Bu çalışma Ulusal Sağlık Enstitüleri hibe WM (CA127574, CA107040 ve CA09071) tarafından desteklenen

1. Fare ile Hasat Xenografts <ol><li> Dulbecco&#39;nun modifiye Eagle orta (DMEM)% 10 fetal buzağı serumu (FCS), penisilin-streptomisin, 200 U / ml kollajenaz tip IV ve 0.6 U / ml dispase ile desteklenmiş hücre disosiasyon tampon hazırlayın. </li><li> Büyük çapı 1 cm den daha az bir subkutan insan pankreas kanseri xenograft barındıran bir atimik (nu + / nu +) fare, karbon dioksit boğulma ve servikal dislokasyon ötenazi. </li><li> Subkutan bir tümör forseps ve makas kullanılarak künt diseksiyon ile fare kanadını hasat ve hemen hücre disosiasyon tampon yerleştirilir. </li><li> Tümörler tartılır ve tümör dokusu 1 gram başına 10 ml hücre disosiasyon tampon içine yerleştirilir. </li></ol> 2. Tek hücreli bir Xenografts Süspansiyon oluşturun <ol><li> Steril bir biyogüvenlik kabini içinde çalışmak, tümör hücre disosiasyon tampon 1 ml ile 100 x 20 mm kültür kaplarına aktarılır. Tümörler mekanik steril Jilet ve forseps kullanılarak kıyılmış. Tümör hücre süspansiyonu, 5 ml serolojik pipetlemeyin ile 10 kez triturated. Tümör adet 5 ml serolojik pipetlemeyin tıkamaz kadar küçük olmalıdır. </li><li> Tümör hücre süspansiyon hücre disosiasyon tampon kalan hacmi (% 50 daha az dolu) içeren 50 ml konik tüp ve 1 dakika boyunca maksimum hızda transfer vortekslenmiş. </li><li> Tümör hücre süspansiyonu daha sonra, 37 ° C&#39;de 2 saat boyunca her 20 dakikada bir 1 dakika aralıklı bir vorteks ile inkübe. </li></ol> 3. Hücre Doku Enkaz Süspansiyon ve Ölü Hücreleri İncelten <ol><li> 2 saatlik inkübasyondan sonra hücre süspansiyonu, 1 dakika boyunca maksimum hızda vortekslenmiş. </li><li> Hücre süspansiyonu daha sonra 70 mikron filtre geçirilir ve yeni bir 50-ml konik tüp içinde toplanan ve nihai bir hacim başına 15 ml tüp şekilde ayarlanabilir. </li><li> , Ölü hücreler ve doku artıkları daha da ayrı bir canlı hücreler, tümör hücre süspansiyonu Ficoll-Paque Plus kullanarak yoğunluğu santrifüj ayrılır. Ficoll-Paque Plus bir underlayment Ayrıca iki kat karıştırmak için dikkatli olmak hücre süspansiyonu altında yavaş yavaş 15 ml Ficoll-Paque pipeting tarafından oluşturulur. </li><li> Hücre süspansiyon ve frenler ile 30 dakika Ficoll katmanları 500x g santrifüj kapalıdır. </li><li> Ficoll Paque Plus ve hücre disosiasyon tampon arayüzü hücreler toplanır ve taze bir 50 mL konik tüp transfer. </li><li> Taze DMEM% 10 FCS ile desteklenmiş sulandırmak için hücre süspansiyonu eklenir 01:03 (10 ml hücre süspansiyonu eklenir örneğin 20 ml taze medya). </li><li> 10 dakika, dekante medya 450x g santrifüj hücreleri toplanır ve hücre pelletini ALDEFLUOR tampon 1 mL yeniden süspanse. </li><li> On mikrolitre hücre süspansiyonu, 10 mcL tripan mavi ve canlı hücreler hemasitometre kullanarak sayılır ile seyreltilir. Bu adım sırasında, ölü hücreler ve doku artıkları çok sayıda not edilir, sonra yoğunluğu santrifüj hücre ayırma (3.7 adımlar 3,3) tekrarlanan olabilir. </li></ol> 4. Floresan Aktif Hücre Sıralama Hücreler Leke <ol><li> Olarak Etiket yedi 12 x 75 mm polistiren test tüpleri aşağıdaki gibidir: <ol><li> Lekesiz </li><li> CD44 APC </li><li> CD24-PE </li><li> fare CD31-biotin + fare soyun-biotin + fare H-2Kd-biotin </li><li> ALDEFLUOR </li><li> ALDEFLUOR + DEAB + IgG 2bκ APC + IgG 2aκ-PE </li><li> ALDEFLUOR + CD44 APC + CD24-PE + fare CD31-biotin + fare soy biotin + fare H-2Kd-biotin. </li></ol></li><li> ALDEFLUOR tampon 2 ml hücre süspansiyonu, 5-10 milyon hücreleri / ml bir final konsantrasyon seyreltilir ve buz üzerinde tutmak. Tüpler # 1, 2, 3, ve 4 hücre süspansiyonu 100 mcL ekleyin ve buz üzerinde saklayın. Tüp # 6 1 mcL DEAB ekleyin ve buz üzerinde tutmak. 1000 seyreltilmiş kalan hücre süspansiyonu ALDEFLUOR reaktif ekle kat (örneğin 1.6 ml hücre süspansiyonu 1.6 mcL ALDEFLUOR reaktif eklemek), iyice karıştırın ve buz üzerinde yer. Tüp # 7 100 tüpler # 5 ve 6 için bu karışımın mcL ve kalan karışımı (1.4 mL) aktarın. Tüm tüpleri 40 dakika boyunca 37 ° C su banyosunda inkübe edin. Hücrelerin tüplerin altına yerleşmesini önlemek için hücre süspansiyonu, her 10 dakikada bir karıştırın. </li><li> Santrifüj 450x g hücreleri ve 4 ° C 10 dakika sonra tampon süzün. # 1 ve 5 100 mcL ALDEFLUOR tampon tüpler pelet yeniden süspanse edin. Tüpler # 2, 3, 4, ve 6 için aşağıdaki gibi seyreltilmiş uygun antikorları içeren ALDEFLUOR tampon 100 mcL ekleyin: IgG 2bΚ-APC, IgG 2aΚ-PE, CD44-APC, ve CD24-PE için 01:20antikorlar için 1:100 fare CD31-biotin, fare soy biotin ve fare H-2Kd-biotin antikorlar. # 7 tüpe 1.4 mL CD44 APC 1:20 seyreltme içeren ALDEFLUOR tampon, ve CD24-PE antikorları ve bir fare 1:100 dilüsyon CD31-biotin, fare soy biotin ve fare H-2Kd-biotin antikorlar. 10 dakika boyunca buz üzerinde hücre süspansiyonları inkübe edin. </li><li> Santrifüj 450x g hücreleri ve 4 ° C 10 dakika sonra tampon süzün. Pelet tüpler # 1, 2, 3, 5, ve (6) 100 mcL ALDEFLUOR tampon tekrar ALDEFLUOR tampon 1:100 dilüsyon streptavidin-PerCP protein içeren 1,5 mL hazırlayın. Tüp # 4 100 mcL ekleyin ve tüp # 7 1,4 ml ekleyin. 10 dakika boyunca buz üzerinde hücre süspansiyonları inkübe edin. </li><li> Santrifüj 450x g hücreleri ve 4 ° C 10 dakika sonra tampon süzün. Pelet tüpler # 1, 2, 3, 4, 5, ve (6) 200 mcL ALDEFLUOR tampon tekrar # 7, 2 mcg / ml propidium iyodür içeren ALDEFLUOR tampon 2,8 ml tüp pelletini tekrar. Tüpleri her zaman buz üzerinde tutun. </li><li> 35 mikron filtre ile süzgeç kapaklar 12 x 75 mm polistiren test tüpleri kullanarak hücrelerin iletin. </li></ol> 5. Floresan Aktive Hücre Sıralama Kanser Kök Hücreler Isolate <ol><li> Bir FACSAria akış sitometresinin hücre sıralama için hazırlanmıştır. </li><li> Tazminat kontrolleri tüpler # 1, 2, 3, 4, ve 5 hücreleri kullanılarak ayarlanır. </li><li> Gates&#39;in hücre mayoları toplamak için ileri ve yan dağılım parametreleri göre oluşturulur. </li><li> Sigara fare (PerCP negatif) ve uygulanabilir (non-propidium iyodür boyanmış) hücreleri kullanarak kapılı FL-3 kanal. </li><li> DEAB-tedavili hücrelerde (tüp # 6) ve FL-1 kanal kullanılarak oluşturulan ALDH + hücrelerin kapıları üzerinde toplanır. </li><li> CD44 + CD24 + hücrelerin kapıları üzerinde toplanan FL-4 ve FL-2 kanallarını kullanarak ALDEFLUOR 2bΚ APC, IgG ve IgG 2aκ-PE (tüp # 6) ile boyanan hücreler kullanılarak oluşturulur. </li><li> Hücreler,% 10 FCS ile desteklenmiş DMEM 1 mL içeren 12 x 75 mm polistiren test tüpleri toplanmıştır. </li><li> Hücreler sıralanmış sonra, süzün 10 dakika, medya 450x g hücre süspansiyonu santrifüj ve 500 mcL taze DMEM hücreleri tekrar süspansiyon% 10 FCS ile desteklenebilir. </li><li> Adım 3.8 &#39;de açıklandığı gibi hemasitometre kullanarak sıralaması hücrelerinin sayısını. </li></ol> 6. Temsilcisi Sonuçlar Bu protokol ALDH + ve CD44 + CD24 + pankreas CSCS izolasyonuna yol açacaktır. Fare kökenli hücrelerin yüzdesi değişkendir, ama biz çoğu insan pankreas kanseri xenografts fare fare kullanılarak CD31-kökenli hücreleri, fare soy kokteyl, ve fare, H-2Kd biotin-konjuge antikor (Şekil 1A 20-60% içerdiğini buldular .) Aynı şekilde farklı xenografts her CSC nüfus sıklığı değişken, ancak genel olarak toplam hücre popülasyonunun% 1-4 ALDH + (Şekil 1B) ve% 0,2-5 CD44 + CD24 + (Şekil 1C) olduğunu bulduk. FACSAria makine sayıları FACSAria tarafından tespit hücresel olmayan parçacıklar nedeniyle sık sık yanlış beri rutin hemasitometre kullanarak elle sıralanmış hücreleri saymak. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="Şekil 1" /> Şekil 1. Flow sitometri arsa pankreas kanseri xenograft belirli toplumlarda betimleyen (A) FL-3 kanal olumlu boyanma Hücreler (propidium iyodür + fare CD31 + fare soy + veya fare H-2Kd +) sadece izole olarak dışlanan uygulanabilir ve non-fare hücreleri türetilmiştir. (B) bir insan pankreas kanseri xenograft ALDH faaliyet ALDH1 inhibitörü diethylamino benzaldehit (DEAB) varlığı ve yokluğu ALDEFLUOR reaktifi kullanılarak flow sitometri ile ölçüldü. Kare DEAB ile muamele edilen hücreler alınarak oluşturulan ve daha sonra tedavi edilmezse hücrelere uygulanan ALDH + hücreleri tasvir kapıları temsil eder . ALDH + hücre yüzdeleri kapısı gösterilmiştir. (C) CD44 + CD24 + kapısı ALDEFLUOR ile boyanan hücreler göre oluşturulur ve IgG 2bκ-APC (CD44 APC için izotipik kontrol) ve IgG 2aκ-PE (CD24-PE için izotipik kontrol) antikorları oldu. CD44 + CD24 + hücrelerin yüzdeleri kapısı gösterilmiştir.

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

Bu protokol, insan xenografts pankreas CSCS izolasyonu açıklamaktadır. Bu yordamı bazı önemli adımlar uygulanabilir CSCS verimi artıracaktır. Pankreas CSCS saf bir nüfusun kurtarma FACSAria sıralama önce hücre süspansiyonu bulunan canlı hücrelerin saflık bağlıdır. En büyük çapı 1 cm&#39;den daha az olan xenografts kullanmak bakımı tümör merkezinde nekrotik doku miktarını azaltmaya yardımcı olur. Ayrıca, Ficoll Paque degrade santrifüj sırasında doku artıkları ve ölü hücreleri hücre süspansiyonu tüketen kritik ve doku artıkları veya cansız hücrelerin çok sayıda hücre hemasitometre sayıldığında tespit edilmesi halinde tekrar edilebilir. Boyama protokol burada ALDH faaliyet yanı sıra fluorofor-konjuge antikorları tespit etmek için belirli bir hücre yüzey antijenleri tespit etmek için ALDEFLUOR reaktif sistemi kullanır nitelendirdi. ALDEFLUOR boyama 37 ° C ve bu nedenle, antikor boyama (buz) 37 gerçekleşebilir antikor sınırını potansiyeli, ° C önlemek için önce tamamlanması gerekmektedir Hücreleri ALDEFLUOR reaktif efflux beri, 4 ALDEFLUOR tampon hücreleri korumak için önemli ° C ALDEFLUOR boyama sonra tamamlanmıştır. ALDEFLUOR tampon ALDEFLUOR içi düzeylerini korumak yardımcı olan bir ilaç dışarı atım inhibitörü içerir. Bu yöntem uygulanabilir pankreas CSCS izole olduklarından, bu hücrelerin fizyolojik fonksiyonu ölçen deneyler bir dizi uygulanabilir. Biz bu hücreler immün sistemi baskılanmış fareler ve in vitro hücre göç / işgali deneyleri kullanarak tümör başlatılması deneyleri için kullandık. Ayrıca, RNA, DNA ve protein, CSC ve non-CSC popülasyonları karşılaştıran analitik çalışmalar, bu hücrelerin toplanabilir.

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		<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

Kanser kök hücreleri (CSCS) malignitelerin giderek artan sayıda tespit edilmiştir ve işlevsel yeteneklerini, kendini yenileme tabi ve farklılaşmış döl 1 üretmek tarafından belirlenir . Bu özellikler, immün sistemi baskılanmış farelere enjekte edildiğinde CSCS orijinal tümör özetlemek için izin verir. CSCS epitelyal malignite içinde meme kanseri ilk açıklanan ve belirli bir hücre yüzey antijen ifade (CD44 + CD24 düşük / -) görüntülemek için 2 . O zamandan beri, CSCS CD44 ve CD24 gibi diğer yüzey antijenlerinin bir dizi kullanarak diğer insan malignitelerin giderek artan sayıda tespit edilmiştir. Fizyolojik özellikleri, aldehit dehidrogenaz (ALDH) aktivite de dahil olmak üzere 3-5 malign dokulardan CSCS izole etmek için kullanılır olmuştur. Son zamanlarda, biz ve diğerleri ALDH aktivite ve hücre yüzeyindeki antijenler CD44 ve CD24 ve CD133 6-8 ifadesi dayalı pankreatik adenokarsinoma CSCS belirledi. Bu son derece tümörojenik nüfus ya da örtüşen ve diğer işlevleri görüntülemek olmayabilir. Olduğunu saptadık ALDH + ve CD44 + CD24 + pankreas CSCS benzer tümörojenik, ama nispeten daha invaziv 8 ALDH + hücreler. Bu protokolde, düşük geçiş insan xenografts 9 canlı pankreas CSCS izole etmek için bir yöntem açıklanmaktadır . Xenografted tümörler farelerden elde edilen hasat ve tek bir hücre süspansiyonu haline getirilmiştir. Doku artıkları ve ölü hücreleri canlı hücrelerden ayrılmış ve daha sonra, CD44 ve CD24 karşı antikor kullanarak ve ALDEFLUOR reaktifi ALDH 10 floresan substrat kullanılarak boyandı. CSCS sonra floresan aktif hücre sıralama tarafından izole edilmiştir. İzole CSCS sonra canlı hücreler gerektiren analitik veya fonksiyonel testleri için kullanılabilir.

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

Kanser kök hücreleri (CSCS) malignitelerin çok sayıda tespit edilmiştir. Bu protokolde, aldehit dehidrogenaz aktivitesi ve CD44 ve CD24 ifade kullanan bir insan pankreas adenokarsinomu xenografts CSCS yalıtmak için akım sitometri yöntemi açıklar. Bu canlı hücreler, sonra fonksiyonel ve analitik çalışmalar kullanılabilir.

İnsan Pankreas Kanseri Xenografts Kök Hücre İzolasyonu

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

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

Research

JoVE Journal

Biology

22.2K Görüntüleme.  Johns Hopkins University School of Medicine. Kanser kök hücreleri (CSCS) malignitelerin çok sayıda tespit edilmiştir. Bu protokolde, aldehit dehidrogenaz aktivitesi ve CD44 ve CD24 ifade kullanan bir insan pankreas adenokarsinomu xenografts CSCS yalıtmak için akım sitometri yöntemi açıklar. Bu canlı hücreler, sonra fonksiyonel ve analitik çalışmalar kullanılabilir.

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.

Plasenta Hematopoetik Kök Hücre İzolasyonu ve Analizi

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. ...

Biyoloji

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.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

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.

İnsan Pankreas Adacık İzolasyonu: Bölüm I: Sindirim ve Pankreas Doku Koleksiyon

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.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

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.

İnsan İsteyerek Pluripotent Kök Hücre seçme ve ayırma Kolonileri Yetişkin Fibroblastlar gelen 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.

Primer Tümörler gelen Bağışıklık Hücre İzolasyonu

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.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

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.

İnsan Lipoaspirates den Yağ-türetilen Kök Hücre Manuel İzolasyon

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.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

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.

Insan serumundan küçük kodlayıcı olmayan RNA&#39;lar izolasyonu

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 ...