1. La décongélation des cellules hES Pré-montage expérimental <ol><li> Un jour avant la décongélation des cellules hES, plaque une couche nourricière de fibroblastes de souris irradiées embryonnaires (MEF), CF1 souche, sur un puits d&#39;une gélatine à revêtement de 6 puits plaque de culture de tissus dans un milieu de culture MEF (D-MEM complété avec un milieu de base 10% de FBS inactivé à la chaleur et 1% de non-acides aminés essentiels). Incuber une nuit à 37 ° C et CO 2 5%. </li><li> Avant de retirer les cellules hES de l&#39;azote liquide, être sûr d&#39;avoir tout l&#39;équipement nécessaire et les réactifs en place et prêt à utiliser pour assurer un dégel efficace et réussie. </li></ol> La décongélation des cellules hES <ol start="3"><li> Retirer un flacon cryogénique de cellules hES du réservoir de stockage d&#39;azote liquide à l&#39;aide des pinces de métal. </li><li> Faites rouler le flacon entre les mains gantées pour les 3-5 secondes pour enlever le givre. </li><li> Enregistrer les informations sur l&#39;étiquette du flacon. </li><li> En utilisant des pinces métalliques, immerger le flacon dans un bain d&#39;eau à 37 ° C. Agiter le flacon doucement et observer les progrès du dégel souvent (mais rapidement!) En tenant le flacon à la lumière pour voir la taille du cristal de glace. Ne pas plonger le bouchon de la fiole dans le bain d&#39;eau car cela pourrait contaminer les cellules. </li><li> Lorsque seul un cristal de glace petite demeure, immerger le flacon entier dans de l&#39;éthanol à stériliser. </li><li> Dans une enceinte de sécurité biologique stérile, transférer le contenu de la fiole cryogénique directement au fond d&#39;un tube de 15 ml conique. </li><li> Lentement ajouter 4 ml de milieu de culture cellulaire hES (D-MEM/F-12 milieu basal complété par le remplacement Knockout 20% de sérum, 1% de non-acides aminés essentiels, soit 1% de L-glutamine, 0,2% β-mercaptoéthanol et 4 ng / ml de bFGF) au tube. Secouez délicatement au mélange en permanence les cellules comme le nouveau média est ajouté dans le tube. </li><li> Centrifuger les cellules pendant 5 minutes à 200 x g. </li></ol> Placage des cellules hES <ol start="11"><li> Alors que les cellules hES sont dans la centrifugeuse, préparez la plaque d&#39;alimentation du MEF par un marquage avec les informations hES cellule appropriée: lignée cellulaire, le numéro de passage, et la date de dégel. </li><li> Quand il ya environ 2 minutes sur le temps de spin, aspirer le milieu de culture MEF et ajoutez 1,0 ml de PBS dans le puits. </li><li> Apportez les cellules hES granulés de retour à l&#39;enceinte de sécurité biologique, et soigneusement aspirer le surnageant. Attention à ne pas aspirer le culot cellulaire, mais enlever autant que possible le surnageant, comme cette solution contient du DMSO dans le milieu de congélation. </li><li> Re-suspendre le culot très doucement en ajoutant 2,5 mL de milieu de culture cellulaire hES en utilisant une pipette 5 ml en verre et de pipetage 3-4 fois. </li><li> Aspirer le PBS à partir du chargeur MEF bien et ajouter lentement 2,5 ml toutes les de la suspension de cellules hES au bien préparée de la plaque de 6 puits. </li><li> Placer la plaque dans l&#39;incubateur à 37 ° C et glissez soigneusement la plaque d&#39;avant en arrière, et latéralement afin de répartir uniformément les cellules à travers le puits. Ne pas remuer le plateau dans un modèle circulaire pour éviter la concentration des colonies dans le centre. </li><li> Laisser les cellules d&#39;attacher à 37 ° C et CO 2 5% durant la nuit. </li><li> Le jour après le dégel, retirez le support (avec toutes les cellules flottantes) et ajoutez 2,5 ml de milieu de culture frais hES cellule pour le bien. </li><li> En option: Le milieu retiré et les cellules peuvent être transférés à une plaque séparée MEF préparés mangeoire. Cette étape génère souvent de nouvelles colonies qui peuvent être cultivées en parallèle avec les cellules du dégel d&#39;origine. </li><li> Incuber les plaques à 37 ° C et CO 2 5% durant la nuit. </li></ol> 2. Congélation des cellules hES <ol><li> Alors que les cellules hES sont dans la centrifugeuse, l&#39;étiquette des flacons cryogéniques à l&#39;intérieur de l&#39;enceinte de sécurité biologique, notamment la lignée cellulaire, le numéro de passage, et la date de congélation. La densité typique de cellules hES congélation est un puits de cellules (à partir d&#39;une plaque à 6 puits) par flacon cryogénique. </li><li> Lorsque les cellules hES sont finis centrifugation, mettre les tubes de retour à l&#39;enceinte de sécurité biologique. Aspirer le surnageant du tube, en faisant attention de ne pas perturber le lâche-emballés culot cellulaire. </li><li> Reprendre le culot cellulaire avec la quantité appropriée de milieu de culture cellulaire hES: 0,5 mL hES milieu de culture cellulaire par flacon cryogénique. Soyez très doux lors du pipetage, comme les cellules hES mieux récupérer quand elle est congelée en morceaux colonie relativement importante. </li><li> Lentement, tout en secouant doucement le tube, ajoutez la quantité appropriée (0,5 ml par cryovial) de 2X milieu de congélation de cellules hES (60% de FBS définis, 20% moyennes de culture de cellules hES, et 20% de DMSO) pour les cellules. Une fois que le milieu de congélation est ajouté, pipette la solution extrême douceur une à deux fois pour mélanger. </li><li> Rapidement mais doucement, ajouter 1 mL de la suspension cellulaire dans chaque flacon cryogénique. </li><li> Transfert des flacons dans un contenant de congélation et les placer dans l&#39;isopropanolune congélateur à -80 ° C la nuit. Les cellules gèle à 1 ° C par minute dans le récipient de congélation isopropanol. </li><li> Le lendemain, transférer rapidement les flacons congelés dans un réservoir de stockage d&#39;azote liquide à l&#39;aide des pinces de métal. </li></ol> Les résultats représentatifs Le premier jour après que les cellules ES humaines sont décongelés, de petites colonies peuvent apparaître transparentes et peuvent être difficiles à voir sous le microscope. Comme les cellules ES nouvellement décongelés ont tendance à proliférer lentement, ils peuvent prendre quelques jours à apparaître comme des colonies établies (figure 1). Figure 1. La récupération et la croissance cellulaire. Cellules hES dégelée de l&#39;azote liquide ont été imagées Jours 1, 7 et 11 après la décongélation.

<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>

La vidéo qui l&#39;accompagne montre une méthode pour la décongélation et la congélation des cellules hES. Cellules congelées dans l&#39;azote liquide doivent être décongelés de bain aussi rapidement que possible afin d&#39;obtenir la meilleure récupération possible. N&#39;oubliez pas d&#39;être extrêmement prudent lors du pipetage cellules dégel: minimiser la manipulation des cellules et la pipette doucement. Un flacon de cellules hES peuvent être étalés sur une ou l&#39;autre puits d&#39;une plaque de 6 puits, ou tous les puits d&#39;une plaque 4-même et immédiatement placés dans l&#39;incubateur. Depuis la culture dans quatre puits séparé réduit la possibilité de perdre toute la culture de la contamination, et il est plus facile de localiser les colonies de cellules hES sur la plus petite surface, la plaque 4-même est recommandé lors de la décongélation des cellules hES pour la première fois. Le premier jour après la décongélation des cellules hES, les colonies sont petites et peuvent être transparents. Reconstituer le support de culture de cellules hES chaque jour, même si les colonies ne sont pas apparents sous le microscope, car il peut prendre quelques jours de culture de cellules hES d&#39;apparaître comme des colonies établies. Il est important d&#39;utiliser une couche fraîchement plaquée MEF alimentation lors de la décongélation des cellules, comme les colonies ne sont souvent pas atteindre une taille appropriée pour repiquage jusqu&#39;au 10 12 jours après le dégel. Lorsque la décongélation d&#39;un flacon passage bas de cellules hES, il est bon d&#39;élargir et de gel des flacons supplémentaires pour maintenir un stock de cellules passage bas pour la culture à venir et des expériences. C&#39;est aussi une bonne idée de geler toute routine &quot;extra&quot; saine cellules hES de maintenir des stocks congelés. Le dégèle plus réussies et les cultures subséquentes ont été gelés en haute qualité, indifférenciée, se divisant activement les colonies de cellules hES. cellules hES récupérer un gel plus efficace s&#39;il est manipulé doucement comme des agrégats de cellules plus grandes. La taille globale finale devrait être similaire à (ou légèrement plus grand que) la taille des agrégats plaqués après un passage de routine.

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
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<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>
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<td>bFGF</td>
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<td>Non-essential Amino Acids</td>
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<td>PBS</td>
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<td>Collagenase IV</td>
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<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
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<td>Gelatin</td>
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<td>Sigma</td>
<td>G1890</td>
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<td>DMSO</td>
<td>&nbsp;</td>
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<td>6-well plates</td>
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<td>Nunc</td>
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<td>4-well plates</td>
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<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
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<td>5 mL glass pipettes</td>
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<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
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<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
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<tr>
<td>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
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<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
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freezing and thawing human embryonic stem cells

Depuis James Thomson<em&gt; Et al</em&gt; A développé une technique en 1998 à isoler et à cultiver hES dans la culture, les cellules de congélation pour une utilisation ultérieure et le dégel et l&#39;expansion des cellules à partir d&#39;un stock congelé sont devenus d&#39;importantes procédures effectuées en culture de cellules hES de routine. Comme les cellules hES sont très sensibles aux contraintes de gel et de dégel, des précautions particulières doivent prendre. Ici nous démontrer la bonne technique pour rapidement dégel cellules hES partir des stocks d&#39;azote liquide, les cellules de souris sur le plaquage d&#39;alimentation embryonnaire, et lentement les congeler pour stockage à long terme.

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

Freezing and Thawing Human Embryonic Stem Cells

Ce protocole démontre comment maintenir en bonne santé, indifférencié souches embryonnaires humaines (ES) cellules.

Congélation et de décongélation cellules souches embryonnaires humaines

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 ...

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45.7K vues.  Stemgent. Ce protocole démontre comment maintenir en bonne santé, indifférencié souches embryonnaires humaines (ES) cellules.

Vidéo: 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.

La congélation des cellules ES humaines

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

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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.

Propagation des souches embryonnaires humaines (ES) Cellules

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.

Dérivation des cellules souches embryonnaires humaines par 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.

Palpage pour activité complexe mitochondrial dans les cellules souches embryonnaires humaines

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.

Congélation, la décongélation, et les cellules d&#39;emballage pour les transports

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.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

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

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

Culture et entretien des cellules souches embryonnaires humaines

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.

Préparation Étaler chromosomique des cellules souches embryonnaires humaines pour le caryotype

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

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

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

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

La différenciation des cellules souches embryonnaires en précurseurs d&#39;oligodendrocytes

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