Menschliche embryonale Stammzellen-Studien in der Teitell Labor werden derzeit von California Institute for Regenerative Medicine (CIRM) Saatgut gewähren RS1-00313 unterstützt. Wir danken Dr. Carla Koehler (UCLA) und Mitglieder der Koehler-Labor für die Beratung und Einblicke in diese und weitere Studien.

Hohe Auflösung Klare nativen Gel Elektrophorese (hrCNE) für Mitochondriale Komplexe In-Gel Aktivitätstests Humane embryonale Stammzellen (hES) wurden auf einem Monolayer von ˠ-bestrahlten oder Mitomycin C-behandelten embryonalen Maus-Fibroblasten (MEFs), dann das Wachstum auf Matrigel für 5 Tage in MEF-konditioniertem Medium unter Standardbedingungen eingeschaltet gepflegt. So entfernen Sie kontaminieren MEFs wurden hESCs auf Matrigel erneut verzinkt und aufgewachsen in MEF-konditioniertem Medium für weitere 3 Tage. Immunfluoreszenzmikroskopie für Pluripotenz Marker Oct-4 und SSEA-4 verwendet wurde, um den Mangel an menschlichen embryonalen Stammzellen die Differenzierung zu bestätigen. <ol><li> Wash-Zellen mit warmem 1x PBS, pH 7,4, durch Trypsinierung (1 ml 1x Trypsin pro Vertiefung einer 6-Well-Platte) für 5 min. </li><li> Fügen Sie dem gleichen Volumen frischen Trypsin Inhibitor (1mg/ml in 1x PBS, pH 7,4). Ernten Sie die Zellen und Zentrifuge bei 200g, 5 min, Raumtemperatur. </li><li> Verwerfen Sie den Überstand und resuspendieren pelletierten Zellen in 0,8 - 1,7 ml (bezogen auf die Größe des Zellpellets) eiskaltem Mitochondrien isoliert Puffer (83mm Saccharose; 6.6mm Imidazol / HCl, pH 7,0) mit frisch zubereiteten 1 &#39;Protease Inhibitor Cocktail. Inkubieren auf Eis für 10 min. </li><li> Dounce Zellen mit einem Homogenisator (~ 40 bis 50 Schläge). Stain einen aliquoten Teil der dounced Zellen mit Trypanblau auf Vollständigkeit der Homogenisierung mit einem Lichtmikroskop zu überprüfen. </li><li> Zentrifuge bei 20.000 g für 10 min bei 4 ° C, um eine rohe mitochondriale Pellet enthält auch Kerne und größere Zellfragmente zu erhalten. Dekantieren und den Überstand verwerfen und wiegen das ausgefallene Rohprodukt mitochondriale Pellet. Das Pellet kann bei -80 ° C gelagert werden folgende Instant Einfrieren in flüssigem Stickstoff, ob dies ein Haltepunkt. HINWEIS: Differential Zentrifugation oder andere Gradienten-basierten Verfahren können zur Isolierung und Sammeln einer reinen mitochondrialen Membranfraktion verwendet werden. Siehe Lit.. 4 für Details. Dieser alternative Ansatz wird Ertrag der mitochondrialen Komplex-Aktivität pro mg des mitochondrialen Proteins statt komplexer Aktivität pro mg Zellen Gewicht oder Zellzahl. Die Konzentration der mitochondrialen Proteine ​​müssen nach der Reinigung auf diesen Vergleich zu machen bestimmt werden. Auch kann eine andere Isolation Puffer für diesen Ansatz erforderlich sein. Für die Solubilisierung der reinen mitochondriale Membran-Fraktion, siehe die Anmerkung folgende Schritt 6 unten. </li><li> Solubilisieren das rohe mitochondriale Pellet auf Eis durch Vortexen mit eiskaltem Solubilisierungspuffer (50 mM NaCl, 50 mM Imidazol / HCl pH 7,0, 1 mM EDTA, 2 mM 6-Aminohexansäure). 6-Aminohexansäure kann mit dem 1x Protease Inhibitor Cocktail ersetzt werden. Verwenden Sie 35μl der Solubilisierungspuffer pro 20mg Tablette Gewicht. Add 20 &amp;mgr; l von 10% w / v Digitonin pro 20mg Tablette Gewicht und mischen flicking die Röhre. Inkubieren für 5-10 min auf Eis. Dodecyl-bD-Maltosid oder Triton X-100 kann für Digitonin ersetzt werden, um die Proteine ​​zu lösen. Die Wahl des Waschmittels und die Menge kann sich auf die Bildung von Superkomplexen oder multimere Formen der Mitochondrien-Komplexen. Weitere Informationen finden Sie unter Ref.1. HINWEIS: Die Menge an Waschmittel benötigt, um eine reine mitochondrialen Membranfraktion solubilisieren entspricht einem Detergenz / Protein Gewichtsverhältnis von 2-6g / g. Das optimale Verhältnis der mitochondrialen Membranen lösen sich 2,5 g / g für Dodecyl-bD-Maltosid, 3g / g für Triton X-100 und 6 g / g für Digitonin. </li><li> Centrifuge das gelöste Pellet bei 100.000 g für 15 min bei 4 ° C. Sammeln Sie die klare Überstand. </li><li> Add-Ladepuffer, um eine endgültige 5% w / v Glycerin und 0,01% w / v Ponceau S (mit einem 50% w / v Glycerin, 0,1% w / v Ponceau S Ladepuffer Stammlösung). </li><li> Laden Gel Brunnen mit einem Volumen, das 20-40mg des rohen mitochondrialen Pellet pro Spur enthält. Verwenden Sie ein Acrylamid Gradienten-Gel (4 -13%) mit einem 3,5% Sammelgel. HINWEIS: Gel-Elektrophorese Vorbereitung und Bedingungen: Für Gelzubereitung, verwenden Sie ein Acrylamid / Bisacrylamid (AA / bisAA) 3-Mix wie folgt: 48g Acrylamid und 1,5 g Bisacrylamid (49,5% T, 3% C, wobei T Gesamtkonzentration von AA und bisAA und C der Anteil der Vernetzer (bisAA) zum Gesamtumsatz Monomer). Gel-Puffer (3 &#39;): 1,5 6-Aminohexansäure; 75mm Imidazol, pH 7,0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td> Gradient Gel: </td><td> 4% </td><td> 13% </td><td> Stacking-Gel: </td><td> 3,5% </td></tr><tr><td> AA / bisAA 3 Mix </td><td> 2 ml </td><td> 6,5 ml </td><td></td><td> 0,6 ml </td></tr><tr><td> Gel-Puffer 3 &#39; </td><td> 8,3 ml </td><td> 8,3 ml </td><td></td><td> 2,7 ml </td></tr><tr><td> Glycerol </td><td></td><td> 5 g </td><td></td><td></td></tr><tr><td> H 2 O </td><td> 14,7 ml </td><td> 5 ml </td><td></td><td> 4,7 ml </td></tr><tr><td> Gesamtvolumen </td><td> 25 ml </td><td> 25 ml </td><td></td><td> 8 ml </td></tr><tr><td> 10% APS </td><td> 139 ul </td><td> 125 ul </td><td></td><td> 67 ul </td></tr><tr><td> TEMED </td><td> 14 ul </td><td> 12,5 ul </td><td></td><td> 6,7 ul </td></tr></tbody></table> Anode Puffer: 25mM Imidazol / HCl, pH 7,0 Cathode Puffer: 50 mM Tricin, 7,5 mm Imidazol / HCl, 0,02% w / v Dodecyl-bD-Maltosid, 0,05% w / v Desoxycholat, pH 7,0 Gießen und führen Gele in einem kalten Raum (4-7 ° C). Die anfängliche Spannung verwendet wird 100V. Wenn die Probe hat das Stacking-Gel eingegeben, wird die Spannung auf 500 V mit dem Strom begrenzt auf 15mA (für 0,15 &#39;14&#39; 14-cm-Gele) angehoben. Die Elektrophorese wird nach 3-4 h, wenn die scharfe Linie des roten Farbstoff Ponceau S das Gel vor Ansätze gestoppt. Alternativ kann ein Gel über Nacht mit konstanter Spannung betrieben werden bei 70-100V. <ol><li> In-Gel-Aktivitäts-Assays. Die Inkubationszeit für jeden Test hängt von der Effizienz des Proteins Solubilisierung und die Menge an Material pro Vertiefung geladen. Komplexe I, IV und V liefern in der Regel stärkere Signale als Komplexe II und III. </li></ol> Komplex I (CI) Inkubieren Sie die Gelscheibe mit CI-Puffer (5 mM Tris-HCl, pH 7,4; 0.1mg/ml NADH; 2.5mg/ml NBT) bei Raumtemperatur für mehrere Stunden. CI-Puffer: € pro 100 ml 5mM Tris-HCl, pH 7,4: 0.01g NADH 0,25 g NBT Fix das Gel in 50% Methanol und 10% Essigsäure für 15-45 min. Die feste Gel wird in 10% Essigsäure erhalten. Komplex II (CII) Inkubieren Sie die Gelscheibe mit CII-Puffer (20 mM Natrium-Succinat; 0,2 mM Phenazin methasulfate; 2.5mg/ml NBT; 5mM Tris-HCl pH 7,4) bei Raumtemperatur für mehrere Stunden. CII-Puffer: € pro 100 ml 5mM Tris-HCl, pH 7,4: 200 ul 1M Natriumsuccinat 80μl Phenazin methasulfate (250mm PMS gelöst in DMSO) 0,25 g NBT Fix das Gel für komplexe beschrieben I Komplexe III (CIII) Inkubieren Sie die Gelscheibe mit CIII-Puffer (50 mM Natriumphosphat, pH 7,2; 0,05% 3,3 &#39;-Diaminobenzidintetrahydrochlorid (DAB)) bei Raumtemperatur für mehrere Stunden. CIII Puffer: € pro 100 ml 50 mM Natrium-Phosphat, pH 7,2: 50mg Diaminobenzidintetrahydrochlorid Fix das Gel für komplexe beschrieben I Komplex IV (CIV) Inkubieren Sie die Gelscheibe mit CIV-Puffer (50 mM Natriumphosphat, pH 7,2; 0,05% 3,3 &#39;-Diaminobenzidintetrahydrochlorid (DAB), 50 &amp;mgr; Pferd Cytochrom c) bei Raumtemperatur für mehrere Stunden. CIV Puffer: € pro 100 ml 50 mM Natrium-Phosphat, pH 7,2: 50mg Diaminobenzidintetrahydrochlorid 0.1g Pferd Cytochrom c Fix das Gel für komplexe beschrieben I Komplexe V (CV) Inkubieren Sie die Gelscheibe mit CV-Puffer (35mm Tris-base, 270mm Glycin, pH 8,3 bei 25 ° C, 14mm MgSO 4, 0,2% w / v Pb (NO 3) 2, 8mm ATP) bei Raumtemperatur für mehrere Stunden. CV Puffer: je 100ml Lösung hinzu: <table border="0"><tbody><tr><td> Tris-base </td><td> 0.424g </td></tr><tr><td> Glycine </td><td> 2.026g </td></tr><tr><td> MgSO 4 </td><td> 0.345g </td></tr><tr><td> Pb (NO 3) 2 </td><td> 0.29g </td></tr></tbody></table> Keine Notwendigkeit, pH-Wert (sollte 8,3 sein) einstellen - nicht mit Säure oder Base. Filter und fügen <table border="0"><tbody><tr><td> ATP </td><td> 0.441g </td></tr></tbody></table> Fix das Gel in 50% Methanol für 30 min und Transfer zum Wasser.

<ol>
	<li>Wittig, I., Braun, H. -. P., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nature Protocols. 1, 418-428 (2006).</li><li>Wittig, I., Karas, M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6.">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. 6.</a> Molecular &amp; Cellular Proteomics. 6, 1215-1225 (2007).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28, 3811-3820 (2007).</li><li>Castle, J. D., Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Organelles+from+Mammalian+Cells.">Purification of Organelles from Mammalian Cells.</a> Current Protocols in Protein Science. 4.2, 4.2.1-4.2.57 (2007).</li></ol>

In diesem Video haben wir die Extraktion von mitochondrialen Protein-Komplexe aus humanen embryonalen Stammzellen, deren Trennung durch hochauflösende klare native Gelelektrophorese (hrCNE), und die anschließende Analyse von in-Gel-katalytische Aktivität beschriebenen Assays. hrCNE löst mitochondriale Membran-Protein-Komplexen mit einer Auflösung vergleichbar mit dem von blue native Gelelektrophorese und ist überlegen in-Gel-Aktivitäts-Assays. Diese Technik kann nicht nur zur mitochondrialen Atmungskette Komplexe IV quantifizieren, a...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>0.5M EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP120-1</td>
<td>adjust pH to 8.0 with NaOH </td>
</tr>
<tr>
<td>1M NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014-500MG</td>
<td>&nbsp;</td>
<tr>
<td>1M Sodium succinate</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S413-500</td>
<td>&nbsp;</td>
<tr>
<td>0.1M Sodium phosphate buffer pH 7.2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP332-1 and BP329-1</td>
<td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td>
</tr>
<tr>
<td>1M Tricine, pH 7.0 (adjusted with NaOH)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>9010</td>
<td>store at 4 C </td>
</tr>
<tr>
<td>1M imidazole/HCl, pH 7.0</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-2399</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>1M Tris-HCl, pH 7.4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP153-1</td>
<td>&nbsp;</td>
<tr>
<td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44205-500MG</td>
<td>Dissolved in water. Store 1 ml aliquots at &#x2013;20 C.</td>
</tr>
<tr>
<td>10% (wt/vol) digitonin (>50% purity, used without recrystallization)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>37008-1G</td>
<td>Dissolved in water. Store 0.1&#x2013;1 ml aliquots at &#x2013;20 C. Should be warmed up to 95 C before use.</td>
</tr>
<tr>
<td>2M 6-aminohexanoic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>07260-100G</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>10x Ponceau S/glycerol stock solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3504 and G6279-1L</td>
<td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td>
</tr>
<tr>
<td>10% w/v sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Fluka </td>
<td>30970-25G</td>
<td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td>
</tr>
<tr>
<td>250 mM phenazine methasulfate (PMS)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>P0083</td>
<td>Dissolved in DMSO and aliquoted. Store at &#x2013;20 C.</td>
</tr>
<tr>
<td>Nitro-blue tetrazolium (NBT)</td>
<td>&nbsp;</td>
<td>Amresco </td>
<td>0329-1G</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride (DAB)</td>
<td>&nbsp;</td>
<td>TCI America </td>
<td>D0078</td>
<td>&nbsp;</td>
<tr>
<td>Pb(NO3)2</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>L62-100</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP213-1</td>
<td>&nbsp;</td>
<tr>
<td>Tris base </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP152-10</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>G45-212</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2019;-triphosphate disodium salt (ATP)</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2383-5G</td>
<td>Grade I, minimum 99%</td>
</tr>
<tr>
<td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>N-8129</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c From horse heart</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C2506-250MG</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP220-212</td>
<td>&nbsp;</td>
<tr>
<td>100x protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P8340</td>
<td>Store at &#x2013; 20 C.</td>
</tr>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D128-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue Stain 0.4%</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>10x Trypsin (0.5%)</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>17075-029</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

probing for mitochondrial complex activity in human embryonic stem cells

Mitochondrien sind Organellen, die zytoplasmatische eine primäre Rolle im Zellstoffwechsel und Homöostase haben, die Regulierung der zellulären Signalübertragung Netzwerk und den programmierten Zelltod. Mitochondrien erzeugen ATP, regulieren die cytoplasmatische Redoxzustand und Ca2 + Balance, katabolisieren Fettsäuren, zu synthetisieren Häm, Nukleotide, Steroidhormone, Aminosäuren, und helfen montieren Eisen-Schwefel-Clustern in Proteinen. Mitochondrien haben auch eine wesentliche Rolle in der Wärmeproduktion. Mutationen des mitochondrialen Genoms verursachen verschiedene Arten von menschlichen Erkrankung. Die Anhäufung von Mutationen der mtDNA korreliert mit dem Alter und steht im Verdacht, eine wichtige Rolle bei der Entstehung von Krebs haben. Aufgrund ihrer äußerst wichtige Rolle in allen Zelltypen, muss die Funktion der Mitochondrien auch entscheidend für die Stammzellen. Key Fortschritte in unserem Verständnis von Stammzellen Lebensfähigkeit, der Vermehrung und Differenzierung Kapazität gemacht. Aber die funktionelle Aktivität von Stammzellen, insbesondere ihre Energie-Status, war noch nicht im Detail untersucht worden. Fast nichts ist über den mitochondrialen Eigenschaften von humanen embryonalen Stammzellen (hES) und ihrer differenzierten Vorläufer Nachkommen bekannt. Ein Weg, um zu verstehen und bewerten die Rolle der Mitochondrien in hESC Funktion und Entwicklungspotential ist die direkte Messung der Aktivität der mitochondrialen Atmungskette Komplexe. Hier beschreiben wir hochauflösende klare native Gelelektrophorese und anschließender in Gel-Aktivität Visualisierung als Methode zur Analyse der fünf Atmungskette Komplexe von HES.

Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

In diesem Video zeigen wir Ihnen, wie der mitochondrialen Atmungskette Komplexe von humanen embryonalen Stammzellen analysiert werden mit in Gel-Aktivitäts-Assays werden.

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 ...

probing-for-mitochondrial-complex-activity-human-embryonic-stem

Research

JoVE Journal

Biology

14.2K Aufrufe.  University of California, Los Angeles. In diesem Video zeigen wir Ihnen, wie der mitochondrialen Atmungskette Komplexe von humanen embryonalen Stammzellen analysiert werden mit in Gel-Aktivitäts-Assays werden.

Serie: Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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 ...

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 ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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.

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.