1. Descongelación hES células Pre-montaje experimental <ol><li> Un día antes de descongelar las células hES, la placa de una capa alimentadora de fibroblastos de ratón irradiados embrionarias (MEF), cepa CF1, en un pocillo de una gelatina con cubierta de 6 y la placa de cultivo de tejidos en el medio de cultivo MEF (D-MEM medio basal suplementado con 10% de SFB inactivado por calor y el 1% no aminoácidos esenciales). Incubar toda la noche a 37 ° C y CO 2 al 5%. </li><li> Antes de extraer las células hES de nitrógeno líquido, asegúrese de tener todo el equipo necesario y los reactivos en su lugar y listo para usar para asegurar un deshielo eficiente y exitosa. </li></ol> Descongelar las células hES <ol start="3"><li> Eliminar un vial criogénico de las células hES desde el tanque de nitrógeno líquido de almacenamiento con unas pinzas de metal. </li><li> Girar el vial entre las manos enguantadas de 3.5 segundos para eliminar la escarcha. </li><li> Registrar la información en la etiqueta del vial. </li><li> Con unas pinzas de metal, sumergir el frasco en un baño de agua a 37 ° C. Agitar suavemente el vial y observar el progreso de la descongelación a menudo (pero rápido!) Manteniendo el frasco a la luz para ver el tamaño de los cristales de hielo. No sumerja la tapa del frasco en el baño de agua, ya que podría contaminar las células. </li><li> Cuando sólo un cristal de hielo pequeños restos, sumergir el frasco entero en etanol para esterilizar. </li><li> En una cabina de seguridad biológica estéril, transferir el contenido del vial criogénico directamente a la parte inferior de un tubo cónico de 15 ml. </li><li> Poco a poco agregue 4 ml de medio de cultivo celular hES (D-MEM/F-12 medio basal suplementado con el reemplazo de 20% de suero golpe de gracia, un 1% no aminoácidos esenciales, 1% de L-glutamina, un 0,2% β-mercaptoetanol y 4 ng / mL bFGF) en el tubo. Agite suavemente para mezclar continuamente las células como el nuevo medio se añade al tubo. </li><li> Centrifugar las células durante 5 minutos a 200 x g. </li></ol> Revestimiento de las células hES <ol start="11"><li> Mientras que las células son hES en la centrífuga, preparar la placa de alimentación MEF, mediante el etiquetado con la información de la célula apropiada hES: la línea celular, el número de pases, y la fecha de deshielo. </li><li> Cuando hay aproximadamente 2 minutos por jugarse en el tiempo de giro, se aspira el medio de cultivo MEF y agregar 1,0 ml de PBS para el bienestar. </li><li> Llevar el granulado células hES de nuevo a la cabina de seguridad biológica, y con mucho cuidado aspirar el sobrenadante. Tenga cuidado de no aspirar el pellet de células, pero eliminar la mayor cantidad sobrenadante como sea posible, ya que esta solución contiene DMSO del medio de congelación. </li><li> Vuelva a suspender el pellet con mucho cuidado por la adición de 2,5 ml de medio de cultivo celular hES usando una pipeta de vidrio de 5 ml y pipeta 3-4 veces. </li><li> Aspirar el PBS del alimentador MEF bien y poco a poco agregar todos los de 2,5 ml de la suspensión celular hES al bien preparado de la placa de 6 pocillos. </li><li> Colocar la placa en la incubadora a 37 ° C y deslice la placa de adelante hacia atrás, de lado a lado para distribuir uniformemente las células a través del pozo. No girar la placa en un patrón circular para evitar la concentración de las colonias en el centro. </li><li> Permiten a las células para conectar a 37 ° C CO 2 y el 5% durante la noche. </li><li> El día después de la descongelación, se elimina el medio (con la presencia de células flotantes) y añadir 2,5 ml de medio de cultivo fresco hES celular a la fuente. </li><li> Opcional: El medio removido y las células pueden ser transferidos a un plato preparado por separado alimentador MEF. Este paso suele generar nuevas colonias que pueden ser cultivados en paralelo con las células del deshielo original. </li><li> Las placas se incuban a 37 ° C CO 2 y el 5% durante la noche. </li></ol> 2. Congelación de las células hES <ol><li> Mientras que las células son hES en la centrífuga, la etiqueta de los viales criogénicos el interior de la cabina de seguridad biológica, incluyendo la línea celular, el número de pases, y la fecha de congelación. La densidad típica para la congelación de las células hES es un bien de las células (de una placa de 6 pocillos) por vial criogénico. </li><li> Cuando las células hES terminado de centrifugación, llevar los tubos de nuevo a la cabina de seguridad biológica. Aspirar el sobrenadante del tubo, teniendo cuidado de no perturbar la suelta llena de sedimento celular. </li><li> Resuspender el botón celular con la cantidad apropiada de medio hES cultivo celular: 0,5 ml de células hES medio de cultivo por vial criogénico. Sea muy cuidadoso al pipetear, como las células hES se recuperan mejor cuando se congela en la colonia piezas relativamente grandes. </li><li> Poco a poco, mientras agitando suavemente el tubo, añada la cantidad apropiada (0,5 ml por criovial) de 2X medio de congelación hES celular (60% de SFB se define, el 20% medio celular hES la cultura, y el 20% DMSO) a las células. Una vez que el medio de congelación, se añade, la solución de pipeta muy suavemente una o dos veces para mezclar. </li><li> Rápidamente, pero suavemente, agregar 1 ml de la suspensión celular a cada vial criogénico. </li><li> La transferencia de los viales en un contenedor de congelación isopropanol y el lugar enun congelador de -80 ° C durante la noche. Las células se congelan a 1 ° C por minuto en el recipiente de congelación de isopropanol. </li><li> Al día siguiente, de forma rápida transferencia de los viales congelados a un tanque de nitrógeno líquido de almacenamiento con unas pinzas de metal. </li></ol> Resultados representante El primer día después de que las células embrionarias humanas se descongelan, pueden aparecer pequeñas colonias transparentes y puede ser difícil de ver bajo el microscopio. Desde recién descongelado células madre embrionarias tienden a proliferar lentamente, pueden tardar varios días en aparecer en las colonias establecidas (Figura 1). Figura 1. La recuperación y el crecimiento celular. HES células descongeladas del nitrógeno líquido son imágenes 1, 7 y 11 días después de la descongelación.

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <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), 1145-1147 (1998).</li></ol>

El video que acompaña muestra un método para la descongelación y la congelación de las células hES. Las células congeladas en nitrógeno líquido debe ser descongelado baño tan pronto como sea posible para obtener la mejor recuperación posible. Recuerde que debe tener mucho cuidado al pipetear células deshielo: minimizar la manipulación de las células y la pipeta suavemente. Un vial de células hES puede ser chapada en cualquiera de los dos pocillos de una placa de 6 pocillos, o todos los pocillos de una placa de 4 bien y colocarse inmediatamente en la incubadora. Desde el cultivo en cuatro pozos separados reduce la posibilidad de perder toda la cultura a la contaminación, y hace más fácil para localizar colonias hES celda en la superficie más pequeña, la placa de 4 y se recomienda descongelar las células hES por primera vez. El primer día después de la descongelación las células hES, las colonias son pequeñas y pueden ser transparentes. Reponer las células hES medio de cultivo todos los días, aunque las colonias no son visibles bajo el microscopio, ya que puede tardar algunos días en la cultura de las células hES para aparecer como las colonias establecidas. Es importante utilizar una capa MEF alimentador recién plateado descongelar las células, como las colonias a menudo no llegan a un tamaño apropiado para los pases hasta el 10 de 12 días después de la descongelación. Cuando se descongela un vial paso bajo de células HES, es una buena práctica para ampliar y la congelación de los viales adicionales para mantener un stock de células de paso bajo para futuros cultivos y experimentos. También es una buena idea de congelar sistemáticamente todos los &quot;extra&quot; las células sanas hES de mantenimiento de existencias congeladas. El deshielo de más éxito y de las culturas posteriores han sido bloqueados por alta calidad, diferenciado, de forma activa división de las colonias de células hES. células hES recuperarse de una congelación de manera más eficiente si se maneja con cuidado, como los agregados de células grandes. El tamaño total final debe ser similar a (o ligeramente más grande que) el tamaño de los agregados plateado después de un viaje de rutina.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF mediumjavascript:void(0);</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
</tr>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
</tr>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2560</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
</tr>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
</tr>		</tbody>
		</table>
		</div>

freezing and thawing human embryonic stem cells

Desde James Thomson<em&gt; Et al</em&gt; Desarrollado una técnica en 1998 para aislar y hacer crecer hES en la cultura, congelar células para su uso posterior y el deshielo y la ampliación de las células de una masa congelada se han convertido en importantes procedimientos realizados en el cultivo de rutina hES celular. Dado que las células hES son muy sensibles a las tensiones de la congelación y descongelación, debe tomar especial cuidado. Aquí se demuestra la técnica adecuada para descongelar rápidamente las células hES de las reservas de nitrógeno líquido, revestimiento de las células de ratón en el alimentador de embriones, y poco a poco de congelación para almacenamiento a largo plazo.

Watch this Scientific Journal Video about Freezing and Thawing Human Embryonic Stem Cells at JoVE.com

Freezing and Thawing Human Embryonic Stem Cells

Desde James Thomson et al desarrollaron una técnica en 1998 para aislar y hacer crecer hES en cultivo, las células de congelación para su uso posterior y el deshielo y la ampliación de las células de un stock congelado se han convertido en importantes procedimientos realizados en el cultivo de rutina hES celular. Dado que las células hES son muy sensibles a las tensiones de la congelación y descongelación, debe tomar especial cuidado. Aquí se demuestra la técnica adecuada para descongelar rápidamente las células hES de las reservas de nitrógeno líquido, revestimiento de las células de ratón en el alimentador de embriones, y poco a poco de congelación para almacenamiento a largo plazo.

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 ...

freezing-and-thawing-human-embryonic-stem-cells

Research

JoVE Journal

Biology

45.7K vistas.  Stemgent. Desde James Thomson et al desarrollaron una técnica en 1998 para aislar y hacer crecer hES en cultivo, las células de congelación para su uso posterior y el deshielo y la ampliación de las células de un stock congelado se han convertido en importantes procedimientos realizados en el cultivo de rutina hES celular. Dado que las células hES son muy sensibles a las tensiones de la congelación y descongelación, debe tomar especial cuidado. Aquí se demuestra la técnic...

Video: Freezing and Thawing Human Embryonic Stem Cells

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Congelar las células madre embrionarias humanas

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Investigación

Biología

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

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 ...

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

Congelación, descongelación, y las Pilas de embalaje para el transporte

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

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

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

La cultura y el mantenimiento de células estaminales embrionarias humanas

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

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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

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

Preparación Extienda los cromosomas de las células estaminales embrionarias para cariotipo

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

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