Agradecemos a los miembros del laboratorio de Meshorer, especialmente Shai Melcer, Alajem Adi, Edupuganti Raghu Ram, Badi Sri Sailaja, Mattout Anna y Biran Alva, por comentarios críticos y ensayos de solución de problemas photobleaching sobre una base diaria. EM es una H. Joseph y Belle R. Braun Profesor titular de Ciencias de la Vida y con el apoyo de la Fundación Ciencias de Israel (ISF 943/09), el Ministerio de Salud de Israel (6007) de la Unión Europea (IRG-206872 y 238176), la Fundación para la Investigación del Cáncer de Israel, las subvenciones internas Aplicativo de Medicina de la Universidad Hebrea y del Instituto de Psicobiología Israel.

1. Revestimiento de las células madre embrionarias T = 0 horas MEF placas <ol><li> Escudo de la imagen en vivo de 8 y μ-Slides (ibidi, Munich, Alemania) con gelatina o en cámaras cubiertas de vidrio (Lab-Tek, Rochester, NY) o en placas de cultivo con fondo de cristal (MatTek, Ashland, MA). Deja de 5-30 min y aspirar lejos de la gelatina sin. </li><li> Semillas de 22.000 MEFs / pocillo en 250 l de volumen total de DMEM [complementado con fetal al 10% de suero bovino (FBS)]. Permiten a las células crecer en un cultivo de tejidos incubadora (37 ° C, 5% CO2). </li></ol> T = 6 horas ES recubrimiento celular <ol start="3"><li> Aspirar DMEM. </li><li> Semillas de cada MEF pozo cubierto con 15.000 células R1 / pocillo en 250 l de volumen total de los medios de comunicación de las células ES [complementado con ESC de grado 10% de suero fetal bovino (FBS), 1 mM piruvato de sodio, 0,1 mM de aminoácidos no esenciales, 0,1 mM de β- mercaptoetanol, y 1000 U / ml de factor inhibidor de leucemia (LIF)], para obtener un 30% a 50% de confluencia al día siguiente. </li></ol> 2. Transfección de las células madre embrionarias T = 24 horas Transfección transitoria <ol><li> Vuelva a colocar los medios de comunicación de las células ES, con 250 l / pocillo de nuevo los medios de comunicación de células ES. </li><li> En un tubo de ensayo 1,5 ml estéril, añadir 100 ml de suero libre de los medios de comunicación [Opti-MEM (Gibco)], a continuación, añadir 10 TransIT-LT1 l reactivo de transfección (Mirus) directamente en los medios de comunicación libres de suero. Mezclar por pipeteo suave e incubar a temperatura ambiente durante 50-20 min. </li><li> Añadir 1,5 g GFP ADN plásmido de fusión (H1E, H1o o HP1) de los gases de tránsito LT1 reactivo. Mezclar por pipeteo suave e incubar a temperatura ambiente durante 15-30 min. </li><li> Añadir 13,5 l / pocillo de la mezcla de transfección. Agitar el 8 y μ-Slides para asegurar una dispersión. Después de 24 horas reemplazar los medios de comunicación ES celulares viejos, con 250 l de nuevo los medios de comunicación de células ES. </li></ol> 3. Realización de FRAP y FLIP T = 48 a 72 hrs <ol><li> El experimento se puede realizar en cualquier microscopio láser confocal de barrido (CLSM) pero como en una normal FRAP / FLIP experimento, muchas imágenes consecutivas son adquiridos, se recomienda el uso de un microscopio confocal de disco giratorio, que permite que la velocidad de adquisición y asegura que no muestra no deseada decoloración se produce después de la inicial de eventos intencionales de blanqueo. En este caso, recomendamos el uso de la Revolución giro de disco del sistema confocal (www.Andor.com), con la Yokogawa CSU-X cabeza del disco giratorio. Este sistema tiene la doble capacidad de photobleach con un módulo especializado FRAPPA con un sistema de puntos de exploración, y cambiar rápidamente la luz de fondo para recoger las imágenes con el disco giratorio. Los tres proteínas fluorescentes más comunes utilizados para los experimentos se photobleaching GFP, YFP y Cherry. Si GFP o YFP se utilizan, a ~ 488 nm láser es necesario. Para Cherry, el uso de un láser ~ 560 nm. En todos los casos, recomendamos el uso de láseres de estado sólido. Tener una etapa automatizado es útil pero no necesario. Dado que las células vivas son imágenes, es fundamental utilizar una cámara de medio ambiente (se puede utilizar un LIS, Suiza), el control de oxígeno, humedad, CO 2 y la temperatura. FRAP se realiza con la intensidad del láser, mientras que el máximo de imágenes se hace con la potencia del láser mínimo requerido (por lo general en el área del 10%, cuando el nivel de fluorescencia es adecuada). Observar las células con luz fluorescente de la longitud de onda apropiada y seleccione una célula que expresa GFP usando un lente de 60x de inmersión en aceite. Asegurar una distribución subcelular correcta. En ocasiones, cuando los niveles de expresión son muy altos, la localización de la proteína puede &quot;derrame&quot; de otros compartimentos como el nucléolo. Estas células no se debe elegir. </li><li> Ahora establezca un protocolo de imagen: recolectar 5.3 cuadros antes que el photobleaching, entonces photobleach en eucromatina o heterocromatina (vistos como focos condensada GFP) y recoger 90 a 120 fotogramas después de la photobleach, con intervalos de 250 a 1.000 ms: H1E-GFP, 1000 ms, H1o y HP1-GFP, 250 ms (intervalo de tiempo los cambios de acuerdo a la dinámica de las proteínas, donde las proteínas altamente dinámicos requieren menor tiempo de intervalo). Normalmente se usan 80 a 100% la intensidad del láser para photobleaching con un pulso láser de μseconds 20-40 (1-2 repeticiones), pero estos números pueden cambiar de acuerdo a la proteína analizados y los niveles de expresión. Cuando photobleaching es apropiado, se debería observar un &quot;agujero negro&quot; en la fluorescencia de GFP. El agujero negro se va re-lleno de recuperación después de la fluorescencia. Aunque el disco giratorio se puede obtener un máximo de alrededor de 60 imágenes por segundo (con buena intensidad de la fluorescencia y el zoom en una sola célula), no se recomienda utilizar el sistema a una velocidad tan alta debido a la baja calidad de imagen y mayor potencial de fototoxicidad. </li><li> Para un experimento de la FLIP, establecer un protocolo de imágenes diferentes: recolectar 5.3 cuadros antes del blanqueo, y comienza a repetir blanqueo en el mismo lugar durante la percepción de imágenes. Para H1E-GFP, lejía cada 5 segundos, para H1o-GFP, cloro cada 2 segundos, y para HP1-GFP cloro cada 1 seg. Bleach und recoger imágenes en repetidas ocasiones a lo largo de todo el experimento. </li><li> Para cualquiera de estas técnicas, repita el proceso en 20-30 células. A efectos estadísticos, repita el experimento 3 veces o más, preferentemente en días diferentes. En poblaciones homogéneas y configuración correcta, la desviación estándar es generalmente baja (&lt;5%). Por tanto FRAP y FLIP, el tamaño y la forma de la zona blanqueada influye en la dinámica de recuperación y debe permanecer constante dentro de un experimento. Además, cuando se comparan dos células, los protocolos debe ser idéntico y las células deben ser analizadas de forma secuencial en el mismo día que la potencia del láser y otras condiciones pueden variar y pueden afectar el resultado del experimento. </li></ol> 4. FRAP y Análisis de Datos FLIP <ol><li> En todos los cuadros recogidos FRAP, medir la intensidad de fluorescencia en el retorno de la inversión (ROI b = área blanqueada), el área de fondo (ROI bg), y el área de no proliferación nuclear, blanqueadas (ROI nb) como una función de tiempo antes y después del blanqueamiento. Cuando la región blanqueada es insignificante todo el núcleo puede ser seleccionado para fines de normalización. </li><li> Por cada punto del tiempo, normalizar los datos de acuerdo con la fórmula: (b ROI - ROI bg) / (ROI nb - ROI bg) / (b pbROI - pbROI bg) / (pbROI nb - pbROI bg), pb indica pre-blanqueado. Para pre-cloro imágenes que debería obtener un valor aproximado de 1. La primera imagen después de que el cloro se indica la profundidad de Bleach. Restar el valor de 1 para el valor de Bleach profundidad real. Repita la operación para todas las células y el promedio de 20 a 30 células de cada experimento. </li><li> En todos los cuadros recogidos FLIP, medir la intensidad de fluorescencia en la zona de no proliferación nuclear, blanqueados, y el área de fondo (ROI nb = no blanquea la zona, el ROI de fondo bg =). Cálculo de los datos de la FLIP es similar a una curva de FRAP, sólo el retorno de la inversión analizada (ROI nb) debe ser diferente de la propia región blanqueada, que no se utiliza para el cálculo: (ROI nb - ROI bg) / (pbROI nb - pbROI bg) . También es posible utilizar una celda contigua (ROI n celular vecino =) para fines de normalización: (ROI nb - ROI bg) / (ROI n - ROI bg) / (pbROI nb - pbROI bg) / (pbROI n - pbROI bg ). Tras la recogida de datos, es posible ajustar los datos experimentales a la simulación por ordenador. Esto permite calcular, con una buena proximidad, la fracción móvil, la fracción de inmovilidad y la mitad del máximo. No vamos a discutir los aspectos matemáticos y estadísticos de análisis FRAP aquí y remitimos al lector a otras publicaciones excelentes 4-9. La profundidad de cloro se refiere a la distancia (en el eje y) entre el pre-cloro (100%) de la señal y la primera imagen después de cloro, la fracción móvil se refiere a la distancia (en el eje y) entre la profundidad de cloro y la señal recuperada cuando la cinética alcanza una meseta, y la fracción inmóvil refiere a la distancia (en el eje y) entre la señal recuperada y la señal de pre-cloro (100%) (ver Figuras 1B y 2B). Más allá de este análisis, hay buenos modelos matemáticos para ajustar los datos. Para un exponente único, la ecuación  <img src="/files/ftp_upload/2696/eq0.jpg" alt="Ecuación" width="80" /> donde T es el tiempo, A es la fracción móvil, 1-A es la fracción inmóvil y fuera de k es la constante de disociación, se puede utilizar para ajustar los datos, y una estimación directa de la tasa de descuento de la unión (k off) se puede obtenidos, así como para el parámetro A, que se puede utilizar para calcular el tipo de asociación. </li></ol> 5. Los resultados representativos: <img src="/files/ftp_upload/2696/2696fig1.jpg" alt="Figura 1" /> Figura 1. A y B muestran las curvas de FRAP representante de HP1 (izquierda), H1o (centro) y H1E (derecha) en las células ES R1. La figura de la simplicidad y claridad 1A muestra los datos en bruto de una sola célula antes de que la normalización y el cálculo. La curva amarilla corresponde a la región blanqueada, la curva de color púrpura se corresponde con el área de no proliferación nuclear, blanqueadas (cuando la región blanqueada es insignificante todo el núcleo puede ser seleccionado para fines de normalización), y la línea roja corresponde a la fluorescencia de fondo, que es mínima en este caso. Flecha vertical representa el tiempo de blanqueo. Los datos normalizados y un promedio se muestra en la Figura 1B. Tenga en cuenta la recuperación más lenta de H1 (azul) en comparación con HP1 (rojo). También la variante H1E (azul oscuro) es más lenta que la variante H1o (azul claro). Fracciones móviles e inmóviles, y la profundidad de Bleach están indicados para HP1. <img src="/files/ftp_upload/2696/2696fig2.jpg" alt="Figura 2" /> Figura 2. A y B muestran las curvas de FRAP representante comparar eucromatina (izquierda) con la heterocromatina (derecha) de HP1 en las células ES R1. Al igual que en la Figura 1, Figura 2A muestra los datos en bruto de una sola célula, la curva amarilla corresponde a la región blanqueada, la curva de color púrpura se corresponde con el área de no proliferación nuclear, blanqueados, y la línea roja corresponde a la fluorescencia de fondo. Flecha vertical representa el tiempo de blanqueo. Los datos normalizados y un promedio se muestra en la Figura 2B. Tenga en cuenta la recuperación más lenta de la heterocromatina (rojo oscuro) en comparación con la eucromatina (luz roja). Fracciones móviles e inmóviles, y la profundidad de Bleach están indicados para la eucromatina. <img src="/files/ftp_upload/2696/2696fig3.jpg" alt="Figura 3" /> Figura 3.. Un experimento típico de la FLIP H1o R1 células madre embrionarias se muestra en la Figura 3 (en bruto, sin normalizar los datos) y B (datos normalizados y medio). En este experimento, la curva de color púrpura se corresponde con el área nuclear no blanqueado, la línea verde corresponde a un núcleo de la célula vecina y la línea roja corresponde a la fluorescencia de fondo.

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	<li>Phair, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+nature+of+dynamic+protein-chromatin+interactions+in+vivo:+three-dimensional+genome+scanning+and+dynamic+interaction+networks+of+chromatin+proteins.">Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.</a> Mol Cell Biol. 24, 6393-6402 (2004).</li><li>Meshorer, E., Girard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging chromatin in embyonic stem cells in StemBook. , (2008).</li><li>Meshorer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdynamic+plasticity+of+chromatin+proteins+in+pluripotent+embryonic+stem+cells.">Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.</a> Dev Cell. 10, 105-116 (2006).</li><li>Bancaud, A., Huet, S., Rabut, G., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photo conversion, and FLIP. , (2009).</li><li>Dundr, M., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+dynamics+of+nuclear+proteins+by+photobleaching.">Measuring dynamics of nuclear proteins by photobleaching.</a> Curr Protoc Cell Biol. Chapter 13, Unit 13-Unit 13 (2003).</li><li>Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+membrane+dynamics+and+reassembly+in+living+cells:+targeting+of+an+inner+nuclear+membrane+protein+in+interphase+and+mitosis.">Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.</a> J Cell Biol. 138, 1193-1206 (1997).</li><li>Lenser, T., Weisshart, K., Ulbricht, T., Klement, K., Hemmerich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+fluctuation+microscopy+to+reveal+3D+architecture+and+function+in+the+cell+nucleus.">Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.</a> Methods Cell Biol. 98, 2-33 (2010).</li><li>Mueller, F., Mazza, D., Stasevich, T. J., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+and+kinetic+modeling+in+the+analysis+of+nuclear+protein+dynamics:+what+do+we+really+know.">FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know.</a> Curr Opin Cell Biol. 22, 403-411 (2010).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modelling+approaches+to+in+vivo+imaging.">Kinetic modelling approaches to in vivo imaging.</a> Nat Rev Mol Cell Biol. 2, 898-907 (2001).</li><li>Poser, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+TransgeneOmics:+a+high-throughput+method+for+exploration+of+protein+function+in+mammals.">BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.</a> Nat Methods. 5, 409-415 (2008).</li><li>Sigal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+fluorescently+labeled+endogenous+protein+library+in+living+human+cells.">Generation of a fluorescently labeled endogenous protein library in living human cells.</a> Nat Protoc. 2, 1515-1527 (2007).</li><li>Cohen, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+proteomics+of+individual+cancer+cells+in+response+to+a+drug.">Dynamic proteomics of individual cancer cells in response to a drug.</a> Science. 322, 1511-1516 (2008).</li></ol>

A diferencia de la mayoría de las técnicas disponibles, que incluyen la cromatina purificada a partir de poblaciones de células o células fijadas, los experimentos FRAP seguir los cambios en la dinámica de las proteínas de la cromatina en las células vivas. Encontramos dinámica de la cromatina proteínas para ser un buen indicador de la plasticidad de la cromatina. Sin embargo, debido a que requiere la fusión del gen de interés con las buenas prácticas agrarias, la adición de la etiqueta fluorescente puede i...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> DMEM </td><td> Sigma </td><td> D5671 </td></tr><tr><td> Gelatina </td><td> Merck </td><td> 1,04078 </td></tr><tr><td> Opti-MEM </td><td> Gibco </td><td> 31985 </td></tr><tr><td> TransIT-LT1 </td><td> Mirus </td><td> MIR2300 </td></tr><tr><td> 8 y μ-Diapositivas </td><td> ibidi </td><td> 80826 </td></tr></tbody></table>

photobleaching assays (frap &amp; flip) to measure chromatin protein dynamics in living embryonic stem cells

La recuperación de fluorescencia después de photobleaching (FRAP) y la pérdida de fluorescencia en photobleaching (FLIP) permiten el estudio de la dinámica de proteínas en las células vivas con una buena resolución espacial y temporal. Aquí se describe cómo llevar a cabo ensayos FRAP y FLIP de proteínas de la cromatina, como H1 y HP1, en madre embrionarias de ratón (ES) las células. En un experimento FRAP, las células son transfectadas, ya sea transitoria o estable, con una proteína de interés fusionada con la proteína verde fluorescente (GFP) o sus derivados (YFP, PPC, cerezo, etc.) En las células transfectadas, fluorescentes, un intenso haz de láser enfocado blanqueadores una región relativamente pequeña de interés (ROI). La longitud de onda se selecciona de acuerdo a la proteína fluorescente utilizado para la fusión. La luz del láser irreversiblemente blanquea la señal fluorescente de moléculas en el retorno de la inversión, e inmediatamente después de blanqueo, la recuperación de la señal fluorescente en la zona blanqueada - mediado por la sustitución de las moléculas de blanqueado con las moléculas de crudo - se vigila por medio de imágenes de lapso de tiempo. Las curvas de fluorescencia generada recuperación de proporcionar información sobre la movilidad de la proteína. Si las moléculas fluorescentes son inmóviles, no hay recuperación de fluorescencia se observó. En un enfoque complementario, la pérdida de fluorescencia photobleaching (FLIP), el rayo láser blanquea el mismo lugar varias veces y la intensidad de la señal se mide en otras partes de la célula de fluorescencia. Experimentos FLIP por lo tanto, medir la descomposición de la señal en lugar de la recuperación de la fluorescencia y son útiles para determinar la movilidad de proteínas, así como proteínas y venir entre los compartimentos celulares. Unión transitoria es una característica común de las proteínas asociadas a la cromatina. Aunque la mayor fracción de cada proteína de la cromatina se une a la cromatina en un momento dado en el estado estacionario, la unión es temporal y la mayoría de las proteínas de la cromatina tienen una alta rotación en la cromatina, con un tiempo de residencia en el orden de segundos. Estas propiedades son cruciales para la generación de alta plasticidad en la expresión del genoma 1. Photobleaching experimentos son particularmente útiles para determinar la plasticidad de la cromatina mediante la fusión GFP-versiones de la cromatina proteínas estructurales, especialmente en las células madre embrionarias, donde el intercambio dinámico de las proteínas de la cromatina (incluyendo la proteína de heterocromatina 1 (HP1), la histona H1 y enlazador histonas) es mayor que en las células diferenciadas 2,3.

Watch this Scientific Journal Video about Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells at JoVE.com

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Se describen los métodos de photobleaching incluyendo recuperación de fluorescencia después de photobleaching (FRAP) y la pérdida de fluorescencia en photobleaching (FLIP) para controlar la dinámica de la cromatina de proteínas en células madre embrionarias (ES) las células. Dinámica de la cromatina proteína, que es considerado como uno de los medios para estudiar la plasticidad de la cromatina, es mayor en las células pluripotentes.

Photobleaching ensayos (FRAP y FLIP) para medir la dinámica de la cromatina de proteínas en células vivas madre embrionarias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

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Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

27.1K vistas.  The Hebrew University of Jerusalem. Se describen los métodos de photobleaching incluyendo recuperación de fluorescencia después de photobleaching (FRAP) y la pérdida de fluorescencia en photobleaching (FLIP) para controlar la dinámica de la cromatina de proteínas en células madre embrionarias (ES) las células. Dinámica de la cromatina proteína, que es considerado como uno de los medios para estudiar la plasticidad de la cromatina, es mayor en las células pluripotentes.

Video: Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

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 ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Los patrones de las células madre embrionarias usando el Flip Chip Bio

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondeo de la actividad mitocondrial complejo en células estaminales embrionarias humanas

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

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 ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

El uso de LysoTracker para detectar la muerte celular programada en embriones y la diferenciación de células madre embrionarias

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Perfiles de Células Madre Embrionarias Humanas individual por RT-PCR cuantitativa

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Usando plusTipTracker Software a Medida microtúbulos dinámica en<em&gt; Xenopus laevis</em&gt; Conos de Crecimiento

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...