><td></td></tr><tr><td> beta-mercaptoetanol </td><td> Sigma </td><td> M7522 </td><td></td></tr><tr><td> PBS </td><td> Gibco, Invitrogen </td><td> 14190-169 </td><td></td></tr><tr><td> DNasa I </td><td> Sigma </td><td> D4527 </td><td></td></tr><tr><td> Mitomicina C </td><td> Roche </td><td> 10107409001 </td><td></td></tr><tr><td> Factor básico de crecimiento de fibroblastos </td><td> PeproTech </td><td> 100-18B </td><td></td></tr><tr><td> Biotinilado Activina Anti-human/mouse/rat Un anticuerpo </td><td> R &amp; D Systems </td><td> BAM3381 </td><td></td></tr><tr><td> Gelatina </td><td> Sigma </td><td> G9391 </td><td></td></tr><tr><td> Humano / ratón /Rata activina un MAb </td><td> R &amp; D Systems </td><td> MAB3381 </td><td></td></tr><tr><td> 0,05% de tripsina-EDTA </td><td> Gibco, Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Extra Fino Tijeras Iris </td><td> FST </td><td> 14088-10 </td><td></td></tr><tr><td> Adicionales Pinza Graefe Bellas </td><td> FST </td><td> 11150-10 </td><td></td></tr><tr><td> Pinzas de fijación Pierse </td><td> FST </td><td> 18155-13 </td><td></td></tr></tbody></table>

preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells

En general, las células madre embrionarias humanas (hESCs) y humanos células madre pluripotentes inducidas (hiPSCs) 1 pueden ser cultivadas bajo condiciones variables. Sin embargo, no es fácil establecer un sistema eficaz para el cultivo de estas células. Dado que las condiciones de cultivo pueden influir en la expresión del gen que confiere la pluripotencia de hESCs y hiPSCs, la optimización y estandarización del método de cultivo es crucial. El establecimiento de líneas de células madre se describió por primera vez mediante MEFs como células alimentadoras y suero bovino fetal (FBS)-que contiene medio de cultivo 2. A continuación, FBS fue reemplazado con el reemplazo de octavos de final del suero (KSR) y FGF2, lo que aumenta la proliferación de hESCs 3. Finalmente, libres de conexión sistemas de cultivo de permitir el cultivo de células en Matrigel placas recubiertas en KSR-contiene medio condicionado (medio condicionado por MEFs) 4. Posteriormente, las condiciones de cultivo de células madre embrionarias humanas han avanzado hacia la alimentación libre de la cultura en la defi químicamenteed 5-7 condiciones. Además, para evitar la contaminación potencial por patógenos y proteínas animales métodos de cultivo utilizando xeno componentes libres se han establecido 8. Para obtener mejores condiciones de células de ratón alimentadoras han sido sustituidos con líneas celulares humanas (por ejemplo, músculo y células de la piel del feto 9, las células adultas de la piel 10, fibroblastos de prepucio 11-12, amnióticas células mesenquimales 13). Sin embargo, la eficacia de mantener hESCs indiferenciados utilizando prepucio humano derivados de fibroblastos capas alimentadoras no es tan alta como la de células alimentadoras de ratón debido al nivel inferior de la secreción de activina A 14. Obviamente, hay una diferencia evidente en la producción del factor de crecimiento por células de ratón y humanos alimentadoras. Los análisis de los transcriptomes de ratón y células humanas de alimentación reveló diferencias significativas entre las células de apoyo y no de apoyo. Exógenos FGF2 es crucial para manteNing auto-renovación de hESCs y hiPSCs, y ha sido identificado como un factor clave en la regulación de la expresión de Tgfβ1, Una activina y Gremlin (un antagonista de BMP) en células alimentadoras. Activina A se ha demostrado que induce la expresión de Oct4, Sox2, y Nanog en hESCs 15-16. A largo plazo la cultura, y hESCs hiPSCs se puede cultivar en mitóticamente inactivados MEFs o bajo condiciones libres de conexión en el MEF-CM (MEF medio condicionado) en Matrigel placas recubiertas para mantener su estado indiferenciado. El éxito de las dos condiciones de cultivo depende totalmente de la calidad de las células de alimentación, ya que afectan directamente el crecimiento de hESCs. A continuación, presentamos un algoritmo optimizado para el aislamiento y cultivo de fibroblastos embrionarios de ratón (MEFs), la preparación de medio condicionado (MC) y ligado a enzimas (ELISA) para evaluar los niveles de activina A en los medios de comunicación.

Watch this Scientific Journal Video about Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells at JoVE.com

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

La calidad de fibroblastos de embriones de ratón (MEFs) está dictada por la cepa derecha de ratón tal como CF-1. Pluripotencia-MEFs de apoyo y medios de comunicación condicionado (CM) obtenido a partir de éstas debe contener concentraciones óptimas de activina A, Gremlin y Tgfβ1 necesarios para la Activina / Nodal y las vías de FGF de manera cooperativa mantener la auto-renovación y pluripotencialidad.

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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90.4K vistas.  Max Planck Institute for Molecular Genetics. La calidad de fibroblastos de embriones de ratón (MEFs) está dictada por la cepa derecha de ratón tal como CF-1. Pluripotencia-MEFs de apoyo y medios de comunicación condicionado (CM) obtenido a partir de éstas debe contener concentraciones óptimas de activina A, Gremlin y Tgfβ1 necesarios para la Activina / Nodal y las vías de FGF de manera cooperativa mantener la auto-renovación y pluripotencialidad.

Video: Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparación Extienda los cromosomas de las células estaminales embrionarias para cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfección y Nucleofecting Humanos células madre pluripotentes inducidas

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...