EQUIPAMENTO <ul><li> Cultura de tecidos incubadora, 37 ° C CO, 5% 2 </li><li> Centrifugador </li><li> Conjunto de micropipettors (100, 100μL) </li><li> Banho-maria 37 ° C </li><li> Banho-maria 90 ° C a ser fonte de vapor de água </li><li> Microscópio de contraste de fase </li></ul> PRIMEIRO PASSO Tratar as células com Colcemid Neste procedimento foi utilizada a linha CTEh H9 cultivadas sobre fibroblastos embrionárias de rato (MEF) inativados com mitomicina C (Sigma) e mantidos em DMEM/F12 (Invitrogen) suplementado com a substituição knockout 20% de soro (KSR, Gibco) e 8ng/mL de fator de crescimento fibroblástico (FGF-2, R &amp; D). A fim de obter um grande número de metáfases se espalha, é recomendado o uso de culturas com alto índice mitótico em que há muitas células em divisão. <ol><li> Pegue o frasco de cultura da incubadora e, no fluxo laminar, adicione Colcemid na concentração final de 0,1 mg / mL. </li><li> Retornar o frasco para a incubadora e esperar 3 horas para que o inibidor pode causar paragem da mitose na metáfase, quando os cromossomos estão bem condensados ​​e podem ser facilmente visualizadas ao microscópio. </li></ol> Fixação das células <ol start="3"><li> Retire e adicione o meio tripsina / EDTA 0,05% suficiente para cobrir a superfície das células. Permitir que as células incubar por 5 minutos em temperatura ambiente e depois inativar a enzima quer pela inclusão de meio de cultura contendo soro ou apenas adicionando soro para as células. NOTA: É importante certificar-se que uma suspensão única célula é conseguido obter bons spreads metáfase. </li><li> Transferência das células para um tubo de centrifugação de 15 mL e centrifugar a 122 g por 5 minutos. </li><li> Desprezar o sobrenadante, deixando cerca de 200 mL, e ressuspender o sedimento delicadamente tocando o tubo, como mostrado no vídeo. Certifique-se que as células estão bem dissociada, então você não pode visualizar qualquer aglomerados na solução. </li><li> Em seguida, você terá que adicionar 5 mL de solução hipotônica (KCl 75mm) pré-aquecido a 37 ° C, lentamente, pela parede do tubo como no seguinte descrição. Primeiro adicione 3 ml, inverter o tubo para a posição horizontal apenas para misturar as células com a solução hipotônica em seguida, adicionar os 2 ml restantes e manter aquecido a 37 ° C por 15 minutos. NOTA: Neste ponto, a solução hipotônica irá causar um aumento no volume celular e ajudam a desembaraçar os cromossomos. O tempo de incubação é importante para adquirir spreads cromossomo bom. Se o tempo é muito longo, a membrana celular pode estourar cedo demais e os cromossomos são perdidos. Se for muito curto, pode ser difícil obter spreads cromossomo porque a membrana celular não pode perturbar. </li><li> Adicionar 3 gotas da solução fixadora (metanol / ácido acético glacial 3:1), inverter o tubo suavemente para misturar a solução fixadora com as células e centrifugar a 122 g por 5 minutos sem intervalo. NOTA: A solução fixadora deve ser preparado. NOTA: É importante o uso de centrifugação, sem interrupção, para evitar o desgaste das células, evitando a perda do material. </li><li> Desprezar o sobrenadante, deixando cerca de 200 mL, e ressuspender o sedimento delicadamente tocando no fundo do tubo. NOTA: Mais uma vez, certifique-se que as células estão bem dissociada, então você não pode visualizar qualquer aglomerados na solução. </li><li> Adicione o primeiro de 3 mL de solução fixadora, lentamente pela parede do tubo, enquanto você gira o tubo. Inverter o tubo para a posição horizontal apenas para misturar as células, e em seguida, adicione mais 2 mL da solução de fixador de parede do tubo para tirar as células para baixo. Estoque a 4 ° C durante a noite. NOTA: As células fixas podem ser armazenados em solução fixadora por meses a 4 ° C. Além de proteger as células em seu estado inchado, a solução fixadora elimina lipídios e proteínas desnatura. Estes eventos fazem a membrana celular frágil e, como resultado, fazer o cromossomo espalhar facilmente. </li></ol> LIMPEZA DO lâminas de vidro Antes de iniciar fazendo espalha o seu cromossomo se certificar de que seus slides estão devidamente limpos, ou você pode perder alguns núcleos e também metáfases. Esta etapa é bem importante se você quiser experimentar qualquer técnica de hibridização 9. <ol><li> Coloque as lâminas num copo contendo HCl 6M por pelo menos 3 h à temperatura ambiente. </li><li> Após este tempo, lavagem das lâminas em água corrente por 10 minutos. </li><li> Enxágüe as lâminas em água destilada e deixar eles secam em temperatura ambiente. </li><li> Agora as lâminas podem ser armazenadas em etanol 96% e secos com um pano imediatamente antes da utilização. </li></ol> NOTA: Para nos métodos de hibridização in situ, as lâminas não pode ser armazenado por mais de dois dias em etanol 96% SEGUNDA ETAPA Lavar as células <ol start="10"><li> No dia seguinte, centrifugar as células em 122 g por 5 minutos sem intervalo. </li><li> Discard o sobrenadante deixando cerca de 200 ml. Ressuspender o sedimento suavemente flicking o tubo, em seguida, adicionar 5 ml de solução fixadora fresco (metanol / ácido acético glacial 3:1), lentamente pela parede do tubo durante a rotação do tubo. Repita este passo de lavagem mais 2 vezes para um total de três lavagens. NOTA: A solução fixadora deve ser preparado. </li></ol> Preparando os slides <ol start="12"><li> Na última etapa de centrifugação deixar quantidade suficiente de solução para homogeneizar o pellet celular e obter uma densidade adequada de células nas lâminas (ver figura 1). </li><li> Adicionar 2-30 mL de suspensão de células sobre uma lâmina limpa e seca, movendo a pipeta muito próximo e paralelo à superfície. Como a evaporação solução fixadora, a superfície da lâmina torna-se granulado. </li><li> Imediatamente, expor o slide, a face para cima, para o vapor de água quente (90 °) por 30 segundos para fazer com que as células explodir. NOTA: Neste momento, o metanol evapora a partir da solução fixadora, resultando em uma concentração de ácido acético aumentou, o que junto com a água estimula o cromossomo se espalhando. </li><li> Olhe para um microscópio de contraste de fase, apenas para verificar se a concentração, bem como a distribuição das células é bom (ver figura 1). </li><li> Agora os slides estão prontos para ser utilizados para verificar o conjunto de cromossomos por citogenética abordagens como bandamento G, SKY FISH (Giemsa), ou CGH. </li></ol> DICAS IMPORTANTES Como escolher uma metáfase adequada a ser analisada Escolhendo metáfases redondo e isolado (figura 2D), você pode evitar considerando aneuploidias falso como o ganho de cromossomos gerados por dois ou mais se espalha misturados entre si ou a perda de cromossomos gerados por um cromossomo lançado. Assim, evitar a escolha de metáfases perto núcleos porque alguns cromossomos pode ser escondida por eles (figura 2A e 2B). Além disso, procurar metáfases redonda e ter certeza de que qualquer cromossomo é separado da metáfase (figura 2C). Células de alimentação e do cariótipo CTEh Para bandamento G e análise SKY, a presença de células alimentadoras na cultura CTEh não interfere com os resultados, porque as células de alimentação não estão sob um estado de divisão (inativados por mitomicina C ou irradiação) e não será uma parte da população metáfase. No entanto, para a aplicação da técnica FISH em núcleos interfásicos, é preferível para separar a população CTEh a partir de células de alimentação por colher as colônias manualmente antes de tripsinização para análise FISH. <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="Figura 1" /> Figura 1: Como conhecer a densidade de célula apropriada nos slides. Imagens de microscópio de contraste de fase na objetiva de 10x. (A) A densidade celular é muito alto. As setas apontam para metáfase se espalha muito próximo de núcleos. (B) Um slide com boa densidade celular. Os círculos mostram metáfases spreads isolado de núcleos ou metáfases outros. Por favor, <a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="Figura 2" /> Figura 2: Como escolher uma metáfase adequada para ser analisado. Cromossomos são corados com DAPI e as imagens obtidas por um microscópio de fluorescência em objetiva de 100x. (A) (B) Evite metáfases perto núcleos (indicado pelas setas) (C) e metáfases não volta que os cromossomos presentes separados (círculo). (D) A rodada é uma metáfase metáfase bom para ser analisado. Por favor, <a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">clique aqui</a> para ver uma versão ampliada da figura 2.

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

The authors have nothing to disclose.

A preparação dos spreads cromossomo é um passo crítico para uma análise bem sucedida do estado genético das células-tronco embrionárias pelo rotineiramente técnicas como bandamento G, e as técnicas mais sofisticadas, como FISH, SKY e CGH. Este procedimento pode ser aplicado para vários tipos de células, variando o período de incubação Colcemid, que depende da duração do ciclo celular. Para colônias de células-tronco embrionárias, vamos esperar três horas enquanto que par...

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chromosomal spread preparation of human embryonic stem cells for karyotyping

Apesar de células estaminais embrionárias humanas (CTEh) foram mostrados para apresentar um cariótipo diplóide estável 1, muitos estudos têm relatado que dependendo das condições de cultura torna-se propenso a adquirir anomalias cromossômicas, como adição de conjunto ou de partes de cromossomos. Com efeito, durante longo prazo de cultura, alterações cariotípicas são observadas quando enzimática ou química de dissociação são usados ​​2,3,4, enquanto dissecção manual de colônias para passaging mantém um cariótipo estável 5. Além disso, as mudanças no ambiente, tais como a remoção de células de alimentação também parecem comprometer a integridade genética das CTEh 3,6. Uma vez que alterações cromossômicas podem afetar a fisiologia celular, a caracterização da integridade genética das CTEh in vitro é CTEh considerando crucial como uma ferramenta essencial em estudos de embriogênese e testes de drogas. Além disso, para o futuro fins terapêuticos alterações cromossômicas são uma preocupação real como ele é freqüentemente associada à carcinogênese. Aqui nós mostramos um método simples e útil para obter spreads cromossomo de alta qualidade para posterior análise do cromossomo definido pelo G-banding, FISH SKY, ou técnicas de CGH 7,8. Recomendamos que verifique o estado cromossômicas rotineiramente com intervalos de 5 passagens a fim de monitorar o aparecimento de translocações e aneuploidias Priscila Britto e Rafaela Sartore contribuíram igualmente para o papel.

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

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Cariótipo é uma técnica simples e útil amplamente utilizado para detectar alterações genéticas. Descrevemos aqui um passo a passo protocolo para a preparação cromossomo disseminação de células estaminais embrionárias humanas para monitorar o estado cromossômica dessas células mantidas em cultura.

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 ...

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

Research

JoVE Journal

Biology

48.6K visualizações.  Federal University of Rio De Janeiro-UFRJ. Cariótipo é uma técnica simples e útil amplamente utilizado para detectar alterações genéticas. Descrevemos aqui um passo a passo protocolo para a preparação cromossomo disseminação de células estaminais embrionárias humanas para monitorar o estado cromossômica dessas células mantidas em cultura.

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

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.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

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.

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 ...

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 ...

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

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

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

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

Preparação de pool de plaquetas Lysate Humanos (pHPL) como um suplemento eficiente para animais de soro livre de culturas de células estaminais humanas

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

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

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 ...

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 ...