1. Descongelamento células-tronco embrionárias humanas Pré-experimental Set-up <ol><li> Um dia antes de as células-tronco embrionárias humanas descongelamento, prato uma camada nutriente de fibroblastos embrionários de ratos irradiados (MEF), CF1 tensão, em um poço de uma gelatina revestidos placa de cultura de tecido de 6 bem no meio de cultura MEF (D-MEM suplementado com meio basal 10% inativado pelo calor FBS e 1% não-aminoácidos essenciais). Incubar overnight a 37 ° CO C e 5% 2. </li><li> Antes de remover as células-tronco embrionárias humanas a partir de nitrogênio líquido, a certeza de ter todos os equipamentos necessários e reagentes no lugar e pronto para uso para assegurar um degelo eficiente e bem sucedida. </li></ol> Descongelar as células-tronco embrionárias humanas <ol start="3"><li> Remover um frasco criogênico de células-tronco embrionárias humanas do tanque de armazenamento de nitrogênio líquido usando uma pinça de metal. </li><li> Role o frasco entre as mãos enluvadas por 3-5 segundos para remover o gelo. </li><li> Registrar as informações no rótulo do frasco. </li><li> Utilizando uma pinça de metal, mergulhe o frasco em banho-maria a 37 ° C. Gire o frasco cuidadosamente e observar a evolução do degelo, muitas vezes (mas rapidamente!), Segurando o frasco contra a luz para ver o tamanho dos cristais de gelo. Não mergulhe a tampa do frasco em banho-maria, pois isso pode contaminar as células. </li><li> Quando apenas um pequeno cristal de gelo permanece submergir, o frasco inteiro em etanol para esterilizar. </li><li> Em uma cabine de segurança biológica estéril, transferir o conteúdo do frasco criogênico diretamente para o fundo de um tubo cônico de 15 mL. </li><li> Lentamente, adicionar 4 mL de meio de cultura de células-tronco embrionárias humanas (D-MEM/F-12 meio basal suplementado com Knockout substituição de 20% de soro, 1% não-aminoácidos essenciais, de 1% L-glutamina, 0,2% β-mercaptoetanol e 4 ng / mL bFGF) ao tubo. Agite levemente para misturar continuamente as células como o novo meio é adicionado ao tubo. </li><li> Centrifugar as células por 5 minutos a 200 x g. </li></ol> Chapeamento as células-tronco embrionárias humanas <ol start="11"><li> Enquanto as células-tronco embrionárias humanas são na centrífuga, prepare o prato alimentador MEF pela rotulagem com as informações apropriadas de células-tronco embrionárias humanas: linha celular, o número de passagem, e data de degelo. </li><li> Quando há cerca de 2 minutos em tempo de rotação, aspirar o meio de cultura MEF e adicionar 1,0 mL de PBS para o bem. </li><li> Trazer as células-tronco embrionárias humanas peletizadas de volta para o gabinete de segurança biológica e aspirar cuidadosamente o sobrenadante. Tenha cuidado para não aspirar o pellet celular, mas remover o máximo possível sobrenadante, já que esta solução contém DMSO do meio de congelamento. </li><li> Re-suspender o pellet muito delicadamente, adicionando 2,5 mL de meio de cultura de células-tronco embrionárias humanas usando uma pipeta de vidro 5 mL e pipetagem 3-4 vezes. </li><li> Aspirar o PBS a partir do alimentador MEF bem e adicionar lentamente todos os 2,5 mL da suspensão de células-tronco embrionárias humanas para o bem preparados da placa de 6 poços. </li><li> Coloque a placa no 37 ° C incubadora e deslize com cuidado a placa para a frente para trás, e para os lados para distribuir uniformemente as células ao longo do poço. Não agite a placa em um padrão circular para evitar a concentração de colônias no centro. </li><li> Permitir que as células para anexar a 37 ° C e 5 CO 2% durante a noite. </li><li> O dia após o degelo, remova a mídia (com as células flutuante) e adicionar 2,5 ml de meio de cultura fresco CTeh para o bem. </li><li> Opcional: O meio removido e as células podem ser transferidos para uma placa de alimentação preparada MEF separado. Este passo muitas vezes gera novas colônias que podem ser cultivadas em paralelo com as células do degelo original. </li><li> Incubar as placas a 37 ° C e 5 CO 2% durante a noite. </li></ol> 2. Congelamento das Células-tronco embrionárias humanas <ol><li> Enquanto as células-tronco embrionárias humanas são na centrífuga, rótulo dos frascos criogênicos dentro do gabinete de segurança biológica, incluindo a linha celular, o número de passagem, e data de congelamento. A densidade típica de células-tronco embrionárias humanas congelamento é um bem de células (a partir de uma placa de 6 poços) por frasco criogênico. </li><li> Quando as células-tronco embrionárias humanas terminar a centrifugação, os tubos de trazer de volta para o gabinete de segurança biológica. Aspirar o sobrenadante do tubo, tomando cuidado para não perturbar o frouxamente cheia de pellet celular. </li><li> Ressuspender o pellet celular com a quantidade adequada de meio de cultura de células-tronco embrionárias humanas: 0,5 mL de cultura de células-tronco embrionárias humanas médio por frasco criogênico. Ser muito gentil quando pipetagem, como as células-tronco embrionárias humanas se recuperar melhor quando congelados em pedaços colônia relativamente grande. </li><li> Lentamente, enquanto agitando o tubo, adicione a quantidade adequada (0,5 mL por cryovial) de 2X médio congelamento CTeh (60% FBS definido, 20% TEh meio de cultura celular, e 20% DMSO) para as células. Uma vez que o meio de congelamento é adicionado, a solução extremamente pipeta suavemente 1-2 vezes para misturar. </li><li> Rapidamente mas com cuidado, adicione 1 mL da suspensão de células a cada frasco criogênico. </li><li> Transferir os frascos em um recipiente de congelamento isopropanol e coloque emum freezer -80 ° C durante a noite. As células irá congelar a 1 ° C por minuto no recipiente de congelamento isopropanol. </li><li> No dia seguinte, transferir rapidamente os frascos congelados para um tanque de nitrogênio líquido de armazenamento usando uma pinça de metal. </li></ol> Resultados representante O primeiro dia depois que as células ES humanas são descongelados, pequenas colônias podem aparecer transparente e pode ser difícil de ver sob o microscópio. Como as células ES recém descongelados lentamente tendem a proliferar, eles podem levar alguns dias para aparecer como colônias estabelecidas (Figura 1). Figura 1. Recuperação e crescimento das células. Células-tronco embrionárias humanas a partir de descongelado nitrogênio líquido foram fotografadas 1, 7 e 11 dias após o descongelamento.

<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>

O vídeo que acompanha demonstra um método para descongelar e congelar células-tronco embrionárias humanas. Células congeladas em nitrogênio líquido devem ser descongeladas banho o mais rápido possível para obter a melhor recuperação possível. Lembre-se de ser extremamente cuidadoso ao pipetar células descongelamento: minimizar o manuseio das células e pipeta suavemente. Um frasco de células-tronco embrionárias humanas podem ser semeados em qualquer um poço de uma placa 6-bem, ou todos os poços de uma placa de 4 bem e imediatamente colocado na incubadora. Uma vez que o cultivo em quatro poços separados reduz a possibilidade de perder toda a cultura à contaminação, e torna mais fácil para localizar as colônias de células-tronco embrionárias humanas na superfície menor, a placa de 4 bem é recomendado quando o descongelamento de células-tronco embrionárias humanas, pela primeira vez. O primeiro dia após o descongelamento de células-tronco embrionárias humanas, as colónias são pequenas e podem ser transparentes. Reabastecer o meio de cultura de células-tronco embrionárias humanas a cada dia, mesmo que as colônias não são aparentes sob o microscópio, pois pode levar alguns dias em cultura de células-tronco embrionárias humanas para aparecer como colônias estabelecidas. É importante usar uma camada de alimentador de recém-banhado MEF quando descongelar as células, como as colônias muitas vezes não chegar a um tamanho adequado para até 10 passaging 12 dias após o degelo. Quando descongelar um frasco de passagem baixa de células-tronco embrionárias humanas, é uma boa prática para expandir e congelar os frascos adicionais para manter um estoque de células passagem baixa para a cultura e as experiências futuras. Também é uma boa idéia rotineiramente congelar todos os &quot;extras&quot; células-tronco embrionárias humanas saudáveis ​​para manter estoques congelados. O degelo de maior sucesso e culturas subseqüentes foram congelados de alta qualidade, indiferenciado, colônias de células-tronco embrionárias humanas dividindo ativamente. células-tronco embrionárias humanas se recuperar de um congelamento de forma mais eficiente se for manuseada com cuidado como agregados maiores celular. O tamanho final global deve ser semelhante (ou ligeiramente maior do que) o tamanho dos agregados banhado depois de uma passagem de rotina.

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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 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>
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<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>
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<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>
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<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
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<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>
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freezing and thawing human embryonic stem cells

Desde que James Thomson<em&gt; Et al</em&gt; Desenvolveu uma técnica em 1998 para isolar e cultivar-tronco embrionárias em cultura, as células de congelamento para uso posterior e descongelamento e expandir as células de um estoque de congelados tornaram-se procedimentos importantes realizados em cultura de rotina CTeh. Como as células-tronco embrionárias humanas são muito sensíveis ao estresse de congelamento e descongelamento, cuidados especiais devem tomadas. Aqui nós demonstrar a técnica adequada para rapidamente descongelamento CTeh de stocks nitrogênio líquido, plaqueamento em células embrionárias de rato de alimentação, e, lentamente, congelá-los para o armazenamento de longo prazo.

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

Freezing and Thawing Human Embryonic Stem Cells

Desde que James Thomson et al desenvolveram uma técnica em 1998 para isolar e cultivar-tronco embrionárias humanas em cultura, as células de congelamento para uso posterior e descongelamento e expandir as células de um estoque de congelados tornaram-se procedimentos importantes realizados em cultura de rotina CTeh. Como as células-tronco embrionárias humanas são muito sensíveis ao estresse de congelamento e descongelamento, cuidados especiais devem tomadas. Aqui nós demonstrar a técnica adequada para rapidamente descongelamento CTeh de stocks nitrogênio líquido, plaqueamento em células embrionárias de rato de alimentação, e, lentamente, congelá-los para o armazenamento de longo prazo.

Congelamento e descongelamento, células estaminais embrionárias 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

45.6K visualizações.  Stemgent. Desde que James Thomson et al desenvolveram uma técnica em 1998 para isolar e cultivar-tronco embrionárias humanas em cultura, as células de congelamento para uso posterior e descongelamento e expandir as células de um estoque de congelados tornaram-se procedimentos importantes realizados em cultura de rotina CTeh. Como as células-tronco embrionárias humanas são muito sensíveis ao estresse de congelamento e descongelamento, cuidados especiais devem tomadas. Aqui n...

Vídeo: 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.

Congelamento células ES

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

Biologia

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.

Propagação de-tronco embrionárias 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.

Derivação de células estaminais embrionárias 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.

Sondagem para a Atividade Complexo mitocondrial em células estaminais embrionárias 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.

Congelamento, descongelamento e Células de embalagem para 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.

Cromatina imunoprecipitação de células estaminais embrionárias 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.

Na rotulagem in vitro de células estaminais embrionárias humanas para Ressonância 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.

Cultura e Manutenção de células estaminais embrionárias 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.

Preparação Spread cromossômica de células estaminais embrionárias humanas para Cariotipagem

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

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.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

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