Los autores agradecen a Janet Rossant para el protocolo original. Este estudio fue apoyado por el Instituto Nacional de Salud de subvención CA106308 de WH.

En este protocolo, se describe la preparación de los blastocistos de ratón y el establecimiento de líneas de células TS. Manipulaciones generales del ratón antes de la recolección de los blastocistos, incluyendo la puesta a punto para el apareamiento natural y la inducción de la superovulación, básicamente, seguir el protocolo estándar ilustrado por Nagy et al. 2003 (pp146-150). Derivación y el mantenimiento de las células TS <ol><li> Configuración de cruzamiento entre ratones de interés. </li><li> Prepare fibroblastos embrionarios de ratón (MEFs) como células alimentadoras. Dos días antes de la recolección de los blastocistos, MEFs (2 x 10 6 células) se sembraron en placas de 100 mm. Al día siguiente, estas células son tratadas con 10 ml de medio de TS que contiene la mitomicina C (20 mg / ml) para un 2 horas de incubación a 37 ° C, 5% de CO 2. Esto es seguido por lavado de las células dos veces con PBS. Las células tratadas con mitomicina C luego se sembraron en placas de 12 pocillos (1 x 10 5 células / pocillo). </li><li> En el día de la recogida de blastocistos (3,5 dpc, conocido como el primer día), media MEF cultura se sustituye por el TS medio plus 1x FGF4/Heparin (0,5 ml / pocillo). </li><li> Preparación para la recogida del útero: la eutanasia con el ratón y localizar el útero (fig. 1A). </li><li> Extirpar el útero, sujetándola con una pinza en el cuello del útero (que se encuentra detrás de la vejiga) y corte en la unión del útero y el cuello (fig. 1B). Tire el útero, el asiento de la membrana y los tejidos grasos de distancia y corta el útero por debajo de la confluencia con el oviducto (fig. 1C). Lugar del cuello (Figura 1 D) en un pequeño volumen (0,2 ml) de medio de TS en una placa de 60 mm. </li><li> Vaciar cada cuerno uterino con 1 ml de medio TS utilizando una jeringa de 1 ml con una aguja de calibre 26. Insertar la aguja en la base del útero y enjuague lentamente en una placa de 60 mm con un nuevo medio de TS ml. El útero debe ser inflado y totalmente extendida durante el lavado. </li><li> Recoger y traspasar los blastocitos cuidadosamente en un recipiente limpio 12 y placa que contiene medio TS con una pipeta de boca controlada. Lavado de blastocistos en tres ocasiones por la transferencia en serie a través de medio TS en placas de 12 pocillos. Coloque un blastocisto por pozo (Figura 2) en una placa de 12 pocillos que contiene la mitomicina C tratados con alimentadores MEF y la cultura a 37 ° C, 5% de CO 2. </li><li> El blastocisto se salen de la zona pelúcida y se unen a los pozos de 24 a 36 horas. Una consecuencia pequeños se les puede observar en el día 3. Alimentar los cultivos con medio 500 l TS fresca más FGF4/Heparin 1x. </li><li> El día 4 ó 5, la consecuencia de blastocisto debe estar listo para la desagregación en función de su tamaño. El tamaño ideal para el desglose de células TS se ilustra en la Figura 3B. Lave las culturas, una vez con 0,5 ml de PBS. Añadir 100 ml de 0,25% de tripsina / EDTA y se incuba durante 5 minutos a 37 ° C, 5% de CO 2. Romper el fruto en pequeños grupos pipeteando arriba y hacia abajo con fuerza con un pipetteman P100. Detener la tripsina mediante la adición de TS-70cm-1.5F4H (media del 30% TS, el 70% MEF-CM más FGF4/Heparin 1,5 x) en el pozo. Cambio de medio 8 horas después de desagregación y seguir alimentando a las células de cada 2 días. </li><li> Entre los días 6 y 10 (muy variable) TS colonias de células pueden ser observadas. Tienen hoja plana, como la morfología del epitelio con un límite claro colonia (Figura 2C). Seguir alimentando la cultura cada 2 días </li><li> Cuando las células TS alcanzar el 50% de confluencia (por lo general entre los días 15 y 20), lavar una vez con PBS y añadir 100 ml 0,25% de tripsina / EDTA. Pipeta hacia arriba y abajo durante la tripsina para asegurar una cerca de una sola célula de la suspensión. Detener la tripsina mediante la adición de TS-70cm-1.5F4H y traslado a las nuevas placas de 6 pocillos o de 60 mm que contiene la mitomicina C tratados con alimentadores MEF (2,5 x 10 5 cells/6-well o 5 x 10 5 cells/60mm placa). </li><li> Cambio de medio de 8 horas tras el paso y seguir alimentando a los cultivos cada 2 días. </li><li> Después de uno o dos pasajes (3-5 días entre pasajes) en los alimentadores del MEF, las células TS se puede cultivar sin ellos. Porque MEFs se adhieren a la placa de cultivo mucho más rápido que las células TS después de tripsina, tal diferencia en la tasa de adherencia pueden ser utilizados para separar las células TS de los alimentadores de MEF. Después de pasar a las células cultivadas, que se incuba a 37 ° C CO, 2 al 5% durante 30 min. La mayoría de MEFs se adhieren a la placa y la población de TS permanece en suspensión. Transferir el sobrenadante a un nuevo plato. Repita este proceso para varios pasajes para obtener una población pura TS. </li><li> Mantener las células en TS TS-70cm-1.5F4H y pasarlos cada 4 días (01:10-1:20 split) o ​​cuando el cultivo alcanza el 70% de confluencia. Que pasa con las células de alta densidad (mayor que dividir 1:5) o su cultivo cuando está muy confluentes puede causar su diferenciación. </li><li> La identidad de las células TS puede ser confirmado por tinción de un marcador de células, Cdx2 (Figura 3). </li></ol> Congelar células TS <ol><li> Prepare 2x medio de congelación (50% de SFB, 30 medianas TS% y 20% DMSO). </li><li> Cocélulas llect TS por tripsinización, seguido por centrifugación a 450 g durante 6 minutos. Resuspender en medio de TS con igual volumen de medio de congelación 2x. </li><li> Congelar las células lentamente usando una cámara de alcohol isopropílico a -70 ° C y la transferencia de nitrógeno líquido después de 24-48 horas. </li><li> Una placa de 60 mm (70% confluente) generalmente se mantiene en 2 viales. </li></ol> Descongelar las células TS <ol><li> Rápido deshielo del vial en 37 ° C, que se acumulan en 10 ml de medios TS mediante centrifugación (450 g, 6 min), y volver a suspender en el 30% TS medios de comunicación, 70cm-1.5F4H. </li><li> Un vial de células se sembraron en una placa de 60 mm. </li></ol> TS diferenciación celular en TGC Diferenciación de las células TS en células gigantes trofoblasto (PGT) puede ser inducida por la retirada de las MEF-CM, FGF4 y heparina. El fenotipo de TGC (grandes núcleos) se pueden detectar cuatro días después de la inducción (Tanaka et al., 1998 y Chiu et al. 2008). Inmunotinción de P450scc detectar la expresión de este marcador TGC en la cultura diferenciadas (Figura 4). Análisis de citometría de flujo de PI (yoduro de propidio) las células teñidas muestra, además, la presencia de células diferenciadas que contienen un mayor contenido de ADN (Figura 4). Estos resultados sugieren que las células se someten TS endorreduplicación para convertirse en PGT poliploides. Resultados representante <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Figura 1" /> Figura 1. Disección del cuello del ratón. (A) Ubicación del cuello es indicado por las flechas. (B) Corte a través de la unión del útero y el cuello como se muestra. (C) Corte del útero justo por debajo del oviducto (OD) por debajo del ovario (OV). (D) de la imagen del cuello. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Figura 2" /> Imágenes la figura 2. Representante ilustrar blastocisto de ratón, consecuencia de blastocisto y de la colonia trofoblasto. (A) Un blastocisto de ratón recogidos en E3.5 se muestra. ICM, la masa celular interna, MT, trophectoderm mural, PT, trophectoderm polar. (B) Una consecuencia del trofoblasto en los alimentadores MEF se muestra. TS, de células madre del trofoblasto. (C) Una madre trofoblasto colonia de células muestra una clara ventaja en los alimentadores MEF. Las flechas indican el límite de la celda. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Figura 3" /> Figura 3. Identificación de las células TS por inmunotinción análisis. TS (AD) y ES (control, EH), las células están sujetos a la identificación de marcadores. Las células son immunostained con anticuerpos para reconocer Oct4 (A, E) o Cdx2 (B, F), y counterstained por DAPI (C, G) se presentan en las imágenes fusionadas (D, H). Barra de escala, de 50 micras. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Figura 4" /> Figura 4. Diferenciación de las células TS in vitro. (A) no diferenciadas (Undiff) o diferenciados (dif) las células en cultivo fueron examinados para la expresión de un marcador de TGC, P450scc (verde), contrastados con DAPI (rojo). (B) el análisis de citometría de flujo de las células diferenciadas, teñidas con PI, las medidas del contenido de ADN (M1, de dos a cuatro copias; M2, más de cuatro copias). La población M2 representa las células trofoblásticas poliploides.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

En este vídeo, que muestran el proceso para recoger E3.5 blastocistos de los procedimientos de útero y experimentales para establecer las líneas de células TS. También describe la condición de mantener la troncalidad de las células TS y para inducir su diferenciación en células diferenciadas. Dos pasos críticos para obtener líneas puras de células TS son el momento para la desagregación consecuencia (paso 9) y el procesamiento del primer paso (paso 11). Se ha informado de que la primitiva endodermo derivado...

Reactivos TS medio: RPMI-1640, 20% de SFB, 1 mM piruvato de sodio, 100 mM ß-mercaptoetanol y 100 mg / ml de penicilina-estreptomicina. Mitomicina C: Disolver 2 mg de mitomicina C (Sigma M-0503) en 2 ml de PBS y añadir esta mezcla de 2 ml a 200 ml de medio TS (10 ng / ml). Hacer 20 alícuotas (10 ml) y se almacena a -20 ° C hasta su uso. Añadir una alícuota por placa de 100 mm. Fibroblastos de embriones de ratón condición media (MEF-CM): Placa MEFs en placas de 100 mm (1 x 10 6 / placa). Al día siguiente, agregar 10 ml de medio de TS por plato y continuar a la cultura durante 48 horas. Recoger el medio de cultivo, filtrarlos (0,45 mm) y se almacena a -20 ° C en alícuotas de 35 ml. Descongele cada alícuota cuando sea necesario, que se pueden almacenar a 4 ° C. El MEFs se puede utilizar para preparar dos lotes más de CM antes de confluir. PBS / BSA: Disolver BSA, fracción V (Sigma A3311, un 0,1% (w / v)) en PBS (10 ml), a través de un filtro de jeringa de filtro de 0,45 mm y hacer alícuotas de 1 ml en tubos de 1,5 ml. Guardar a -70 ° C y descongelar un tubo cuando sea necesario para preparar FGF4. FGF4 (1000x): Resuspender liofilizado FGF4 (Sigma F8424, 25 mg) con 1 ml de la solución de PBS / BSA. Mezclar bien y hacer alícuotas de 10 (100 ml) en tubos de 1,5 ml y almacenar a -70 ° C. Descongele cada tubo cuando sea necesario y guarde el resto a 4 ° C, no volver a congelar. Diluir 1000 veces en el medio de TS para obtener 1x FGF4 (25 ng / ml). Heparina (1000x): Volver a suspender la heparina (Sigma H3149, 10.000 unidades) en PBS a una concentración final de 1 mg / ml (1000x). Hacer alícuotas de 100 ml en tubos de 1,5 ml y almacenar a -70 ° C. Alícuotas de deshielo cuando sea necesario y guarde el resto en 4 ° C, no volver a congelar. Diluir 1000 tiempo en el medio de TS para obtener 1x heparina (1 ng / ml).

derivation of mouse trophoblast stem cells from blastocysts

Especificación de la trophectoderm es uno de los eventos tempranos de diferenciación de desarrollo de los mamíferos. El linaje trofoblasto derivado de la implantación trophectoderm media y genera la parte fetal de la placenta. Como resultado, el desarrollo de este linaje es esencial para la supervivencia de los embriones. Derivación de trofoblasto del tallo (TS) las células de los blastocistos de ratón fue descrito por primera vez por Tanaka<em&gt; Et al.</em&gt; 1998. La capacidad de las células TS para preservar la propiedad del trofoblasto específicas y su expresión de tipos específicos de etapa y marcadores de células después de la estimulación adecuada proporciona un sistema modelo valioso para investigar el desarrollo del trofoblasto linaje por el que recapitula los primeros eventos placentación. Además, las células del trofoblasto son uno de los pocos tipos de células somáticas sometidos a la amplificación del genoma natural. A pesar de las vías moleculares que subyacen a poliploidización trofoblasto han empezado a desentrañar el papel fisiológico y las ventajas de la amplificación del genoma trofoblasto permanece en gran parte difícil de alcanzar. El desarrollo de las células madre en células diploides trofoblasto poliploides en la cultura hace que este<em&gt; Ex vivo</em&gt; El sistema una excelente herramienta para dilucidar el mecanismo de regulación de la replicación del genoma y la inestabilidad en la salud y la enfermedad. A continuación se describe un protocolo basado en informes anteriores, con la modificación publicada en Chiu<em&gt; Et al.</em&gt; 2008.

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

En este vídeo, se demuestra el aislamiento de los blastocistos de ratón y la derivación de células madre del trofoblasto de los blastocistos. También describe las condiciones para el mantenimiento de la propiedad de células madre, así como la inducción de diferenciación en la cultura.

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 ...

derivation-of-mouse-trophoblast-stem-cells-from-blastocysts

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JoVE Journal

Biology

18.1K vistas.  University of Rochester. En este vídeo, se demuestra el aislamiento de los blastocistos de ratón y la derivación de células madre del trofoblasto de los blastocistos. También describe las condiciones para el mantenimiento de la propiedad de células madre, así como la inducción de diferenciación en la cultura.

Video: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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

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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Aislamiento de células madre de la retina del ojo del ratón

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

El aislamiento de células primarias de ratón trofoblasto y ensayo de invasión trofoblasto

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

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 ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimiento de serie de la madre de espermatogonias y células progenitoras (SSC) en Cultura para la derivación de largo plazo de ratones adultos Líneas SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...