Diese Arbeit wurde durch Zuschüsse der National Institutes of Health (CA127574, CA107040 und CA09071) zu WM unterstützt

1. Ernte Xenografts von Mice <ol><li> Bereiten Sie die Zelle Dissoziationspuffer: Dulbecco modifiziertem Eagle Medium (DMEM) mit 10% fötalem Kälberserum (FCS), Penicillin-Streptomycin, 200 U / ml Collagenase Typ IV, und 0,6 U / ml Dispase ergänzt. </li><li> Ein athymischen (nu + / nu +) Maus beherbergt eine subkutane humanen Pankreaskarzinom Xenotransplantat, dass weniger als 1 cm in größter Durchmesser ist durch Kohlendioxid Ersticken und Genickbruch getötet. </li><li> Die subkutane Tumor ist von der Flanke der Maus durch stumpfe Präparation mit einer Pinzette und Schere geerntet und legte sofort in die Zelle Dissoziationspuffer. </li><li> Tumore werden gewogen und wieder in 10 mL Zelle Dissoziationspuffer pro 1 Gramm Tumorgewebe platziert. </li></ol> 2. Generieren Sie eine Einzel-Zell-Suspension aus dem Xenografts <ol><li> Arbeiten in einem sterilen Biosicherheitswerkbank ist der Tumor auf eine 100 x 20 mm Kulturschale mit 1 ml der Zell-Dissoziation Puffer übertragen. Tumoren sind mechanisch zerkleinert mit sterilen Rasierklingen und Pinzetten. Die Tumorzelle Suspension wird durch ein 5-ml-serologischen Pipette 10 mal verrieben. Der Tumor Stücke sollten klein genug, um nicht verstopfen die 5-mL serologischen Pipette. </li><li> Die Tumorzelle Suspension wird auf einem 50-mL konischen Rohr mit dem restlichen Volumen der Zelle Dissoziationspuffer (gefüllt weniger als 50%) überführt und verwirbelt bei maximaler Geschwindigkeit für 1 Minute. </li><li> Der Tumor Zellsuspension wird dann bei 37 ° C für 2 Stunden mit intermittierenden Vortexen für 1 Minute alle 20 Minuten inkubiert. </li></ol> 3. Führen Zellsuspension von Gewebereste und toten Zellen <ol><li> Nach der 2-stündigen Inkubation der Zellsuspension wird bei maximaler Geschwindigkeit für 1 Minute gevortext. </li><li> Die Zellsuspension wird dann durch ein 70-um-Filter geleitet und in einem frischen 50-mL konischen Rohr und angepasst, um ein Endvolumen von 15 ml pro Röhrchen. </li><li> Zur weiteren separaten lebensfähigen Zellen von toten Zellen und Gewebereste, wird der Tumor Zellsuspension durch Dichtegradientenzentrifugation mit Ficoll-Paque Plus-getrennt. Eine Unterlage von Ficoll-Paque Plus ist durch Pipettieren 15 ml Ficoll-Paque Plus-langsam unter der Zellsuspension man aufpassen, nicht zu den beiden Schichten vermischen erstellt. </li><li> Die Zellsuspension und Ficoll Schichten sind bei 500x g für 30 Minuten mit den Bremsen zentrifugiert ausgeschaltet. </li><li> Cells an der Schnittstelle von der Ficoll-Paque Plus und Zelle Dissoziationspuffer werden gesammelt und in ein frisches 50-mL konischen Rohr. </li><li> Frische DMEM mit 10% FCS zu der Zellsuspension gegeben, um es zu verdünnen 1:3 (zB 20 ml frisches Medium zugesetzt, um 10 ml Zellsuspension). </li><li> Die Zellen werden durch Zentrifugation bei 450x g für 10 Minuten, die Medien dekantiert gesammelt, und das Zellpellet in 1 ml ALDEFLUOR resuspendiert. </li><li> Zehn Mikroliter der Zellsuspension mit 10 l Trypanblau und lebensfähigen Zellen gezählt werden unter Verwendung eines Hämocytometers verdünnt. Wenn eine große Anzahl von toten Zellen oder Gewebe Fremdkörper in diesem Schritt werden zur Kenntnis genommen, dann Zellseparation durch Dichtezentrifugation kann wiederholt werden (Schritte von 3,3 bis 3,7) werden. </li></ol> 4. Stain Cells für Fluorescence Activated Cell Sorting <ol><li> Label sieben 12 x 75 mm Polystyrol Reagenzgläser wie folgt: <ol><li> Unbefleckt </li><li> CD44-APC </li><li> CD24-PE </li><li> Maus CD31-Biotin + Mauslinie-Biotin + Maus H-2KD-Biotin </li><li> ALDEFLUOR </li><li> ALDEFLUOR + DEAB + IgG 2bκ-APC + IgG 2aκ-PE </li><li> ALDEFLUOR + CD44-APC + CD24-PE + Maus CD31-Biotin + Mauslinie-Biotin + Maus H-2KD-Biotin. </li></ol></li><li> Verdünnen der Zellsuspension in 2 ml ALDEFLUOR Puffer auf eine Endkonzentration von 5-10 Millionen Zellen / ml und halten auf dem Eis. 100 l der Zellsuspension in Röhrchen Nr. 1, 2, 3 und 4 und halten sie auf dem Eis. Add 1 ul DEAB zu Rohr Nr. 6 und halten auf dem Eis. Add ALDEFLUOR Reagenz, um die verbleibenden Zellsuspension verdünnt 1000 - fach (zB add 1,6 ul ALDEFLUOR Reagenz zu 1,6 ml Zellsuspension), gut mischen und auf Eis stellen. Transfer 100 ul dieser Mischung Rohre Nr. 5 und 6, und das verbleibende Gemisch (1,4 ml) mit dem Rohr Nr. 7. Inkubieren alle Rohre in einem 37 ° C Wasserbad für 40 Minuten. Mischen Sie die Zellsuspension alle 10 Minuten, um die Zellen absetzen, um den Boden der Rohre zu verhindern. </li><li> Zentrifugieren Sie die Zellen bei 450x g und 4 ° C für 10 Minuten und dann dekantieren den Puffer. Resuspendieren der Pellets in Rohren Nr. 1 und 5 in 100 ul ALDEFLUOR Puffer. Um Rohre Nr. 2, 3, 4 und 6, 100 l von ALDEFLUOR Puffer mit den entsprechenden Antikörpern wie folgt verdünnt: 1:20 für IgG 2bΚ-APC, IgG 2aΚ-PE, CD44-APC und CD24-PEAntikörper, 1:100 für Maus CD31-Biotin-, Maus-Linie-Biotin und Maus H-2KD-Biotin-Antikörpern. Um Rohr Nr. 7 hinzufügen, 1,4 ml ALDEFLUOR Puffer mit einer 1:20 Verdünnung des CD44-APC und CD24-PE-Antikörper und eine 1:100 Verdünnung des Maus CD31-Biotin-, Maus-Linie-Biotin und Maus H-2KD-Biotin Antikörper. Inkubieren Sie die Zellsuspensionen auf Eis für 10 Minuten. </li><li> Zentrifugieren Sie die Zellen bei 450x g und 4 ° C für 10 Minuten und dann dekantieren den Puffer. Resuspendieren der Pellets in Rohren Nr. 1, 2, 3, 5 und 6 in 100 ul ALDEFLUOR Puffer. Bereiten Sie 1,5 ml ALDEFLUOR Puffer mit einer 1:100 Verdünnung von Streptavidin-PerCP Protein. 100 l bis Rohr Nr. 4 und fügen 1,4 ml bis Rohr Nr. 7. Inkubieren Sie die Zellsuspensionen auf Eis für 10 Minuten. </li><li> Zentrifugieren Sie die Zellen bei 450x g und 4 ° C für 10 Minuten und dann dekantieren den Puffer. Resuspendieren der Pellets in Rohren Nr. 1, 2, 3, 4, 5 und 6 in 200 ul ALDEFLUOR Puffer. Das Pellet in Rohr Nr. 7 in 2,8 ml ALDEFLUOR Puffer mit 2 pg / mL Propidiumiodid. Keep Röhrchen auf Eis zu allen Zeiten. </li><li> Pass-Zellen durch 35-um-Filter mit 12 x 75 mm Polystyrol Reagenzgläser mit Sieb caps. </li></ol> 5. Isolieren Cancer Stem Cells durch Fluorescence Activated Cell Sorting <ol><li> Ein FACSAria Durchflusszytometer ist für Zellsortierung vorbereitet. </li><li> Compensation Kontrollen werden mit Hilfe von Zellen aus Rohren Nr. 1, 2, 3, 4 und 5. </li><li> Tore werden auf der Basis nach vorne und seitlich Streuparameter geschaffen, um Zelle Singuletts zu sammeln. </li><li> Non-Maus (PerCP-negativ) und lebensfähigen (non-Propidiumiodid gefärbten Zellen) sind gated mit dem FL-3-Kanal. </li><li> ALDH +-Zellen werden auf der Grundlage Tore gesammelt erstellt mit DEAB-behandelten Zellen (Tube # 6) und die FL-1 Kanal. </li><li> CD44 + CD24 +-Zellen werden auf der Grundlage Tore gesammelt erstellt unter Verwendung von Zellen mit ALDEFLUOR, IgG 2bΚ-APC und IgG 2aκ-PE (Tube # 6) gefärbt mit dem FL-4 und FL-2-Kanäle. </li><li> Die Zellen werden in 12 x 75 mm Polystyrol-Röhrchen mit 1 ml DMEM mit 10% FCS gesammelt. </li><li> Nach Zellen sortiert wurden, Zentrifuge die Zellsuspension bei 450x g für 10 Minuten, giesst die Medien und die Zellen in 500 ul frisches DMEM, ergänzt mit 10% FCS. </li><li> Zählt die Anzahl der sortierten Zellen mit einer Zählkammer, wie in Schritt 3.8 beschrieben. </li></ol> 6. Repräsentative Ergebnisse Dieses Protokoll wird auf die Isolierung von ALDH + und CD44 + CD24 + Pankreas CSCs führen. Der Anteil der Maus-abgeleiteten Zellen ist variabel, aber wir haben festgestellt, dass die meisten menschlichen Bauchspeicheldrüsenkrebs Xenotransplantate 20-60% enthalten Maus-abgeleiteten Zellen mit der Maus CD31, Mauslinie Cocktail und Maus H-2KD Biotin-konjugierte Antikörper (Abbildung 1A ). Auch die Häufigkeit der einzelnen CSC Bevölkerung aus verschiedenen Xenotransplantate ist variabel, aber generell haben wir festgestellt, dass 1-4% der gesamten Zellpopulation ALDH + (Abbildung 1B) und 0,2-5% ist CD44 + CD24 + (Abbildung 1C). Wir routinemäßig manuell zählen sortierten Zellen mit einer Zählkammer seit FACSAria Maschine zählt oft ungenau durch nicht-zelluläre Partikel durch die FACSAria erkannt. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Durchflusszytometrie Handlung darstellt bestimmte Bevölkerungsgruppen aus einem Pankreaskarzinom Xenotransplantat. (A) Zellen Färbung positiv in die FL-3-Kanal (Propidiumiodid +, Maus CD31 + Mauslinie + oder Maus H-2KD +) sind ausgeschlossen, so dass nur isolieren lebensfähigen und nicht-Maus-abgeleiteten Zellen. (B) ALDH Aktivität in einer humanen Pankreaskarzinom Xenograft wurde mittels Durchflusszytometrie mit dem ALDEFLUOR Reagens in Gegenwart und Abwesenheit der ALDH1 Inhibitor diethylamino-benzaldehyd (DEAB) gemessen. Die Frames darstellen Tore, ALDH + Zellen, basierend auf Zellen mit DEAB behandelt erstellt wurden und dann auf unbehandelten Zellen darzustellen. Der Anteil der ALDH + Zellen im Tor gezeigt. (C) Die CD44 + CD24 + Tor wurde basierend auf Zellen mit ALDEFLUOR gebeizt erstellt, und die IgG 2bκ-APC (Isotypiekontrolle für CD44-APC) und IgG 2aκ-PE (Isotypiekontrolle für CD24-PE)-Antikörpern. Der prozentuale Anteil der CD44 + CD24 +-Zellen sind in das Tor gezeigt.

<ol>
	<li>Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells--perspectives+on+current+status+and+future+directions:+AACR+Workshop+on+cancer+stem+cells.">Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells.</a> Cancer Res. 66, 9339-9344 (2006).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 3983-3988 (2003).</li><li>Ginestier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALDH1+Is+a+Marker+of+Normal+and+Malignant+Human+Mammary+Stem+Cells+and+a+Predictor+of+Poor+Clinical+Outcome.">ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome.</a> Cell stem cell. 1, 555-567 (2007).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+clonotypic+B+cells+in+classic+Hodgkin+lymphoma.">Circulating clonotypic B cells in classic Hodgkin lymphoma.</a> Blood. 113, 5920-5926 (2009).</li><li>Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+multiple+myeloma+progenitors,+stem+cell+properties,+and+drug+resistance.">Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance.</a> Cancer Res. 68, 190-197 (2008).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pancreatic+cancer+stem+cells.">Identification of pancreatic cancer stem cells.</a> Cancer Res. 67, 1030-1037 (2007).</li><li>Hermann, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+cancer+stem+cells+determine+tumor+growth+and+metastatic+activity+in+human+pancreatic+cancer.">Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.</a> Cell stem cell. 1, 313-323 (2007).</li><li>Rasheed, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+tumorigenic+cells+with+mesenchymal+features+in+pancreatic+adenocarcinoma.">Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.</a> J Natl Cancer Inst. 102, 340-351 (2010).</li><li>Rubio-Viqueira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+platform+for+translational+drug+development+in+pancreatic+cancer.">An in vivo platform for translational drug development in pancreatic cancer.</a> Clin Cancer Res. 12, 4652-4661 (2006).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+aldehyde+dehydrogenase+in+viable+cells.">Assessment of aldehyde dehydrogenase in viable cells.</a> Blood. 85, 2742-2746 (1995).</li></ol>

Dieses Protokoll beschreibt die Isolierung von Pankreas-CSCs aus menschlichem Xenotransplantate. Mehrere wichtige Schritte in diesem Verfahren wird die Ausbeute von lebensfähigen CSCs. Die Wiederherstellung einer reinen Population von Pankreas-CSCs ist abhängig von der Reinheit der lebensfähigen Zellen in der Zellsuspension vor dem Sortieren auf dem FACSAria. Darauf zu achten Xenotransplantate, dass weniger als 1-cm in größter Durchmesser verwenden hilft, die Menge von nekrotischem Gewebe in der Mitte des Tumors zu reduzieren. Zusätzlich zum Abbau der Zellsuspension von Gewebereste und toten Zellen während der Ficoll-Paque Gradientenzentrifugation ist kritisch und kann wiederholt werden, wenn eine große Anzahl von Gewebereste oder nicht lebensfähigen Zellen erkannt werden, wenn die Zellen auf die Zählkammer gezählt werden. Die Färbung hier beschriebene Protokoll nutzt die ALDEFLUOR Reagenz System ALDH Aktivität sowie Fluorophor-konjugierten Antikörpern an spezifische Oberflächenantigene erkennen. ALDEFLUOR Färbung ist bei 37 ° C und daher muss abgeschlossen sein, bevor Antikörper-Färbung (auf Eis) das Potential für die Antikörper-Deckelung, die bei 37 ° C auftreten können verhindern, dass sein Da Zellen des ALDEFLUOR Reagenz Efflux kann, ist es wichtig, die Zellen in ALDEFLUOR Puffer bei 4 ° C zu halten, nachdem ALDEFLUOR Färbung ist abgeschlossen. Die ALDEFLUOR Puffer enthält eine Efflux-Inhibitor, der Aufrechterhaltung intrazellulären ALDEFLUOR hilft. Da diese Methode isoliert lebensfähig Pankreas CSCs können sie in einer Reihe von Experimenten, die die physiologische Funktion dieser Zellen beantragt werden. Wir haben diese Zellen für die Tumor-Initiation Assays immungeschwächten Mäusen und in vitro Zell-Migration / Invasion Assays verwendet. Darüber hinaus kann RNA, DNA und Protein aus diesen Zellen für analytische Studien zum Vergleich von CSC und Nicht-CSC Bevölkerung gesammelt werden.

<!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>DMEM </td>
<td>&nbsp;</td>
<td>Invitrogen (Carlsbad, CA) </td>
<td>11965-118 </td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum </td>
<td>&nbsp;</td>
<td>Harlan (Indianapolis, IN)</td>
<td>BT-9501</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15140-122 </td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type IV </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>17104-019 </td>
<td>&nbsp;</td>
<tr>
<td>Dispase </td>
<td>&nbsp;</td>
<td>Sigma (St. Louis, MO) </td>
<td>D4818 </td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm culture dish </td>
<td>&nbsp;</td>
<td>BD Biosciences (San Jose, CA) </td>
<td>353003 </td>
<td>&nbsp;</td>
<tr>
<td>70-&mu;m filter </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352350 </td>
<td>&nbsp;</td>
<tr>
<td>50-mL conical tube </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352070 </td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque Plus </td>
<td>&nbsp;</td>
<td>GE Healthcare (Upsala, Sweden) </td>
<td>17-1440 </td>
<td>&nbsp;</td>
<tr>
<td>ALDEFLUOR reagent system </td>
<td>&nbsp;</td>
<td>Stem Cell Technologies (Vancouver, BC, Canada) </td>
<td>01700 </td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15250-061 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352054 </td>
<td>&nbsp;</td>
<tr>
<td>CD44-APC (clone G44-26) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>559942 </td>
<td>&nbsp;</td>
<tr>
<td>CD24-PE (clone ML5) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555428 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse CD31-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553371 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse lineage-biotin </td>
<td>&nbsp;</td>
<td>Miltenyi Biotec (Auburn, CA) </td>
<td>130-092-613 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse H-2Kd-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553564 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2b&#x03BA;-APC</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555745 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2a&#x03BA;-PE</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555574 </td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin-PerCP protein </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>554064 </td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4170 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes with strainer caps </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352235 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of stem cells from human pancreatic cancer xenografts

Krebsstammzellen (CSCS) haben in einer wachsenden Zahl von bösartigen Erkrankungen festgestellt worden und werden funktionell durch ihre Fähigkeit zur Selbst-Erneuerung unterzogen und produzieren differenzierte Nachkommenschaft 1 definiert. Diese Eigenschaften ermöglichen CSCs dem ursprünglichen Tumor rekapitulieren, wenn sie in immungeschwächten Mäusen injiziert. CSCs innerhalb eines epithelialen Malignomen wurden zunächst bei Brustkrebs beschrieben und festgestellt, dass spezifische Zelloberflächen-Antigen-Expression (CD44 + CD24 low / -) Display 2. Seitdem haben CSCs in einer wachsenden Zahl von anderen menschlichen Tumoren mit CD44 und CD24 sowie eine Reihe weiterer Oberflächenantigene identifiziert worden. Physiologische Eigenschaften, einschließlich Aldehyd-Dehydrogenase (ALDH) Aktivität, wurden ebenfalls verwendet, um CSCs von malignen Geweben 3-5 isolieren. Kürzlich haben wir festgestellt, und andere CSCs von Pankreaskarzinomen auf ALDH Aktivität und der Ausdruck der Zelloberflächen-Antigene CD44 und CD24 und CD133 6-8. Diese hoch tumorigenen Populationen kann oder auch nicht überlappen und Anzeige weiterer Funktionen. Wir fanden, dass ALDH + und CD44 + CD24 + Pankreas CSCs sind ähnlich tumorigenen, aber ALDH + Zellen sind relativ invasive 8. In diesem Protokoll beschreiben wir eine Methode, um lebensfähige Pankreas CSCs von Low-Passage menschlichen Xenotransplantate 9 zu isolieren. Xenotransplantiert Tumoren von Mäusen geerntet und zu einem Einzel-Zell-Suspension. Gewebereste und tote Zellen von lebenden Zellen getrennt und dann gefärbt mit Antikörpern gegen CD44 und CD24 und mit dem ALDEFLUOR Reagenz, ein fluoreszierendes Substrat der ALDH 10. CSCs werden dann mittels Fluoreszenz-activated cell sorting isoliert. Isolierte CSCs kann dann für analytische oder funktionellen Assays erfordern lebensfähigen Zellen verwendet werden.

Watch this Scientific Journal Video about Isolation of Stem Cells from Human Pancreatic Cancer Xenografts at JoVE.com

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Krebsstammzellen (CSCS) haben in einer Reihe von Tumoren identifiziert worden. In diesem Protokoll beschreiben wir eine durchflusszytometrische Methode unter Verwendung Aldehyd-Dehydrogenase-Aktivität und CD44 und CD24-Expression auf CSCs aus menschlichen Pankreaskarzinom Xenotransplantate isolieren. Diese entwicklungsfähigen Zellen können dann in funktionelle und analytische Untersuchungen verwendet werden.

Isolierung von Stammzellen aus menschlichen Bauchspeicheldrüsenkrebs Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

isolation-of-stem-cells-from-human-pancreatic-cancer-xenografts

Research

JoVE Journal

Biology

22.2K Aufrufe.  Johns Hopkins University School of Medicine. Krebsstammzellen (CSCS) haben in einer Reihe von Tumoren identifiziert worden. In diesem Protokoll beschreiben wir eine durchflusszytometrische Methode unter Verwendung Aldehyd-Dehydrogenase-Aktivität und CD44 und CD24-Expression auf CSCs aus menschlichen Pankreaskarzinom Xenotransplantate isolieren. Diese entwicklungsfähigen Zellen können dann in funktionelle und analytische Untersuchungen verwendet werden.

Serie: Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolierung und Analyse von hämatopoetischen Stammzellen aus der Plazenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Forschung

Biologie

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 ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part I: Verdauung und Sammlung von Pankreasgewebe

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selektieren und Isolieren Colonies of Human induzierten pluripotenten Stammzellen aus adulten Fibroblasten Umprogrammierte

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolierung von Immunzellen aus Primärtumoren

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Manuelle Isolierung von Fettgewebe gewonnene Stammzellen aus menschlichen Lipoaspirates

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolierung von Kleine-kodierenden RNAs von Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...