Besonderer Dank geht an Dr. Boris Greber für die Einrichtung des Activin A ELISA-Protokoll wie in Greber et al. 2007 veröffentlicht. Wir sind besonders dankbar, dass Frau Monica Shevack für die Vorbereitung der grafischen Übersicht. Wir sind sehr dankbar, dass Dr. Heiko Fuchs für alle Hilfe und wertvolle Anregungen vor und während der Dreharbeiten. Wir möchten alle Mitglieder der Adjaye Labor, vor allem Elisabeth Socha zur Aufrechterhaltung einer konstanten Zufuhr von MEFs und CM danken. Wir erkennen auch unsere Kollegen in der Tierhaltung des MPIMG für ihre ständige Unterstützung. Diese Arbeit wurde teilweise von der Max-Planck-Gesellschaft und der Finanzierung [BMBF, Förderkennzeichen 0315717A], Partner der ERASysBio + Initiative im Rahmen des EU-ERA-NET-Plus im Rahmen des RP7 unterstützt.

1. Isolierung von embryonalen Maus-Fibroblasten (MEFs) Die folgenden beiden Schritte werden unter nicht-aseptischen Bedingungen durchgeführt. <ol><li> Opfere eine schwangere Maus (CF1, Harlan, USA) bei 13 oder 14 DPC (Tag post-coitum) durch Genickbruch. </li><li> Präparieren Sie die Uterushörner, kurz in 70% (v / v) Ethanol und Stelle gründlich in ein Falcon-Röhrchen mit PBS ohne Ca 2 + Mg 2 + (Gibco, Invitrogen). </li></ol> Die folgenden Schritte werden in einem Gewebekulturabzug unter aseptischen Bedingungen durchgeführt und unter Verwendung von sterilen Instrumenten. <ol start="3"><li> Platzieren Sie Uterushörner in eine Petrischale und trennen Sie die einzelnen Embryo von seiner Plazenta und embryonalen sac. </li><li> Sezieren Kopf und roten Organen, in PBS waschen und legen Sie alle Embryonen in einem sauberen Petrischale. Fein Blatt das Gewebe unter Verwendung einer sterilen Rasierklinge bis es möglich wird zu pipettieren. </li><li> 1 ml 0,05% Trypsin / EDTA (Gibco, Invitrogen), IncluDing 100 Kunitz-Einheiten DNase I (USB), pro Embryo. </li><li> Übertragen Sie das Gewebe in einem 50 ml-Falcon-Röhrchen und inkubieren Sie für 15 min bei 37 ° C Nach jeweils 5 min Inkubation, distanzieren Zellen durch Auf-und Abpipettieren gründlich. </li><li> Inaktivierung des Trypsin durch Zugabe von ca. 1 Volumen an frisch zubereiteten MEF Medium. MEF Kulturmedium (Komponenten zu 500 ml Medium zu machen, mischen Sie alle Komponenten und Filter): 450 ml DMEM, 50 ml FBS (10% (v / v)), 5 ml von 200 mM L-Glutamin (1/100 (v / v)), 5 ml Penicillin-Streptomycin (1/100 (v / v)). </li><li> Zentrifugieren Sie die Zellen mit Low-Speed ​​(300 xg), 5 min, entfernen Sie vorsichtig den Überstand und resuspendieren Zellpellet in warmen MEF Medium. </li><li> Tafel etwa eine Anzahl von Zellen, die äquivalent zu 3-4 Embryonen in jeder T150 (TPP)-Kolben mit 0,2% Gelatine (Gelatine aus Rinderhaut, Typ B, Sigma) für 2 Stunden beschichtet ist. Die Fibroblasten (P0, Kanal 0) sind die einzigen Zellen, die die Fähigkeit der Gelatine-beschichteten Kolben legen haben. </li&gt; <li> Im Idealfall sind die Zellen 80-90% konfluent nach 24 Stunden und in dieser Phase ein großer Teil der P0-Zellen ist für die zukünftige Verwendung eingefroren. </li><li> Erweitern Sie die restlichen T150 Flasche (n) der Zellen P0 bis P3 oder P4, dann inaktiviert und die Verwendung als Zubringer zu hESCs replate oder konditioniertem Medium (CM) zu produzieren. </li></ol> 2. Inaktivierung und Plating MEFs (Feeder-Zellen Vorbereitung) Alle Schritte werden in einer Gewebekultur Haube unter aseptischen Bedingungen durchgeführt. <ol><li> Coat T150 Flaschen mit 0,2% Gelatine und inkubieren bei RT für mindestens 2 Stunden. </li><li> Verdünnte Mitomycin C in PBS (1 mg / ml) und filtriert. </li><li> Saugen Sie Medien aus MEFs und waschen mit PBS ohne Ca 2 + Mg 2 +. </li><li> 20 ml Medium, enthaltend 10 ug / ml Mitomycin C auf MEFs. </li><li> Inkubieren für 2 Stunden bei 37 ° C mit Mitomycin C-haltigem Medium dann zweimal mit PBS gewaschen, trypsinieren, Zentrifuge (für 5 min bei 300 × g) und resuspendieren Zellen in warmes Medium. </li><li>Zählen von Zellen und Platten in einer Dichte von 56,000 Zellen / cm 2 im T150 Flaschen und verwenden Sie für CM-Produktion für die folgenden 6 Tage. </li></ol> 3. Konditioniertem Medium (CM) Herstellung Alle Schritte werden in einer Gewebekultur Haube unter aseptischen Bedingungen durchgeführt. <ol><li> Der Tag nach dem Ausplattieren inaktivierten MEFs bei einer Dichte von 56,000 Zellen / cm 2 ersetzen die MEF-Medium mit hESC Medium (UM, unbedingte Medium) (0,5 ml / cm 2) ergänzt frisch mit 4 ng / ml FGF2. </li><li> Sammle CM von Feeder-Flaschen nach 24 h Inkubation und fügen frische hES-Medium mit 4 ng / ml FGF2 zu den Verteilern. </li><li> Wiederholen Sie diesen Vorgang für die nächsten 6 Tage. Jeden Tag Shop gesammelt CM bei -20 ° C </li><li> Nach 6 Tagen Mischen Sie alle Aliquots von mittel-und Filter (Corning, 0,22 um, PAS). Machen Sie 50 ml Aliquots und lagern bei -80 ° C </li><li> Beilage CM mit zusätzlichen 4 ng / ml vor der Zugabe zu FGF2 HES auf Matrigel gezüchtet. &lt;/ Li&gt; </ol> 4. Die Messung von Activin A in konditionierten Medien (ELISA) 15 <ol><li> Bringen Sie alle Proben und Reagenzien auf Raumtemperatur. </li><li> Verdünnen Sie erfassen Antikörper (Human \ Mouse \ Ratte Activin einem mAb, R &amp; D Systems) in PBS mit 1% BSA, zu ergänzen Mikrotiterplatten (100 ul / Well) und Inkubation über Nacht bei RT. </li><li> Nach 24 Stunden verwerfen, die Vertiefungen dreimal mit PBST (PBS mit 0,05% Tween 20) (300 ul / Vertiefung) dann wird der Block (1% BSA / PBS, 300 ul / Well) für 1 h bei RT. </li><li> In dieser Zeit bereiten eine Activin A (R &amp; D Systems) Standardkurve, darunter 7 Verdünnungen (Konzentration von weniger als 30 ng / ml) und Blindprobe. Die linearen Arbeitsbereich der Activin A zwischen 0,25 und 32 ng / ml. </li><li> Fügen Sie Duplikate von Standards und Proben in die Vertiefungen (100 ul / Well) und Inkubation für 2 h bei RT. </li><li> Die Vertiefungen dreimal (300 ul PBST). </li><li> Fügen Sie die sekundäre (biotinylierten) Antikörper (Human / Mouse / Rat Biotinylierte Activin A MAb, R &amp; D Systems) (0.25 ug / ml in 1% BSA / PBS) und Inkubation für 2 h bei RT. </li><li> Die Vertiefungen dreimal (300 ul PBST), fügen Streptavidin-HRP (verdünnt in 1% BSA / PBS, R &amp; D Systems) und Inkubation für 20 min bei RT. </li><li> Die Vertiefungen dreimal (300 ul PBST), fügen Sie 100 ul Substratlösung (Quantikine, R &amp; D Systems) und Inkubation für 30 min bei RT im Dunkeln. </li><li> Geben Sie 100 ul Stop-Lösung (Quantikine, R &amp; D Systems) in jede Vertiefung geben und vorsichtig mischen. </li><li> Set Mikroplatten-Reader (Molecular Devices Spectra Max 250, Global Medical Instrumentation, Inc., Minnesota) bis 450 nm, mit Korrektur für Wellenlängen von 540 oder 570 nm, um die optische Dichte jeder Vertiefung zu bestimmen. </li></ol> 5. Repräsentative Ergebnisse Die allgemeine Systematik der Isolation ist in Abbildung 1 dargestellt. Die typische Morphologie von HES und hiPSCs unter verschiedenen Bedingungen kultiviert wird in Abbildung 2 dargestellt. Die Morphologie<html> MEFs und inaktivierten Feeder-Zellen verwendet werden, um CM vorbereiten wird in Abbildung 3 dargestellt. Im Allgemeinen sollten Zellen sein konfluenten 24 Stunden nach der Isolierung und bereit, gefroren oder erweitert werden. Aber manchmal kann es dauern 2-3 Tage vor Erhalt konfluenten Kulturen. Der CM sollte von Zellen bei Passage 4 und nicht später vorbereitet werden. Dies ist entscheidend, da Primärzellen kann nur für 4-5 Passagen vor dem Einsetzen der Seneszenz erweitert werden. Das Zytokin, Activin A wird als der wichtigste Faktor, der durch Feeder-Zellen für die Unterstützung von undifferenzierten Wachstum von pluripotenten Zellen 14 sezerniert betrachtet. Die Messung des Pegels von Activin A CM (4) ist ein sehr praktisch quantitative Bestimmung der Qualität der MEFs überwachen. <img alt="1" src="/files/ftp_upload/3854/3854fig1.jpg" /> 1. Eine schematische Darstellung des MEFs Isolierungsverfahren. NHALT &quot;&gt; <img alt="2" src="/files/ftp_upload/3854/3854fig2.jpg" /> 2. Die typische Morphologie von undifferenzierten Zellkulturen in Gegenwart von (A) Feeder-Zellen, (B) konditioniertem Medium und (C) definierten Medium kultiviert. Die typische Morphologie hiPSCs in Gegenwart von (D, E) Feeder-Zellen kultiviert. <img alt="Abbildung 3" src="/files/ftp_upload/3854/3854fig3.jpg" /> Abbildung 3. Die typische Morphologie der embryonalen Maus-Fibroblasten (MEFs). (A) Passage 0 (P0) 2 Tage nach dem Ausplattieren / Isolation, (B) inaktivierten Feeder-Schicht bei einer Dichte von 56,000 Zellen / cm. 2 <img alt="Abbildung 4" src="/files/ftp_upload/3854/3854fig4.jpg" /> 4. Enzyme-linked Immunosorbent Assay (ELISA)-basierten Messungen der Konzentration von Activin A in konditioniertem Medium (CM) prepared mit embryonale Maus-Fibroblasten aus dem CF1 Maus-Stamm abgeleitet. CM wurde 6 Tage lang gesammelt und dann vereinigt. CM &quot;1&quot; und CM &quot;2&quot; beziehen sich auf verschiedene Chargen von Medien und UM zu unbedingte Medien. Da die Funktion des Aufbereitungsprozesses ist Activin A Sekretion in das Medium durch MEFs Activin A ist fast nicht nachweisbar in UM.

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Die Isolierung MEFs hier vorgestellte Verfahren ermöglicht die Etablierung eines einheitlichen Kultur Voraussetzung für hESCs und hiPSCs. Darüber hinaus ist die ELISA-System verwendet werden, um Cytokin-Produktion durch Feeder-Zellen zu bewerten, ist ein nützlicher Indikator für die Qualität der MEF-abgeleiteten konditionierten Medien. Die laufende Instandhaltung des Mausstamm (CF1) bietet Unterstützung Fibroblasten ist notwendig, um von Charge zu Charge Variation von Kulturmedien zu vermeiden. Darüber hinaus empfiehlt es sich, mehrere Embryonen aus Mäusen isolieren gleichzeitig eine gleichbleibende Qualität der Zellen zu erhalten. Sicherlich frisch isolierten MEFs kann gefroren bei P0 und P1 werden. Auch inaktiviert MEFs kann in Aliquots eingefroren entsprechender Mengen je nach den Anforderungen für die Zellkultur werden. In der Regel ca. 250.000 Zellen in einer einzigen Vertiefung einer 6-Well-Platte zur Kultur hESCs und iPS-Zellen plattiert werden. Die etabliert und optimiert Verfahren kann routinemäßig verwendet werden, um die Variabilität zu minimieren zwischen unterschiedmieten Experimente.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> DMEM (High Glucose) </td><td> Gibco, Invitrogen </td><td> 41966-052 </td><td></td></tr><tr><td> FBS </td><td> Biochrom </td><td> S0115 </td><td></td></tr><tr><td> L-Glutamin </td><td> Gibco, Invitrogen </td><td> 25030-024 </td><td></td></tr><tr><td> Penicillin-Streptomycin </td><td> Gibco, Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Vakuum-Filter-System </td><td> Corning </td><td> 431097 </td><td> 500 ml, 0,22 um, PAS </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650 </td><td></td></tr><tr><td> Knockout DMEM </td><td> Gibco, Invitrogen </td><td> 10829-018 </td><td></td></tr><tr><td> Knockout SerUM-Ersatz </td><td> Gibco, Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> Gibco, Invitrogen </td><td> 11140-035 </td><td></td></tr><tr><td> Beta-Mercaptoethanol </td><td> Sigma </td><td> M7522 </td><td></td></tr><tr><td> PBS </td><td> Gibco, Invitrogen </td><td> 14190-169 </td><td></td></tr><tr><td> DNase I </td><td> Sigma </td><td> D4527 </td><td></td></tr><tr><td> Mitomycin C </td><td> Roche </td><td> 10107409001 </td><td></td></tr><tr><td> Basischer Fibroblasten-Wachstumsfaktor </td><td> PeproTech </td><td> 100-18B </td><td></td></tr><tr><td> Biotinyliertes Anti-human/mouse/rat Activin A Antikörper </td><td> R &amp; D Systems </td><td> BAM3381 </td><td></td></tr><tr><td> Gelatine </td><td> Sigma </td><td> G9391 </td><td></td></tr><tr><td> Mensch / Maus /Ratte Activin A MAb </td><td> R &amp; D Systems </td><td> MAB3381 </td><td></td></tr><tr><td> 0,05% Trypsin-EDTA </td><td> Gibco, Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Extra dünn Iris Schere </td><td> FST </td><td> 14088-10 </td><td></td></tr><tr><td> Extra Fine Graefe Pinzette </td><td> FST </td><td> 11150-10 </td><td></td></tr><tr><td> Pierse Fixierpinzette </td><td> FST </td><td> 18155-13 </td><td></td></tr></tbody></table>

preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells

Im allgemeinen menschlichen embryonalen Stammzellen (HES) und humanen induzierten pluripotenten Stammzellen (hiPSCs) 1 kann unter variablen Bedingungen kultiviert werden. Jedoch ist es nicht einfach, ein wirksames System zur Kultivierung dieser Zellen zu etablieren. Da die Kulturbedingungen der Genexpression, die Pluripotenz in hES und hiPSCs verleiht beeinflussen können, ist die Optimierung und Standardisierung der Kultur-Methode von entscheidender Bedeutung. Die Einrichtung von HES-Linien wurde zuerst unter Verwendung MEFs als Feeder-Zellen und fötalem Rinderserum (FBS) enthaltenden Kulturmedium 2 beschrieben. Als nächstes wurde FBS mit Knockout-Serum-Ersatz (KSR) und FGF2, die Proliferation von hESCs 3 erweitert ersetzt. Schließlich ermöglichen Feeder-freien Kultur-Systemen Kultivierung von Zellen auf Matrigel-beschichteten Platten in KSR-haltige konditionierte Medium (Medium durch MEFs konditioniert) 4. Anschließend haben hESCs Kultur in Richtung Feeder-freien Kultur in chemisch defin bewegted Bedingungen 7.5. Darüber hinaus wurden die möglichen Kontaminierung durch Krankheitserreger und tierische Proteine ​​Kultur Methoden unter Verwendung von xeno-freien Komponenten zu vermeiden, wurden 8 hergestellt. Um bessere Bedingungen erhalten Maus Feeder-Zellen wurden mit humanen Zelllinien (zB fetale Muskel-und Hautzellen 9, erwachsenen Hautzellen 10, Vorhautfibroblasten 11-12, Fruchtwasser Mesenchymzellen 13) ersetzt worden. Jedoch ist die Effizienz der Wartung undifferenzierten HES mit humaner Vorhaut-Fibroblasten-Feeder-Schichten abgeleiteten nicht so hoch wie die von Maus-Feeder-Zellen aufgrund der niedrigeren Ebene der Sekretion von Activin A 14. Offensichtlich gibt es einen offensichtlichen Unterschied in Wachstumsfaktor durch Maus und menschlichen Feeder-Zellen. Die Analyse der Transkriptome Maus und Mensch-Feeder-Zellen ergab signifikante Unterschiede zwischen unterstützende und-Stützzellen. Exogene FGF2 ist entscheidend für den UnterhaltNing Selbsterneuerung von hES und hiPSCs, und hat sich als entscheidender Faktor für die Regulation der Expression von TGFß1, Activin A und Gremlin (eine BMP-Antagonist) in Feeder-Zellen identifiziert worden. Activin A wurde gezeigt, dass die Expression von Oct4, Sox2 und NANOG im HES 15-16 induzieren. Für Langzeit-Kultur, kann HES und hiPSCs auf mitotisch inaktiviert MEFs oder unter Feeder-freien Bedingungen in MEF-CM (MEF-konditioniertem Medium) auf Matrigel-beschichteten Platten gezüchtet werden, um undifferenzierten Zustand aufrecht zu erhalten. Erfolg beider Kulturbedingungen vollständig abhängig von der Qualität der Feeder-Zellen, da sie Auswirkungen auf das Wachstum von HES. Hier präsentieren wir ein optimiertes Verfahren zur Isolierung und Kultivierung von embryonalen Maus-Fibroblasten (MEFs), Vorbereitung von konditioniertem Medium (CM) und Enzyme Linked Immuno Assay (ELISA) auf das Niveau von Activin A in den Medien zu beurteilen.

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Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Die Qualität von embryonalen Maus-Fibroblasten (MEFs) durch die rechte Mausstamm wie CF-1 bestimmt. Pluripotenz-unterstützende MEFs und konditionierte Medien (CM) aus diesen gewonnenen Erkenntnisse sollten den optimalen Konzentrationen von Activin A, Gremlin und TGFß1 für die Activin / Nodal und FGF Signalwege erforderlich, um kooperativ zu halten Selbsterneuerung und Pluripotenz enthalten.

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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90.6K Aufrufe.  Max Planck Institute for Molecular Genetics. Die Qualität von embryonalen Maus-Fibroblasten (MEFs) durch die rechte Mausstamm wie CF-1 bestimmt. Pluripotenz-unterstützende MEFs und konditionierte Medien (CM) aus diesen gewonnenen Erkenntnisse sollten den optimalen Konzentrationen von Activin A, Gremlin und TGFß1 für die Activin / Nodal und FGF Signalwege erforderlich, um kooperativ zu halten Selbsterneuerung und Pluripotenz enthalten.

Serie: Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent 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 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 ...

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 ...

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 ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...