EQUIPO <ul><li> De cultivo de tejidos incubadora a 37 º C, 5% de CO 2 </li><li> Centrífugo </li><li> Juego de micropipetas (100, 100μL) </li><li> Baño de agua 37 ° C </li><li> Baño de agua a 90 ° C para ser fuente de vapor de agua </li><li> Microscopio de contraste de fase </li></ul> PRIMER PASO Tratar las células con colcemid En este procedimiento se utilizó la línea de células madre cultivadas H9 en fibroblastos embrionarios de ratón (MEF) inactivados con mitomicina C (Sigma) y se mantuvieron en DMEM/F12 (Invitrogen), complementado con el reemplazo de octavos de final 20% de suero (KSR, Gibco) y de 8ng/mL factor de crecimiento fibroblástico (FGF-2, R &amp; D). Con el fin de obtener un gran número de los diferenciales de metafases, se recomienda el uso de cultivos con alto índice mitótico en el que hay muchas células que se dividen. <ol><li> Tome el frasco de cultivo de la incubadora y, en el flujo laminar, añadir Colcemid a una concentración final de 0,1 mg / ml. </li><li> Volver el frasco a la incubadora y esperar 3 horas para que el inhibidor puede causar un paro mitótico en metafase, cuando los cromosomas están bien condensada y se pueden visualizar fácilmente bajo el microscopio. </li></ol> La fijación de las células <ol start="3"><li> Saque el medio y agregar tripsina / EDTA 0,05% suficiente para cubrir la superficie de las células. Permitir que las células se incuban durante 5 minutos a temperatura ambiente y luego inactivar la enzima, ya sea por la adición de medio de cultivo que contiene suero o con sólo añadir suero de las células. NOTA: Es importante asegurarse de que una única suspensión de células se realiza para obtener una buena propaga metafase. </li><li> Transferencia de las células a un tubo de centrífuga de 15 ml y centrifugar a 122 g durante 5 minutos. </li><li> Descartar el sobrenadante, dejando alrededor de 200 ml, y resuspender el precipitado suavemente tocando el tubo, como se muestra en el video. Asegúrese de que las células están bien disociado, por lo que no se puede visualizar cualquier grupo en la solución. </li><li> A continuación, tendrá que añadir 5 ml de solución hipotónica (KCl 75 mm) pre-calentado a 37 ° C, lentamente, por la pared del tubo como en la siguiente descripción. Primero se debe agregar 3 ml, invertir el tubo a una posición horizontal justo para mezclar las células con la solución hipotónica a continuación, añadir el resto de 2 ml y mantener caliente a 37 ° C durante 15 minutos. NOTA: En este punto la solución hipotónica se producirá un aumento en el volumen celular y ayudan a desenredar los cromosomas. El tiempo de incubación es importante para adquirir una buena extiende cromosoma. Si el tiempo es demasiado tiempo, la membrana celular puede explotar muy pronto y los cromosomas se han perdido. Si es demasiado corto, puede ser difícil de obtener porque se extiende el cromosoma de la membrana celular no puede alterar. </li><li> Añadir 3 gotas de la solución fijadora (metanol / ácido acético glacial 3:1), invertir el tubo suavemente para mezclar la solución de fijación con las células y se centrifuga a 122 g durante 5 minutos sin interrupción. NOTA: La solución de fijación debe estar recién hecho. NOTA: Es importante la utilización de la centrifugación sin descanso para evitar el desgaste células, evitando la pérdida del material. </li><li> Descartar el sobrenadante, dejando alrededor de 200 ml, y resuspender el precipitado suavemente tocando el fondo del tubo. NOTA: Una vez más, asegúrese de que las células están bien disociado, por lo que no se puede visualizar cualquier grupo en la solución. </li><li> Añadir los primeros 3 ml de solución fijadora, lentamente por la pared del tubo, mientras se gira el tubo. Invierta el tubo a una posición horizontal justo para mezclar las células, y luego añadir más 2 ml de la solución de fijación a la pared del tubo para tomar las células hacia abajo. De archivo a 4 ° C durante la noche. NOTA: Las células fijadas se pueden almacenar en la solución fijadora durante meses a 4 ° C. Además de proteger las células en su estado hinchado, la solución fijadora elimina los lípidos y las proteínas se desnaturaliza. Estos hechos hacen que la frágil membrana de la célula y, en consecuencia, que el cromosoma propaga fácilmente. </li></ol> LIMPIEZA DE LA portaobjetos de vidrio Antes de iniciar la toma se extiende a su cromosoma asegurarse de que las diapositivas se limpian correctamente o es posible que pierda algunos núcleos y también metafases. Este paso es también importante si quieres probar alguna técnica de hibridación 9. <ol><li> Coloque los portaobjetos en un vaso que contiene HCl 6M durante al menos 3 horas a temperatura ambiente. </li><li> Después de este tiempo, lavar los portaobjetos con agua corriente durante 10 minutos. </li><li> Enjuague los portaobjetos en agua destilada y dejar que se sequen a temperatura ambiente. </li><li> Ahora las diapositivas se pueden almacenar en 96% de etanol y se seca con un paño inmediatamente antes del uso. </li></ol> NOTA: Para los métodos de hibridación in situ, las diapositivas no se pueden almacenar durante más de 2 días en etanol al 96% SEGUNDO PASO Lavado de las células <ol start="10"><li> Al día siguiente, centrifugar las células a 122 g durante 5 minutos sin interrupción. </li><li> Discard el sobrenadante dejando alrededor de 200 ml. Resuspender el precipitado agitando suavemente el tubo a continuación, añadir 5 ml de solución fijadora fresca (metanol / ácido acético glacial 3:1), lentamente por la pared del tubo mientras se gira el tubo. Repita este paso de lavado dos veces más para un total de tres lavados. NOTA: La solución de fijación debe estar recién hecho. </li></ol> Preparación de las diapositivas <ol start="12"><li> En la última etapa de centrifugación dejar una cantidad suficiente de solución para homogeneizar el sedimento celular y obtener una densidad adecuada de las células en las diapositivas (ver figura 1). </li><li> Añadir 20-30 l de suspensión de células en un portaobjetos limpio y seco, moviendo la pipeta muy cerca y en paralelo a la superficie. Medida que se evapora la solución de fijación, la superficie de deslizamiento se convierte en granulado. </li><li> Inmediatamente, exponer la diapositiva, boca arriba, en el vapor de agua caliente (90 °) durante 30 segundos para hacer que las células explotan. NOTA: En este momento, el metanol se evapora de la solución de fijación, lo que resulta en una mayor concentración de ácido acético, que junto con el agua estimula la difusión de los cromosomas. </li><li> Mira a un microscopio de contraste de fase sólo para comprobar si la concentración, así como la distribución de las células es buena (ver figura 1). </li><li> Ahora las diapositivas están listos para ser utilizados para comprobar el juego de cromosomas mediante enfoques como la citogenética de bandas G (Giemsa), FISH, SKY y CGH. </li></ol> CONSEJOS IMPORTANTES ¿Cómo elegir una metafase adecuados para ser analizados La elección de metafases redondas y aisladas (figura 2D), es posible evitar la consideración de aneuploidías falsa como la ganancia de los cromosomas generados por dos o más se extiende mezclados entre sí o la pérdida de cromosomas generados por un cromosoma en marcha. Por lo tanto, evitar la elección de metafases cerca de los núcleos, ya que algunos cromosomas pueden estar ocultos por ellos (Figura 2A y 2B). Además, busca metafases ronda y estar seguros de que cualquier cromosoma se separa de la metafase (figura 2C). Células alimentadoras y el cariotipo células madre De bandas G y análisis SKY, la presencia de células alimentadoras en la cultura células madre no interferir con los resultados, porque las células de alimentación no están bajo un estado de división (inactivada por la mitomicina C o irradiación) y no será parte de la población de la metafase. Sin embargo, para la aplicación de la técnica de FISH en núcleos en interfase, es preferible separar a la población de células madre a las células de alimentación por la recolección de las colonias manualmente antes de tripsinización para el análisis de FISH. <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="Figura 1" /> Figura 1: ¿Cómo saber la densidad celular apropiada en las diapositivas. Las imágenes de microscopio de contraste de fase en el objetivo de 10x. (A) La densidad celular es muy alta. Las flechas indican la metafase se propaga muy cerca de los núcleos. (B) Una diapositiva con una buena densidad de las células. Los círculos muestran los diferenciales metafases aislado de núcleos o metafases otros. Por favor, <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1512/1512fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 1. <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="Figura 2" /> Figura 2: ¿Cómo elegir una metafase adecuados para ser analizados. Los cromosomas son teñidos con DAPI y las imágenes obtenidas por un microscopio de fluorescencia en el objetivo de 100x. (A) (B) Evitar metafases cerca de los núcleos (indicado por las flechas) (C) y no metafases ronda que los cromosomas presentan separados (círculo). (D) A la metafase ronda es una metafase bueno para ser analizados. Por favor, <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1512/1512fig2.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 2.

<ol>
	<li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

La preparación de los diferenciales de los cromosomas es un paso crítico para el éxito de un análisis de la situación genética de las células madre embrionarias mediante técnicas de rutina como el G-bandas, y las técnicas más sofisticadas como el pescado, SKY y CGH. Este procedimiento se puede aplicar a muchos tipos de células, variando el período de incubación colcemid, que depende de la duración del ciclo celular. Para las colonias de células madre embrionarias, que esperar 3 horas, mientras que para los cuerpos embrioides (EB), este tiempo puede ser de hasta 6 horas. En el caso de trabajar con agregados de células (como los cuerpos embrioides) tendrá que disocian muy bien con el fin de obtener una solución única célula. En este caso, después de la tripsina y la disociación mecánica, puede hacer uso de un filtro de nylon de células 40μm (BD Biosciences) y luego dar la secuencia del procedimiento (desde el paso 4 del tema de procedimientos). En el procedimiento descrito aquí vale la pena señalar que la suspensión de células se ha caído &quot;de cerca&quot; a las diapositivas con el fin de evitar una excesiva difusión de los cromosomas y, por consiguiente generación de artefactos. Creemos que este protocolo es una herramienta útil y sencilla para ser aplicados de manera rutinaria, no sólo por los laboratorios que trabajan con células madre embrionarias, pero por otros investigadores interesados ​​en estudiar la inestabilidad cromosómica en células madre de otros.

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chromosomal spread preparation of human embryonic stem cells for karyotyping

Aunque las células madre embrionarias (células madre) se ha demostrado que presentan un cariotipo diploide estable 1, muchos estudios han reportado que en función de las condiciones de cultivo se convierten propensos a adquirir las anomalías cromosómicas como la suma de la totalidad o partes de los cromosomas. De hecho, durante el cultivo prolongado, alteraciones del cariotipo se observan cuando la disociación enzimática o química se utilizan 2,3,4, mientras que la disección manual de las colonias de pases conserva un cariotipo estable 5. Además, los cambios en el medio ambiente tales como la eliminación de las células de alimentación también parecen poner en peligro la integridad genética de las células madre a 3,6. Una vez que las alteraciones cromosómicas pueden afectar la fisiología celular, la caracterización de la integridad genética de las células madre in vitro células madre es esencial considerar como una herramienta esencial en los estudios de la embriogénesis y la prueba de la droga. Por otra parte, para el futuro con fines terapéuticos cambios cromosómicos son una preocupación real, ya que se asocia frecuentemente a la carcinogénesis. Aquí mostramos un método sencillo y útil para obtener alta calidad se extiende cromosoma para su posterior análisis del cromosoma establecidos por bandas G, FISH, SKY o técnicas de CGH 7,8. Se recomienda comprobar el estado cromosómico de rutina, con intervalos de 5 pasajes a fin de vigilar la aparición de las translocaciones y aneuploidías Priscila Britto y Sartore Rafaela contribuido igualmente a la de papel.

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

El cariotipo es una técnica sencilla y útil ampliamente utilizado para la detección de alteraciones genéticas. A continuación se describe paso a paso el protocolo para la preparación de los cromosomas propagación de células madre embrionarias humanas para controlar el estado cromosómico de estas células se mantienen en cultivo.

Preparación Extienda los cromosomas de las células estaminales embrionarias para cariotipo

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

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Nanyang Technological University

Research

JoVE Journal

Biology

48.6K vistas.  Federal University of Rio De Janeiro-UFRJ. El cariotipo es una técnica sencilla y útil ampliamente utilizado para la detección de alteraciones genéticas. A continuación se describe paso a paso el protocolo para la preparación de los cromosomas propagación de células madre embrionarias humanas para controlar el estado cromosómico de estas células se mantienen en cultivo.

Video: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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 ...

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 ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

En el etiquetado in vitro de células estaminales embrionarias humanas de Resonancia Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

La cultura y el mantenimiento de células estaminales embrionarias humanas

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

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparación de combinados lisado plaquetario humano (pHPL) como un suplemento eficaz de los animales libres de suero humano culturas de células madre

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

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