EQUIPMENT <ul><li> Gewebekultur-Inkubator bei 37 ° C, 5% CO 2 </li><li> Zentrifuge </li><li> Set von micropipettors (100, 100 &amp;mgr;) </li><li> Wasserbad 37 ° C </li><li> Wasserbad 90 ° C bis Dampfquelle Wasser </li><li> Phasenkontrast-Mikroskop </li></ul> FIRST STEP Die Behandlung der Zellen mit Colcemid In diesem Verfahren verwendeten wir die hESC line H9 auf embryonalen Maus-Fibroblasten (MEF) mit Mitomycin C (Sigma) inaktiviert und gepflegt in DMEM/F12 (Invitrogen) kultiviert, ergänzt mit 20% Knockout Serumersatz (KSR, Gibco) und 8ng/mL der Fibroblasten-Wachstumsfaktor (FGF-2, R &amp; D). Um eine große Anzahl von Metaphasen Spreads zu erhalten, empfiehlt es sich, Kulturen mit hohem Mitoseindex in denen es viele gibt sich teilenden Zellen zu verwenden. <ol><li> Nehmen Sie die Kulturflasche aus dem Inkubator und, in der laminaren Strömung, fügen Colcemid in einer Endkonzentration von 0,1 pg / mL. </li><li> Bringen Sie den Kolben in den Inkubator und warten 3 Stunden so der Inhibitor mitotischen Arrest auf Metaphase, wenn sich die Chromosomen und werden kondensiert und können leicht sichtbar unter dem Mikroskop verursachen können. </li></ol> Fixierung der Zellen <ol start="3"><li> Nehmen Sie das Medium und fügen tripsin / EDTA 0,05% ausreichen, um die Zellen bedecken. Lassen Zellen für 5 Minuten bei Raumtemperatur inkubieren und dann inaktiviert das Enzym entweder durch Zugabe Kulturmedium mit Serum oder einfach nur mit Serum zu den Zellen. HINWEIS: Es ist darauf zu achten, dass eine einzige Zellsuspension erreicht ist, gute Metaphase Spreads zu erhalten. </li><li> Übertragen Sie die Zellen in ein 15 mL Zentrifugenröhrchen und Zentrifuge bei 122 g für 5 Minuten. </li><li> Überstand verwerfen, so dass etwa 200 ml, und das Pellet vorsichtig durch Antippen der Röhre, wie in dem Video zu sehen. Achten Sie darauf, die Zellen sind gut dissoziiert, so kann man nicht visualisieren alle Klümpchen in der Lösung. </li><li> Weiter benötigen Sie, um 5 ml hypotonische Lösung (KCl 75mm) vorgewärmt auf 37 ° C, langsam, durch die Rohrwand als in der folgenden Beschreibung. Hinzufügen Fügen Sie zunächst 3 ml, das Röhrchen in eine horizontale Position, nur um die Zellen mit dem hypotonischen Lösung dann mischen fügen Sie die restlichen 2 ml und halten bei 37 ° C erwärmen für 15 Minuten. HINWEIS: Zu diesem Zeitpunkt der hypotonischen Lösung wird ein Anstieg auf die zelluläre Volumen führen und dazu beitragen, die Chromosomen entwirren. Die Inkubationszeit ist wichtig, gute Chromosom Spreads zu erwerben. Wenn das Timing zu lang ist, kann die Zellmembran platzen zu früh und Chromosomen verloren gehen. Wenn er zu kurz, kann es schwierig sein, Chromosom-Spreads zu erhalten, da die Zellmembran nicht stören kann. </li><li> 3 Tropfen der Fixierlösung (Methanol / Eisessig 3:1), das Röhrchen vorsichtig, um die Fixierlösung mit den Zellen und Zentrifuge bei 122 g für 5 Minuten ohne Pause zu mischen. HINWEIS: Die Fixierlösung sollte frisch zubereitet werden. HINWEIS: Es ist wichtig, Zentrifugation, ohne zu brechen, um Zellen Abrieb zu vermeiden verwenden, verhindert Verlust des Materials. </li><li> Überstand verwerfen, so dass etwa 200 ml, und das Pellet vorsichtig durch Antippen der Boden des Röhrchens. HINWEIS: Auch hier stellen Sie sicher, die Zellen sind gut dissoziiert, so kann man nicht visualisieren keine Klumpen in der Lösung. </li><li> Fügen Sie die ersten 3 ml Fixierlösung, langsam durch die Rohrwand, während Sie das Rohr zu drehen. Drehen Sie die Röhre in eine horizontale Position, nur um die Zellen zu mischen, und fügen Sie dann weitere 2 ml der Fixierlösung durch die Rohrwand an die Zellen take down. Lager bei 4 ° C über Nacht. HINWEIS: Die fixierten Zellen können in Fixierlösung für Monate bei 4 ° C gelagert Neben dem Schutz Zellen in ihrem gequollenen Zustand entfernt die Fixierlösung Lipide und Proteine ​​denaturiert. Diese Ereignisse machen die Zellmembran zerbrechlich und, als Folge, zu dem Chromosom Verbreitung einfach. </li></ol> REINIGUNG Die Glasträger Bevor Ihre Chromosomen verteilt sicherstellen, dass Ihre Folien ordnungsgemäß gereinigt oder Sie können einige Kerne verlieren und auch Metaphasen. Dieser Schritt ist ebenso wichtig, wenn Sie keine Hybridisierungstechnik 9 Ausprobieren wollen. <ol><li> Die Objektträger in ein Becherglas mit 6M HCl für mindestens 3 h bei Raumtemperatur. </li><li> Nach dieser Zeit, waschen Dias in fließendem Leitungswasser für 10 Minuten. </li><li> Spülen Sie die Folien in destilliertem Wasser und lassen sie bei Raumtemperatur trocknen. </li><li> Nun ist die Dias können in 96% Ethanol gelagert und getrocknet werden mit einem fusselfreien unmittelbar vor der Verwendung. </li></ol> Hinweis: Für in-situ-Hybridisierung Methoden können die Folien nicht mehr als 2 Tage in 96% Ethanol gelagert werden ZWEITER SCHRITT Waschen der Zellen <ol start="10"><li> Am nächsten Tag, Zentrifuge die Zellen bei 122 g für 5 Minuten ohne Pause. </li><li> Discard der Überstand bis auf ca. 200 ml. Das Pellet durch vorsichtiges flicking das Rohr dann mit 5 ml frischer Fixierlösung (Methanol / Eisessig 3:1), langsam durch die Rohrwand beim Drehen des Rohres. Wiederholen Sie diesen Waschschritt 2 weitere Male für insgesamt drei Wäschen. HINWEIS: Die Fixierlösung sollte frisch zubereitet werden. </li></ol> Vorbereitung der Objektträger <ol start="12"><li> Bei der letzten Zentrifugationsschritt ein ausreichender Menge Lösung, um das Zellpellet homogenisieren und erhalten eine ausreichende Dichte der Zellen in der Folien (siehe Abbildung 1). </li><li> Add 20-30 ul Zellsuspension auf einen sauberen trockenen Objektträger, indem Sie die Pipette sehr nahe und parallel zur Oberfläche. Als Fixierlösung verdunstet, wird die Oberfläche der Folie körnig. </li><li> Sofort setzen die Folie, nach oben, in den Dampf von heißem Wasser (90 °) für 30 Sekunden, um zu bewirken die Zellen platzen. HINWEIS: Zu diesem Zeitpunkt das Methanol aus der Fixierlösung verdampft, was zu einer erhöhten Essigsäure-Konzentration, die zusammen mit dem Wasser stimuliert das Chromosom zu verbreiten. </li><li> Schauen Sie sich ein Phasenkontrast-Mikroskop nur zu prüfen, ob die Konzentration sowie die Zell-Verteilung ist gut (siehe Abbildung 1). </li><li> Nun ist die Dias sind bereit, verwendet werden, um den Satz von Chromosomen durch Zytogenetik Ansätze als G-banding (Giemsa), FISH, SKY oder CGH zu überprüfen. </li></ol> WICHTIGE HINWEISE Wie wählen Sie den passenden Metaphase zu analysierenden Die Auswahl um und isoliert Metaphasen (Abbildung 2d), können Sie vermeiden, angesichts falsche Aneuploidien wie die Verstärkung der Chromosomen von zwei oder mehr erzeugt breitet sich untereinander gemischt oder den Verlust von Chromosomen von einem Start-Chromosom erzeugt. So vermeiden Wahl Metaphasen in der Nähe Kerne, weil einige von ihnen Chromosomen (Abb. 2A und 2B) versteckt sein können. Außerdem für runde Metaphasen aussehen und sicher sein, dass jedes Chromosom aus der Metaphase (Abb. 2C) getrennt ist. Feeder-Zellen und die hESC Karyotyp Für G-banding und SKY-Analyse, ist die Anwesenheit von Feeder-Zellen in hESC Kultur mit dem Ergebnis nicht stören, weil Feeder-Zellen nicht unter eine Trennungslinie Zustand (inaktiviert durch Mitomycin C oder Bestrahlung) und wird nicht Teil der Metaphase Bevölkerung. Doch für die Anwendung der FISH-Technik in Interphase-Kernen, ist es bevorzugt, die hESC Bevölkerung von Feeder-Zellen getrennt durch Ernten der Kolonien manuell vor Trypsinierung für FISH-Analyse. <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="Abbildung 1" /> Abbildung 1: Wie wissen die entsprechenden Zelldichte in den Folien. Bilder von Phasenkontrast-Mikroskop in 10-fach Objektiv. (A) Die Zelldichte ist zu hoch. Die Pfeile zeigen die Spreads zu nahe der Kerne Metaphase. (B) Eine Folie mit guter Zelldichte. Die Kreise zeigen Metaphasen breitet sich von Kernen oder anderen Metaphasen isoliert. Bitte <a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen. <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="Abbildung 2" /> Abbildung 2: Wie man eine angemessene Metaphase zu analysierenden wählen. Chromosomen sind mit DAPI und die Bilder durch ein Fluoreszenzmikroskop in 100x-Objektiv erhalten gefärbt. (A) (B) Vermeiden Sie Metaphasen in der Nähe Kerne (angedeutet durch die Pfeile) (C) und Metaphasen nicht rund vorhandenen Chromosomen (Kreis) getrennt. (D) Ein runder Metaphase ist ein guter Metaphasen analysiert werden. Bitte <a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 2 zu sehen.

<ol>
	<li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

Die Vorbereitung des Chromosoms verbreitet ist ein wichtiger Schritt für eine erfolgreiche Analyse des genetischen Status von embryonalen Stammzellen durch die routinemäßig Techniken wie G-banding, und anspruchsvollere Techniken wie FISH, SKY und CGH. Dieses Verfahren kann für viele Zelltypen durch Variation der Zeit der Colcemid Inkubation, die auf der Dauer des Zellzyklus hängt angewendet werden. Für Kolonien von embryonalen Stammzellen, warten wir 3 Stunden, während für Embryoidk?...

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<td>15 ml centrifuge tube</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>91015</td>
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<td>Colcemid Karyo MAX</td>
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<td>Glacial acetic acid</td>
<td>&nbsp;</td>
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<td>100</td>
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<td>Methanol</td>
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<td>208</td>
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chromosomal spread preparation of human embryonic stem cells for karyotyping

Obwohl humanen embryonalen Stammzellen (hES) haben gezeigt worden, um eine stabile diploide Karyotyp 1 vorliegt, haben viele Studien berichteten, dass je nach Kulturbedingungen sie anfällig für chromosomale Anomalien wie Addition von ganzen oder Teile von Chromosomen zu erwerben sein. In der Tat, während der langfristigen Kultur, karyotypischen Veränderungen beobachtet werden, wenn enzymatische oder chemische Spaltung 2,3,4 verwendet werden, während die manuelle Zerlegung der Kolonien für die Passage behält eine stabile Karyotyp 5. Neben Veränderungen in der Umgebung wie die Entfernung von Feeder-Zellen scheinen auch die genetische Integrität der hESC 3,6 Kompromiss. Sobald chromosomalen Veränderungen beeinflussen könnten zelluläre Physiologie, ist die Charakterisierung der genetischen Integrität der hESC in vitro entscheidend angesichts hESC als wesentliches Instrument in der Embryogenese Studien und Drogentests. Darüber hinaus für zukünftige therapeutische Zwecke chromosomalen Veränderungen sind ein echtes Problem, wie es häufig zur Krebsentstehung verbunden. Hier zeigen wir eine einfache und nützliche Methode, um qualitativ hochwertige Chromosom Spreads für die anschließende Analyse des Chromosoms von G-banding, FISH, SKY oder CGH-Techniken 7,8 eingestellt zu erhalten. Wir empfehlen die Überprüfung der chromosomalen Status routinemäßig mit Intervallen von 5 Passagen, um das Auftreten von Translokationen und Aneuploidien Monitor Priscila Britto und Rafaela Sartore trugen zu gleichen Teilen auf das Papier.

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Karyotypisierung ist eine einfache und nützliche Technik allgemein zum Nachweis genetischer Veränderungen verwendet. Hier beschreiben wir Schritt für Schritt-Protokoll für das Chromosom verteilt Vorbereitung von menschlichen embryonalen Stammzellen für die Überwachung der chromosomalen Status dieser Zellen in Kultur gehalten.

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 ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.6K Aufrufe.  Federal University of Rio De Janeiro-UFRJ. Karyotypisierung ist eine einfache und nützliche Technik allgemein zum Nachweis genetischer Veränderungen verwendet. Hier beschreiben wir Schritt für Schritt-Protokoll für das Chromosom verteilt Vorbereitung von menschlichen embryonalen Stammzellen für die Überwachung der chromosomalen Status dieser Zellen in Kultur gehalten.

Serie: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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 ...

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

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

In-vitro-Markierung von humanen embryonalen Stammzellen für Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

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 ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Vorbereitung der gepoolten menschlichen Thrombozyten-Lysat (PHPL) als effiziente Ergänzung zum tierischen Serum-freien menschlichen Stammzell-Kulturen

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

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 ...