Questo lavoro è stato sostenuto dalle concessioni dal National Institutes of Health (CA127574, CA107040 e CA09071) a WM

1. Xenotrapianti raccolta da topi <ol><li> Preparare la cellula dissociazione buffer: Dulbecco modificato (DMEM) supplementato con 10% di siero fetale bovino (FCS), la penicillina-streptomicina, 200 U / mL Collagenasi tipo IV, e 0,6 U / mL dispasi. </li><li> Un atimici (+ nu / nu +) topo ospitare un essere umano per via sottocutanea xenotrapianto cancro al pancreas che è meno di 1 cm di diametro è eutanasia per asfissia anidride carbonica e dislocazione cervicale. </li><li> Il tumore sottocutaneo viene raccolto dal fianco del mouse per via smussa con pinze e forbici e collocato immediatamente nel buffer cellula dissociazione. </li><li> I tumori sono pesati e posti di nuovo in 10 ml di cellule dissociazione tampone per 1 grammo di tessuto tumorale. </li></ol> 2. Generare una singola cella di sospensione dal xenotrapianti <ol><li> Lavorare in un armadio sterile biosicurezza, il tumore viene trasferito in un piatto x 100 20 mm con la cultura 1 ml di tampone delle cellule dissociazione. I tumori sono meccanicamente tritata con lame di rasoio sterile e pinze. La sospensione delle cellule tumorali viene triturato con un 5-mL pipetta sierologica 10 volte. I pezzi del tumore dovrebbe essere abbastanza piccolo da non intasare il 5-mL pipetta sierologica. </li><li> La sospensione delle cellule tumorali viene trasferito in un tubo da 50 ml conica contenente il volume residuo della cellula dissociazione buffer (riempito meno del 50%) e in agitazione alla massima velocità per 1 minuto. </li><li> La sospensione delle cellule tumorali è quindi incubate a 37 ° C per 2 ore con vortex intermittente per 1 minuto ogni 20 minuti. </li></ol> 3. Riducono sospensione di cellule di detriti dei tessuti e le cellule morte <ol><li> Dopo le 2 ore di incubazione, la sospensione cellulare è in agitazione alla massima velocità per 1 minuto. </li><li> La sospensione cellulare viene poi passato attraverso un filtro di 70 micron e raccolti in un nuovo tubo da 50 ml conica e portato a un volume finale di 15 ml per provetta. </li><li> Per ulteriori celle separate vitali dalle cellule morte e detriti dei tessuti, la sospensione delle cellule tumorali viene separata per centrifugazione densità con Ficoll-Paque Plus. Un sottostrato di Ficoll-Paque Plus è creato da pipeting 15 ml di Ficoll-Paque In più lentamente sotto la sospensione cellulare facendo attenzione a non mescolare i due strati. </li><li> La sospensione cellulare e gli strati Ficoll si è centrifugato a 500x g per 30 minuti con i freni spento. </li><li> Cellule a livello di interfaccia di Ficoll-Paque Plus e cellule tampone dissociazione sono raccolti e trasferiti ad un nuovo 50-ml tubo conico. </li><li> Fresco DMEM supplementato con 10% FCS si aggiunge alla sospensione cellulare di diluirlo 1:3 (es. 20 ml di mezzi freschi aggiunto a 10 ml di sospensione cellulare). </li><li> Le cellule vengono raccolte mediante centrifugazione a 450x g per 10 minuti, i media decantato, e il pellet cellulare viene risospeso in 1 ml di tampone ALDEFLUOR. </li><li> Dieci microlitri di sospensione cellulare viene diluito con 10 L trypan blu e cellule vitali vengono conteggiati utilizzando un emocitometro. Se un gran numero di cellule morte o frammenti di tessuto sono noti in questa fase, poi separazione cellulare mediante centrifugazione densità può essere ripetuto (passi 3,3-3,7). </li></ol> 4. Macchia Celle per fluorescenza ordinamento cellulare attivo <ol><li> Etichetta sette 12 x 75 mm provette di polistirene come segue: <ol><li> Senza macchia </li><li> CD44-APC </li><li> CD24-PE </li><li> topo CD31-biotina + mouse lignaggio-biotina + mouse H-2Kd-biotina </li><li> ALDEFLUOR </li><li> ALDEFLUOR DEAB + + IgG 2bκ-APC + IgG 2aκ-PE </li><li> ALDEFLUOR + CD44 + CD24-APC-PE + mouse + CD31-biotina-biotina lignaggio del mouse + mouse H-2Kd-biotina. </li></ol></li><li> Diluire la sospensione di cellule in 2 ml di tampone ALDEFLUOR ad una concentrazione finale di 5-10 milioni di cellule / ml e tenere in ghiaccio. Aggiungere 100 microlitri della sospensione cellulare ai tubi # 1, 2, 3 e 4 e tenerli su ghiaccio. Aggiungere 1 DEAB microlitri per tubo # 6 e tenere in ghiaccio. Aggiungere il reagente ALDEFLUOR alla sospensione cellulare residua diluita 1000 - volte (ad esempio aggiungere 1,6 microlitri di reagente ALDEFLUOR a 1,6 ml di sospensione cellulare), mescolare bene, e posto sul ghiaccio. Trasferire 100 ml di questa miscela a tubi # 5 e 6, e la miscela rimanente (1,4 ml) al tubo # 7. Incubare tutte le provette in un bagno d&#39;acqua a 37 ° C per 40 minuti. Mescolare la sospensione cellulare ogni 10 minuti per evitare che le cellule di assestamento al fondo dei tubi. </li><li> Centrifugare le cellule a 450x g e 4 ° C per 10 minuti e poi decantare il buffer. Risospendere il pellet in tubi # 1 e 5 in 100 microlitri di buffer ALDEFLUOR. Per tubi # 2, 3, 4 e 6, aggiungere 100 ml di tampone ALDEFLUOR contenente gli anticorpi adeguati diluita come segue: 1:20 per IgG 2bΚ-APC, IgG 2aΚ-PE, CD44-APC, e CD24-PEanticorpi; 1:100 per mouse CD31-biotina, mouse lignaggio-biotina, mouse e H-2Kd-biotina anticorpi. Per tubo # 7 aggiungere 1,4 ml di tampone ALDEFLUOR contenente una diluizione 1:20 di CD44-APC, e anticorpi CD24-PE e una diluizione 1:100 del mouse CD31-biotina, mouse lignaggio-biotina, e mouse H-2Kd-biotina anticorpi. Incubare le sospensioni delle cellule in ghiaccio per 10 minuti. </li><li> Centrifugare le cellule a 450x g e 4 ° C per 10 minuti e poi decantare il buffer. Risospendere il pellet in tubi # 1, 2, 3, 5, e 6 in 100 microlitri di buffer ALDEFLUOR. Preparare 1,5 mL di tampone ALDEFLUOR contenente una diluizione 1:100 di streptavidina-PerCP proteine. Aggiungere 100 ul a tubo # 4 e aggiungere 1,4 ml di tubo # 7. Incubare le sospensioni delle cellule in ghiaccio per 10 minuti. </li><li> Centrifugare le cellule a 450x g e 4 ° C per 10 minuti e poi decantare il buffer. Risospendere il pellet in tubi # 1, 2, 3, 4, 5, e 6 in 200 microlitri di buffer ALDEFLUOR. Risospendere il pellet in tubo # 7 in 2,8 mL di tampone ALDEFLUOR contenente 2 mg / ml di ioduro di propidio. Sì che le tubature su ghiaccio in ogni momento. </li><li> Passano attraverso le cellule di 35 micron con filtro 12 x 75 mm provette di polistirene con tappi filtro. </li></ol> 5. Isolate cellule staminali del cancro da fluorescenza ordinamento cellulare attivo <ol><li> Un citofluorimetro FACSAria è pronto per l&#39;ordinamento delle cellule. </li><li> Controlli di compensazione sono impostate utilizzando cellule di tubi # 1, 2, 3, 4 e 5. </li><li> Gates vengono creati in base al futuro e side-scatter parametri per la raccolta delle cellule singoletti. </li><li> Senza mouse (PerCP-negativo) e vitali (non macchiato ioduro di propidio) le cellule sono gated utilizzando il FL-3 canali. </li><li> ALDH cellule + sono raccolti sulla base di cancelli creati utilizzando DEAB cellule trattate (tubo # 6) e la FL-1 canale. </li><li> Cellule CD44 + CD24 + sono raccolti sulla base di cancelli creati utilizzando cellule colorate con ALDEFLUOR, IgG 2bΚ-APC, e IgG 2aκ-PE (tubo # 6) utilizzando il FL-4 e FL-2 canali. </li><li> Le cellule sono raccolte in 12 x 75 mm in polistirene tubi di prova contenente 1 ml di DMEM supplementato con 10% FCS. </li><li> Dopo che le cellule sono state ordinate, centrifuga la sospensione cellulare a 450x g per 10 minuti, decantare i media, e sospendere nuovamente le cellule in 500 microlitri DMEM fresca supplementato con 10% FCS. </li><li> Contare il numero di celle ordinate usando un emocitometro come descritto al punto 3.8. </li></ol> 6. Rappresentante Risultati Questo protocollo porterà all&#39;isolamento di ALDH + e CD44 + CD24 + pancreas CSC. La percentuale di cellule di topo derivati ​​è variabile, ma abbiamo trovato che la maggior parte xenotrapianti cancro al pancreas umano contiene 20-60% di cellule di topo derivate usando il mouse CD31, cocktail lignaggio del mouse, mouse e H-2Kd biotina anticorpi coniugati (Figura 1A ). Allo stesso modo la frequenza di ogni popolazione CSC da diversi xenotrapianti è variabile, ma in generale abbiamo trovato che il 1-4% della popolazione totale di cellule è ALDH + (Figura 1B) e 0,2-5% è CD44 + CD24 + (Figura 1C). Noi abitualmente manualmente contare le celle ordinate usando un emocitometro quanto i conteggi macchina FACSAria sono spesso imprecisi a causa della non-cellulari particelle rilevato dal FACSAria. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="Figura 1" /> Figura 1. Citometria a flusso trama raffigurante popolazioni specifiche di trapianto di cancro al pancreas. (A) Cellule colorazione positivamente nella FL-3 canali (propidio ioduro +, CD31 + + topo lignaggio del mouse, o il mouse H-2Kd +) sono esclusi in modo da isolare solo vitali e non topo cellule derivate. (B) l&#39;attività ALDH in caso di trapianto di cancro pancreatico umano è stato misurato mediante citometria di flusso utilizzando il reagente ALDEFLUOR in presenza e in assenza di inibitore ALDH1 dietilammino-benzaldeide (DEAB). I telai rappresentano cancelli che raffigurano le cellule + ALDH che sono stati creati sulla base di cellule trattate con DEAB e poi applicata a cellule non trattate. Le percentuali di cellule + ALDH sono riportati nella porta. (C) La CD44 + CD24 + cancello è stato creato sulla base di cellule colorate con ALDEFLUOR, e IgG 2bκ-APC (controllo isotipico per CD44-APC) e IgG 2aκ-PE (controllo isotipico per CD24-PE) gli anticorpi. Le percentuali di cellule CD44 + CD24 + sono mostrati in porta.

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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>

Nessun conflitto di interessi dichiarati.

Questo protocollo descrive l&#39;isolamento di pancreas da CSC xenotrapianti umani. Diversi passaggi chiave di questa procedura sarà migliorare il rendimento di validi CSC. Il recupero di una popolazione pura di pancreas CSC dipende dalla purezza delle cellule vitali presenti nella sospensione cellulare prima di ordinare sul FACSAria. Avendo cura di utilizzare xenotrapianti che sono meno di 1 cm di diametro aiuta a ridurre la quantità di tessuto necrotico nel centro del tumore. Inoltre, riducono la sospensione di cellule di detriti tessuti e cellule morte durante Ficoll-Paque centrifugazione gradiente è critica e può essere ripetuto se un gran numero di detriti dei tessuti o cellule non vitali vengono rilevati quando le cellule si contano sulle emocitometro. Il protocollo di colorazione descritta qui utilizza il sistema reagente ALDEFLUOR per rilevare l&#39;attività ALDH così come fluoroforo anticorpi coniugati per rilevare specifici antigeni di superficie delle cellule. ALDEFLUOR colorazione è eseguita a 37 ° C e, quindi, deve essere completata prima della colorazione degli anticorpi (su ghiaccio) per evitare il rischio di anticorpi tappatura, che può avvenire a 37 ° C. Dato che le cellule possono efflusso il reagente ALDEFLUOR, è importante mantenere le cellule in ALDEFLUOR tampone a 4 ° C dopo la colorazione ALDEFLUOR è stata completata. Il buffer ALDEFLUOR contiene un inibitore di efflusso di farmaco che aiuta a mantenere i livelli intracellulari di ALDEFLUOR. Poiché questo metodo praticabile isola pancreatica CSC possono essere applicate in un certo numero di esperimenti che misurano la funzione fisiologica di queste cellule. Abbiamo usato queste cellule per tumore-iniziazione test utilizzando topi immunocompromessi e in vitro delle cellule migrazione / invasione saggi. Inoltre, RNA, DNA e proteine ​​possono essere raccolti da queste cellule per studi analitici confronto CSC e non CSC popolazioni.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<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>
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isolation of stem cells from human pancreatic cancer xenografts

Cellule staminali tumorali (CSC), sono stati identificati in un numero crescente di tumori maligni e sono funzionalmente definite dalla loro capacità di subire auto-rinnovamento e produrre progenie differenziata 1. Queste proprietà consentono CSC di ricapitolare il tumore originale quando iniettato in topi immunocompromessi. CSC all&#39;interno di un tumore epiteliale sono stati descritti nel cancro al seno e ha trovato per visualizzare specifiche espressione sulla superficie delle cellule antigene (CD44 + CD24 bassa / -) 2. Da allora, CSC sono stati identificati in un numero crescente di altre neoplasie umane usando CD44 e CD24, nonché un certo numero di antigeni di superficie. Proprietà fisiologiche, tra cui aldeide deidrogenasi (ALDH) attività, sono stati utilizzati anche per isolare dal CSC tessuti maligni 3-5. Recentemente, abbiamo identificato e altri CSC da adenocarcinoma pancreatico sulla base dell&#39;attività ALDH e l&#39;espressione degli antigeni di superficie della cellula CD44 e CD24 e CD133 6-8. Queste popolazioni altamente cancerogeno può o non può essere sovrapposti e visualizzare altre funzioni. Abbiamo scoperto che ALDH + e CD44 + CD24 + pancreas CSC sono ugualmente cancerogeni, ma ALDH cellule + sono relativamente più invasiva 8. In questo protocollo si descrive un metodo per isolare praticabile pancreas CSC a basso passaggio xenotrapianti umani 9. Tumori xenotrapiantati vengono raccolti dai topi e reso in una cella singola sospensione. Detriti tessuti e le cellule morte sono separate da cellule vive e quindi colorato utilizzando anticorpi diretti contro CD44 e CD24 e usando il reagente ALDEFLUOR, un substrato fluorescente di ALDH 10. CSC vengono poi isolate dalla fluorescenza separazione delle cellule attivate. Isolato CSC può quindi essere utilizzata per le prove di analisi o funzionali che richiedono cellule vitali.

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

Cellule staminali tumorali (CSC), sono stati identificati in una serie di tumori maligni. In questo protocollo si descrive un metodo di citometria a flusso utilizzando aldeide deidrogenasi attività e CD44 e CD24 espressione di isolare dal CSC xenotrapianti adenocarcinoma pancreatico umano. Queste cellule vitali possono poi essere utilizzati in studi funzionali e di analisi.

Isolamento di cellule staminali da xenotrapianti di cancro pancreatico umano

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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JoVE Journal

Biology

22.2K Views.  Johns Hopkins University School of Medicine. Cellule staminali tumorali (CSC), sono stati identificati in una serie di tumori maligni. In questo protocollo si descrive un metodo di citometria a flusso utilizzando aldeide deidrogenasi attività e CD44 e CD24 espressione di isolare dal CSC xenotrapianti adenocarcinoma pancreatico umano. Queste cellule vitali possono poi essere utilizzati in studi funzionali e di analisi.

Video: 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.

Isolamento e analisi delle cellule staminali ematopoietiche dalla 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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Biologia

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.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

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.

Isolamento delle isole pancreatiche umane: Parte I: Digestione e Collezione di tessuto pancreatico

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.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

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.

Selezione e isolamento di colonie di cellule pluripotenti indotte umane staminali riprogrammato da fibroblasti adulti

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.

Isolamento di cellule immunitarie di tumori primari

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 Adult Stem Cells isolamento da colture primarie di fibroblasti umani

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.

Isolamento Manuale di cellule staminali derivate da tessuto adiposo da lipoaspirato umani

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.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

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.

Isolamento dei Piccoli RNA non codificanti dal siero umano

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 ...