Agradecemos ao Alvin J. Siteman Cancer Center, em Washington University School of Medicine e Barnes-Jewish Hospital, em St Louis, MO, para o uso da Alta Velocidade Núcleo Sorter Cell, que forneceu equipamentos de citometria de fluxo e serviços de classificação. Este trabalho foi financiado pelo National Institutes of Health Grants R01 HL082549 e K02 HL084179 (NRY). O Siteman Cancer Center é suportado, em parte, por um suporte NCI Centro de Câncer Grant P30 CA91842.

1. PREPARAÇÃO DE REAGENTES <ol><li> Prepare estéril soluções estoque de 1000x Fms relacionados tirosina quinase 3 (FLT-3) ligando, estimulante de colônias de granulócitos / macrófagos factor (GM-CSF), fator de células tronco (SCF), trombopoietina (TPO), interleucina (IL) -3 e IL-6 de acordo com instruções do fabricante. Preparar a solução estoque estéril Retronectin (1mg/ml) também de acordo com instruções do fabricante. Dividir estas soluções estoque em pequenas alíquotas e manter a -20 ° C para evitar repetidos de congelamento e descongelamento. </li><li> Prepare FBS 2% em IMDM. </li><li> Prepare IMDM médio completo, com concentrações finais de FBS 20%, 2 mM de glutamina, 100 unidades / ml penicilina / estreptomicina (PS). Adicione o seguinte citocinas imediatamente antes da utilização: 100 ng / ml ligante FLT-3, 20 ng / ml GM-CSF, 100 ng / ml SCF, 100 ng / ml TPO, 50 ng / ml de IL-3, e 100 ng / ml IL-6. </li><li> Prepare BSA 1% em HBSS (Ca e Mg-livre, sem vermelho de fenol) e BSA 2% em D-PBS (Ca e Mg-free). Esterilizar as soluções, passando por filtros de 0,22 mm e seringa de armazenamento a 4 ° C. </li><li> Prepare EDTA 0,02% em HBSS e armazenar a 4 ° C. </li><li> Prepare-GALV pseudotyped retrovírus codificação do DNA de interesse e armazenamento em alíquotas a -80 ° C. Este procedimento está fora do escopo deste artigo, mas é descrita em outra parte (1-3). </li></ol> Para coloração Giemsa <ol start="7"><li> Imediatamente antes da utilização, filtrar a solução Wright / Giemsa duas vezes através de 1 mícron papéis de filtro dentro de uma garrafa de vidro limpa. </li><li> Imediatamente antes do uso, prepare Wright / Giemsa tampão pH 6,4, através da mistura de 50 ml da solução filtrada Wright / Giemsa e 200 ml de solução tampão fosfato, pH 6.4 - Giordano fórmula. </li><li> Preparar a solução tampão fosfato, pH 6,0, dissolvendo 27,3 g de KH 2 PO 4 e 4,62 g de Na 2 HPO 4 em 3,5 LH 2 O. </li><li> Prepare metanol a 50% em H 2 O. </li></ol> 2. PROCEDIMENTO Descongelamento e pré-ativação de células CD34 + <ol><li> Descongelar um frasco de congelados células CD34 + rapidamente a 37 ° C agitando até que um pequeno cristal de gelo última é para a esquerda e transferir a suspensão celular (1 ml) para um tubo de 50 ml. Suavemente enxaguar as células restantes do frasco com 1 ml de temperatura ambiente 2% FBS / IMDM e adicioná-lo gota a gota, ao tubo de 50 ml enquanto agita suavemente. Aguarde 3 minutos. Em seguida, adicionar lentamente 2 ml de 2% FBS / IMDM, enquanto misturando suavemente, e espere por 3 minutos. Repita o procedimento, adicionando 2% FBS / IMDM que é o mesmo volume da suspensão diluída de células em intervalos de 3 min, rodando suavemente entre adições, até que o volume final chega a 32 ml. </li><li> Centrifugar a 250 xg por 10 minutos em temperatura ambiente e remover o sobrenadante deixando para trás cerca de 0,5 ml. </li><li> Lave o pellet, suspendendo em 20 ml de 2% FBS / IMDM e centrifugação a 200 xg por 8 minutos em temperatura ambiente. </li><li> Remover o sobrenadante e suspender as células em meio IMDM completa em aproximadamente 0,5 x 10 6 células / ml. Tomar 10 ml da suspensão de células em um microtubo mix, com o mesmo volume de solução de azul de Trypan (0,4%) e contar com um hemocitômetro. </li><li> Diluir as células a 0,1-0,15 X 10 6 células / ml, adicionando médio IMDM completo suplementado com citocinas (ver 1.3) </li><li> Cultura as células em um umidificado 37 ° C incubadora com 5% de CO 2 para 2 dias. </li></ol> Transdução retroviral por vírus pré-carregamento <ol start="7"><li> No dia de transdução, contagem de células antes de iniciar a pré-carga de vírus para determinar o número de poços a ser preparada. </li><li> Diluir a solução estoque Retronectin a 25 mg / ml e adicionar 400 mL em cada poço de uma placa de 24 poços de cultura de tecidos não-tratados. Incubar por 2 horas em temperatura ambiente na capa de biossegurança de fluxo laminar para o revestimento de pré-. </li><li> Remover a solução Retronectin e adicionar 400 mL de BSA 2% em D-PBS a cada poço. Incubar por 30 minutos em temperatura ambiente. </li><li> Descongelar soluções estoque de vírus e manter em gelo. Remover a solução BSA, adicionar 0,5 ml da preparação de vírus a cada poço, e centrifugar a 2200 xg, a 4 ° C por 15 minutos. </li><li> Remover o vírus solução de poços e repita o carregamento vírus descrito no 2,10 mais três vezes. </li><li> Enxágüe bem com o frio cada 0,5 ml HBSS (+ Ca e Mg) ou IMDM. </li><li> Recolher o pré-ativado células CD34 + por centrifugação a 200 xg por 8 minutos em temperatura ambiente. </li><li> Volte a suspender as células em 0,1-0,15 x 10 6 células por 1,5 ml em meio IMDM completo com citocinas. </li><li> Adicione 1,5 ml de suspensão celular a cada poço e incubar em um umidificado 37 ° C incubadora com 5% de CO 2 para 2 dias. </li></ol> Separação de células e recolha de células transduzidas <ol start="16"><li> Suave mas completamente suspender as células do vírus-carregado acima da placas e filtrar através de um filtro de 50 mm de células CellTrics em um tubo cônico. Tomar 10 ml da suspensão de células em um microtubo mix, com um volume igual de solução de azul de Trypan e contar com um hemocitômetro. Enxágüe bem com cada 0,8 ml de frio 0,02% EDTA / HBSS (Ca e Mg-livre sem vermelho de fenol) e adicionar ao tubo de coleta mesma através de um filtro 50 mm de células CellTrics. </li><li> Centrifugar as células a 200 xg por 8 minutos a 4 ° C e lavar o pellet com frio HBSS (Ca e Mg-livre sem vermelho de fenol) por centrifugação a 200 xg por 8 minutos a 4 ° C. </li><li> Ressuspender o sedimento em 1% BSA / HBSS (Ca e Mg-livre sem vermelho de fenol) para uma concentração de 15 X 10 6 ml / ou um volume mínimo de 0.250 ml para separação de células. Manter as células no gelo no escuro. Prepare um tubo contendo 20% FBS frio / IMDM / Glutamina / PS médio para a coleta das células classificadas. Triagem é realizada no núcleo de alta velocidade Sorter Celular do J. Alvin Siteman Cancer Center, em Washington University School of Medicine utilizando um MoFlo classificador de alta velocidade (Dako, Glostrup, Dinamarca). </li></ol> CFC ensaio <ol start="19"><li> Descongelar o número necessário de 3 ml alíquotas de mídia ensaio CFC. Vortex vigorosamente para misturar e deixar os tubos de ficar pelo menos 5 min para que as bolhas existentes a subir para a superfície antes de adicionar células. </li><li> Para obter a concentração de células para cada amostra classificados, dividir o número de células fornecidas pela instalação de classificação pelo volume aproximado de células de suspensão. Tome-vírus 3000 transduzidas, células classificadas em um microtubo estéril contendo frio de 2% FBS / IMDM. Pré-ajustar o volume de 2% FBS / IMDM para que o volume final será de suspensão de aproximadamente 0,3 ml. </li><li> Suspender as células e transferência de toda a 0,3 ml de suspensão celular a uma alíquota de 3 ml de Methocult GF + H4435, que consiste em metilcelulose 1%, 30% FBS, BSA 1%, 10 -4 M 2-mercaptoetanol, 2 mM L-glutamina, 50 ng / ml SCF, 20 ng / ml GM-CSF, 20 ng / ml de IL-3, 20 ng / ml de IL-6, 20 ng / ml G-CSF, e 3 unidades / ml em IMDM eritropoietina. Enriquecido Humanos Metilcelulose Media (R &amp; D Systems), que consiste de metilcelulose 1,3%, FBS 25%, BSA 1%, 5 x 10 -5 M 2-mercaptoetanol, 2 mM L-glutamina, 50 ng / ml SCF, 20 ng / ml GM-CSF, 20 ng / ml de IL-3, 20 ng / ml de IL-6, 20 ng / ml G-CSF, e 3 unidades / ml em IMDM eritropoietina podem ser usados ​​também. </li><li> Vortex vigorosamente para fazer a mistura ascensão e queda total de 3-4 vezes. Deixe o tubo de ficar parado por 3 min. </li><li> Anexar uma agulha calibre 16 blunt-end para uma seringa de 3 ml e elaborar 2,2 ml. Não elaborar grandes bolhas; expulsá-los no início, empurrando para fora um par de vezes. Empurrar para fora 1,1 ml cada em dois 30 prato não tratados mm e espalhar a mistura uniformemente girando. </li><li> Coloque placas duplicadas em um prato de 100 mm, juntamente com um prato de água contendo 3 ml de água estéril. Cultura para 14-17 dias. </li><li> Caracterizar e marcar as colônias de acordo com sua morfologia com um microscópio invertido com ampliação de 40x em uma placa de cultura com uma grelha de pontuação. Para efeitos do nosso ensaio, as colônias são classificadas em três categorias: eritróide pura, mielomonocítica e mistos. Veja as instruções do fabricante de subclassificação colônia mais. </li><li> A placa de ensaio inteiro CFC pode ser digitalizado sem ampliação usando um scanner regular em 600 dpi, e de baixa potência (40x) fotomicrografias das colônias podem ser tomadas através de um microscópio invertido equipado com uma câmera de cor. </li><li> Para posterior análise da diferenciação e proliferação, as células da placa inteira CFC ensaio são recuperados através da suspensão de vários volumes de temperatura ambiente 2% FBS / IMDM. Após centrifugação a 300 xg por 10 min a 4 ° C, as células são ressuspendidas em 2% FBS / IMDM, contados, e quer coradas com anticorpos por citometria de fluxo ou transferidos para slides usando uma centrífuga para Cytospin Giemsa. </li></ol> Coloração de anticorpos e citometria de fluxo <ol start="28"><li> Células Centrifugar a 200 xg por 8 min a 4 ° C e ressuspender em soro de rato frio 50% normal em HBSS (Ca e Mg-livre sem vermelho de fenol). </li><li> Coloque cerca de 5 x 10 5 células em 80 mL por tubo (12 x 75 mm de fundo redondo de tubos) e manter em gelo. Adicione a mistura de anticorpos (aproximadamente 20 mL mistura ou solução de controle negativo para anticorpos não-controles) e misturar batendo levemente os tubos. Prepare os tubos de controle de calibração sem anticorpos ou com um anticorpo anti-CD45 para cada fluorocromo. Colocar os tubos no gelo, no escuro e incubar por 20 minutos. Mix batendo levemente tubos após os primeiros 10 minutos. Após a incubação de 20 minutos, encher os tubos com HBSS fria e centrifugação a 200 xg por 8 min a 4 ° C. Ressuspender o sedimento em 0,5% paraformaldeído frio / HBSS. Seguintes misturas de anticorpos são normalmente utilizados para nossa análise. Para a diferenciação mielóide: CD11b (ficoeritrina conjugada clone ICRF44) / CD33 (allophycocyanin conjugada clone WM53) / CD45 (ficoeritrina-Cy7-congjugated clone HI30). Para a diferenciação eritróide: CD71 (ficoeritrina conjugada clone M-A712) / CD235a (allophycocyanin conjugada clone GA-R2) / CD45 (ficoeritrina-Cy7-congjugated clone HI30). </li><li> Citometria de fluxo é realizada no núcleo de alta velocidade Sorter Celular do J. Alvin Siteman Cancer Center, em Washington University School of Medicine em um citômetro de fluxo FACScan atualizado para 5 cores e dois lasers (BD Biosciences) e analisados ​​utilizando CellQuest (BD Biosciences) ou programa FlowJo v7.2.4 (Árvore Star, Inc., Ashland, OR, EUA) software. </li></ol> Giemsa <ol start="31"><li> Cerca de 30.000 células recuperado de placas CFC ensaio descrito acima são suspensas em 0,3 ml de 2% FBS / IMDM e transferidos para um slide usando uma centrífuga Cytospin a 1.000 rpm por 10 min em temperatura ambiente. </li><li> Configurar nove navios coloração contendo soluções na seguinte ordem. <ol><li> Metanol absoluto </li><li> Wright / Giemsa solução estoque </li><li> Wright / Giemsa tampão, pH 6,4 </li><li> Metanol 50% em H 2 O </li><li> H 2 O </li><li> H 2 O </li><li> Tampão fosfato, pH 6,0 </li><li> H 2 O </li><li> H 2 O </li></ol></li><li> Coloque os slides em uma transportadora de slide e mergulho em metanol absoluto (# 1) por 2 min e blot off metanol em excesso. </li><li> Imediatamente, mergulhe a transportadora de slides em Wright / Giemsa solução estoque (# 2) por 5 min. </li><li> Transferência do transportador em Wright / Giemsa, pH 6.4 (# 3) e incubar por 10 min. </li><li> Mergulhe a transportadora duas vezes em metanol a 50% (# 4), 10 vezes em H 2 O (# 5), mais 10 vezes em H 2 O (# 6), e depois 5 vezes em tampão fosfato, pH 6.0 (# 7) . </li><li> Transferência do transportador em H 2 O (# 8) e incubar por 2 min. Repita o lava-H 2 O (# 9). </li><li> Permitir que as lâminas ao ar secar completamente no suporte. Retire cada slide e limpe a parte de trás da lâmina com metanol embebido Kimwipes para remover manchas. Coloque uma gota de Cytoseal 60 e uma tampa de vidro para selar. </li><li> A contagem diferencial de 500 células é realizada para cada slide corados pelo Giemsa usando um microscópio Olympus BX51. As células são divididas em cinco categorias: células primitivas incluem explosões e promielócitos; células mielóides intermediária incluem mielócitos e metamielócitos; células mielóides maduras incluem bandas, neutrófilos segmentados, monócitos e macrófagos, células intermediárias eritróide incluem células com hemoglobinization intermediária e células maduras eritróide incluem células com hemoglobinization completo. Fotomicrografias são tomadas com uma câmera Olympus DP71 com um objetivo de petróleo 60x. </li></ol> 3.0) Os resultados representativos: <ol><li> Ensaio CFC: Alguns da população celular de entrada irão proliferar, diferenciar e formam colônias no meio semi-sólido durante o período de incubação de 14-17-dia. Neste experimento, é evidente que a expressão do oncogene NUP98-HOXA9 causou a formação de vermelho proeminente (eritróide colônias) (Figura 1) (3). </li><li> Coloração de anticorpos e citometria de fluxo: imunofenotipagem por citometria de fluxo de células coletadas de placas CFC ensaio fornece informações sobre o estado de diferenciação da população celular. Pelo uso combinado de anticorpos contra certos marcadores de diferenciação, a linhagem eo grau de maturação das células pode ser avaliado. Neste caso CD235a foi utilizado para identificar células eritróides. Conforme mostrado na Figura 2A, NUP98-HOXA9 causou um aumento da percentagem de células CD235a + eritróide, mas o brilho de expressão CD235a por estas células foi diminuído em comparação com controles, indicando inibição da maturação eritróide. Na Figura 2B, células mielóides são identificados em virtude de sua positividade CD33. NUP98-HOXA9 causou uma diminuição no número de células CD33 +, com uma diminuição no brilho da expressão CD11b, consistente com a inibição da maturação mielóide (3). Assim, os resultados mostram que a citometria de fluxo NUP98-HOXA9 causou uma hiperplasia eritróide com a inibição da maturação tanto mielóide e eritróide. </li><li> Coloração Giemsa: análise morfológica de células coletadas de placas CFC ensaio fornece informações sobre a linhagem eo grau de maturação da população celular. As conclusões obtidas morfologicamente podem diferir daqueles obtidos por estudos de citometria de fluxo e, portanto, estes métodos são complementares entre si (3). O experimento é repetido pelo menos 3 vezes, e cada vez que uma contagem diferencial de 500 células é realizada para distinguir cinco categorias de células, como descrito acima. Exemplos dessas células são apontados na Figura 3. Os resultados são tabulados e analisados, a fim de determinar se existem diferenças estatisticamente significativas entre o oncogene-transduzidas amostra e do controle. Neste caso, os resultados mostraram que NUP98-HOXA9 causou um aumento global do números de células, com hiperplasia eritróide e inibição de ambos os eritróide e maturação mielóide (3) (não mostrados). </li></ol> LEGENDS FIGURA <img src="/files/ftp_upload/2195/2195fig1.jpg" alt="Figura 1" /> Figura 1: Os efeitos da NUP98-HOXA9 na morfologia CFC humana. Humanos primários células CD34 + foram retrovirally transduzidas com qualquer controle de vetores MSCV-IRES-GFP ou vetor expressando NUP98-HOXA9, e as células foram classificadas para a positividade GFP. Mil células foram semeadas em cada uma das duas placas duplicadas para ensaio de CFC eo experimento foi repetido 3 4 tempos independentes. Placas representante sem ampliação (à esquerda) e fotomicrografias de baixa potência do representante colônias eritróides (direita) são mostrados (3). <img src="/files/ftp_upload/2195/2195fig2.jpg" alt="Figura 2" /> Figura 2: A citometria de fluxo mostra interrupção do humano CD34 + principal diferenciação celular por NUP98-HOXA9. (A) A citometria de fluxo para a diferenciação eritróide: Células das placas CFC (ver Figura 1) foram colhidos e corados com anticorpos para CD45 e CD235a. O CD235a + portão foi plotado em um (painel direito) histograma para mostrar a expressão de CD235a relação às células controle (B) A citometria de fluxo para a diferenciação mielóide: Células das placas CFC foram colhidos e corados com CD45 e CD33. O CD33 + portão foi plotado em um histograma para mostrar expressão CD11b comparado ao grupo controle (painel direito). As porcentagens de células caindo dentro de cada porta são mostradas (3). <img src="/files/ftp_upload/2195/2195fig3.jpg" alt="Figura 3" /> Figura 3: A morfologia mostra rompimento de diferenciação por NUP98-HOXA9 relação ao controle de vetores. Manchas Cytospin foram preparados a partir de placas CFC e corados com Giemsa. Fotomicrografias foram tiradas de áreas representativas com um objetivo de petróleo 60x. B: explosão; MM: mielóides maduras, IM: intermediário mielóide, ME: eritróide maduro, IE: eritróide intermediária (3).

<ol>
	<li>Takeda, A., Goolsby, C., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXA9+induces+long-term+proliferation+and+blocks+differentiation+of+primary+human+CD34++hematopoietic+cells.">NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.</a> Cancer Res. , 66-6628 (2006).</li><li>Yassin, E. R., Abdul-Nabi, A. M., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+NUP98-DDX10+oncogene+on+primary+human+CD34++cells:+role+of+a+conserved+helicase+motif.">Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif.</a> Leukemia. , (2010).</li><li>Yassin, E. R., Sarma, N. J., Abdul-Nabi, A. M., Dombrowski, J., Han, Y., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+transformation+of+primary+human+hematopoietic+cells+by+the+oncogene+NUP98-HOXA9.">Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.</a> PLoS One. 4, e6719-e6719 (2009).</li><li>Nissen-Druey, C., Tichelli, A., Meyer-Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hematopoietic+colonies+in+health+and+disease.">Human hematopoietic colonies in health and disease.</a> Acta Haematol. 113, 5-96 (2005).</li><li>Pereira, C., Clarke, E., Damen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cell+assays.">Hematopoietic colony-forming cell assays.</a> Methods Mol Biol. 407, 177-208 (2007).</li><li>Coulombel, L. <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+hematopoietic+stem/progenitor+cells:+strength+and+drawbacks+of+functional+assays.">Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.</a> Oncogene. 23, 7210-7222 (2004).</li><li>Hogge, D. E., Lansdorp, P. M., Reid, D., Gerhard, B., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection,+maintenance,+and+differentiation+of+primitive+human+hematopoietic+cells+in+cultures+containing+murine+fibroblasts+engineered+to+produce+human+steel+factor,+interleukin-3,+and+granulocyte+colony-stimulating+factor.">Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.</a> Blood. 88, 3765-3773 (1996).</li></ol>

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O ensaio de CFC tem sido amplamente utilizado para determinar a proliferação e os padrões de diferenciação de células progenitoras hematopoéticas e estudar os efeitos dos oncogenes (4, 5). Tem a vantagem sobre culturas líquidas de ser um ensaio clonal, de tal forma que as colônias representam a progênie de um único progenitor e podem ser removidos individualmente para análise posterior. A limitação do ensaio CFC é que ele não é adequado para a detecção de células progenitoras imaturas ou mais célula...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>12440</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>06150</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin (PS)</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>FLT-3 ligand</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-19</td>
<td>&nbsp;</td>
<tr>
<td>GM-CSF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-03</td>
<td>&nbsp;</td>
<tr>
<td>SCF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-07</td>
<td>&nbsp;</td>
<tr>
<td>TPO</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-18</td>
<td>&nbsp;</td>
<tr>
<td>IL3</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-03</td>
<td>&nbsp;</td>
<tr>
<td>IL6</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-06</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP118</td>
<td>&nbsp;</td>
<tr>
<td>Retronectin</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>T100A</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14200</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue solution (0.4%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Methocult GF+ H4435</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>04445</td>
<td>&nbsp;</td>
<tr>
<td>Human Methylcellulose Enriched Media</td>
<td>&nbsp;</td>
<td>R &amp; D Systems</td>
<td>HSC005</td>
<td>&nbsp;</td>
<tr>
<td>Wright/ Giemsa stain</td>
<td>&nbsp;</td>
<td>Harleco</td>
<td>64571</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Solution, pH 6.4 - Giordano formula</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>1450</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A412-4</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 60</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>8310</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum </td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals</td>
<td>D208</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD11b phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555388</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD33 allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551378</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD71 phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555537</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD235a allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551336</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>24-well non-treated tissue culture plates</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-1147</td>
<td>&nbsp;</td>
<tr>
<td>30 mm non-treated dish</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27150 </td>
<td>&nbsp;</td>
<tr>
<td>100 mm tissue culture dish </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27500</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2097</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2070</td>
<td>&nbsp;</td>
<tr>
<td>Syringes 3 ml </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28240</td>
<td>&nbsp;</td>
<tr>
<td>16 gauge blunt-end, 1&frac12; inch needle </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28110</td>
<td>&nbsp;</td>
<tr>
<td>50 &mu;m CellTrics cell filter</td>
<td>&nbsp;</td>
<td>Partec</td>
<td>04-004-2327</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0267110</td>
<td>&nbsp;</td>
<tr>
<td>TPX sample chambers</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>A78710018</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Superfrost/Plus Microscope Slides, Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>Shandon filter cards</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>5991022</td>
<td>&nbsp;</td>
<tr>
<td>Shandon cytospin slide holder</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>59920063</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Complete Staining Assembly 100</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark Corporation</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>1 &mu;m filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28307-134</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Diaphot</td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>DS-F11</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX51 </td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DP71</td>
<td>&nbsp;</td>
<tr>
<td>Scanner</td>
<td>&nbsp;</td>
<td>Microtek</td>
<td>Scanmaker 4</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-812</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator</td>
<td>&nbsp;</td>
<td>Sanyo</td>
<td>MCO-18AIC</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>&nbsp;</td>
<td>Shandon</td>
<td>Cytospin 2</td>
<td>&nbsp;</td>
<tr>
<td>Bench-top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5810-R</td>
<td>&nbsp;</td>
<tr>
<td>Water purification system</td>
<td>&nbsp;</td>
<td>Barnstead</td>
<td>Nanopure-Diamond</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colony forming cell (cfc) assay for human hematopoietic cells

Humano-tronco hematopoiéticas / progenitoras células são normalmente obtidas da medula óssea, sangue do cordão umbilical ou sangue periférico e são usados ​​para estudar a hematopoiese e leukemogenesis. Eles têm a capacidade de se diferenciar em linhagens linfóide e mielóide. O ensaio de células formadoras de colônia (CFC) é utilizado para estudar o padrão de proliferação e diferenciação de células progenitoras hematopoéticas por sua capacidade de formar colônias em meio semi-sólido. O número ea morfologia das colônias formadas por um número fixo de células de entrada fornecer informações preliminares sobre a capacidade dos progenitores para se diferenciar e proliferar. As células podem ser colhidas a partir de colônias individuais ou de toda a placa para melhor avaliar os seus números e os estados de diferenciação por citometria de fluxo e avaliação morfológica de Giemsa-manchada slides. Este ensaio é útil para avaliar a diferenciação mielóide, mas não linfóides. O mielóide termo neste contexto é usada em seu sentido mais amplo para abranger linhagens granulocítica, monocítica, eritróide e megacariocítica. Temos utilizado neste ensaio para avaliar os efeitos de oncogenes na diferenciação de humanos primários células CD34 + derivadas do sangue periférico. Para este fim são as células transduzidas com qualquer construção de controle retroviral ou uma construção expressando o oncogene de interesse, neste caso NUP98-HOXA9. Nós empregamos um vetor retroviral comumente usados, MSCV-IRES-GFP, que expressa um mRNA bicistronic que produz o gene de interesse e um marcador GFP. Células são pré-ativado pelo crescimento na presença de citocinas por dois dias antes de transdução retroviral. Após mais dois dias, as células GFP + são isolados por fluorescência-activated cell sorting (FACS) e misturado com um meio de metilcelulose contendo semi-sólido suplementado com citocinas e incubados até colônias aparecem na superfície, geralmente 14 dias. O número e morfologia das colônias são documentados. Células são então removidas das placas, lavados, contados, e submetido a citometria de fluxo e análise morfológica. Citometria de fluxo com anticorpos específicos para os marcadores de superfície celular, expressas durante a hematopoiese fornece informações sobre a linhagem e maturação. Estudos morfológicos de células individuais sob um microscópio após Wright-Giemsa coloração fornecer informações adicionais em relação a linhagem e maturação. Comparação das células transduzidas com controle de vetores vazios aos transduzidas com um oncogene revela os efeitos do oncogene na diferenciação hematopoiética.

Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

O ensaio de células formadoras de colônia (CFC) é um ensaio in vitro em que células progenitoras hematopoéticas formar colônias em meio semi-sólido. Uma combinação de morfologia da colônia, morfologia celular e citometria de fluxo são utilizados para avaliar a capacidade dos progenitores a proliferar e se diferenciar ao longo dos diferentes linhagens hematopoiéticas.

Formadoras de Colônias celular Assay (CFC) para células hematopoiéticas humanas

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

colony-forming-cell-cfc-assay-for-human-hematopoietic-cells

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

35.5K visualizações.  Washington University School of Medicine. O ensaio de células formadoras de colônia (CFC) é um ensaio in vitro em que células progenitoras hematopoéticas formar colônias em meio semi-sólido. Uma combinação de morfologia da colônia, morfologia celular e citometria de fluxo são utilizados para avaliar a capacidade dos progenitores a proliferar e se diferenciar ao longo dos diferentes linhagens hematopoiéticas.

Vídeo: Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Imunologia e Infecção

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA-Ig Baseado Artificial células apresentadoras de antígenos para a eficiente Ex vivo Ampliação da CTL Humanos

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

O uso de interferão-γ Enzyme-linked Assay immunospot Caracterizar Novel epitopos de células T de papilomavírus humano

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac modificação do sistema Transposon de células T primárias Humanos

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitativo de alta capacidade ensaio de citotoxicidade de célula única para as células T.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

A identificação de DNA Mutações em Purificada-Tronco Hematopoéticas / células progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Cultura de macrófagos macrófagos Colony estimulante fator diferenciado Humano monócitos derivados

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Um ensaio de citotoxicidade baseados em citometria de fluxo para a avaliação da atividade das células NK humana

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...