Este trabalho foi financiado por subvenções dos Institutos Nacionais de Saúde (CA127574, CA107040 e CA09071) para WM

1. Xenoenxertos colheita de Ratos <ol><li> Prepare a célula de dissociação buffer: Dulbecco modificado Eagle (DMEM) suplementado com 10% de soro fetal bovino (FCS), a penicilina-estreptomicina, 200 U / mL de colagenase tipo IV, e 0,6 dispase U / mL. </li><li> Um atímicos (nu + / nu +) do mouse abrigar um subcutânea xenoenxerto de câncer humano de pâncreas que é menos de 1 cm de maior diâmetro é sacrificados por asfixia de dióxido de carbono e deslocamento cervical. </li><li> O tumor é colhida subcutâneo do flanco do mouse por dissecção romba usando uma pinça e tesouras e colocado imediatamente no buffer de células dissociação. </li><li> Tumores são pesados ​​e colocados de volta em 10 mL de células dissociação tampão por 1 grama de tecido do tumor. </li></ol> 2. Gerar uma suspensão de células isoladas da xenoenxertos <ol><li> Trabalhando em um estéril biossegurança gabinete, o tumor é transferido para uma placa de cultura 100 x 20 mm com 1 mL de tampão de células dissociação. Tumores são mecanicamente picada usando lâminas de barbear estéreis e fórceps. A suspensão de células tumorais é triturado através de uma pipeta de 5 mL sorológicos 10 vezes. As peças tumor deve ser pequeno o suficiente para não entupir a pipeta de 5 mL sorológicos. </li><li> A suspensão de células tumorais é transferido para um tubo cônico de 50 mL contendo o volume restante de células dissociação buffer (cheio menos de 50%) e centrifugadas a uma velocidade máxima por 1 minuto. </li><li> A suspensão de células tumorais é, então, incubados a 37 ° C por 2 horas com vórtex intermitente por 1 minuto a cada 20 minutos. </li></ol> 3. Destroem suspensão de células de restos de tecido e células mortas <ol><li> Após a incubação de 2 horas da suspensão de células é agitadas na velocidade máxima durante 1 minuto. </li><li> A suspensão de células é então passado através de um filtro de 70 mm e coletado em um tubo de 50 mL fresco cônica e ajustado para um volume final de 15 mL por tubo. </li><li> Para separar ainda mais células viáveis ​​a partir de células mortas e restos de tecido, a suspensão de células tumorais é separado por centrifugação de densidade Ficoll-Paque Plus. Um underlayment de Ficoll-Paque Plus é criado por pipetar 15 mL de Ficoll-Paque Plus lentamente abaixo da suspensão de células com cuidado para não misturar as duas camadas. </li><li> A suspensão de células e camadas de Ficoll estão é centrifugado a 500x g por 30 minutos com os freios desligado. </li><li> Células na interface de o Plus Ficoll-Paque e buffer de células dissociação são coletados e transferidos para um tubo de 50 mL fresco cônico. </li><li> Frescos DMEM suplementado com 10% SFB é adicionado à suspensão de células para o diluir 1:3 (por exemplo, 20 mL de mídia fresco adicionado a 10 mL de suspensão de células). </li><li> As células são coletadas por centrifugação a 450X g por 10 minutos, a mídia decantado, eo pellet celular é ressuspenso em 1 mL de tampão ALDEFLUOR. </li><li> Dez microlitros da suspensão de células é diluído com 10 mL de tripan azul e células viáveis ​​são contadas usando um hemocitômetro. Se um grande número de células mortas ou restos de tecido são anotados durante esta etapa, a separação das células por centrifugação de densidade podem ser repetidos (passos 3,3-3,7). </li></ol> 4. Células mancha para separação de células de fluorescência Activated <ol><li> Rótulo sete 12 x 75 mm tubos de poliestireno teste da seguinte forma: <ol><li> Imaculado </li><li> CD44-APC </li><li> CD24-PE </li><li> rato CD31-biotina + mouse linhagem-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 + mouse linhagem-biotina + mouse H-2Kd-biotina. </li></ol></li><li> Diluir a suspensão de células em 2 mL de tampão ALDEFLUOR para uma concentração final de 5-10 milhões de células / mL e manter em gelo. Adicionar 100 mL da suspensão de células de tubos # 1, 2, 3 e 4 e mantê-los no gelo. Adicionar 1 mL para tubo de deab # 6 e manter em gelo. Adicionar reagente ALDEFLUOR à suspensão de células restantes diluídas 1000 - fold (por exemplo, adicionar 1,6 mL de reagente ALDEFLUOR para 1,6 mL suspensão celular), misturar bem e colocar no gelo. Transferir 100 mL dessa mistura para tubos de # 5 e 6, ea mistura restante (1,4 mL) ao tubo n º 7. Incubar todos os tubos em banho-maria a 37 ° C por 40 minutos. Misture a suspensão celular a cada 10 minutos a fim de impedir as células de sedimentação para o fundo dos tubos. </li><li> Centrifugar as células a 450X g e 4 ° C por 10 minutos e decantar o buffer. Volte a suspender as pelotas em tubos de # 1 e 5 em cada 100 mL de buffer ALDEFLUOR. Para tubos # 2, 3, 4 e 6, adicionar 100 mL de ALDEFLUOR buffer que contém os anticorpos adequados diluído da seguinte forma: 1:20 para IgG 2bΚ-APC, IgG 2aΚ-PE, CD44-APC, e CD24-PEanticorpos; 1:100 para mouse CD31-biotina, mouse linhagem-biotina e mouse H-2Kd-biotina anticorpos. Ao tubo # 7 adicionar 1,4 mL de tampão contendo ALDEFLUOR uma diluição 1:20 do CD44-APC, e CD24-PE anticorpos e uma diluição de 1:100 do mouse CD31-biotina, mouse linhagem-biotina e mouse H-2Kd-biotina anticorpos. Incubar as suspensões de células no gelo por 10 minutos. </li><li> Centrifugar as células a 450X g e 4 ° C por 10 minutos e decantar o buffer. Volte a suspender as pelotas em tubos de # 1, 2, 3, 5, e 6 em 100 mL de buffer ALDEFLUOR. Prepare 1,5 mL de tampão contendo ALDEFLUOR uma diluição de 1:100 de estreptavidina-PerCP proteína. Adicionar 100 mL para o tubo n º 4 e adicionar 1,4 mL para tubo de # 7. Incubar as suspensões de células no gelo por 10 minutos. </li><li> Centrifugar as células a 450X g e 4 ° C por 10 minutos e decantar o buffer. Volte a suspender as pelotas em tubos de # 1, 2, 3, 4, 5 e 6 em 200 mL de buffer ALDEFLUOR. Ressuspender o sedimento em tubo de 7 em 2,8 mL de tampão contendo 2 ALDEFLUOR mcg / mL de iodeto de propídio. Mantenha os tubos no gelo em todos os momentos. </li><li> Passe as células através de 35 mícrons filtro com 12 x 75 mm tubos de ensaio com tampas de poliestireno filtro. </li></ol> 5. Isolar células-tronco cancerosas por separação de células de fluorescência Activated <ol><li> Um citômetro de fluxo FACSAria está preparado para separação de células. </li><li> Controles de compensação são definidas usando as células dos tubos n º 1, 2, 3, 4 e 5. </li><li> Portões são criados com base em declarações de lado e dispersão de parâmetros para coletar singlets celular. </li><li> Mouse-sem (PerCP-negativos) e viáveis ​​(não coradas propidium iodeto), as células são fechadas através do canal de FL-3. </li><li> Células ALDH + são coletados com base em portas criado usando deab células tratadas (tubo # 6) e do canal-1 FL. </li><li> CD44 + CD24 + células são coletadas com base em portas criado usando células coradas com ALDEFLUOR, IgG 2bΚ-APC, e IgG 2aκ-PE (tubo # 6) usando o FL-4 e FL-2 canais. </li><li> As células são coletadas em 12 x 75 mm tubos de ensaio contendo poliestireno 1 mL de DMEM suplementado com 10% SFB. </li><li> Depois que as células foram ordenadas, centrifugar a suspensão de células em 450X g por 10 minutos, decantar a mídia, e ressuspender as células em 500 mL DMEM fresco suplementado com 10% SFB. </li><li> Contar o número de células classificadas utilizando um hemocitômetro como descrito no passo 3.8. </li></ol> 6. Resultados representante Este protocolo vai levar ao isolamento da ALDH + e CD44 + CD24 + CSCs pancreático. A porcentagem de células derivadas do mouse é variável, mas descobrimos que xenoenxerto de câncer pancreático mais humano contêm 20-60% de células de rato derivados usando o mouse CD31, mouse cocktail linhagem, e mouse H-2Kd biotina anticorpos conjugados (Figura 1A ). Da mesma forma a freqüência de cada população CSC de xenoenxertos diferentes é variável, mas geralmente descobrimos que 1-4% da população total de células é ALDH + (Figura 1B) e 0,2-5% é CD44 + CD24 + (Figura 1C). Rotineiramente manualmente contagem de células classificados usando um hemocitômetro desde a máquina de contagem de FACSAria são muitas vezes imprecisas devido à não-celular partículas detectadas pelo FACSAria. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="Figura 1" /> Figura 1. Enredo citometria de fluxo retratando populações específicas de um xenoenxerto de câncer pancreático. (A) Células coradas positivamente no canal de FL-3 (propidium iodeto +, mouse CD31 + + lineage mouse, ou mouse H-2Kd +) são excluídos de forma a isolar apenas viáveis ​​e não do rato células derivadas. (B) ALDH atividade em um xenoenxerto de câncer pancreático humano foi medido por citometria de fluxo usando o reagente ALDEFLUOR na presença e ausência do inibidor ALDH1 dietilamino-benzaldeído (deab). Os quadros representam portões que retratam células + ALDH que foram criadas com base em células tratadas com deab e depois aplicada a células não tratadas. As porcentagens de células + ALDH são mostrados no portão. (C) O CD44 + CD24 + portão foi criado a partir de células coradas com ALDEFLUOR, eo IgG 2bκ-APC (controle isotípico para CD44-APC) e IgG 2aκ-PE (controle isotípico para CD24-PE) anticorpos. As porcentagens de células CD44 + CD24 + são mostrados no portão.

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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>

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Este protocolo descreve o isolamento de pâncreas CSCs de xenoenxertos humanos. Várias etapas fundamentais neste procedimento irá melhorar o rendimento de CSCs viável. A recuperação de uma população pura de pâncreas CSCs depende da pureza de células viáveis ​​presentes na suspensão celular antes de classificar na FACSAria. Tendo o cuidado de usar xenoenxertos que são menos de 1 centímetro de diâmetro maior ajuda a reduzir a quantidade de tecido necrosado no centro do tumor. Além disso, esgotando a suspensão de células de restos de tecido e células mortas durante a centrifugação Ficoll-Paque gradiente é crítico e pode ser repetido se um grande número de restos de tecido ou de células não-viáveis ​​são detectados quando as células são contadas sobre o hemocitômetro. O protocolo de coloração descrito aqui utiliza o sistema de reagentes para detectar atividade ALDEFLUOR ALDH bem como fluoróforos anticorpos para detectar antígenos de superfície específicos de células. ALDEFLUOR coloração é realizada a 37 ° C e, portanto, precisa ser concluído antes de coloração de anticorpos (no gelo) para evitar a possibilidade de nivelamento de anticorpos, o que pode acontecer a 37 ° C. Como as células podem efluxo o reagente ALDEFLUOR, é importante para manter as células em ALDEFLUOR tampão a 4 ° C após coloração ALDEFLUOR foi concluída. O buffer ALDEFLUOR contém um inibidor de efluxo de drogas que ajuda a manter os níveis intracelulares de ALDEFLUOR. Uma vez que este método isola viável pancreático CSCs eles podem ser aplicados em uma série de experimentos que medem a função fisiológica dessas células. Nós temos usado essas células de tumor de iniciação ensaios utilizando camundongos imunodeprimidos e em células in vitro de migração / invasão ensaios. Além disso, RNA, DNA e proteínas podem ser coletados a partir dessas células para estudos analíticos comparando CSC e não-CSC populações.

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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

Células-tronco cancerosas (CSCs) foram identificados em um número crescente de doenças malignas e são funcionalmente definidas por sua habilidade de sofrer auto-renovação e produzir descendentes diferenciados 1. Essas propriedades permitem que CSCs recapitular o tumor original quando injetadas em ratos imunodeficientes. CSCs dentro de um tumor maligno epitelial foi primeiramente descrito no câncer de mama e está em exibição específico de células a expressão do antígeno de superfície (CD44 + CD24 / low -) 2. Desde então, CSCs foram identificados em um número crescente de outras doenças malignas humanas usando CD44 e CD24, assim como um número de antígenos de superfície. Propriedades fisiológicas, incluindo a atividade de aldeído desidrogenase (ALDH), também têm sido usados ​​para isolar CSCs de tecidos malignos 3-5. Recentemente, nós e outros identificados CSCs de adenocarcinoma do pâncreas com base na atividade ALDH ea expressão dos antígenos de superfície celular CD44 e CD24, CD133 e 6-8. Estas populações altamente tumorigénico podem ou não podem ser sobrepostas e exibir outras funções. Descobrimos que ALDH + e CD44 + CD24 + pâncreas CSCs são igualmente tumorigénico, mas as células ALDH + são relativamente mais invasivos 8. Neste protocolo, descrevemos um método para isolar viável pancreático CSCs de passagem de baixa xenoenxertos humanos 9. Xenografted tumores são colhidos a partir de camundongos e transformado em uma suspensão de uma única célula. Restos de tecido e células mortas são separados a partir de células vivas e, em seguida, coradas utilizando anticorpos contra CD44 e CD24 e usando o reagente ALDEFLUOR, um substrato fluorescente de ALDH 10. CSCs são então isolados por separação de células de fluorescência ativado. Isolado CSCs pode então ser usado para ensaios analíticos ou funcionais que requerem células viáveis.

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

Células-tronco cancerosas (CSCs) foram identificados em uma série de tumores malignos. Neste protocolo, descrevemos um método de citometria de fluxo utilizando atividade aldeído desidrogenase e CD44 e CD24 expressão para isolar CSCs de xenoenxertos adenocarcinoma pancreático humano. Estas células viáveis ​​podem então ser utilizados em estudos funcionais e analíticos.

Isolamento de células-tronco de xenoenxertos Humano do Câncer de pâncreas

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

Nanyang Technological University

Research

JoVE Journal

Biology

22.3K visualizações.  Johns Hopkins University School of Medicine. Células-tronco cancerosas (CSCs) foram identificados em uma série de tumores malignos. Neste protocolo, descrevemos um método de citometria de fluxo utilizando atividade aldeído desidrogenase e CD44 e CD24 expressão para isolar CSCs de xenoenxertos adenocarcinoma pancreático humano. Estas células viáveis ​​podem então ser utilizados em estudos funcionais e analíticos.

Vídeo: 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 Análise de Células-Tronco Hematopoéticas da 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. ...

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.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

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 Islet humana Pancreatic: Parte I: Digestão e Coleta de tecido pancreático

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 Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Isolamento de Células-Tronco da retina do olho do rato

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up 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 Cultura de Células-Tronco Adultas epitelial da pele humana

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.

Seleção e isolamento de colônias de células-tronco pluripotentes induzidas a partir de fibroblastos adultos reprogramado

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 de células imunes de tumores primários

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&#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 de culturas primárias de fibroblastos humanos

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 manual de células-tronco adiposo-derivadas de Lipoaspirates Humanos

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