Rotulagem de manganês de células estaminais embrionárias humanas <ol><li> Trypsinize as hastes embrionárias humanas que foram cultivadas no alimentador sem condições por 5 minutos. Neutralizar a tripsina, adicionando meios de cultura. </li><li> Girar as células por centrifugação a 800 rpm por 5 minutos a 20 ° Celsius. Após re-suspensão de células do sedimento, contar o número de células por trypan coloração azul. Com base na contagem de células obtidas, divide as células em quatro alíquotas de 3 milhões de células em tubos cônicos. Agregar as células novamente por centrifugação. </li><li> Pouco antes de iniciar a rotulagem de manganês, dissolver o cloreto de manganês fresco com solução 0,9% de cloreto de sódio para fazer uma solução de cloreto de 0,1 mM de manganês. </li><li> , A fim de observar a atividade dos canais de cálcio, quatro amostras foram preparadas. A primeira amostra é de nosso controle que contém células incubadas em cloreto de sódio 0,9% sozinho. A segunda amostra contém células incubadas em 0,1 mM MnCl 2. Amostra 3 e 4 contêm células incubadas em 0,1 mM MnCl 2 com qualquer um dos 5 mM de Ca 2 + canais agonista, (s )-(-)- Bay K8644, ou 250 mM de Ca 2 + canais antagonista, verapamil. Incubar as quatro amostras a 37 ° Celsius com 5% de CO 2 por 30 minutos. </li><li> Após 30 minutos, girar as células a 800 rpm por 5 minutos a 20 ° Celsius. Em seguida, aspire o sobrenadante e lavar a células marcadas duas vezes com PBS. Após a lavagem, re-suspender cada pellet com 200 mL de PBS e transferir para tubos de PCR. Essas pelotas são geralmente brancos. </li></ol> Rotulagem de ferro de células estaminais embrionárias humanas <ol><li> Mix clínico-grade sulfato de protamina com água destilada para obter uma solução estoque 1 mg / mL. </li><li> Em um tubo contendo-tronco embrionárias humanas meios de cultura de células, adicionar ferumoxides a 100 mcg / mL seguido pela adição de 12 mcg / mL sulfato de protamina. Misturar a solução vigorosamente por cinco minutos. </li><li> Adicionar solução mista para a mesma quantidade de meios de cultura celular para obter uma concentração final de 50 mcg / mL de ferumoxides e 6 mcg / ml do sulfato de protamina para as células rótulo. Incubar as células nessa solução por 12 horas. </li><li> Lave as células duas vezes com PBS, com a última lavagem contendo heparina 10U/ml de dissolver o complexo de sulfato extracelular ferumoxides-protamina. Após a lavagem, trypsinize células para obter uma suspensão única célula. </li><li> Contar as células e alíquotas para o número necessário de amostras. Após a lavagem, re-suspender cada pellet com 200 mL de PBS e transferir para tubos de PCR. Estas pelotas deve ser laranja escuro na cor marrom. </li></ol> Realizar Ressonância Magnética Cellular <ol><li> Faça um fantasma, a fim de estabilizar os tubos contendo os pellets celulares e também para evitar artefatos a partir do ar circundante durante a digitalização. Para isso, mistura 0,8% de ágar e 1% de sulfato de cobre em água destilada e trazê-la para ferver por microondas por 5-7 minutos. Legal essa mistura durante pelo menos 1 hora antes de digitalizar para atingir um gel. </li><li> Os tubos contendo os pellets celulares rotulados são colocados no fantasma para a digitalização. Celulares in vitro MRI é realizada em Signa HDX 3T MRI (GE Medical Systems), utilizando bobina de joelho clínico. </li><li> Varredura as células de manganês rotulados para cada uma das três amostras utilizando uma seqüência spin echo: TR, tempo de repetição = 800 ms. TE, tempo de eco = mínimo, FOV, campo de visão = 12 x 12 cm; matriz = 256x256, NEX 1. </li><li> Varredura as células de ferro rotulados usando uma seqüência gradiente-eco. Usamos os seguintes parâmetros para esta imagem: TR = 100 ms; TE = 10 ms, FA; ângulo de inclinação = 30 °; FOV = 12 x 12 cm; matriz = 256 x 256, NEX 1. </li></ol> Analisando os resultados da RM <ol><li> Analisar imagens de ressonância magnética de 4 diferentes amostras de células-manganês rotulados: <ul><li> Amostra n º 1 é nosso controle que contém células incubadas em cloreto de sódio 0,9% sozinho. </li><li> Amostra # 2 contém células incubadas em 0,1 mM MnCl 2. </li><li> # 3 da amostra contém células incubadas em 0,1 mM MnCl 2 com 5 mM de Ca 2 + canal-agonista, (s )-(-)- Bay K8644. </li><li> Amostra # 4 contém células incubadas em 0,1 mM MnCl 2 com 250 mM de Ca 2 + canal-antagonista, verapamil. </li></ul></li><li> Como previsto, as diferenças na intensidade do sinal de manganês células marcadas são visíveis. Em comparação com as células rotuladas puramente com o manganês, a intensidade do sinal claramente aumenta na presença do agonista dos canais de cálcio e diminui na presença de antagonista dos canais de cálcio. A área onde a intensidade do sinal aumentou de manganês células marcadas são medidos usando Image J (NIH, Bethesda, MD, EUA). O mesmo software é usado para analisar a área de sinal fora de ressonância escuro de ferro células marcadas. O tamanho do sinal escura do ferro-rotulados células depende do número de células ou o número de partículas de ferro contido pelas células. Neste caso, Um milhão de células mostra uma área menor sinal escura em comparação com o sinal a partir dos três milhões de células. </li></ol>

<ol>
	<li>Bruvold, M., Nordhoy, W., Anthonsen, H. W., Brurok, H., Jynge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-calcium+interactions+with+contrast+media+for+cardiac+magnetic+resonance+imaging:+a+study+of+manganese+chloride+supplemented+with+calcium+gluconate+in+isolated+Guinea+pig+hearts.">Manganese-calcium interactions with contrast media for cardiac magnetic resonance imaging: a study of manganese chloride supplemented with calcium gluconate in isolated Guinea pig hearts.</a> Invest Radiol. 40, 117-125 (2005).</li><li>Aoki, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+labeling+for+magnetic+resonance+imaging+with+the+T1+agent+manganese+chloride.">Cell labeling for magnetic resonance imaging with the T1 agent manganese chloride.</a> NMR Biomed. 19, 50-59 (2006).</li><li>Arbab, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Arbab, A. S., Frank, 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=Cellular+MRI+and+its+role+in+stem+cell+therapy.">Cellular MRI and its role in stem cell therapy.</a> Regen Med. 3, 199-215 (2008).</li></ol>

Estes resultados indicam que o manganês não entra através do canal de cálcio voltagem-dependentes, e manganês pode ser usado como agente de contraste para IRM, que também pode mostrar a viabilidade celular. Portanto, sugerimos que o manganês guiada ressonância magnética pode ser útil para monitorar a viabilidade das células após o transplante de células estaminais embrionárias humanas no miocárdio.

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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>Manganese chloride tetrahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M8054</td>
<td>&nbsp;</td>
<tr>
<td>(S)-(-)-Bay K8644 </td>
<td>Reagent</td>
<td>Sigma</td>
<td>B133</td>
<td>&nbsp;</td>
<tr>
<td>(+)-Verapamil hydrochloride, minimum 99.0% titration </td>
<td>Reagent</td>
<td>Sigma</td>
<td>V4629</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride 0.85-0.90% W/V</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW 3257-1</td>
<td>&nbsp;</td>
<tr>
<td>Feridex I.V. </td>
<td>Reagent</td>
<td>Berlex Laboratories</td>
<td>NDC 59338-7035-5</td>
<td>ferumoxides injectable solution 11.2mg iron/mL</td>
</tr>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>American Pharmaceutical Partners</td>
<td>NDC 63323-229-05 </td>
<td>50 mg (10 mg/mL)</td>
</tr>
<tr>
<td>Knockout SR (Serum replacement for ES cells)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12 (1:1)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>2- mercaptoethanol 98+%</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M 3148</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 200mM 100x</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomyocin 10,000 units/ml</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>MEM Non-Essential Amino Acids 100x solution</td>
<td>Reagent</td>
<td>Gibco </td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>Signa HDx 3T MRI</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>clinical Knee coil</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro labeling of human embryonic stem cells for magnetic resonance imaging

Células estaminais embrionárias humanas (CTEh) demonstraram a capacidade de restaurar o miocárdio lesado. A ressonância magnética (MRI) emergiu como uma das modalidades de imagem predominante para avaliar a recuperação do miocárdio lesado. Além disso, os agentes ex-vivo rotulagem, tais como nanopartículas de óxido de ferro, têm sido empregados para rastrear e localizar as células-tronco transplantadas. No entanto, este método não monitora uma propriedade biologia celular fundamental em relação à viabilidade das células transplantadas. Tem sido conhecido que o cloreto de manganês (MnCl 2) entra nas células através de voltagem dependentes de cálcio (Ca 2 +) canais quando as células são biologicamente ativos, e acumula intracelularmente para gerar T efeito encurtamento 1. Portanto, sugerimos que o manganês guiada ressonância magnética pode ser útil para monitorar a viabilidade das células após o transplante de CTEh no miocárdio. Neste vídeo, vamos mostrar como rotular CTEh com MnCl 2 e como essas células podem ser claramente vistos usando MRI in vitro. Ao mesmo tempo, a atividade biológica de Ca 2 +-channels será modulada utilizando tanto Ca 2 + canal-agonistas e antagonistas para avaliar as alterações do sinal concomitante.

Watch this Scientific Journal Video about In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging at JoVE.com

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

Neste vídeo, estamos mostrando como rotular células estaminais embrionárias humanas (CTEh) com cloreto de manganês (MnCl<sub&gt; 2</sub&gt;), Que pode entrar nas células através dos canais de cálcio quando as células são biologicamente ativos. Além disso, mostramos o uso de MnCl<sub&gt; 2</sub&gt; Como agente de contraste celular ressonância magnética para determinar a viabilidade in vitro de CTEh.

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 ...

in-vitro-labeling-human-embryonic-stem-cells-for-magnetic-resonance

Research

JoVE Journal

Biology

10.1K visualizações.  Stanford University. Neste vídeo, estamos mostrando como rotular células estaminais embrionárias humanas (CTEh) com cloreto de manganês (MnCl 2), Que pode entrar nas células através dos canais de cálcio quando as células são biologicamente ativos. Além disso, mostramos o uso de MnCl 2 Como agente de contraste celular ressonância magnética para determinar a viabilidade in vitro de CTEh.

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

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 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 ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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 ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro e in vivo de imagens Gene Reporter bioluminescência de células estaminais embrionárias humanas

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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 ...

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 ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análise de Crescimento Humano embrionárias Colony Stem Cell

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...