1. Auftauen hES-Zellen Pre-experimentellen Set-up <ol><li> Einen Tag vor dem Auftauen hES-Zellen, Platte eine Feeder-Schicht von bestrahlten embryonalen Maus-Fibroblasten (MEF), CF1-Stamm, auf der einen sowie einer Gelatine-beschichteten 6-well Gewebekulturplatten in MEF Kulturmedium (D-MEM Basalmedium, ergänzt mit 10% Hitze-inaktiviertes FBS und 1% nicht-essentiellen Aminosäuren). Inkubieren über Nacht bei 37 ° C und 5% CO 2. </li><li> Vor dem Entfernen der hES-Zellen aus flüssigem Stickstoff, achten Sie darauf, alle notwendigen Geräte und Reagenzien verfügen und bereit zu verwenden, um eine effiziente und erfolgreiche Tauwetter zu gewährleisten. </li></ol> Das Auftauen der HES-Zellen <ol start="3"><li> Entfernen eines Kryodosen von hES-Zellen aus dem flüssigen Stickstoff Lagerbehälter mit Metall Pinzette. </li><li> Rollen Sie die Durchstechflasche zwischen behandschuhten Händen für 3-5 Sekunden, um den Frost zu entfernen. </li><li> Notieren Sie sich die Angaben auf dem Etikett der Durchstechflasche. </li><li> Mit Metall-Pinzette, tauchen Sie das Fläschchen in eine 37 ° C Wasserbad. Schwenken Sie die Durchstechflasche vorsichtig und beobachten den Fortschritt der Schneeschmelze oft (aber schnell!), Indem Sie die Flasche gegen das Licht, um die Größe der Eiskristalle zu sehen. Tauchen Sie den Deckel der Flasche im Wasserbad, da dies die Zellen verunreinigen könnten. </li><li> Wenn nur ein kleiner Eiskristall bleibt, tauchen die ganze Flasche in Ethanol zu sterilisieren. </li><li> In einer sterilen Sicherheitswerkbank, übertragen Sie die Inhalte der Kryodosen direkt auf den Boden eines 15 ml konischen Rohr. </li><li> Langsam 4 ml hES-Zell-Kulturmedium (D-MEM/F-12 Basalmedium mit 20% Knockout Serumersatz, 1% nicht-essentiellen Aminosäuren, 1% L-Glutamin, 0,2% β-Mercaptoethanol und 4 ng / ergänzt mL bFGF) auf das Rohr. Schütteln Sie ständig mischen die Zellen als das neue Medium in das Röhrchen gegeben wird. </li><li> Zentrifugieren Sie die Zellen für 5 Minuten bei 200 x g. </li></ol> Plating der HES-Zellen <ol start="11"><li> Während die hES-Zellen in der Zentrifuge werden, bereiten die MEF Feeder Platte durch Kennzeichnung mit der entsprechenden hES-Zell-Informationen: Zell-Linie, Passage-Nummer und Tauwetter date. </li><li> Wenn es etwa 2 Minuten auf die Spin-Zeit bleibt, saugen Sie den MEF Kulturmedium und 1,0 ml PBS, um den Brunnen. </li><li> Bringen Sie die pelletierten hES-Zellen zurück, um die biologische Sicherheitswerkbank und sorgfältig absaugen des Überstandes. Achten Sie darauf, das Zellpellet absaugen, sondern entfernen Sie so viel Überstand wie möglich, da diese Lösung enthält DMSO aus dem Einfriermedium. </li><li> Re-suspend das Pellet sehr sanft durch Zugabe von 2,5 mL der hES-Zell-Kulturmedium mit einer 5 ml Glaspipette und Pipettieren 3-4 mal. </li><li> Saugen Sie das PBS aus dem MEF feeder gut und langsam alle 2,5 mL der hES-Zell-Suspension auf die gut vorbereitet an den 6-Well-Platte. </li><li> Legen Sie die Platte in die 37 ° C Inkubator und vorsichtig die Platte nach vorne nach hinten und seitlich zur gleichmäßigen Verteilung der Zellen im ganzen gut. Nicht wirbeln die Platte in einem kreisförmigen Muster zu vermeiden, konzentriert sich die Kolonien in der Mitte. </li><li> Lassen Sie die Zellen bei 37 ° C und 5% CO 2 über Nacht befestigen. </li><li> Am Tag nach der Schneeschmelze, entfernen Sie das Medium (mit allen schwimmenden Zellen) und 2,5 ml frischer hES Zellkulturmedium zum Brunnen. </li><li> Optional: Der entfernte Medium und Zellen können in eine separate vorbereitet MEF Feeder Platte übertragen werden. Dieser Schritt führt häufig zu neuen Kolonien, die parallel mit Zellen aus dem ursprünglichen Tauwetter kultiviert werden können. </li><li> Die Inkubation bei 37 ° C und 5% CO 2 über Nacht. </li></ol> 2. Das Einfrieren der HES-Zellen <ol><li> Während die hES-Zellen in der Zentrifuge werden, beschriften Sie die Kryoröhrchen im biologischen Sicherheitsschrank, einschließlich der Zell-Linie, Passage-Nummer und das Einfrieren date. Die typische Dichte für das Einfrieren von hES-Zellen ist eine gut von Zellen (aus einer 6-Well-Platte) pro Kryodosen. </li><li> Wenn der hES-Zellen fertig sind Zentrifugieren, bringen die Rohre zurück in den biologischen Sicherheitswerkbank. Saugen Sie den Überstand aus der Tube, dass Sie nicht die lose verpackt Zellpellet stören. </li><li> Zellpellet mit der entsprechenden Menge von hES-Zell-Kulturmedium: 0,5 mL hES-Zell-Kulturmedium pro Kryodosen. Seien Sie sehr vorsichtig vorgehen, wenn Pipettieren, wie die hES-Zellen besser zu erholen, als in relativ große Kolonie Stücke eingefroren. </li><li> Langsam, unter leichtem Schütteln der Röhre, fügen Sie die entsprechende Menge (0,5 ml pro Kryoröhrchen) von 2X hES-Zell-Einfriermedium (60% definiert FBS, 20% hES-Zell-Kulturmedium und 20% DMSO) zu den Zellen. Sobald die Einfriermedium zugegeben, die Lösung pipet äußerst schonend 1.59 mal zu mischen. </li><li> Schnell, aber vorsichtig, geben Sie 1 ml der Zellsuspension auf jeweils Kryodosen. </li><li> Übertragen Sie die Flaschen in einem Isopropanol Gefrierbehälter und ineiner -80 ° C Tiefkühltruhe über Nacht. Die Zellen werden bei 1 ° C pro Minute in das Isopropanol Gefrierbehälter einfrieren. </li><li> Am folgenden Tag, schnelle Übertragung der eingefrorenen Ampullen zu einem flüssigen Stickstoff Lagerbehälter mit Metall Pinzette. </li></ol> Repräsentative Ergebnisse Am ersten Tag nach menschlichen ES-Zellen aufgetaut werden, können kleine Kolonien erscheinen transparent und schwierig sein kann, unter dem Mikroskop zu sehen. Da neu aufgetaut ES-Zellen zu langsam vermehren neigen, können sie ein paar Tage dauern als etablierte Kolonien (Abbildung 1) erscheinen. Abbildung 1. Erholung der Zellen und Wachstum. HES-Zellen aus flüssigem Stickstoff aufgetaut wurden abgebildet 1, 7 und 11 Tage nach dem Auftauen.

<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>

Das dazugehörige Video zeigt ein Verfahren zum Auftauen und Einfrieren hES-Zellen. Zellen in flüssigem Stickstoff eingefroren werden sollten, Bad so schnell wie möglich aufgetaut werden, um die bestmögliche Verwertung zu erreichen. Denken Sie daran, äußerst vorsichtig sein, beim Pipettieren Auftauen von Zellen: Minimierung Handling der Zellen und Pipette vorsichtig. Eine Durchstechflasche mit hES-Zellen können entweder auf eine Vertiefung einer 6-Well-Platte, oder alle Wells einer 4-Well-Platte beschichtet werden und sofort in den Inkubator gestellt. Da Kultivierung in vier getrennten Wannen reduziert die Möglichkeit des Verlusts der gesamten Kultur, um eine Kontamination und macht es einfacher, hES-Zell-Kolonien auf der kleineren Fläche zu finden, ist das 4-Well-Platte empfohlen, wenn Auftauen hES-Zellen für die erste Zeit. Der erste Tag nach dem Auftauen hES-Zellen werden die Kolonien klein und kann transparent sein. Tanken die hES-Zell-Kulturmedium jeden Tag, auch wenn die Kolonien nicht unter dem Mikroskop sichtbar, da es ein paar Tage in Kultur für hES-Zellen kann als etablierte Kolonien erscheinen. Es ist wichtig, ein frisch vergoldet MEF Feeder-Schicht zu verwenden, wenn Auftauen von Zellen, wie die Kolonien, oft nicht erreichen eine angemessene Größe für die Passage bis 10 12 Tage nach der Schneeschmelze. Beim Auftauen einen niedrigen Gang Fläschchen hES-Zellen, ist es gute Praxis zu erweitern und zu frieren zusätzliche Fläschchen auf einen Bestand von niedrigen Durchgang Zellen für zukünftige Kultur und Experimente zu erhalten. Es ist auch eine gute Idee, regelmäßig freeze all &quot;extra&quot; gesunde hES-Zellen eingefroren Aktien zu halten. Die erfolgreichsten auftaut und nachfolgende Kulturen wurden als hochwertige, undifferenzierten, sich aktiv teilenden hES-Zell-Kolonien eingefroren. hES-Zellen aus einem Einfrieren effizienter wiederherzustellen, wenn sanft wie größere Zellaggregate behandelt. Die endgültige Körnung sollte ähnlich (oder etwas größer als) die Größe der Aggregate nach einer Routine-Passage überzogen.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF mediumjavascript:void(0);</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
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<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
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<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final</td>
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<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
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<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>
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<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2560</td>
<td>For freezing medium</td>
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<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
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<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
</tr>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
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freezing and thawing human embryonic stem cells

Seit James Thomson<em&gt; Et al</em&gt; Eine Technik entwickelt, im Jahr 1998 zu isolieren und zu wachsen hES in Kultur, haben Einfrieren von Zellen für die spätere Verwendung und Auftauen und Ausbau von Zellen aus einem gefrorenen Lager zu wichtigen Verfahren in der Routine-hES-Zell-Kultur durchgeführt. Da hES-Zellen sehr empfindlich auf die Belastungen von Einfrieren und Auftauen sind, ist besondere Sorgfalt. Hier zeigen wir die richtige Technik für die schnelle Auftauen hES-Zellen aus flüssigem Stickstoff Aktien, Plattieren sie auf embryonalen Feeder-Zellen, und langsam Einfrieren für die langfristige Lagerung.

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

Freezing and Thawing Human Embryonic Stem Cells

Seit James Thomson et al eine Technik entwickelt, im Jahr 1998 zu isolieren und zu wachsen hES in Kultur, haben Einfrieren von Zellen für die spätere Verwendung und Auftauen und Ausbau von Zellen aus einem gefrorenen Lager zu wichtigen Verfahren in der Routine-hES-Zell-Kultur durchgeführt. Da hES-Zellen sehr empfindlich auf die Belastungen von Einfrieren und Auftauen sind, ist besondere Sorgfalt. Hier zeigen wir die richtige Technik für die schnelle Auftauen hES-Zellen aus flüssigem Stickstoff Aktien, Plattieren sie auf embryonalen Feeder-Zellen, und langsam Einfrieren für die langfristige Lagerung.

Einfrieren und Auftauen humanen embryonalen Stammzellen

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

freezing-and-thawing-human-embryonic-stem-cells

Research

JoVE Journal

Biology

45.7K Aufrufe.  Stemgent. Seit James Thomson et al eine Technik entwickelt, im Jahr 1998 zu isolieren und zu wachsen hES in Kultur, haben Einfrieren von Zellen für die spätere Verwendung und Auftauen und Ausbau von Zellen aus einem gefrorenen Lager zu wichtigen Verfahren in der Routine-hES-Zell-Kultur durchgeführt. Da hES-Zellen sehr empfindlich auf die Belastungen von Einfrieren und Auftauen sind, ist besondere Sorgfalt. Hier zeigen wir die richtige Technik für die schnelle Auftauen hES-Zelle...

Serie: 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.

Einfrieren menschlicher ES-Zellen

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

Forschung

Biologie

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.

Vermehrung von humanen embryonalen Stammzellen (ES-Zellen)

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.

Gewinnung menschlicher embryonaler Stammzellen durch 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.

Probing für Mitochondriale komplexe Aktivität in humanen embryonalen Stammzellen

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.

Einfrieren, Auftauen und Packaging Cells für Verkehr

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.

Chromatin Immunopräzipitation von humanen embryonalen Stammzellen

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.

Kultur und Wartung von humanen embryonalen Stammzellen

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.

Chromosomale Spread-Vorbereitung von humanen embryonalen Stammzellen für die Karyotypisierung

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.

Isolation und Ableitung von embryonalen Maus-Keimzellen

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.

Differenzierung von embryonalen Stammzellen in Oligodendrozyten Precursors

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