Ce travail a été soutenu par des subventions des National Institutes of Health (CA127574, CA107040 et CA09071) à WM

1. Récolte de xénogreffes Souris <ol><li> Préparer la cellule tampon de dissociation: Eagle modifié par Dulbecco (DMEM) supplémenté avec 10% sérum de veau foetal (FCS), la pénicilline-streptomycine, 200 U / ml de collagénase de type IV, et 0,6 U / ml dispase. </li><li> Un athymiques (nu + / nu +) abritant un sous-cutanée de souris xénogreffe humaine du cancer du pancréas qui est inférieur à 1 cm de plus grand diamètre est euthanasié par asphyxie de dioxyde de carbone et de dislocation cervicale. </li><li> La tumeur sous-cutanée est récoltée à partir du flanc de la souris par dissection à l&#39;aide de pinces et de ciseaux et placé immédiatement dans le tampon dissociation cellulaire. </li><li> Les tumeurs sont pesés et placés de nouveau dans 10 ml de tampon de dissociation cellulaire pour 1 gramme de tissu tumoral. </li></ol> 2. Générer une suspension monocellulaire de la xénogreffes <ol><li> Travailler dans une enceinte de sécurité biologique stérile, la tumeur est transféré à un 100 x 20 mm boîte de culture avec 1 ml de cellules tampon de dissociation. Les tumeurs sont mécaniquement hachée à l&#39;aide de lames de rasoir stérile et une pince. La suspension de cellules tumorales est triturée à travers une pipette de 5 ml sérologiques 10 fois. Les pièces tumeur doit être assez petit pour ne pas obstruer la pipette de 5 ml sérologique. </li><li> La suspension de cellules tumorales est transféré dans un tube conique de 50 ml contenant le volume restant de la cellule tampon de dissociation (rempli au moins 50%) et vortexés à vitesse maximale pendant 1 minute. </li><li> La suspension de cellules tumorales est ensuite incubée à 37 ° C pendant 2 heures avec vortex intermittente pendant 1 minute toutes les 20 minutes. </li></ol> 3. Appauvrissent suspension cellulaire de débris tissulaires et des cellules mortes <ol><li> Après l&#39;incubation de 2 heures la suspension cellulaire est vortexé à vitesse maximale pendant 1 minute. </li><li> La suspension cellulaire est ensuite passée à travers un filtre de 70 um et recueilli dans un endroit frais 50 ml tube conique et ajusté à un volume final de 15 ml par tube. </li><li> Afin de mieux séparer les cellules viables des cellules mortes et les débris tissulaires, la suspension de cellules tumorales est séparé par centrifugation de densité utilisant Ficoll-Paque Plus. Une sous-couche de Ficoll-Paque Plus est créé par pipetage 15 ml de Ficoll-Paque plus lentement en dessous de la suspension cellulaire en faisant attention de ne pas mélanger les deux couches. </li><li> La suspension de cellules et les couches sont Ficoll est centrifugé à 500x g pendant 30 minutes avec les freins mis hors tension. </li><li> Les cellules à l&#39;interface de le Plus Ficoll-Paque et dissociation cellulaire tampon sont recueillies et transférées à une nouvelle de 50 ml tube conique. </li><li> Frais DMEM supplémenté avec 10% de SVF est ajouté à la suspension de cellules pour le diluer 1:3 (par exemple 20 ml de milieu frais ajoutés à 10 ml de suspension cellulaire). </li><li> Les cellules sont collectées par centrifugation à 450x g pendant 10 minutes, les médias décanté et le culot cellulaire est resuspendu dans 1 ml de tampon ALDEFLUOR. </li><li> Dix microlitres de la suspension cellulaire est dilué avec 10 uL bleu trypan et les cellules viables sont comptées en utilisant un hématimètre. Si un grand nombre de cellules mortes ou des débris tissulaires sont notées au cours de cette étape, puis la séparation des cellules par centrifugation de densité peut être répétée (étapes 3.3 à 3.7). </li></ol> 4. Colorer des cellules pour le tri cellulaire activé par fluorescence <ol><li> Étiquette sept 12 x 75 mm tubes en polystyrène de test comme suit: <ol><li> Sans tache </li><li> CD44-APC </li><li> CD24-PE </li><li> la souris CD31-biotine + souris de lignée biotine + souris H-2Kd-biotine </li><li> ALDEFLUOR </li><li> ALDEFLUOR + + IgG DEAB 2bκ-APC + IgG 2aκ-PE </li><li> ALDEFLUOR + CD44 + CD24-APC-PE + souris CD31-biotine + souris de lignée biotine + souris H-2Kd-biotine. </li></ol></li><li> Diluer la suspension cellulaire dans 2 mL de tampon ALDEFLUOR à une concentration finale de 5-10 millions de cellules / ml et de garder sur la glace. Ajouter 100 ul de la suspension de cellules dans des tubes n ° 1, 2, 3 et 4 et les garder sur la glace. Ajouter une DEAB uL dans le tube n ° 6 et de garder sur la glace. Ajouter le réactif ALDEFLUOR à la suspension cellulaire diluée restantes 1000 - pli (par exemple, ajouter 1,6 uL ALDEFLUOR réactif à 1,6 ml de suspension cellulaire), bien mélanger, et placer sur la glace. Transférer 100 ul de ce mélange dans des tubes n ° 5 et 6, et le reste du mélange (1,4 ml) pour le tube n ° 7. Incuber tous les tubes dans un bain d&#39;eau à 37 ° C pendant 40 minutes. Mélanger la suspension de cellules toutes les 10 minutes afin d&#39;empêcher les cellules de se fixer au fond des tubes. </li><li> Centrifuger les cellules à 450x g et 4 ° C pendant 10 minutes et décanter ensuite le tampon. Resuspendre les boulettes dans des tubes # 1 et 5, 100 pi de tampon ALDEFLUOR. Pour les tubes n ° 2, 3, 4 et 6, ajouter 100 ul de tampon ALDEFLUOR contenant les anticorps appropriés dilué comme suit: 1h20 pour les IgG 2bΚ-APC, IgG 2aΚ-PE, CD44-APC, et CD24-PEanticorps; 1:100 pour souris CD31-biotine, la souris de lignée biotine, et la souris H-2Kd-biotine. Dans le tube n ° 7 ajouter 1,4 mL de tampon ALDEFLUOR contenant une dilution 1:20 de CD44-APC, et CD24-PE anticorps et une dilution à 1:100 de souris CD31-biotine, la souris de lignée biotine, et la souris H-2Kd-biotine anticorps. Incuber les suspensions cellulaires sur la glace pendant 10 minutes. </li><li> Centrifuger les cellules à 450x g et 4 ° C pendant 10 minutes et décanter ensuite le tampon. Resuspendre les boulettes dans des tubes n ° 1, 2, 3, 5 et 6 dans 100 ul de tampon ALDEFLUOR. Préparer 1,5 ml de tampon contenant ALDEFLUOR une dilution à 1:100 de streptavidine-PerCP protéines. Ajouter 100 uL dans le tube n ° 4 et ajouter 1,4 ml au tube n ° 7. Incuber les suspensions cellulaires sur la glace pendant 10 minutes. </li><li> Centrifuger les cellules à 450x g et 4 ° C pendant 10 minutes et décanter ensuite le tampon. Resuspendre les boulettes dans des tubes n ° 1, 2, 3, 4, 5 et 6 dans 200 ul de tampon ALDEFLUOR. Reprendre le culot dans le tube n ° 7 à 2,8 ml de tampon contenant ALDEFLUOR 2 pg / ml d&#39;iodure de propidium. Garder les tubes sur la glace en tout temps. </li><li> Passez à travers les cellules de 35 um filtre à l&#39;aide de 12 x 75 mm tubes en polystyrène de test avec des bouchons passoire. </li></ol> 5. Isoler les cellules souches du cancer par le tri cellulaire activé par fluorescence <ol><li> Un cytomètre en flux FACSAria est préparé pour le tri cellulaire. </li><li> Contrôle de rémunération sont fixés en utilisant des cellules provenant de tubes n ° 1, 2, 3, 4 et 5. </li><li> Gates sont créés en fonction de l&#39;avant et latérales de dispersion des paramètres à collecter maillots cellule. </li><li> Non-souris (PerCP-négatif) et viable (teinté d&#39;iodure de propidium non), les cellules sont déclenchées en utilisant le canal FL-3. </li><li> ALDH cellules + sont collectés basée sur des portails créés en utilisant DEAB cellules traitées (tube n ° 6) et le FL-1 canal. </li><li> CD44 + CD24 + cellules sont collectées basée sur des portails créés en utilisant des cellules colorées par ALDEFLUOR, IgG 2bΚ-APC, et IgG 2aκ-PE (tube n ° 6) en utilisant le FL-4 et FL-2 canaux. </li><li> Les cellules sont collectées dans 12 tubes x 75 mm à essai en polystyrène contenant 1 mL de DMEM supplémenté avec 10% de FCS. </li><li> Après les cellules ont été triées, centrifuger la suspension cellulaire à 450x g pendant 10 minutes, décanter les médias, et remettre en suspension les cellules dans 500 ul DMEM frais complété avec 10% de FCS. </li><li> Comptez le nombre de cellules triées à l&#39;aide d&#39;un hémocytomètre comme décrit dans l&#39;étape 3.8. </li></ol> 6. Les résultats représentatifs Ce protocole permettra à l&#39;isolement de l&#39;ALDH + et CD44 + CD24 + pancréas CSC. Le pourcentage de cellules de souris dérivée est variable, mais nous avons constaté que la plupart des humains xénogreffes de cancer du pancréas contiennent 20-60% des cellules de souris dérivées en utilisant la souris CD31, cocktail lignée de souris et la souris H-2Kd biotine conjugué anticorps (figure 1A ). De même la fréquence de chaque population à partir du SCC xénogreffes différents est variable, mais généralement, nous avons constaté que 1-4% de la population cellulaire totale est ALDH + (figure 1B) et 0,2-5% CD44 + CD24 est + (figure 1C). Nous avons régulièrement compter manuellement les cellules triées à l&#39;aide d&#39;un hémocytomètre depuis le compte machine à FACSAria sont souvent inexactes en raison du non-cellulaire des particules détectées par le FACSAria. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="Figure 1" /> Figure 1. La cytométrie en flux parcelle représentant des populations spécifiques d&#39;une xénogreffe cancer du pancréas. (A) Cellules coloration positive dans le FL-3 canaux (iodure de propidium +, CD31 + + souris lignée de souris, souris ou H-2Kd +) sont exclus de manière à isoler seulement viables et non des cellules dérivées de souris. (B) l&#39;activité ALDH dans une xénogreffe de cancer pancréatique humain a été mesurée par cytométrie en flux utilisant le réactif ALDEFLUOR en présence et en absence de l&#39;inhibiteur ALDH1 diéthylamino-benzaldéhyde (DEAB). Les cadres représentent portes qui dépeignent les cellules + ALDH qui ont été créés à partir de cellules traitées avec DEAB puis appliquée à des cellules non traitées. Les pourcentages de cellules + ALDH sont présentés dans la porte. (C) Le CD44 + CD24 + porte a été créé à base de cellules colorées au ALDEFLUOR, et les IgG 2bκ-APC (contrôle isotypique pour le CD44-APC) et des IgG 2aκ-PE (contrôle isotypique pour le CD24-PE) anticorps. Les pourcentages de cellules CD44 + CD24 + sont présentés dans la porte.

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	<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>

Ce protocole décrit l&#39;isolement de la CSC à partir du pancréas xénogreffes humaines. Plusieurs étapes clés de cette procédure permettra d&#39;améliorer le rendement de la viabilité CSC. Le rétablissement d&#39;une population pure de pancréas CSC est dépendante de la pureté de cellules viables présentes dans la suspension cellulaire avant le tri sur le FACSAria. Prendre soin d&#39;utiliser les xénogreffes qui sont moins de 1 cm de plus grand diamètre permet de réduire la quantité de tissus nécrosés au centre de la tumeur. En outre, les appauvrissant la suspension cellulaire de débris de tissus et cellules mortes pendant la centrifugation de gradient de Ficoll-Paque est critique et peut être répétée si un grand nombre de débris de tissus ou de cellules non viables sont détectés lorsque les cellules sont comptées sur l&#39;hématimètre. Le protocole de coloration décrit ici utilise le système de réactifs pour détecter l&#39;activité ALDEFLUOR ALDH ainsi que fluorophore conjugué anticorps spécifiques pour détecter les antigènes de surface cellulaire. Coloration ALDEFLUOR est effectuée à 37 ° C et, par conséquent, doit être achevée avant la coloration des anticorps (sur glace) pour éviter les risques de bouchage des anticorps, ce qui peut arriver à 37 ° C. Comme les cellules peuvent efflux le réactif ALDEFLUOR, il est important de maintenir les cellules en ALDEFLUOR tampon à 4 ° C après coloration ALDEFLUOR a été achevé. Le tampon ALDEFLUOR contient un inhibiteur de l&#39;efflux de médicament qui aide à maintenir des niveaux intracellulaires de ALDEFLUOR. Puisque cette méthode isole du pancréas viables CSC ils peuvent être appliqués dans un certain nombre d&#39;expériences qui mesurent la fonction physiologique de ces cellules. Nous avons utilisé ces cellules pour la tumeur d&#39;initiation dosages utilisant des souris immunodéprimées et in vitro de cellules de migration / invasion des dosages. Par ailleurs, l&#39;ARN, d&#39;ADN et de protéines peuvent être collectées à partir de ces cellules pour des études analytiques en comparant le SCC et la non-CSC populations.

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		<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

Les cellules souches cancéreuses (CSC) ont été identifiés dans un nombre croissant de cancers et sont fonctionnellement définis par leur capacité à subir une auto-renouvellement et de produire une descendance différenciée. Ces propriétés permettent de récapituler les CSC de la tumeur d&#39;origine lorsqu&#39;il est injecté dans des souris immunodéprimées. CSC au sein d&#39;une tumeur maligne épithéliale ont d&#39;abord été décrite dans le cancer du sein et trouvé d&#39;affichage spécifique l&#39;expression des antigènes de surface des cellules (CD44 + CD24 faible / -) 2. Depuis lors, les CSC ont été identifiées dans un nombre croissant d&#39;autres tumeurs malignes humaines CD44 et CD24 à l&#39;aide ainsi que d&#39;un certain nombre d&#39;antigènes de surface autres. Propriétés physiologiques, y compris l&#39;aldéhyde déshydrogénase (ALDH) l&#39;activité, ont également été utilisés pour isoler les CSC à partir des tissus malins 3-5. Récemment, nous avons identifié les CSC et les autres à partir adénocarcinome pancréatique basé sur l&#39;activité ALDH et l&#39;expression des antigènes de surface cellulaire CD44 et CD24, CD133 et 6-8. Ces populations très tumorigènes peuvent ou non se chevaucher et afficher d&#39;autres fonctions. Nous avons constaté que l&#39;ALDH + et CD44 + CD24 + pancréas CSC sont également tumorigène, mais ALDH cellules + sont relativement plus invasives 8. Dans ce protocole, nous décrivons une méthode pour isoler les CSC du pancréas viables à faible passage de xénogreffes humaines 9. Tumeurs xénogreffées sont récoltées à partir des souris et faites en une suspension à cellule unique. Débris de tissus et cellules mortes sont séparées de cellules vivantes et colorées à l&#39;aide des anticorps contre CD44 et CD24 et en utilisant le réactif ALDEFLUOR, un substrat fluorescent de l&#39;ALDH 10. Les CSC sont ensuite isolés par tri cellulaire activé par fluorescence. Isolée CSC peut ensuite être utilisé pour les essais analytiques ou fonctionnelles nécessitant des cellules viables.

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

Les cellules souches cancéreuses (CSC) ont été identifiées dans un certain nombre de tumeurs malignes. Dans ce protocole, nous décrivons une méthode de cytométrie en flux utilisant l&#39;activité aldéhyde déshydrogénase et CD44 et CD24 expression d&#39;isoler les CSC de xénogreffes humaines adénocarcinome pancréatique. Ces cellules viables peuvent ensuite être utilisés dans des études fonctionnelles et analytiques.

Isolement des cellules souches de l&#39;homme xénogreffes de cancer du pancréas

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

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22.2K vues.  Johns Hopkins University School of Medicine. Les cellules souches cancéreuses (CSC) ont été identifiées dans un certain nombre de tumeurs malignes. Dans ce protocole, nous décrivons une méthode de cytométrie en flux utilisant l'activité aldéhyde déshydrogénase et CD44 et CD24 expression d'isoler les CSC de xénogreffes humaines adénocarcinome pancréatique. Ces cellules viables peuvent ensuite être utilisés dans des études fonctionnelles et analytiques.

Vidéo: 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.

Isolement et analyse de cellules souches hématopoïétiques à partir du 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. ...

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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.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

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.

Isolement îlots pancréatiques humaines: Partie I: La digestion et la perception des tissus pancréatiques

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.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

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.

Colonies sélectionner et isoler de l&#39;homme cellules souches pluripotentes induites à partir de fibroblastes adultes Reprogrammed

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.

Isolement des cellules immunitaires des tumeurs primitives

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.

Naïve cellules souches adultes Isolation de cultures de fibroblastes humains primaires

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.

Isolation Manuel des cellules souches du tissu adipeux de Lipoaspirates humaines

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.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

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.

Isolation des petits ARN non codant de sérum humain

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 ...