1. Entretien: Cultures observant au microscope <ol><li> Enlever une plaque de cellules hES de l&#39;incubateur à 37 ° C et porter à la loupe. </li><li> Observez les colonies à faible puissance, en utilisant un grossissement de 2X ou 5X, pour évaluer la qualité globale de la culture. Notez les points suivants: la couleur du milieu normal, absence de toute contamination visible, taille de la colonie d&#39;ensemble, la qualité et la densité, et d&#39;autres observations. </li><li> Observer les changements de couleur moyenne. En raison du changement de pH et de l&#39;épuisement des nutriments dans le milieu après une nuit d&#39;incubation avec les cellules hES, le milieu va changer à partir d&#39;une teinte rouge à un &quot;paille&quot; de couleur. Si le support semble violet, un changement de pH basique est survenue. Si le milieu apparaît extrêmement jaune, le milieu a acidifié. Milieu très jaune peut être le résultat de colonies extrêmement surpeuplées dans le puits ou la contamination. Le milieu doit être limpide en tout temps. Bien qu&#39;un certain niveau de débris cellulaires dans le milieu est normal, il ne doit jamais apparaître nuageux. Si un niveau élevé de débris cellulaires sont observées, la culture n&#39;est pas sain et peut être contaminée. </li><li> Si les cultures ne nécessitent pas d&#39;entretien ou de repiquage, elles peuvent être prises à l&#39;enceinte de sécurité biologique d&#39;être nourri (voir section 2). </li><li> Si les cultures contiennent plus de 10% environ différencier les colonies, les cellules doivent être «nettoyé» en supprimant manuellement les zones différenciées (voir section 3). </li><li> Si les cultures contiennent de grandes colonies ou dense, ou la couche nourricière MEF est de plus de 12 jours, les cellules doivent être repiquées (voir section 4). </li></ol> 2. Nourrir cellules hES <ol><li> Dans une enceinte de sécurité biologique stérile, retirez le couvercle de la boîte de culture et d&#39;aspirer le milieu épuisé. Ne laissez pas la pointe de la pipette pour toucher toute partie de la plaque autre que directement à l&#39;intérieur du puits. S&#39;il n&#39;y a aucun souci de la contamination, le changement vers une nouvelle pipette stérile. </li><li> En utilisant une pipette en verre stérile, ajouter la quantité appropriée de milieu de culture hES réchauffé cellules dans chaque puits. Pour les cultures dans une plaque de 6 puits, ajouter 2,5 ml de milieu par puits. </li><li> Retour à la plaque de l&#39;incubateur de rester à 37 ° C et 5% de CO 2. </li></ol> 3. Retrait de différenciation de cultures de cellules hES <ol><li> Transformez 9 pouces en verre Pipettes Pasteur en instruments de ramassage par moulage des conseils sur la flamme d&#39;un brûleur contrôlé alcool. Soyez sûr que la pointe de la pipette est scellé pour empêcher la contamination et arrondi pour éviter de rayer le plastique de la boîte de culture. Stériliser les instruments de ramassage avant de l&#39;utiliser en utilisant la source de lumière UV dans l&#39;enceinte de sécurité biologique ou la cueillette enclos. </li><li> Retirez les zones différenciées. Différenciation se produit souvent dans une partie seulement d&#39;une colonie de cellules hES, généralement le long des bords ou comme des points isolés dans le centre. Si seulement une section d&#39;une colonie est différenciant, seule la zone différenciée doit être retiré. Différenciation peut souvent soulever la plaque dans un &quot;collant&quot; feuille, et il est utile de commencer par séparer les cellules différenciées du reste de la colonie en traçant une ligne à travers la colonie avec la fin de l&#39;outil de cueillette. </li><li> Une fois les pièces de la colonie sont séparées, glissent doucement l&#39;outil de cueillette le long de la plaque pour détacher les cellules indésirables. Prenez soin de ne pas gratter trop de la couche nourricière MEF entre les colonies dans ce processus. </li><li> Lorsque toutes les cellules différenciées sont retirés de la plaque (et flottant dans le milieu), retour à la culture à l&#39;enceinte de sécurité biologique. Aspirer le milieu contenant des cellules différenciées et de remplacer à moyen hES culture fraîche de cellules. </li></ol></ol> 4. Le repiquage cellules hES Incubation enzymatique <ol><li> Dans une enceinte de sécurité biologique stérile, retirez le couvercle de la boîte de culture de 6 puits et aspirer le milieu épuisé par les puits pour être repiquées. Ne laissez pas la pointe de la pipette pour toucher toute partie de la plaque autre que directement à l&#39;intérieur du puits. </li><li> En utilisant une pipette en verre stérile, ajouter 1 ml de collagénase IV réchauffé à chaque puits pour être repiquées. </li><li> Retour de la plaque à l&#39;étuve à 37 ° C pendant 5 minutes. </li></ol> Grattage et mise en commun des cellules hES <ol start="4"><li> Après incubation des cellules avec de la collagénase, observer les colonies sous le microscope. L&#39;enzyme devrait entraîner un changement subtil mais observables dans les bords colonie. </li><li> Dans le cabinet de sécurité biologique, doucement aspirer l&#39;enzyme de chaque puits et la remplacer par 2,0 mL hES milieu de culture cellulaire. </li><li> Astuce de la plaque de culture légèrement vers vous et prendre la moyenne 2,0 ml dans le premier puits avec une pipette de verre de 5 ml. Tenir la pipette perpendiculairement au fond de la plaque, glissent doucement l&#39;embout de la pipette sur le bien tout en libérant lentement du milieu. </li&gt; <li> Répétez le raclage et le pipetage fois le mouvement jusqu&#39;à ce que toutes 3-4 des colonies ont été retirés du puits. Pipet délicatement pour éviter de casser les colonies dans trop peu de pièces. &nbsp; Lorsque les cellules ont été retirés du premier puits, laisser le contenu dans le puits et commencer à gratter les cellules hors de la suivante aussi. </li><li> Quand tous les puits d&#39;être repiquées ont été grattées, piscine au milieu contenant les morceaux colonie de chaque puits dans un tube de 15 ml stériles conique. </li><li> Rincer les puits gratté en ajoutant 1 mL de milieu de culture cellulaire hES à chaque puits. Recueillir ce rinçage et le transfert au tube conique. </li><li> Pipet les cellules doucement dans le tube pour briser les morceaux colonie jusqu&#39;à la taille désirée. </li><li> Centrifuger les cellules pendant 5 minutes à 200 x g. </li></ol> Placage des cellules de cellules hES <ol start="12"><li> Alors que les cellules hES sont dans la centrifugeuse, préparez une nouvelle plaque de 6 puits d&#39;alimentation du MEF. Étiquette de la plaque avec les informations hES cellule appropriée: lignée cellulaire, le numéro de nouveau passage, et la date de passage. Aspirer le MEF moyennes des puits et ajoutez 1,0 ml de PBS dans chaque puits. Remuer doucement le tampon autour des puits et aspirer le lavage PBS. Ajouter 1,5 mL hES milieu de culture cellulaire dans chaque puits et mis la plaque de côté. </li><li> Lorsque les cellules hES sont finis centrifugation, amener le tube de retour à l&#39;enceinte de sécurité biologique. Aspirer le surnageant, en faisant attention de ne pas perturber le lâche-emballés culot cellulaire. </li><li> Resuspendre les cellules dans le culot avec support suffisant pour 1 ml hES milieu de culture cellulaire par puits d&#39;être plaqué. Lorsque se diviser en six nouveaux puits, resuspendre le culot dans 6 mL de milieu. </li><li> Doucement et uniformément ajouter 1 mL de la suspension cellulaire préparée à chaque puits de la plaque d&#39;alimentation du MEF. </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. </li><li> Laisser les cellules d&#39;attacher à 37 ° C pendant la nuit. </li></ol> 5. Les résultats représentatifs: Cellules hES indifférenciées sont petits, serrés, et se composent généralement de propagation plus grandes, plus les cellules. La différenciation peut se produire dans une colonie (figure 1A) ou entre les colonies (figure 3B). Morhopologies différents peuvent être vus sous un faible grossissement au microscope. Une bonne morphologie cellulaire, comme on le voit à la figure 2A, contient de petites cellules serrées qui poussent en monocouche. Les cellules doivent avoir propre, bords définis, avec peu ou pas de différenciation. Les cellules montré dans la figure 2B sont prêts pour repiquage, et les cellules montré dans la figure 2C sont surpeuplées. <img src="/files/ftp_upload/1427/1427fig1.jpg" alt="Figure 1" /> Figure 1. Différenciation dans les cultures hES. (A) la différenciation d&#39;une colonie (B) la différenciation entre les colonies. <img src="/files/ftp_upload/1427/1427fig2.jpg" alt="Figure 2" /> Figure 2. Morphologies vu dans les cultures hES. (A) la morphologie cellulaire bonne (B) des cellules hES qui sont prêts pour repiquage (C) des cellules surpeuplées.

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li></ol>

Stérilité doit être maintenue en tout temps lorsque vous travaillez avec des cellules hES. Nettoyer et stériliser l&#39;enceinte de sécurité biologique et de tous les équipements avant l&#39;utilisation. Tous les réactifs doivent être filtrés en utilisant un filtre de 0,22 um taille des pores avant l&#39;utilisation. L&#39;utilisation de antiboiotics en culture de cellules hES n&#39;est pas nécessaire et devrait être évitée. Un environnement distinct devrait être mis en place comme une station de cueillette désigné. L&#39;enceinte stérile pour la station peut être une enceinte statique (comme un poste de travail PCR) avec une source de lumière UV, une hotte à flux laminaire, ou une enceinte de sécurité biologique. Un microscope à dissection à l&#39;intérieur du poste de cueillette est nécessaire pour observer les colonies comme des zones différenciées sont supprimés. Le panneau de verre avant d&#39;une enceinte statique ou une enceinte de sécurité biologique doit être modifié pour permettre les oculaires du microscope à s&#39;étendre à travers le panneau, sans compromettre une bonne ventilation et la stérilité. Pour entretenir la santé des cultures de cellules hES, les cellules doivent être passées au moment optimal, généralement tous les 4-7 jours. A cette époque les colonies ont atteint leur taille maximale et peut être commencent à fusionner. Fusionné colonies peuvent augmenter le taux de différenciation dans la culture. Ratios de Split tombent généralement 1:03-1:05, selon le nombre de colonies en plaqué, le taux d&#39;expansion, et les conditions de culture. Overplated colonies (rapport de division trop faible) sera probablement fusionner prématurément et ont besoin d&#39;être repiquées avant qu&#39;ils atteignent leur taille maximale. Cultures sur une couche nourricière MEF qui est plus de 2 semaines doit être passée à nouveau FAE qui soutiendront la croissance et la prolifération indifférenciée. Depuis une différenciation se produit naturellement dans les cultures de cellules hES, une petite quantité est attendue. Différentiation fréquente ou excessive peut se produire si les cultures ne reçoivent pas les soins appropriés. Les cellules doivent être nourris tous les jours, la prolifération entre les passages doivent être évités. Toujours utiliser des réactifs frais. Tous les médias, les FAE et les réactifs doivent être utilisés dans les 14 jours afin d&#39;éviter la croissance de cellules hES indifférenciées. Les nouveaux lots de réactifs, comme le remplacement Knockout Sérum, FBS et FAE, devraient être examinés avant toute utilisation. N&#39;oubliez pas que toute les cellules différenciées pas enlevé avant repiquage sera transférée aux nouvelles cultures. Aussi, essayez de maintenir les cellules à 37 ° C dès que possible. Minimisez le temps passé à l&#39;extérieur des cellules de l&#39;incubateur (par exemple sur la platine du microscope ou dans l&#39;enceinte de sécurité biologique.) Une platine de microscope chauffée peut être très bénéfique si les cellules seront de l&#39;incubateur pour de longues périodes de temps. En observant les cultures de cellules hES sous le microscope, d&#39;abord évaluer la qualité globale et la taille des colonies vue en utilisant une faible puissance (2x ou 5x) objectif. Si les cellules potentiellement différenciés sont observés à faible puissance, de confirmer la morphologie différenciée en utilisant un objectif plus fort grossissement (10x). Immédiatement après avoir enlevé la différenciation, les bords des colonies peuvent apparaître dentelées ou endommagés. Incuber les colonies pendant la nuit dans un milieu frais pour permettre aux cellules indifférenciées qui restent à recouvrer. Sans l&#39;influence de la différenciation dans la culture, ces colonies restantes continueront à proliférer normalement. Si possible, commencer des expériences utilisant faible passage de cellules hES. Caryotype des cellules avant et après toutes les expériences majeures.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF medium</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout&trade; Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
</tr>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final
</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
</tr>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-20D</td>
<td>9&#x201D; glass</td>
</tr>		</tbody>
		</table>
		</div>

culture and maintenance of human embryonic stem cells  

Souches embryonnaires humaines (hES) les cellules doivent être surveillés et soignés afin de maintenir en bonne santé, cultures indifférenciées. Au minimum, les cultures doivent être nourris chaque jour en effectuant un changement de milieu complet à reconstituer les éléments nutritifs perdus et de maintenir les cultures libres de facteurs de différenciation indésirable. Même si une petite quantité de différenciation est normal et prévu dans les cultures de cellules souches, la culture doit être systématiquement nettoyé en supprimant manuellement, ou «cueillette» des zones différenciées. Identifier et éliminer l&#39;excès de différenciation de cultures de cellules hES sont des techniques essentielles dans le maintien d&#39;une population saine de cellules.

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

Culture and Maintenance of Human Embryonic Stem Cells

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

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 ...

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Research

JoVE Journal

Biology

Culture and Maintenance of Human Embryonic Stem Cells  

47.7K vues.  Stemgent. Ce protocole démontre comment maintenir en bonne santé, indifférencié souches embryonnaires humaines (ES) cellules.

Vidéo: Culture and Maintenance of Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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

Propagation des souches embryonnaires humaines (ES) Cellules

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Biologie

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.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

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.

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 ...

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 ...

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 ...

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.

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 ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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

Cartographie sort des cellules souches embryonnaires humaines par la formation de tératome

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

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

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

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

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

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