La diferenciación de células madre embrionarias <ol><li> Recoger un frasco de cerca confluente de células madre embrionarias (ESC) a través de un tratamiento con tripsina ESC. </li><li> Resuspender las células en 5 ml de medio de diferenciación y de traslado a un T25 recubierto de gelatina. Se incuba a 37 ° C durante 45 minutos. Recuperar el sobrenadante y recoger las células por centrifugación. Nota: fibroblastos embrionarios de ratón (MEFs) de las culturas ESC conectar fácilmente a la T25 no recubiertos de gelatina y por lo tanto se agotan de la cultura durante esta etapa de revestimiento. Si usted no es el cultivo de la ESC en MEFs, puede omitir la placa de pre-. </li><li> Resuspender MEF-agotado ESC en los medios de diferenciación en 333.333 cells/50 ml. Esta concentración resulta en 100 ml ESC/15. </li><li> El uso de múltiples canales pipeta, la placa de ESC a 100 ml cells/15 caída de 15 cm 2 placas de petri. Con una pipeta de 8 canales, debe ser capaz de encajar unos 18-22 filas de gotas por la placa (unos 5 ml por placa). Nota: Para este protocolo, 2-5 15 cm 2 platos de gotas será más que suficiente y debe producir 2.5 x 10 6 seis días de deriva EB-células. </li><li> Estirar suavemente las placas para invertir gotas. Se incuba a 37 ° C 5% CO 2 durante 48 horas. </li><li> Piscina cae girando suavemente las placas y la transferencia a 15 ml tubo cónico. Enjuagar las placas con 4-6 ml de PBS y añadir a la misma cónico de 15 ml. Nota: Es posible que la piscina en hasta cinco placas de gota. El EBS crecerá a medida que continúan diferenciar y si son muy concentrados que tienden a formar grupos grandes. </li><li> Permitir agrupados EBs de resolver por la gravedad (aproximadamente 10 minutos). Aspirar el sobrenadante sin perturbar EBS. Resuspenda suavemente en 10 ml de medio de diferenciación y de transferencia de hasta 10 cm no adherentes placa de Petri. </li><li> Lugar de Petri placa en placa de agitación a 50 rpm y se incuba a 37 ° C 5% de CO 2. </li><li> 24 horas más tarde (de cuatro días de diferencia), las placas de remolino para concentrarse EBs en el centro de placa de Petri. Cuidado de cambio del 50% de los medios de comunicación (5 ml) con 5 ml de medio de diferenciación fresco. Vuelva a colocar a la incubadora durante dos días más. </li></ol> EB disociación y la infección con MSCV-HOXB4-IRES-GFP <ol><li> En el sexto día de la diferenciación, la transferencia de EBs de tubo cónico de 15 ml. Se deja sedimentar por gravedad. </li><li> Aspirar los medios de comunicación y resuspender en 10 ml de PBS. Permitir EBs para reasentar por la gravedad. Aspirar PBS. </li><li> Añadir 250 ml de mezcla de enzimas disociación y 1 ml de PBS. Incubar en baño de agua 37 ° C durante 20 minutos, girando el tubo para mezclar las enzimas y EBS. </li><li> Añadir 8 ml de la enzima sin buffer de disociación. </li><li> Triturar la mezcla con 5 ml pipeta hasta EBs son totalmente disociados (alrededor de 10 veces; pipeteo excesivo aumento de la muerte en el EB-derivados de preparación de células). <ol><li> Mezcla será nublado con células como EBS se disocian. </li><li> La colocación de punta de la pipeta contra la parte inferior del tubo cónico durante la trituración se creará una fuerza de corte que facilitará en gran medida la disociación. </li></ol></li><li> Recoger las células por centrifugación. </li><li> Resuspender las células derivadas de EB en 5 ml de 10% y el recuento de IMDM con azul de tripan exclusión Nota: Entre los días cuatro y seis días de diferenciación, EBs cavitación que se traduce en una gran cantidad de apoptosis y death.Thus celular, en el sexto día, más del 30% de EB-derivados de las células puede manchar con azul tripán. </li><li> Resuspender las células derivadas de EB a 100.000 células / 2 ml de 10% IMDM + sobrenadante viral tal que una MOI de 5.10 es achieved.Add sulfato de protamina para una concentración final de 8 mg / ml. Nota: Prepare suficiente EB / mezcla de sobrenadante viral a la placa de cuatro placas de 6 pocillos (suponiendo 2 ml / pocillo). </li><li> Placa de 2 ml EB / mezcla de sobrenadante viral por pozo de cuatro placas de 6 pocillos pre-plateado con OP9 células del estroma (ver más abajo). </li><li> Centrifugar las placas a 2500 rpm a temperatura ambiente durante 90 minutos. Transferencia de las placas a la incubadora a 37 ° C 5% de CO 2. </li><li> 4-6 horas más tarde, la cosecha sobrenadante de las placas y recoger las células que quedan en suspensión potencial a través de centrifugación. </li><li> Resuspender pellet (puede ser pequeño) en 48 ml IMDM 10% + citoquinas. Prescindir de 2 ml / pocillo de la resuspensión de original de cuatro placas de 6 pocillos en los que la infección se realizó y el sobrenadante se recogió. Nota: Siempre prepare un 10% IMDM + citoquinas fresco en el momento de su uso. </li><li> Incubar las placas a 37 ° C 5% CO 2 durante siete días. Las colonias deben ser evidentes después de la infección por cuatro días y grandes y bien formados por siete días. Una fuerte expansión debe ceder 40-60 colonias / y el día siete. </li><li> El séptimo día después de la infección, recoger todas las células y la piscina (incluyendo OP9 estroma) de todos los pozos por tratamiento con tripsina. Nota: NO desechar el sobrenadante o PBS se lava las empleadas durante la tripsina treatment.At este punto de la cultura, algunas colonias pueden ser de baja adherencia y hay muchas células floating en suspension.Collect y la piscina todos los lavados y el sobrenadante para asegurarse de que no hay células potencialmente valiosos se descartan. </li><li> Resuspender las células en 8 ml IMDM fresco 10% + citoquinas. Distribución de 2 ml / frasco en cuatro frascos T75. Añadir otros 13 mL 10% las citoquinas + IMDM a cada frasco. Se incuba a 37 ° C 5% de CO 2 durante tres días (un total de diez días después de la infección). </li></ol> La cultura y de las planchas de OP9 estroma <ol><li> Mantener OP9 estroma de acuerdo a los protocolos estándar en un 20%-MEM. OP9 células cambiar las propiedades cuando se cultivan hasta la confluencia. Siempre se divide en un 80% de confluencia en no más de 1:03 de división. </li><li> 24 horas antes de la infección de los Seis Días derivados EB-células con MSCV-HOXB4-IRES-GFP, placa 25000 OP9 células / pocillo de cuatro placas de 6 pocillos en un 20% al MEM. </li></ol> Recolección, fraccionamiento y el trasplante <ol><li> Recoger células expandidas en OP9 estroma durante diez días a través de un tratamiento con tripsina. Recuento de células. Nota: NO desechar el sobrenadante o PBS se lava las empleadas durante el tratamiento con tripsina. En este punto de la cultura, algunas colonias pueden ser de baja adherencia y hay muchas células flotando en la piscina suspension.Collect y todos los lavados y el sobrenadante para asegurarse de que no hay células potencialmente valiosos se descartan. Nota: Una buena expansión debería producir entre 40-50 x 10 6 células / original de 6 pocillos infectados, para un total de 160 a 200 x 10 6 células. </li><li> Las células pueden ser fraccionado a través de la selección con bolas magnéticas o FACS de acuerdo con las técnicas estándar en este momento en el protocolo. </li><li> Para el trasplante, proporcionar células a través de retro-orbital o de inyección de vena de la cola a los destinatarios Rag-2/gc deficiente (con un peso entre 15-22 gramos) sometidos a una dosis fraccionada de 9.25 Gy de irradiación 2,5 horas. de distancia. Nota: Es muy importante que el peso de los animales se encuentran entre 15 a 22 gramos en el momento de la irradiación. Si son mayores de 22 gramos, 9,25 gy no será una suficiente dosis de radiación para asegurar la ablación adecuado y las células no pueden mediar rescate de irradiación letal y / o injertar el compartimiento hematopoyético. Si el trasplante de animales más grandes, la dosis de irradiación adecuada debe determinarse experimentalmente. </li><li> Una dosis mínima de 5.2 x 10 6 células / animal está obligado a garantizar de rescate de la irradiación letal. Si las células de fraccionamiento, inyectar 2.5 x 10 6 células equivalentes (por ejemplo, si la población de interés representa el 10% de la piscina no fraccionada de las células, inyectar 5.2 x 10 5 células / animal). Nota: Si la inyección&gt; 2 x 10 6 células, SIEMPRE se inyectan a través de vena de la cola para evitar la formación de teratoma en órbita, que tiene lugar cuando un gran número de células se entregan retro-orbital. </li><li> Mantener Rag-2 - / - / gc - / - deficientes en receptores de jaulas autoclave. Dependiendo de la &quot;limpieza&quot; de las instalaciones de animales, después del trasplante los animales pueden requerir la administración de agua acidificada o Trimethiprim Sulfasoxazole (Septra) para las 5 semanas después de la radiación letal. </li><li> Espera&gt; GFP 90% + ES derivados de leucocitos en sangre periférica a las 3 semanas después del trasplante. El injerto debe ser multi-linaje, aunque se sabe que los linfocitos silencio retroviral HOXB4-IRES-GFP. </li></ol>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

Una célula madre se define como una celda con la capacidad de los auto-renovación y generar progenie diferenciada múltiples. Las células madre embrionarias (ESC) se derivan del blastocisto del embrión temprano y son pluripotentes en la capacidad de diferenciación. Su potencial de diferenciación amplia ha convertido en el foco de muchas investigaciones se centraron en deducir cómo persuadirlos para generar tipos celulares de utilidad clínica. La derivación con éxito de células madre hematopoyéticas (HSC) de la ESC de ratón ha sido recientemente realizado y se puede visualizar en este protocolo de vídeo. HSC, podría decirse que la población clínicamente más explotadas de células, se utilizan para tratar una gran variedad de cánceres hematopoyéticos y trastornos. Sin embargo, muchos pacientes que podrían beneficiarse de la terapia de HSC no tienen acceso a los donantes adecuados. ESC podría proporcionar una fuente alternativa de células madre hematopoyéticas para estos pacientes. El siguiente protocolo establece una línea de base que ESC-HSC puede estudiar e informar a los esfuerzos para aislar a HSC de humano ESC. En este protocolo, el CES se diferencian como cuerpos embrioides (EBS) durante 6 días en suero disponible en el mercado pre-seleccionados para la diferenciación hematopoyética óptima. EBs luego se disocia y se infectaron con HOXB4 retroviral. Infectados procedentes EB-células son colocadas en OP9 estroma, una línea de células del estroma de médula ósea derivada de la bóveda craneal de M-CSF-/ - ratones, y co-cultivadas en presencia de la hematopoyesis la promoción de las citoquinas durante diez días. Durante este co-cultivo, las células infectadas ampliar en gran medida, lo que resulta en la generación de un grupo heterogéneo de 100s de millones de células. Estas células se pueden utilizar para rescatar a los ratones irradiados letalmente y reconstituir.

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Este protocolo detalles de la derivación de células madre hematopoyéticas trasplantables a partir de células madre embrionarias de ratón (ESC) y su posterior inyección en ratones receptores irradiados letalmente. En pocas palabras, el CES se diferencian como cuerpos embrioides, que luego se infectan con HOXB4 retrovirales y co-cultivadas con células del estroma y OP9 citocinas hematopoyéticas.

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 ...

derivation-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

13.2K vistas.  Harvard Medical School. Este protocolo detalles de la derivación de células madre hematopoyéticas trasplantables a partir de células madre embrionarias de ratón (ESC) y su posterior inyección en ratones receptores irradiados letalmente. En pocas palabras, el CES se diferencian como cuerpos embrioides, que luego se infectan con HOXB4 retrovirales y co-cultivadas con células del estroma y OP9 citocinas hematopoyéticas.

Video: Derivation of Hematopoietic Stem Cells from Murine Embryonic 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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Aislamiento y trasplante de células madre hematopoyéticas (HSC)

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 Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Aislamiento y análisis de las células madre hematopoyéticas de la placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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 ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

El aislamiento de las primeras células madre hematopoyéticas murinas saco vitelino y AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. 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 tumorigenicity6-8 (see a schematic in Fig. 1A). 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 problematic9-11. 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 molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

El análisis fenotípico y aislamiento de células madre hematopoyéticas murinas y progenitores comprometidos Lineage

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...