1. Wartung: Observing Kulturen unter dem Mikroskop <ol><li> Entfernen einer Platte von hES-Zellen aus dem 37 ° C Inkubator und bringen dem Mikroskop. </li><li> Beachten Sie die Kolonien bei niedriger Leistung, mit 2X oder 5fache Vergrößerung, um die gesamte Kultur Qualität zu beurteilen. Beachten Sie die folgenden: normal mittel Farbe, das Fehlen von sichtbaren Verunreinigungen insgesamt Kolonie Größe, Qualität und Dichte, und andere Beobachtungen. </li><li> Beachten Medium Farbe ändert. Durch die Veränderung des pH und die Erschöpfung der Nährstoffe in das Medium nach einer Inkubation über Nacht mit der hES-Zellen wird das Medium von einem roten Farbton zu einem &quot;Stroh&quot; Farbe zu ändern. Wenn das Medium erscheint lila, hat einen basischen pH-Verschiebung stattgefunden. Wenn das Medium erscheint extrem gelb, hat das Medium angesäuert. Sehr gelb Medium kann das Ergebnis der extrem überfüllten Kolonien in den Brunnen oder Verunreinigungen sein. Das Medium erscheinen soll zu jeder Zeit klar. Obwohl ein gewisses Maß an Zelltrümmer in das Medium ist normal, sollte es nie erscheinen trübe. Wenn ein hohes Maß an Zelltrümmer beobachtet wird, ist die Kultur nicht gesund und können kontaminiert sein. </li><li> Wenn die Kulturen nicht gewartet werden müssen oder die Passage, können sie um die biologische Sicherheit Schrank eingespeist (siehe Abschnitt 2) werden entnommen werden. </li><li> Wenn die Kulturen mehr als etwa 10% Differenzierung Kolonien enthalten, sollten die Zellen &quot;aufgeräumt&quot; werden durch manuelles Entfernen der differenzierten Bereiche (siehe Abschnitt 3). </li><li> Wenn die Kulturen groß oder dichten Kolonien enthalten, oder die MEF Feeder-Schicht ist mehr als 12 Tage alt sind, sollten die Zellen passagiert (siehe Abschnitt 4). </li></ol> 2. Feeding hES-Zellen <ol><li> In einer sterilen Sicherheitswerkbank, entfernen Sie den Deckel der Kulturschale und saugen die verbrauchte Medium. Nicht in die Spitze der Pipette auf einen Teil der Platte außer direkt in die Vertiefungen berühren. Wenn es irgendeine Sorge der Kontamination, um eine neue, sterile Pipette ändern. </li><li> Mit einem sterilen Glaspipette, fügen Sie die entsprechende Menge erwärmt hES-Zell-Kulturmedium in jede Vertiefung. Für Kulturen in einer 6-Well-Platte, fügen Sie 2,5 ml Medium pro Vertiefung. </li><li> Bringen Sie die Platte in den Inkubator bei 37 ° C und 5% CO 2 bleiben. </li></ol> 3. Entfernen Differenzierung von hES-Zell-Kulturen <ol><li> Transform 9-Zoll-Glas Pasteur Pipetten in Kommissionierung Werkzeuge durch Formen der Spitzen über die kontrollierte Flamme eines Spiritusbrenner. Achten Sie darauf, dass die Spitze der Pipette verschlossen ist, um eine Kontamination zu verhindern und abgerundeten um Kratzer zu vermeiden Kunststoff der Kulturschale. Sterilisieren die Kommissionierung Werkzeuge vor dem Einsatz mit den UV-Lichtquelle in der biologischen Sicherheitswerkbank oder Kommissionierung Gehäuse. </li><li> Entfernen Sie die differenzierte Bereiche. Differenzierung tritt oft nur in einem Teil eines hES-Zell-Kolonie, in der Regel an den Rändern oder als isolierte Flecken in der Mitte. Wenn nur ein Abschnitt einer Kolonie ist differenziert, muss nur die differenzierte Bereich entfernt werden. Differenzierung kann oft abheben der Platte in eine &quot;klebrige&quot; Blatt, und es ist hilfreich, zunächst getrennt die differenzierten Zellen aus dem Rest der Kolonie durch Ziehen einer Linie durch die Kolonie mit dem Ende der Kommissionierung-Tool. </li><li> Sobald die Teile der Kolonie getrennt sind, sanft gleiten die Kommissionierung-Tool auf der Platte, um die unerwünschten Zellen zu lösen. Achten Sie darauf, nicht zu kratzen weg zu viel von der MEF Feeder-Schicht zwischen den Kolonien in diesem Prozess. </li><li> Wenn alle differenzierten Zellen von der Platte (und variabel im Medium) entfernt werden, wieder die Kultur die biologische Sicherheitswerkbank. Saugen Sie das Medium mit den differenzierten Zellen und ersetzen mit frischen hES-Zell-Kulturmedium. </li></ol> 4. Passagierung hES-Zellen Enzyminkubation <ol><li> In einer sterilen Sicherheitswerkbank, entfernen Sie den Deckel des 6-well Kulturschale und saugen die verbrauchte Medium aus den Vertiefungen zu passagiert werden. Nicht in die Spitze der Pipette auf einen Teil der Platte außer direkt in die Vertiefungen berühren. </li><li> Mit einem sterilen Glaspipette, 1 mL erwärmt Kollagenase IV für jeden gut zu passagiert werden. </li><li> Bringen Sie die Platte um die 37 ° C Inkubator für 5 Minuten. </li></ol> Kratzen und Pooling der HES-Zellen <ol start="4"><li> Nach Inkubation der Zellen mit Kollagenase, beobachten die Kolonien unter dem Mikroskop. Das Enzym sollte zu einem feinen, aber beobachtbare Veränderung in der Kolonie Kanten. </li><li> In der biologischen Sicherheitswerkbank, sanft saugen das Enzym aus jedem Well und ersetzen mit 2,0 mL hES-Zell-Kulturmedium. </li><li> Tipp der Kulturplatte leicht in Ihre Richtung und nehmen die 2,0 ml Medium in die erste Bohrung mit einer 5 ml Glaspipette. Halten Sie die Pipette senkrecht zur Unterseite der Platte, sanft gleiten die Pipettenspitze über die gut, während langsam die Freigabe der Mittel. </li><li> Wiederholen Sie die Schaben und pipetting Bewegung 3-4 mal, bis alle Kolonien aus dem Brunnen entfernt. Pipettieren vorsichtig, um zu vermeiden Aufbrechen der Kolonien in zu kleine Stücke. Wenn die Zellen aus der ersten Bohrung entfernt worden sind, lassen Sie den Inhalt in den Brunnen und beginnen Abschaben der Zellen aus der nächsten gut. </li><li> Wenn alle Brunnen passagiert werden wurden abgekratzt, Pool das Medium mit der Kolonie Stücke aus jeder Vertiefung in ein steriles 15 ml konischen Rohr. </li><li> Spülen Sie den abgeschabt Brunnen durch Zugabe von 1 ml hES-Zell-Kulturmedium in jede Vertiefung. Sammeln Sie diese gründlich und Transfer zum konischen Rohr. </li><li> Pipettieren Sie die Zellen vorsichtig in das Rohr zum Aufbrechen der Kolonie Stück bis die gewünschte Größe. </li><li> Zentrifugieren Sie die Zellen für 5 Minuten bei 200 x g. </li></ol> Plating der hES-Zell-Cells <ol start="12"><li> Während die hES-Zellen in der Zentrifuge sind, bereiten eine neue 6-well MEF Feeder Platte. Beschriften Sie die Platte mit den entsprechenden hES-Zell-Informationen: Zell-Linie, neue Passage-Nummer und Datum Durchgang. Saugen Sie das MEF Medium aus dem Brunnen und 1,0 ml PBS in jede Vertiefung. Vorsichtig schwenken die Puffer um den Brunnen und saugen Sie den PBS waschen. Fügen Sie 1,5 ml hES-Zell-Kulturmedium in jede Vertiefung und stellte den Teller beiseite. </li><li> Wenn der hES-Zellen fertig sind Zentrifugieren, bringen die Röhre zurück, um die biologische Sicherheitswerkbank. Den Überstand aspirieren, darauf achten, daß die lose verpackt Zellpellet stören. </li><li> Resuspendieren der Zellen in das Pellet mit genügend Medium für 1 mL hES-Zell-Kulturmedium pro Vertiefung zu plattiert werden. Bei der Aufteilung in 6 neue Brunnen, das Pellet in 6 ml Medium. </li><li> Sanft und gleichmäßig 1 mL der Zellsuspension auf jeweils gut vorbereitet der MEF Feeder 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. </li><li> Lassen Sie die Zellen bei 37 ° C über Nacht befestigen. </li></ol> 5. Repräsentative Ergebnisse: Undifferenzierte hES-Zellen sind klein, dicht aneinander, und in der Regel von größeren, ausbreiten Zellen bestehen. Differenzierung kann innerhalb einer Kolonie (Abbildung 1A) oder zwischen Kolonien (Abbildung 3B) auftreten. Verschiedene morhopologies unter geringer Vergrößerung unter dem Mikroskop gesehen werden. Eine gute Zellmorphologie, wie in Abbildung 2A zu sehen, enthält kleine, dicht gepackte Zellen, die in einer Monoschicht wachsen. Zellen sollten sauber, definierte Kanten, mit wenig bis gar keine Differenzierung. Die Zellen in Abbildung 2B dargestellt sind bereit für die Passage, und die Zellen in der Abbildung 2C dargestellt sind überfüllt. <img src="/files/ftp_upload/1427/1427fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Differenzierung in hES Kulturen. (A) Differenzierung von einer Kolonie (B) Unterscheidung zwischen Kolonien. <img src="/files/ftp_upload/1427/1427fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Morphologien in hES Kulturen gesehen. (A) gute Zellmorphologie (B) hES-Zellen, die bereit sind für die Passage (C) überfüllten Zellen sind.

<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>

Sterilität muss jederzeit gewährleistet sein, wenn die Arbeit mit hES-Zellen. Reinigung und Sterilisation der biologischen Sicherheitswerkbank und alle Geräte vor dem Einsatz. Alle Reagenzien müssen gefiltert durch ein 0,22 um Filter mit einer Porengröße vor dem Gebrauch werden. Die Verwendung von antiboiotics in hES-Zell-Kultur ist nicht notwendig und sollte vermieden werden. Eine separate Umwelt sollte als Designated Kommissionierplatz eingestellt werden. Die sterile Umhüllung für die Station kann eine statische Gehäuse (z. B. PCR-Workstation) mit einer UV-Lichtquelle, einer Sterilbank oder einer biologischen Sicherheitswerkbank werden. Ein Binokular in der Kommissionierung Station wird benötigt, um die Kolonien zu beobachten, wie die differenzierte Bereiche entfernt werden. Die Frontscheibe eines statischen Gehäuse oder einer biologischen Sicherheitswerkbank muss geändert werden, um für die Okulare des Mikroskops ermöglichen, um durch die Fenster, ohne eine ausreichende Belüftung und Sterilität zu verlängern. Um gesund zu erhalten hES-Zell-Kulturen, müssen die Zellen zum optimalen Zeitpunkt passagiert werden, in der Regel alle 4-7 Tage. In dieser Zeit der Kolonien haben ihre maximale Größe erreicht und kann beginnen zu verschmelzen. Zusammengeführt Kolonien kann die Geschwindigkeit der Differenzierung in der Kultur zu erhöhen. Split-Verhältnisse in der Regel fallen 1.03 bis 01.05 Uhr, abhängig von der Anzahl der Kolonien plattiert, Expansionsrate und Kulturbedingungen. Overplated Kolonien (Split-Verhältnis zu niedrig) wird wahrscheinlich vorzeitig fusionieren und müssen passagiert werden, bevor sie ihre maximale Größe erreicht. Kulturen auf einer MEF Feeder-Schicht, die mehr als 2 Wochen alt ist, muss auf neue MEFs dass undifferenzierte Wachstum und die Vermehrung Unterstützung passagiert werden. Da die Differenzierung von Natur aus in hES-Zell-Kulturen, wird eine kleine Menge erwartet. Häufige oder starke Differenzierung kann auftreten, wenn die Kulturen nicht erhalten eine angemessene Pflege. Die Zellen zugeführt werden muß jeden Tag sein sollte Überwucherung zwischen Passagen vermieden werden. Verwenden Sie stets frischen Reagenzien. Alle Medien, MEFs und Reagenzien sollten innerhalb von 14 Tagen genutzt werden, um undifferenzierte hES-Zell-Wachstum zu vermeiden. New viele Reagenzien, wie Knockout Serum Replacement, FBS und MEFs, sollte vor gescreent werden, um zu verwenden. Denken Sie daran, dass alle differenzierten Zellen nicht vor Passagierung entfernt, um den neuen Kulturen übertragen werden. Versuchen Sie auch, die Zellen bei 37 ° C, wann immer möglich zu halten. Minimieren Sie die Zeit der Zellen verbringen außerhalb des Inkubators (zB am Mikroskop der Bühne oder im biologischen Sicherheitsschrank.) Ein beheizter Mikroskoptisch kann sehr vorteilhaft, wenn die Zellen des Inkubators für längere Zeit sein. Bei der Betrachtung der hES-Zell-Kulturen unter dem Mikroskop, zunächst zu prüfen, die Qualität und Größe der Kolonien Blick mit einem Low-Power-(2x oder 5x) Ziel. Wenn potentiell differenzierten Zellen bei geringer Leistung beobachtet werden, bestätigen die differenzierte Morphologie mit einer höheren Vergrößerung Objektiv (10-fach). Sofort nach dem Entfernen Differenzierung, können die Ränder der Kolonien gezackt oder beschädigt. Inkubieren Sie die Kolonien über Nacht in frisches Medium, damit die verbleibenden undifferenzierte Zellen sich zu erholen. Ohne den Einfluss der Differenzierung in der Kultur, werden diese verbleibenden Kolonien weiterhin normal vermehren. Wenn möglich, beginnen Experimente mit low-Passage hES-Zellen. Karyotyp der Zellen vor und nach alle wichtigen Experimente.

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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  

Humane embryonale Stammzellen (hES-Zellen) müssen überwacht und gepflegt werden, um gesunde, undifferenzierten Kulturen zu erhalten. Zumindest müssen die Kulturen täglich gefüttert, indem er umfassende Medium ändern, um verlorenen Nährstoffe wieder aufzufüllen und die Kulturen frei von unerwünschten Differenzierungsfaktoren zu halten. Obwohl eine kleine Menge der Differenzierung ist normal und erwartet in Stammzell-Kulturen, sollte die Kultur regelmäßig gereinigt werden, um manuell entfernen oder &quot;picking&quot; differenzierte Bereiche. Erkennen und Entfernen von überschüssigem Differenzierung von hES-Zell-Kulturen sind wesentliche Techniken in der Erhaltung einer gesunden Population von Zellen.

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

Dieses Protokoll zeigt, wie man gesunde, undifferenzierte humane embryonale Stammzellen (ES-Zellen) zu halten.

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 ...

culture-and-maintenance-of-human-embryonic-stem-cells

Research

JoVE Journal

Biology

Culture and Maintenance of Human Embryonic Stem Cells  

47.7K Aufrufe.  Stemgent. Dieses Protokoll zeigt, wie man gesunde, undifferenzierte humane embryonale Stammzellen (ES-Zellen) zu halten.

Serie: 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.

Vermehrung von humanen embryonalen Stammzellen (ES-Zellen)

Forschung

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.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

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.

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 ...

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 ...

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 ...

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.

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 ...

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 ...

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.

Fate Mapping von humanen embryonalen Stammzellen durch Teratom Formation

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.

Adulten und embryonalen Skelettmuskel Microexplant Kultur und Isolation der Skelettmuskulatur 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 ...