Dank Tim Doyle, PhD und der Stanford Center for In Vivo Imaging für die Unterstützung bei Biolumineszenz-Bildgebung. Auch dank Ngan Huang, PhD für die gemeinsame Nutzung ihrer Technik auf Stammzell-Co-Injektion mit Matrix-Lösung. Schließlich Felt dank Steve, Ph.D. für die Unterstützung bei tierärztliche Pflege der Tiere.

<ol><li> Bau von Double Fusion Reporter Gene <ol><li> Um Biolumineszenz-Bildgebung des menschlichen embryonalen Stammzellen durchzuführen, müssen Sie zunächst Zellen, die stabil exprimieren, einem Luciferase-Reportergen wie Glühwürmchen-Luciferase durch einen konstitutiven Promotor wie Ubiquitin oder EF1A getrieben zu erhalten. </li><li> Der Schwerpunkt dieses Protokoll ist auf Reportergen-Anwendungen, so detaillierten Verfahren nicht vorgesehen sind hier. Allerdings ist unser Labor die allgemeine Strategie zu einer doppelten Fusionskonstrukt Vektor, der Glühwürmchen-Luciferase (fluc) und Enhanced Green Fluorescent Protein (eGFP) durch einen Abstandhalter in pCDNA 3,1 + getrennt enthält. </li><li> Kurz gesagt, unsere Doppel-Fusionsgen wurde ursprünglich nach dem Cytomegalovirus-Promotor in pCDNA 3.1 (+) gelegen, so das 3,3 kbp-Fragment wurde unter Verwendung NdeI und NotI Verdauung und blunt-end in die multiple Klonierungsstelle eines selbst-inaktivierenden (SIN ligiert ) lentiviralen Vektor, unter der Kontrolle eines Ubiquitin-Promotor. </li></ol></li><li> Lentivirale Transduktion <ol><li> hESCs kann transduziert werden 3-5 Tage nach dem Durchgang bei einer Multiplizität der Infektion (MOI) von 10 (virale Titer von ca. 107 mit 106 Zellen inkubiert). </li><li> Die aufgetauten virale Lager direkt an die frische hESC Medien hinzugefügt werden. </li><li> Aktualisieren Sie die Medien 12 und 24 Stunden später. </li><li> Nach 48 h kann Transduktionseffizienz von qualitativ beurteilt mittels Fluoreszenzmikroskopie. Anschließend kann FACS verwendet werden, um infizierte Zellen zu isolieren. </li></ol></li><li> Die Kultivierung hESCs und Einführung in die Imaging Set-up <ol><li> Kultur Ihrer Luciferase positive hESCs in Feeder-freien Bedingungen. Wir in der Regel folgt dem Standard WiCell Protokoll und verwenden 6-well Platten in growth factor reduziert Matrigel beschichtet, entweder mit konditionierten Medien oder kommerzielle, Feeder-freien Medien. </li><li> Zum Nachweis der Luciferase-positiven Zellen werden zu können, muss der Reporter-Sonde D-Luciferin bereits vorbereitet. Um dies zu tun, aliquoten Luciferin in 1-1.5 ml Vorbereitungen auf eine Endkonzentration von 45 mg / mL. Halten Sie die Aliquots bei -20 ° C, wenn nicht in Gebrauch ist und zur Vermeidung von Lichteinfall durch Abdecken mit einem Papiertuch, wenn sie nicht in der Lagerung. </li><li> Um die Luciferase-positiven Zellen sichtbar zu machen, verwenden wir IVIS 50 und IVIS 200 Imaging Systems (Xenogen Corporation, Alameda, CA). Letzteres enthält eine integrierte isoflourane Apparate-und Induktions-Kammer für temporäre Anästhesie bei Kleintieren. </li></ol></li><li> In-vitro-Biolumineszenz-Bildgebung von Zellkulturen <ol><li> Für In-vitro-Biolumineszenz-Bildgebung, ist es wichtig, sterilen Bedingungen zu halten. Daher sollte der Imaging-System steril sein, vorzugsweise in einer Zellkultur Raum. </li><li> Vor Bildgebung, entfernen Sie die Zelle Medien und fügen gerade genug PBS zu den Zellen zu decken. Zum Beispiel, 1ml PBS in jede Vertiefung einer 6-Well-Platte mit Ihren hESC Kulturen. </li><li> Das Verhältnis von D-Luciferin zu PBS sollte 1:100 sein, so fügen Sie 10 ul aufgetauten D-Luciferin in jede Vertiefung der 6-Well-Platte. </li><li> Warten Sie eine Minute, dann nehmen Sie ein Bild mit einer Belichtungszeit von 1 Sekunde. Wenn das Signal schwach ist, erhöhen Sie die Belichtung Intervall und versuchen Sie es erneut. </li><li> Die Biolumineszenz-Signal spiegelt Zellzahl, so Quantifizierung Assays durchgeführt werden kann, dass korrelieren Signal mit unterschiedlichen Zellzahlen. </li></ol></li><li> Vorbereitung der Maus und Injizieren Zellen in vivo Bildgebung <ol><li> Reporter-Gen Imaging bietet eine High-Throughput-, kostengünstig und sensitive Methode für die Verfolgung von Zellen nach der Transplantation über Tage, Wochen und sogar Monate. Dies ist besonders wichtig, da transplantierte Stammzellen häufig Teratome zu bilden, werden durch das Immunsystem des Wirtes abgelehnt, oder einfach nur sterben. </li><li> Die einfachste Methode zur Bildgebung Teratom-Bildung in vivo ist hESCs, dass die Glühwürmchen-Luciferase Reporter-Gen exprimieren, in den Rücken von SCID-Mäusen und erwerben Biolumineszenz Bilder mit dem IVIS System zu injizieren. </li><li> Wenn Sie bereit sind, um die Zellen Transplantation sind, verwenden Dispase oder Kollagenase, um die Zellen zu lösen, waschen mehrmals, und hängen sie in einer 1:1-Mischung von Wachstumsfaktoren reduzierten Matrigel und DMEM. In der Regel haben wir erste Mix der Zellen mit DMEM gekühlt und fügen Sie dann in der Matrigel. Für jede subkutanen Injektionsstelle injizieren wir 200.000 Zellen in ein 50-100 ul Volumen der Matrigel / DMEM suspendiert. Die Zellzahl kann je nach Ihrer Anwendung werden. Denken Sie daran, alles auf Eis vor der Injektion zu halten. </li><li> Sobald die Zellen bereit sind, betäuben die Maus, indem Sie es in der Induktion Box mit Isofluran flow eingeschaltet. Nach 1-5 Minuten ist die Maus schläft und kann auf dem OP-Tisch mit stufenloser Isofluran platziert werden. </li><li> Da Fell natürlich auto-fluoresziert und die Biolumineszenz Bild zu verdunkeln können, benötigen wir, um das Haar von der Rückseite der Maus zu entfernen. Der einfachste Weg, dies zu tun ist, um einen elektrischen Rasierer verwenden, aber Sie können auch Haarentfernung Gele. Wischen Sie mit Alkohol zu sterilisierendie Haut hinterher </li><li> Als nächstes erstellen Sie Ihre Zellsuspension in einer Spritze. 23 bis 27 Gauge-Nadeln funktionieren am besten, da sie nicht verstopfen mit Zellen. Wenn die Nadel zu groß ist (&lt;23 Gauge) die Nadel-Darm-Trakt, sollten Sie ein großes Loch, durch das Zellen wieder austreten kann. </li><li> Spritzen Sie die Zellen unter die Haut in die Maus ist wieder da. Da die Haut ist ganz locker, mit dem Daumen und Zeigefinger klemmen und strecken Sie den gewünschten Bereich zu injizieren. Inject direkt unter die Haut, dabei nicht zu durchstechen zu tief. Versuchen Sie auch, um die Nadel ein Herausrutschen aus der Injektionsstelle während Herunterdrücken des Kolbens zu halten: Dadurch wird verhindert, ein Loch in die Haut, durch die den Zellen austreten, wenn Sie wieder zu injizieren ausprobieren können. </li><li> Nachdem die Zellen injiziert werden, ein paar Stunden warten, damit die Maus zu wecken und toben, bevor erneut anästhesiert für Biolumineszenz-Bildgebung. Dies vermeidet Isofluran Toxizität. </li></ol></li><li> In-vivo-Biolumineszenz-Bildgebung der transplantierten Zellen <ol><li> Für ganze Tier Biolumineszenz-Bildgebung haben wir eine intraperitoneale Injektion von D-Luciferin bei 375 mg / kg Körpergewicht. Wiegen Sie das Tier, um die Dosierung vor der Injektion zu berechnen. </li><li> Spritzen Sie die D-Luciferin in das Bauchfell, direkt an der Mittellinie. Seien Sie vorsichtig, um nicht zu tief, oder Sie können innere Organe beschädigt werden. Wenn die Maus beginnt zu erwachen, setzen Sie ihn wieder in die Ko-Box und warten Sie eine Minute oder zwei. Bei Mäusen, die maximale Biolumineszenz tritt in der Regel 15-40 Minuten nach der Injektion. </li><li> Denken Sie daran, matte schwarze Papier in der Imaging-box Ort, um Hilfe zu absorbieren kein Licht nicht durch die hESCs emittiert. Platzieren Sie die Maus (Rückseite nach oben) in der Imaging-Kammer mit Isofluran, auf der Oberseite des schwarzen Papiers. Beginnen Sie mit einer Belichtungszeit von 10 Sekunden. Wenn das Signal gesättigt ist, versuchen die Verringerung der Belichtungszeit, und wenn zu schwach, erhöhen Sie die Belichtung. </li><li> Sobald das Signal Belichtungszeit ist für Sie mit der Maus optimiert wurde, beginnt, die Bilder jede Minute, bis das Signal ein Maximum erreicht. Dies geschieht am besten, indem Sie die Imaging-Software auf &quot;regions of interest (ROI)&quot;, dass Ihre Injektionsstellen Abdeckung wählen getan. Durch die Überwachung der Signalstärke bei jedem ROI können Sie leicht feststellen, wenn das Signal zu verringern beginnt, was darauf hinweist, dass die maximale Signalstärke erreicht wurde. Die maximale Signal sollte als endgültige Daten verwendet werden. </li><li> Wenn Sie mit Ihren Bildern zufrieden sind, entfernen Sie die Maus von Isofluran und lassen Sie ihn aufwachen in seinem Käfig. Normalerweise sollten die Maus aufwachen innerhalb von 15 Minuten. </li><li> Biolumineszenz Reportergen Bildgebung kann wiederholt werden, täglich, wöchentlich und monatlich werden, solange die Zellkulturen in das Tier überleben. Signal aus einem Entwicklungsland Teratom wird exponentiell mit der Zeit, in der Regel in der Größenordnung von Wochen, als die hESCs zu wuchern beginnen. </li><li> Nachdem der gewünschte zeitliche Verlauf der Biolumineszenz Bilder erworben wird, kann das Tier geopfert werden und Gewebeschnitten für die Histologie verwendet. </li></ol></li></ol>

<ol>
	<li>Passier, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cardiomyocyte+differentiation+from+human+embryonic+stem+cells+in+serum-free+cultures.">Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.</a> Stem Cells. 23, 772-780 (2003).</li><li>Laflamme, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature biotechnology. 25, 1015-1024 (2007).</li><li>Baharvand, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+from+human+embryonic+stem+cells+in+a+defined+adherent+culture+conditi.">Neural differentiation from human embryonic stem cells in a defined adherent culture conditi.</a> Int J Dev Biol. 51, 371-378 (2007).</li><li>Schulz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neuronal+differentiation+of+human+embryonic+stem+cells.">Directed neuronal differentiation of human embryonic stem cells.</a> BMC Neurosci. 4, (2007).</li><li>Jiang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Insulin-producing+Islet-like+Clusters+from+Human+Embryonic+Stem+Cells.">Generation of Insulin-producing Islet-like Clusters from Human Embryonic Stem Cells.</a> Stem Cells. 17, 17 (2007).</li><li>Swijnenburg, R. J., van der Bogt, K. E. A., Sheikh, A. Y., Cao, F., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+hurdles+for+the+transplantation+of+cardiomyocytes+derived+from+human+embryonic+stem+cells:+role+of+molecular+imaging.">Clinical hurdles for the transplantation of cardiomyocytes derived from human embryonic stem cells: role of molecular imaging.</a> Current Opinion in Biotechnology. 18, 38-45 (2007).</li><li>Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging:+looking+at+problems,+seeing+solutions.">Molecular imaging: looking at problems, seeing solutions.</a> Science. 302, 605-608 (2003).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature medicine. 4, 245-247 (1998).</li><li>Bhaumik, S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+Renilla+luciferase+reporter+gene+expression+in+living+mice.">Optical imaging of Renilla luciferase reporter gene expression in living mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 377-382 (2002).</li><li>Tisi, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+thermostable+firefly+luciferase.">Development of a thermostable firefly luciferase.</a> Analyica Chimica Acta. 457, 115-123 (2002).</li><li>Ray, P., Tsien, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+validation+of+improved+triple+fusion+reporter+gene+vectors+for+molecular+imaging+of+living+subjects.">Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.</a> Cancer Res. 67, 3085-3093 (2007).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> . Circulation. 113, 1005-1014 (2006).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Embryonic+Stem+Cell+Misbehavior+and+Suicide+Gene+Ablation.">Molecular Imaging of Embryonic Stem Cell Misbehavior and Suicide Gene Ablation.</a> Cloning and Stem Cells. 9, 107-117 (2007).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Wu, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+reporter+genes+for+molecular+imaging+of+transplanted+embryonic+stem+cells.">Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells.</a> Proteomics. 6, 6234-6249 (2006).</li><li>Eiges, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Mohr, J. C., de Pablo, J. J., Palecek, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+human+embryonic+stem+cells:+Small+and+macromolecule+loading+and+DNA+transfection.">Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection.</a> Biotechnol Prog. 22, 825-834 (2006).</li><li>Siemen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Lakshmipathy, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Ma, Y., Ramezani, A., Lewis, R., Hawley, R. G., 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=High-Level+Sustained+Transgene+Expression+in+Human+Embryonic+Stem+Cells+Using+Lentiviral+Vectors.">High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors.</a> Stem Cells. 21, 111-117 (2003).</li><li>Menendez, P., Wang, L., Chadwick, K., Li, L., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+hematopoietic+cells+differentiated+from+human+embryonic+stem+cell-derived+CD45(neg)PFV+hemogenic+precursors.">Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors.</a> Molecular therapy. 10, 1109-1120 (2004).</li><li>Thyagarajan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+engineered+human+embryonic+stem+cell+lines+using+phiC31+integrase.">Creation of engineered human embryonic stem cell lines using phiC31 integrase.</a> Stem cells. 26, 119-126 (2008).</li><li>Liew, C. -. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transient and Stable Transgene Expression in Human Embryonic Stem Cell. , .</li></ol>

Im Vergleich zu anderen Verfahren wie PET und MRI hat Biolumineszenz begrenzte räumliche Auflösung und reduzierten Gewebepenetration aufgrund der relativ schwachen Energie der emittierten Photonen (2-3 eV); aus diesen Gründen hat es bislang nicht anwendbar in großen Tieren. Allerdings hat Biolumineszenz den Vorteil, dass Low-Cost, High-Throughput-und non-invasive, so dass es sehr wünschenswert, in vivo Stammzellen Tracking bei Kleintieren. Non-Biolumineszenz Reporter-Gene wie PET und Fluoreszenz-Konstrukte können in Verbindung mit Luc...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
</tr>
<tr>
<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

Die Entdeckung von menschlichen embryonalen Stammzellen (hES) hat sich dramatisch die Werkzeuge zur Verfügung, um Mediziner in der regenerativen Medizin interessiert erhöht. Allerdings hat die direkte Injektion von hESCs, und Zellen aus hES differenziert, in lebende Organismen bisher durch erhebliche Zelltod, Teratom-Bildung und Host Immunabwehr behindert. Das Verständnis der in vivo hESC Verhalten nach der Transplantation erfordert neuartige bildgebende Verfahren in Längsrichtung Monitor hESC Lokalisation, Ausbreitung und Lebensfähigkeit. Die molekulare Bildgebung hat Ermittler einen hohen Durchsatz, kostengünstig und empfindliche Methode für die Verfolgung von in vivo Proliferation über Tage, Wochen und sogar Monate. Dieser Fortschritt hat sich das Verständnis der räumlich-zeitlichen Kinetik hESC Engraftment, Proliferation und Teratom-Bildung in lebenden Probanden erhöht. Ein wichtiger Fortschritt in der molekularen Bildgebung hat die Ausweitung der nicht-invasiven Reportergen-Assays von Molekular-und Zellbiologie in in vivo multimodalen Bildgebung Plattformen. Diese Reporter-Gene unter der Kontrolle von technischen Promotoren und Enhancer, die die Vorteile der Wirtszelle s Transkriptionsmaschinerie sind in Zellen mit einer Vielzahl von Vektor-und Nicht-Vektor-Methoden eingeführt. Einmal in der Zelle kann Reportergene entweder konstitutiv oder nur unter bestimmten biologischen oder zellulären Bedingungen transkribiert werden, abhängig von der Art des verwendeten Promotor. Transkription und Übersetzung von Reporter-Genen in bioaktive Proteine ​​wird dann mit empfindlichen, nicht-invasive Instrumente (zB CCD-Kameras) mit Signal-Erzeugung Sonden wie D-Luciferin detektiert. Um zu vermeiden, die Notwendigkeit für exzitatorische Licht zur Gewinnung von Stammzellen in vivo verfolgen, wie es für die Fluoreszenz-Bildgebung erforderlich, für Biolumineszenz Reportergen Imaging-Systeme nur eine exogen verabreichtes Sonde Lichtemission zu induzieren. Firefly Luciferase aus dem Leuchtkäfer Photinus pyralis abgeleitet, kodiert ein Enzym, das D-Luciferin katalysiert die optisch aktiven Metaboliten, Oxyluciferin. Optische Aktivität kann dann mit einem externen CCD-Kamera überwacht werden. Stabil transduzierten Zellen, dass der Reporter tragen im Rahmen ihrer chromosomalen DNA-Konstrukt passieren das Reporter-Konstrukt DNA auf Tochterzellen, so dass für die Längs-Überwachung hESC Überleben und Proliferation in vivo. Darüber hinaus, weil die Expression des Reportergens Produkt zur Signalerzeugung erforderlich ist, wird einzig gangbare Eltern und Tochterzellen erzeugen Biolumineszenzsignal; apoptotischen oder tote Zellen nicht. In diesem Video werden die spezifischen Materialien und Methoden für die Verfolgung von Stammzellen die Proliferation und Teratom-Bildung mit Biolumineszenz-Bildgebung erforderlich beschrieben werden.

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Mit dem wachsenden Interesse an Stammzelltherapien, sind molekulare Bildgebungsverfahren ideal für die Überwachung Stammzellen Verhalten nach der Transplantation. Luciferase Reporter Gene nicht-invasive, repetitive Beurteilung der das Überleben der Zelle, den Standort und Verbreitung in vivo ermöglicht. Dieses Video wird gezeigt, wie man hESC Proliferation in einer lebenden Maus zu verfolgen.

In vitro und in vivo Biolumineszenz Reporter Gene Imaging von humanen embryonalen Stammzellen

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

in-vitro-vivo-bioluminescence-reporter-gene-imaging-human-embryonic

Research

JoVE Journal

Biology

21.7K Aufrufe.  Stanford University School of Medicine. Mit dem wachsenden Interesse an Stammzelltherapien, sind molekulare Bildgebungsverfahren ideal für die Überwachung Stammzellen Verhalten nach der Transplantation. Luciferase Reporter Gene nicht-invasive, repetitive Beurteilung der das Überleben der Zelle, den Standort und Verbreitung in vivo ermöglicht. Dieses Video wird gezeigt, wie man hESC Proliferation in einer lebenden Maus zu verfolgen.

Serie: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

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 ...

Forschung

Biologie

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 ...

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 ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Reprogrammierung menschlichen Körperzellen in induzierte pluripotente Stammzellen (IPSCs) mit retroviralen Vektoren mit GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...