O protocolo para a geração de células CD40-activated B da CMSP é dividido em duas partes: Parte A demonstra a preparação de CD40 ligante expressar NIH/3T3 células, que serão usados ​​como células da placa-bound alimentador. Parte B descreve a cultura CD40-B real. A. Preparação de células alimentadoras (tCD40L NIH/3T3) O NIH/3T3 tCD40L é uma linha de células aderentes murino de fibroblastos, que nunca deve tornar-se completamente confluentes. As células são, portanto, dividido por duas vezes por semana. Cultura há mais de seis semanas não é recomendado. <ol><li> Remova o meio de idade a partir da cultura primária com uma pipeta estéril e lavar as células com 10 mL de PBS 1x. Aspirar o PBS após a lavagem. </li><li> Adicionar 4 mL Tripsina / EDTA em um frasco de 75 cm 2 por 5-10 minutos a 37 ° C. Use tocando suave para retirar as células. </li><li> Adicionar 10 mL de meio de tipo selvagem e giratória suavemente. </li><li> Transferir a suspensão de células em um tubo de 50 mL com uma pipeta estéril e girar a células para baixo a 225 xg por 5 min. </li><li> Remover o sobrenadante e ressuspender o sedimento em 10 mL de meio de tipo selvagem. Conte o número de células de uma alíquota da suspensão de células e preparar três tubos de 50 mL com o número adequado de células: <ol><li> 1,5 x 10 6 células para subcultura </li><li> 0,2 x 10 6 células / poço de irradiação utilizadas para a cultura de células CD40-B </li><li> restante para congelar (se necessário). </li></ol></li><li> Spin the células para baixo de 225 xg por 5 min. </li><li> Remover o sobrenadante. <ol><li> Para subcultura: ressuspender 1,5 x 10 6 células em 10 mL de médio tipo selvagem em um frasco de 75 centímetros de cultura de células 2 (densidade celular de 1,5 x 10 5 células / mL), adicione G-418 [0,7 mg / mL] e incubar as células em 37 ° C com 5% de CO 2. Dividir as células duas vezes por semana. </li><li> Para a cultura de células CD40-B: Você precisa de 1,2 x 10 6 células para uma placa de 6 poços. Ressuspender as células em meio de tipo selvagem com uma densidade de 0,1 x 10 6 células / mL e irradiar-los em 78 Gy. Placa de 2 mL da suspensão de células em cada poço e incube-os a 37 ° C com 5% de CO 2. Utilize este placas preparado para a estimulação de células B, quando tCD40L NIH/3T3 células são aderente (pelo menos 4 horas: verificar a aderência com o microscópio, não espere mais de 24 h para iniciar a estimulação de células B). (Continue com B.) </li></ol></li></ol> B. CD 40-B de cultura de células I. Elaboração de PBMCs para CD40-estimulação (dia 0): Por favor note: antes de continuar determinar que as células de alimentação são aderentes. Sempre adicionar novas soluções de interleucina-4 e ciclosporina A para o meio de crescimento imediatamente antes da utilização. <ol><li> Tome PBMCs, fresco ou descongelado adequadamente. Ressuspender PBMCs duas vezes em 50mL de PBS 1x para lavá-los e spin down primeira vez em 265 xg por 7 min e uma segunda vez em xg 190 para 7 min para remover outras células. Elimine o sobrenadante e ressuspender as células em 20 mL de PBS. Determinar o número de células em uma alíquota da suspensão de células. </li><li> Spin down quantidade necessária de células em 225 xg por 5 min. Para uma placa de 6 poços 4 x 10 6 células / poço são necessários, portanto, 24 x 10 6 células por placa. </li><li> Remover o sobrenadante e ressuspender o CMSP em 1 x 10 6 células / mL em CD40-B meio de cultura fresco suplementado com 50 U / mL de IL-4 como fator de crescimento e 0,63 ciclosporina mcg / mL para evitar A conseqüência de células T (concentrações indicados referem-se a um meio de cultura mL!). </li><li> Remover o sobrenadante de 6 bem-placa pré-incubadas com tCD40L NIH/3T3 células. </li><li> Lavar o aderente tCD40L NIH/3T3 células com 2 mL de PBS por poço em primeiro lugar e em uma segunda etapa com 2 mL de CD40-B médio de lavar roupa. </li><li> Delicadamente adicionar 4 mL de suspensão PBMC (1 x 10 6 células / mL) a cada poço da placa de 6 poços. </li><li> Incubar as células a 37 ° C com 5% de CO 2. </li><li> No dia 7, reculture as células (Continue com o 3.2.). </li></ol> II. Recultivo de CD40 células-B (7 º dia e depois a cada 3-4 dias): <ol><li> Clusters de colheita de células CD40-B a partir de 6 bem-chapa pela ressuspensão com uma pipeta de 10 mL e piscina-los em um tubo de 50 mL. </li><li> Girar a 225 xg por 7 minutos e substituir completamente o sobrenadante com CD40-B médio de lavar roupa. Contando a quantidade de células de uma alíquota, gire a células para baixo a 225 xg por 5 minutos. Ressuspender as células CD40-CD40-B em B meio de cultura a uma concentração de 1 x 10 6 células / mL. </li><li> Adicionar novas soluções de interleucina-4 em uma concentração de 50 U / mL e 0,63 ciclosporina mcg / mL A ao meio. </li><li> Remover o sobrenadante de uma placa de 6 poços de pré-incubadas com tCD40L NIH/3T3 células. </li><li> Lave as células aderentes com 2 mL de PBS por poço em primeiro lugar e em uma segunda etapa com 2 mL de CD40-B médio de lavar roupa. </li><li&gt; Gentilmente adicione 4 mL (1 x 10 6 células / mL) de suspensão CD40-B a cada poço da placa de 6 poços. </li><li> Incubar as placas a 37 ° C com 5% de CO 2. </li><li> Células subcultura novamente a cada 3-4 dias para acabar com alta pureza células CD40-B ativadas após um total de 14 dias. </li></ol> C. Resolução de problemas - O que se as células CD40-B não crescem? <ol><li> Você tem verificado para CD40 ligante expressão de células de alimentação? </li><li> A placa-bound células alimentadoras usado para a estimulação não deve ser mais de 24h? </li><li> É uma possível contaminação com micoplasma? </li><li> Foi a solução interleucina-4 utilizada para a suplementação recém descongeladas e tiveram a atividade biológico adequado? </li><li> Ciclosporina A foi adicionado na concentração correta? </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. Por favor, <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">clique aqui</a> para uma versão maior da figura 1. <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="Figura 2" /> Figura 2. Por favor, <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">clique aqui</a> para uma versão maior da figura 2.

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. 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+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15, 955-965 (2008).</li></ol>

A. Preparação de Media: <table border="1"cellspacing="0"><tbody><tr><td colspan="2"> Alimentador celular médio do tipo selvagem </td><td colspan="2"> Alimentador meio de seleção de célula </td></tr><tr><td> Reagente </td><td> Concentração </td><td> Reagente </td><td> Concentração </td></tr><tr><td> DMEM-Ham / F12 </td><td></td><td> DMEM-Ham / F12 </td><td></td></tr><tr><td> L-Glutamina </td><td> 365 mcg / mL </td><td> L-Glutamina </td><td> 365 mcg / mL </td></tr><tr><td> FBS </td><td> 10% </td><td> FBS </td><td> 10% </td></tr><tr><td> HEPES </td><td> 10 mM </td><td> HEPES </td><td> 10 mM </td></tr><tr><td> Gentamicina </td><td> 15 mcg / mL </td><td> Gentamicina </td><td> 15 mcg / mL </td></tr><tr><td></td><td></td><td> G-418 </td><td> 0,7 mg / mL </td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> CD40-B médio de lavar </td><td colspan="2"> CD40-B meio de cultura </td></tr><tr><td> Reagente </td><td> Concentração </td><td> Reagente </td><td> Concentração </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L-Glutamina </td><td> 584 mcg / mL </td><td> L-Glutamina </td><td> 584 mcg / mL </td></tr></tbody><tbody><tr><td> HEPES </td><td> 25 mM </td><td> HEPES </td><td> 25 mM </td></tr></tbody><tbody><tr><td> Gentamicina </td><td> 15 mcg / mL </td><td> Gentamicina </td><td> 15 mcg / mL </td></tr></tbody><tbody><tr><td> </td><td> </td><td> rh Transferrina </td><td> 50 mg / mL </td></tr></tbody><tbody><tr><td> </td><td> </td><td> Insulina rh </td><td> 5 mg / mL </td></tr></tbody><tbody><tr><td> </td><td> </td><td> AB-Humanserum </td><td> 10% </td></tr></tbody></table> B. Diversos Reagentes: <table cellspacing="0" border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Despacho n. </td></tr><tr></tr><tr><td> F12 DMEM / Ham </td><td> PAA </td><td> Nenhum gato: E15-813 </td></tr><tr><td> IMDM </td><td> Invitrogen Gibco ® </td><td> REF 21980-032 </td></tr><tr><td> PBS Dulbecco (10x) </td><td> PAA </td><td> Cat Não H15-011 </td></tr><tr><td> AB-soro humano </td><td> Invitrogen </td><td> Cat Não 34005100 </td></tr><tr><td> holo-transferrina humana </td><td> Sigma </td><td> Cat Não T0665 </td></tr><tr><td> Insulina humana </td><td> Sigma </td><td> Cat Não I2643 </td></tr><tr><td> Gencin ® </td><td> Selecione Delta </td><td> Arte Sem 7395800 </td></tr><tr><td> Interleucina-4 humana recombinante </td><td> Immunotools </td><td> Cat Não 11130045 </td></tr><tr><td> Ciclosporina A </td><td> Novartis </td><td> Cat Não NDC 0078-0109-01 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Cat Não DE14-802C </td></tr><tr><td> Tampão HEPES </td><td> PAA </td><td> Cat Não S11-001 </td></tr><tr><td> G-418 Sulfato </td><td> PAA </td><td> Cat Não P02-012 </td></tr><tr><td> Tripsina / EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table> Fontes C. Diversos: <table cellspacing="0" border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Despacho n. </td></tr><tr></tr><tr><td> Pipeta estéril dicas </td><td> Sarstedt </td><td></td></tr><tr><td> 6-bem placa </td><td> NUNC </td><td> Não Cat 140675 </td></tr><tr><td> 50mL tubo cônico </td><td> BD Falcon ™ </td><td> Não Cat 352070 </td></tr><tr><td> Frasco de cultura de tecidos </td><td> SARSTEDT </td><td> Cat Não 83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

Células CD40-activated B (CD40-células B) foram identificados como uma fonte alternativa de imuno-estimulatórias células apresentadoras de antígenos (APC) para imunoterapia de câncer 1-3. Em comparação com as células dendríticas (DCs), a melhor caracterizada APC, as células CD40-B têm várias propriedades biológicas distintas e técnicos. Semelhante ao DCs, as células B mostram um aumento da expressão de MHC e moléculas co-estimulatórias (Fig.1b), apresentam uma forte capacidade migratória e apresentação de antígenos presentes de forma eficiente para células T, após a estimulação com a interleucina-4 e CD40 ligante (CD40L). No entanto, em contraste com DCs imaturas ou maduro, as células CD40-B expressar a tríade completa nó de linfa homing consistindo de CD62L, CCR7/CXCR4 e função antígeno leucocitário-1 (LFA1, CD11a/CD18), necessário para homing para órgãos linfóides secundários (Fig.1a) 3. Células CD40-B pode ser gerado sem dificuldades a partir de quantidades muito pequenas de sangue periférico que pode ser expandida in vitro de quantidades muito grandes de alta-pura-B CD40 células de câncer (&gt; 10 9 células por paciente) de doadores saudáveis, bem como pacientes (Figura 1C, d) 1,4. Neste protocolo que demonstrar como obter totalmente ativado CD40-B a partir de células PBMC humana. Moléculas-chave para a cultura de células são CD40 ligante, a interleucina-4 (IL-4) e ciclosporina A. (CsA), que são reabastecidos em um ciclo de 3-4 dias de cultura Para fins de laboratório CD40-estimulação é fornecida por células que expressam NIH/3T3 recombinante ligante CD40 humano (tCD40L NIH/3T3) 5. Para evitar a contaminação com os não-transfectadas células, a expressão do ligante CD40 humano no transfectants tem de ser verificados regularmente (Fig.2). Após 14 dias CD40-B culturas de células consistem de mais de 95% de células B puro e uma expansão de células CD40-B com mais de 65 dias é frequentemente possível, sem qualquer perda de função de 1, 4. Células CD40-B de forma eficiente recolher, processar e apresentar antígenos às células T 6. Eles não só naϊve prime, mas também expandir células T de memória 7,8. Células CD40-B ativado pode ser usado para estudo de células B ativação, diferenciação e função. Além disso, eles representam uma ferramenta promissora para a vacinação preventivo ou terapêutico contra tumores 9.

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells at JoVE.com

Generation of Human CD40-activated B cells

Neste vídeo, apresentamos a<em&gt; Ex vivo</em&gt; Geração e expansão de células CD40-activated B (CD40-B) a partir de células mononucleares do sangue periférico (PBMC) de estimulação com CD40 ligante e interleucina-4.

Geração de humanos CD40-activated células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

generation-of-human-cd40-activated-b-cells

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JoVE Journal

Biology

15.1K visualizações.  University Hospital of Cologne, Department I of Internal Medicine. Neste vídeo, apresentamos a Ex vivo Geração e expansão de células CD40-activated B (CD40-B) a partir de células mononucleares do sangue periférico (PBMC) de estimulação com CD40 ligante e interleucina-4.

Vídeo: Generation of Human CD40-activated B cells

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparação Spread cromossômica de células estaminais embrionárias humanas para Cariotipagem

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Biologia

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos humanos com o Lentivirus TF Stemgent Humanos Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Geração robusto de Hepatócito-like Células de populações humanas células-tronco embrionárias

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caracterização fenotípica e funcional das células endoteliais Formadoras de Colônias derivado do sangue do cordão umbilical humano

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Geração de Células-tronco pluripotentes induzidas a partir de sangue periférico usando o STEMCCA Lentivirus Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Geração de Disperso Presomitic mesoderme culturas de células for Imaging da Segmentação Relógio Zebrafish em células individuais

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Geração de cardiomiócitos Humanos: Um Protocolo de Diferenciação de Feeder livres Humanos células-tronco pluripotentes induzidas

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Geração e cultura de sangue Conseqüência células endoteliais do sangue periférico humano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...