1. Mantenimiento: Los cultivos de observación bajo el microscopio <ol><li> Quitar una placa de células hES de la incubadora a 37 ° C y llevar al microscopio. </li><li> Observe las colonias a baja potencia, con aumento de 2X o 5 veces, para evaluar la calidad de la cultura en general. Tenga en cuenta lo siguiente: color medio normal, la ausencia de cualquier tipo de contaminación visible, tamaño de la colonia en general, la calidad y la densidad, y cualquier otra observación. </li><li> Observar los cambios de color medio. Debido al cambio en el pH y el agotamiento de los nutrientes en el medio después de una noche de incubación con las células hES, el medio va a cambiar de un tono rojo a la &quot;paja&quot; de color. Si el medio aparece púrpura, un cambio de pH básico se ha producido. Si el medio aparece muy amarillo, que el medio se acidifica. Medio muy amarilla puede ser el resultado de colonias muy hacinados en el pozo o la contaminación. El medio debe ser transparente en todo momento. A pesar de un cierto nivel de restos celulares en el medio es normal, que nunca debe aparecer nubes. Si un alto nivel de los desechos celulares se observa, la cultura no es saludable y puede estar contaminada. </li><li> Si los cultivos no requieren mantenimiento o pases, que pueden ser llevados a la cabina de seguridad biológica a ser alimentado (ver sección 2). </li><li> Si las culturas contienen más de un 10% diferenciar las colonias, las células deben ser &quot;limpiado&quot; por la eliminación manual de las áreas diferenciadas (ver sección 3). </li><li> Si las culturas contienen grandes colonias o denso, o la capa de alimentación MEF es más de 12 días, las células deben ser pasados ​​(ver sección 4). </li></ol> 2. Las células de alimentación hES <ol><li> En una cabina de seguridad biológica estéril, retire la tapa de la placa de cultivo y se aspira el medio empleado. No permita que la punta de la pipeta toque ninguna parte de la placa que no sea directamente en el interior de los pozos. Si hay alguna preocupación de la contaminación, el cambio de una pipeta nueva y estéril. </li><li> Con una pipeta de vidrio estéril, añada la cantidad apropiada de medio de cultivo calentado hES celular a cada pocillo. Para las culturas en una placa de 6 pocillos, agregar 2,5 ml de medio por pocillo. </li><li> Devolver la placa a la incubadora para permanecer a 37 ° C y 5% CO 2. </li></ol> 3. La eliminación de la diferenciación de cultivos celulares hES <ol><li> Transformar de 9 pulgadas de cristal pipetas Pasteur en instrumentos de recogida mediante moldeo por los consejos sobre la llama controlada de un mechero de alcohol. Asegúrese de que la punta de la pipeta está sellado para evitar la contaminación y redondeados para evitar rayar el plástico de la placa de cultivo. Esterilizar los instrumentos de recogida antes de su uso con la fuente de luz UV en la cabina de seguridad biológica o recoger recinto. </li><li> Eliminar las áreas diferenciadas. Diferenciación ocurre a menudo en sólo una parte de una colonia de células HES, por lo general a lo largo de los bordes o en forma de manchas aisladas en el centro. Si sólo una parte de una colonia es la diferenciación, sólo el área diferenciada debe ser eliminado. Diferenciación a menudo puede levantar de la placa en una &quot;pegajosa&quot; de la hoja, y es útil primero separar las células diferenciadas del resto de la colonia, trazando una línea a través de la colonia con el fin de la herramienta de selección. </li><li> Una vez que las piezas de la colonia se separan, se deslizan suavemente la herramienta de selección a lo largo de la placa para separar las células no deseadas. Tenga cuidado de no raspar demasiado de la capa de alimentación MEF entre las colonias en este proceso. </li><li> Cuando todas las células diferenciadas se retira de la placa (y flotan en el medio), el retorno de la cultura a la cabina de seguridad biológica. Aspirar el medio que contiene las células diferenciadas y reemplazar con medio de cultivo fresco hES celular. </li></ol></ol> 4. Pases hES células Enzima de incubación <ol><li> En una cabina de seguridad biológica estéril, retire la tapa de la placa de cultivo de 6 pocillos y aspirar el medio gastado de los pozos a ser pasados. No permita que la punta de la pipeta toque ninguna parte de la placa que no sea directamente en el interior de los pozos. </li><li> Con una pipeta de vidrio estéril, agregar 1 ml de colagenasa IV calentado a cada pocillo para pasarse. </li><li> Devolver la placa a la incubadora a 37 ° C durante 5 minutos. </li></ol> Raspado y la agrupación de las células hES <ol start="4"><li> Después de incubar las células con colagenasa, observar las colonias bajo el microscopio. La enzima que causa un cambio sutil, pero se observa en los bordes de la colonia. </li><li> En la cabina de seguridad biológica, suavemente aspire a la enzima de cada bien y reemplazar con 2,0 ml de medio de cultivo hES celular. </li><li> La punta de la placa de cultivo ligeramente hacia usted y tome el medio de 2,0 ml en el primer pocillo con una pipeta de vidrio de 5 ml. La celebración de la pipeta perpendicular a la parte inferior de la placa, se deslizan suavemente la punta de la pipeta a través del bien, mientras que poco a poco soltando el medio. </li&gt; <li> Repita el raspado y pipetear 4.3 veces el movimiento hasta que todas las colonias se han retirado del pozo. Pipeta con cuidado para no romper las colonias en muy pequeña de las piezas. &nbsp; Cuando las células se han quitado del primer pozo, dejar el contenido en el pozo y comenzar a raspar las células fuera de la próxima también. </li><li> Cuando todos los pozos a ser pasados ​​han sido raspadas, la piscina del medio que contiene las piezas de cada colonia y en un tubo estéril de 15 mL cónico. </li><li> Lave los pocillos raspada por la adición de 1 ml de medio de cultivo celular hES a cada pocillo. Recoge este enjuague y traslado al tubo cónico. </li><li> Pipeta suavemente las células en el tubo para romper las piezas colonia hasta el tamaño deseado. </li><li> Centrifugar las células durante 5 minutos a 200 x g. </li></ol> Revestimiento de las células de la célula hES <ol start="12"><li> Mientras que las células son hES en la centrífuga, preparar una nueva placa de 6 pocillos de alimentación MEF. Etiqueta de la placa con la información de la célula apropiada hES: la línea celular, el número de pases nuevos, y la fecha de aprobación. Aspirar el medio MEF de los pozos y añadir 1,0 ml de PBS en cada pocillo. Remover con suavidad la seguridad alrededor de los pozos y aspirar el lavado con PBS. Añadir 1,5 ml de células hES medio de cultivo para cada bien y dejar a un lado del plato. </li><li> Cuando las células hES terminado de centrifugación, llevar el metro de vuelta a la cabina de seguridad biológica. Aspirar el sobrenadante, teniendo cuidado de no perturbar la suelta llena de sedimento celular. </li><li> Resuspender las células en el sedimento con un medio suficiente para 1 ml de medio de cultivo celular por hES así a ser plateado. Cuando se divide en 6 nuevos pozos, resuspender el precipitado en un medio de 6 ml. </li><li> Suave y uniformemente agregar 1 ml de la suspensión celular a cada bien preparado de la placa de alimentación MEF. </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. </li><li> Permiten a las células para conectar a 37 ° C durante la noche. </li></ol> 5. Los resultados representativos: Indiferenciado de células hES son pequeños, bien repleto, y por lo general consisten en mayor extensión, más por las células. La diferenciación puede ocurrir dentro de una colonia (Figura 1) o entre las colonias (Figura 3B). Morhopologies diferente puede ser visto con una lupa de baja bajo el microscopio. Una buena morfología de las células, como se ve en la Figura 2A, contiene células pequeñas, apretadas que crecen en una monocapa. Las células deben tener bordes limpios y definidos, con poca o ninguna diferenciación. Las células se muestra en la Figura 2B están listos para los pases, y las células se muestra en la Figura 2C están saturados. <img src="/files/ftp_upload/1427/1427fig1.jpg" alt="Figura 1" /> Figura 1. Diferenciación en las culturas hES. (A) la diferenciación de una colonia (B) la diferenciación entre las colonias. <img src="/files/ftp_upload/1427/1427fig2.jpg" alt="Figura 2" /> Figura 2. Morfologías visto en las culturas hES. (A) morfología de las células buenas (B) Las células hES que están listos para pases (C) celdas superpobladas.

<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></ol>

La esterilidad se debe mantener en todo momento cuando se trabaja con células hES. Limpiar y esterilizar la cabina de seguridad biológica y todo el equipo antes de su uso. Todos los reactivos deben ser filtrados mediante un filtro de 0,22 micras de tamaño de poro antes de su uso. El uso de antiboiotics en cultivo celular hES no es necesario y debe ser evitado. Un ambiente separado se debe configurar como una estación de recogida designados. El recinto estéril para la estación puede ser una caja estática (como una estación de trabajo PCR) con una fuente de luz UV, una campana de flujo laminar, o una cabina de seguridad biológica. Un microscopio de disección dentro de la estación de recolección es necesario para observar las colonias de las zonas diferenciadas se eliminan. El panel de cristal delantero de un recinto estática o una cabina de seguridad biológica debe ser modificada para permitir los oculares del microscopio se extienden a través del panel sin comprometer la correcta circulación del aire y la esterilidad. Para mantener sanos los cultivos celulares hES, las células deben ser pasados ​​en el momento óptimo, por lo general cada 4-7 días. En este momento, las colonias han alcanzado su tamaño máximo y puede ser empezando a unirse. Colonias combinado puede aumentar la tasa de diferenciación en la cultura. Coeficientes de distribución en general, la caída 01:03-01:05, en función del número de colonias plateado, la tasa de expansión, y las condiciones de cultivo. Colonias Overplated (relación de división demasiado baja) probablemente se fusionarán antes de tiempo y necesitan ser pasados ​​antes de alcanzar su tamaño máximo. Culturas en una capa de alimentación MEF que es más de 2 semanas de edad deben ser pasados ​​al nuevo MEFs que apoyar el crecimiento y la proliferación indiferenciada. Ya que la diferenciación se produce naturalmente en los cultivos celulares hES, una pequeña cantidad que se espera. Diferenciación frecuente o excesiva puede ocurrir si los cultivos no reciben la atención adecuada. Las células deben ser alimentados todos los días, el crecimiento excesivo entre los pasajes deben ser evitados. Siempre use reactivos nuevos. Todos los medios de comunicación, MEFs y los reactivos deben ser utilizados dentro de 14 días para evitar el crecimiento indiferenciado de células hES. Nuevos lotes de reactivos, tales como la sustitución de golpe de gracia en suero, FBS y MEFs, deben ser examinados antes de su uso. Recuerde que las células diferenciadas no se elimina antes de que pases serán transferidos a las nuevas culturas. Además, trate de mantener a las células a 37 ° C siempre que sea posible. Minimizar el tiempo de las células pasan fuera de la incubadora (por ejemplo, en la platina del microscopio o en la cabina de seguridad biológica.) Una platina del microscopio con calefacción puede ser muy beneficioso si las células se de la incubadora durante largos períodos de tiempo. Cuando se observan los cultivos celulares hES bajo el microscopio, en primer lugar evaluar la calidad global y el tamaño de las colonias de vista con una potencia baja (2x o 5x) objetivo. Si las células potencialmente diferenciadas se observan a baja potencia, confirmar la morfología diferenciada utilizando un objetivo de mayor aumento (10X). Inmediatamente después de la eliminación de la diferenciación, los bordes de las colonias pueden aparecer irregulares o dañadas. Incube las colonias durante la noche en un nuevo medio para que el resto de células no diferenciadas para recuperarse. Sin la influencia de la diferenciación en la cultura, las colonias restantes seguirán proliferando con normalidad. Si es posible, comenzar los experimentos con células de paso bajo hES. Cariotipo de las células antes y después de todos los experimentos más importantes.

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		<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 medium</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&trade; 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>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 pipets</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>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-20D</td>
<td>9&#x201D; glass</td>
</tr>		</tbody>
		</table>
		</div>

culture and maintenance of human embryonic stem cells  

Madre embrionarias humanas (HES), las células deben ser supervisados ​​y atendidos con el fin de mantener los cultivos sanos, indiferenciado. Como mínimo, las culturas deben ser alimentados todos los días llevando a cabo un cambio de medio completo para reponer los nutrientes perdidos y mantener los cultivos libres de factores de diferenciación no deseados. Aunque una pequeña cantidad de diferenciación es normal y esperable en cultivos de células madre, la cultura debe ser limpiados en forma rutinaria por la eliminación manual o &quot;picking&quot; áreas diferenciadas. Identificar y eliminar el exceso de diferenciación de los cultivos celulares hES son técnicas esenciales en el mantenimiento de una población saludable de las células.

Watch this Scientific Journal Video about Culture and Maintenance of Human Embryonic Stem Cells at JoVE.com

Culture and Maintenance of Human Embryonic Stem Cells

Este protocolo demuestra cómo mantener sano, indiferenciada madre embrionarias humanas (ES) las células.

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 ...

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Research

JoVE Journal

Biology

Culture and Maintenance of Human Embryonic Stem Cells  

47.7K vistas.  Stemgent. Este protocolo demuestra cómo mantener sano, indiferenciada madre embrionarias humanas (ES) las células.

Video: Culture and Maintenance of Human 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 ...

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 ...

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 ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

El destino de la asignación de células estaminales embrionarias humanas mediante la formación de teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...