This work was supported by grants funded by the British Heart Foundation (BHF), Dinosaur Trust, McAlpine Foundation, Fondation Leducq, Fight for Sight, the Cambridge Biomedical Research Centre, National Institute of Health Research including (i) the BHF Oxbridge Centre of Regenerative Medicine [RM/13/3/30159], (ii) the BHF Cambridge Centre of Research Excellence, (iii) Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust and (iv) Papworth Hospital NHS Foundation Trust, and supported the Cambridge NIHR BRC Cell Phenotyping Hub. MLO is funded by a BHF Intermediate Fellowship. FNK is funded by a BHF PhD Studentship.

Um esquema do protocolo de geração BOEC é mostrado na Figura 1. 1. Sangue Coleta e centrifugação em gradiente de densidade <ol><li> Para cada doador, adicionar 3 ml de citrato de sódio para cada um dos dois tubos de centrífuga de 50 mL cónicos. Recolha de 60 ml de sangue por punção venosa e adicionar 30 ml a cada tubo. Inverta suavemente 2-3 vezes para misturar. Use tão pouco quanto 40 ml de sangue no protocolo, com volumes de citrato ajustados em conformidade. NOTA: É essencial que as amostras de sangue são processados ​​dentro de 2 horas da coleta. Processamento de sangue resulta em atraso de uma redução acentuada no rendimento de crescimento de colónias. </li><li> Preparar novos tubos cónicos contendo meio de centrifugação por gradiente de densidade primeiro invertendo o frasco várias vezes para misturar. Em um capuz estéril, adicione 15 ml de meio de centrifugação em gradiente de densidade por tubo de 50 ml (1 tubo para cada 10 ml de sangue, 6 tubos por doador). </li><li> Dilui-se o sangue 1: 1 com fosfato de Dulbecco-Buffered Saline (DPBS). Inclinar o tubo contendo o meio de centrifugação em gradiente de densidade para um ângulo de 20 ° com a superfície de trabalho. Usando uma pipeta de ajuda e uma pipeta serológica 25 ml, camada lentamente 21 ml de sangue diluído em cima do meio por adição gradual de sangue na parede do tubo inclinado. NOTA: Se o fizer, irá minimizar a mistura do sangue com a camada de gradiente de densidade e irá produzir um revestimento mais apertado Buffy, bem como um rendimento melhorado. </li><li> Centrifugar as amostras a 400 xg durante 35 min à temperatura ambiente com o acelerador e o travão de fora. </li><li> Durante este período de centrifugação, iniciar o revestimento de colagénio detalhados nos passos 2.1 - 2.4. Garantir que essas etapas foram concluídas antes de prosseguir para a etapa 3.1. </li></ol> 2. O colágeno Revestimento de T-75 Flask e Preparação de Meio de Cultura NOTA: Execute as seguintes etapas em uma capa de cultura de células. <ol><li> Prepara-se uma solução de colagénio / ml 50 ug por diluição da Type colagénio I em 10 ml de 0,02 M de ácido acético. Exemplo: Para estoque de colagénio a uma concentração de 4,05 mg / ml, adicionar 123,5 mL de colagénio de 10 ml de ácido acético 0,02 M. Suspensão de colagénio Filtrar em condições estéreis antes da utilização. </li><li> Usando uma pipeta serológica, adicionar 7,5 ml de solução de colagénio a um frasco de cultura de células T-75. Este volume dá 5 ug de colagénio / cm2 (ou 375 ug / balão). Revestimento balão durante 1 h à TA. </li><li> Prepara-se o meio de crescimento endotelial usado para geração BOEC suplementando o meio basal endotelial com os suplementos de factor de crescimento fornecidos pelo fabricante. Adicionar todos os suplementos, mas não adicionar soro. Para preparar 15 ml de meio de geração BOEC, adicionar 2,5 ml de soro de bovino fetal definido (FBS) a 12,5 ml de meio de crescimento endotelial contendo os suplementos de factor de crescimento. </li><li> Prossiga com as etapas descritas na Seção 3. Após o período de revestimento de 1 hora, aspirar a solução de colágeno e lavar o ácido acético residual por pipetagemem 10 ml de DPBS. Aspirar fora do DPBS e repita esta etapa de lavagem mais uma vez. Se a suspensão de células obtidas durante o passo 3.3 não está pronto para plaqueamento a este tempo, adicionar 5 ml de meio de geração BOEC ao frasco para manter o revestimento de colagénio à secagem. </li></ol> 3. Recolha e chapeamento de PBMNCs <ol><li> Após a centrifugação de gradiente de densidade descrito na etapa 1.4, cuidadosamente recolher a camada buffy coat usando uma pipeta de transferência de plástico estéril. Recolha de buffy coat deve produzir aproximadamente 20 ml de suspensão de células e no plasma de cada tubo. Evitar a transferência do meio de gradiente de densidade. </li><li> Dilui-se a suspensão de células mononucleares de 1: 1 em DPBS e invertido para misturar. Centrifuga-se a TA durante 20 min a 300 xg, com freio e do acelerador no máximo. </li><li> Após a centrifugação, aspirar o sobrenadante e ressuspender as células por adição de 1 ml de meio de geração BOEC a cada sedimento e pipetando para cima e para baixo várias vezes. Célula suspensões uma piscinad top-se volume total de 10 ml com o meio remanescente. </li><li> Para obter uma estimativa do número total de células, a amostra 10 ul de suspensão de células e diluir com 490 50x ul de solução de Turk, que irá lisar as células vermelhas do sangue. Contagem de uma amostra de 10 uL da suspensão de células diluídas utilizando um hemocitómetro. NOTA: 60 mL de sangue deve render cerca de 100-150 x 10 6 células brancas do sangue para o chapeamento. </li><li> Placa suspensão célula inteira em um único frasco T-75, revestidos com colagénio, topo-se o volume para 15 mL meio / balão e cultura a 37 ° C em atmosfera humidificada contendo um 5% de CO 2. Células plaqueadas neste momento representam passagem 0. NOTA: É possível congelar os PBMNCs isolado antes do isolamento BOEC, a fim de derivar os BOECs em um momento posterior, ou a fim de transportar os PBMNCs para um laboratório diferente onde os BOECs pode ser gerado. No entanto, é importante notar que PBMNCs congelação poderia reduzir a eficiência do isolamento BOEC. SEE secção 8 a seguir para o método. </li></ol> 4. Cultura de Células de longo prazo <ol><li> Mudança de meio a cada 2 dias, adicionando 15 ml de meio de geração BOEC fresco (mistura 5: 1 de meio de crescimento de células endoteliais e definido de FBS). Para fins de semana, as mudanças de meio pode ser retardada a cada 3 dias. NOTA: colônias conseqüência deve aparecer entre 7 e 14 dias de cultura. </li><li> Monitorar a garrafa de cultura BOEC nos dias 7-14 para o aparecimento de colônias conseqüência. Identificar colônias como grupos circulares de células que exibem uma morfologia de paralelepípedos clássico endotelial. </li><li> Uma vez que uma colónia ou colónias múltiplas são identificados no balão, continuar com as mudanças de meio e permitir que as colónias a crescer para aproximadamente 1000 a 2000 células por colónia antes Passaging. Determinar o número de células por colónia através de uma estimativa visual aproximada. NOTA: Como um guia, é habitual a passagem colônias conseqüência iniciais de uma semana após a identificação da primeira colônia conseqüência. </li> &lt;li&gt; células da passagem por enxaguamento frascos duas vezes com 10 ml de DPBS. Adicionar 5 ml de tripsina-EDTA 1X e incubar numa incubadora a 37 ° C durante 5 min. Após 5 min, neutralizar a tripsina com 10 mL de meio (contendo FBS) e trazer as células em suspensão com pipetagem repetida. </li><li> Centrifugar a suspensão a 300 xg durante 5 min, ressuspender em 15 ml de meio fresco e geração BOEC placa suspensão célula inteira para um novo frasco de T-75 (representando uma passagem, P1). Não é necessário o revestimento de colagénio. </li><li> Continuar as mudanças de meio, como descrito acima até que as células estão confluentes (cerca de 3-5 x 10 6 culas por frasco). Uma vez confluentes, as células de passagem tal como descrito na etapa 4.3. NOTA: A partir de passagem 2 em diante, as células já não necessitam de forma geração BOEC e podem ser cultivadas em meio de crescimento de células endoteliais e 10% de FBS inactivado pelo padrão de calor. Revestimento de colagénio pode ser usado como um passo opcional daqui para a frente, como não há evidência que sugira que a presença de colagénio pode aumentar a prolifer BOECtaxas ation. </li><li> Para passaging contínuo, placa de nada menos do que 750.000 células por balão T-75 como densidades celulares baixas podem fazer com que os BOECs para parar a proliferar. </li></ol> 5. Congelamento e descongelamento culturas BOEC <ol><li> Tripsinizar as células como descrito na secção 4.3 e centrifugar a 300 xg durante 5 min. Aspirar o sobrenadante e ressuspender em 10 ml de meio. Isto não requer meio de crescimento celular endotelial; DMEM com 10% de FBS padrão será suficiente. Recolher uma amostra de 10 uL da suspensão de células para realizar uma contagem de células manual usando um hemocitómetro. </li><li> Centrifugar novamente e ressuspender a 2x10 6 células / ml em meio de criopreservação em gelo (40% de DMEM com 50% de FBS e 10% de DMSO). Adicionar 0,5 ml (10 6 células) para cada frasco, frascos em um lugar isopropanol frio navio criopreservação de gelo e coloque em um freezer -80 ° C durante pelo menos 2 horas antes da transferência para nitrogênio líquido. Não deixar as células a -80 ° C durante mais de 24 horas. </li&gt; <li> Para descongelar células, adicionar 10 ml de meio de pré-aquecido a um tubo de centrífuga de 15 ml cônico. NOTA: Quando descongelando células passagem 1, descongelar em meio de crescimento celular endotelial suplementado com FBS a 20% definido para os primeiros 2 dias após a descongelação. Isto leva a um isolamento de células mais estável no futuro. </li><li> Remover as células a partir de azoto líquido e descongelamento num banho de água a 37 C ° com agitação suave até que apenas um pequeno cristal de gelo é deixado no frasco. Adicionar o conteúdo do frasco, gota a gota ao tubo de centrífuga cónico e girar a 300 xg durante 5 min. </li><li> Aspirar o sobrenadante e ressuspender o sedimento celular em 10 ml de EGM-2 mV + 10% de FBS. Adicionar a T-75 frasco e meio superior até 15 ml. </li></ol> 6. Caracterização de BOECs por citometria de fluxo <ol><li> Uma vez que as colônias BOEC descritos na Seção 4 foram autorizados a se expandir, Trypsinize células como descrito na secção 4.4 e ressuspender a uma concentração de 10 6 células por ml em tampão de coloração (DPBS sagacidadeh 2% de FBS e 2 mM de EDTA). </li><li> Combine 10 5 células com anticorpos de fluorocromo conjugado dirigidos contra CD45, CD14, CD34, CD31 (use toda a diluição 1:20) ou VEGFR2 (diluir 1:10) (ver Tabela 1 para mais detalhes). Incubar durante 30 min a 4 ° C no escuro. </li><li> Lavar as células com 1 ml de tampão e centrifuga-se a coloração a 300 xg durante 5 min. Aspirar o sobrenadante e ressuspender em 400 ul de tampão de coloração para análise. Manter as células a 4 ° C e protegidas da luz antes da análise. </li><li> Realizar a análise de citometria de um citómetro equipado para detectar os anticorpos conjugados fluorescentes utilizadas no ensaio. </li><li> Usar uma amostra BOEC não corado para identificar a população de células no citómetro ajustando para a frente e tensões de dispersão lateral, assim como as tensões para os canais FITC e APC. </li><li> Determinar gating limiares utilizando células Isótipo manchado para cada fluorocromo. Avaliar a positividade para cada marcador da superfície celular com base na presence de um deslocamento de pico na intensidade de fluorescência versus controlo do isotipo para o fluorocromo a ser analisado. </li></ol> 7. Caracterização de BOECs por microscopia imunofluorescente <ol><li> BOECs placa numa cavidades 24, do tecido placa de cultura de fundo plano, sem lamelas a uma densidade de 5 x 10 4 células em 500 ul de meio de crescimento de células endoteliais + 10% de FBS por poço e deixar a aderir e crescer a 37 inactivado por calor ° C, 5% de CO 2 até que as células cobrir, pelo menos, 70-80% da área de superfície de cada poço (estimativa de confluência por inspecção visual sob uma luz microsope). </li><li> Lave as células em cada poço durante 5 minutos com 1 ml de DPBS. </li><li> Fix células o / n a 4 ° C com 500 ul de solução de paraformaldeído a 4% em DPBS. </li><li> Lave as células durante 3 x 5 min com 1 ml de DPBS por poço. Adicionar e remover os DPBS usando uma pipeta e um aspirador, respectivamente. </li><li> Permeabilizar as células durante 3 x 10 min, com 0,2% de polissorbato 20 em DPBS. </li&gt; <li> Incubar as células durante 1 h à TA em tampão contendo 10% de FBS em DPBS bloqueio. </li><li> Células da mancha na 4 ° CO / N em 300 ul de tampão de diluição de anticorpo (0,1% de albumina de soro bovino em DPBS) contendo anticorpos primários dirigidos contra CD34 (10 ug / ml), CD144 (1: 300) ou vWF (1: 250) . Ver Tabela 1 para obter mais detalhes. </li><li> Lavar anticorpos primários não ligados para 5 x 10 min com DPBS. </li><li> Incubar as células durante 1 h à temperatura ambiente com o anticorpo secundário fluorescente marcado adequado, como detalhado na Tabela 1 (todos a 1: 200). </li><li> Lavar anticorpos secundários não ligadas por 5 x 10 min com DPBS. </li><li> Incubar as células com 1 ug / ml de 4 &#39;, 6-diamidino-2-fenilindol (DAPI) em DPBS durante 10 min à TA. </li><li> Lavar as células com DPBS durante mais 3 x 5 min. </li><li> Células de imagens em 100x, utilizando um microscópio invertido equipado com uma fonte de luz externa para excitação de fluorescência, e capturar imagens usando uma anexaçãocâmera digital para posterior análise ed offline. </li></ol> 8. congelamento e descongelamento PBMNCs <ol><li> No final da etapa 3.2, após a centrifugação, aspirar o meio e interromper manualmente o sedimento celular com pipetagem repetida cima e para baixo utilizando uma ponta de pipeta P1000. Adicionar 1 ml de meio de congelação, 90% de soro e 10% de DMSO, certificando-se a agitar suavemente para produzir uma suspensão de células. Transferir a suspensão celular para um cryovial e congelar e descongelar conforme descrito na Seção 5. </li></ol>

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The authors have nothing to disclose.

We present a detailed protocol that allows for the robust and efficient derivation of BOECs from adult peripheral blood mononuclear cells (PBMNCs). Our protocol includes two important refinements that represent advances on previous methods of BOEC isolation.14-16 These include the absence of heparin in the initial PBMNC culture medium and the use of defined, embryonic stem cell-qualified serum. This latter refinement is of particular importance. Embryonic stem cell (ESC)-qualified serum is a more consistent grade of serum and, although it is not known yet what component(s) are enriched in the serum that benefit BOEC isolation, the impact of this defined serum on the efficiency of BOEC generation is clear in our hands. In addition, we have also had success in isolating BOECs using human serum, thereby allowing for the generation of BOECs for clinical translation. In our hands, this refined protocol results in the successful isolation of stable BOEC cultures from greater than 90% of donors, making it one of the most reliable BOEC generation methods reported thus far. Although the use of particular sera is critical to BOEC generation, it also represents a primary limitation of the current protocol. Future improvements to the technique could include the generation of these cells in serum-free, defined culture conditions.
Critical Steps in the protocol include processing blood samples as soon as possible after collection, complete harvesting of the buffy coat cells after density gradient centrifugation and the timely passaging of initial colonies from P0 to P1. This passaging step is critical to establishment of a stable isolation. Like other endothelial cells, BOECs appear to be very sensitive to plating density. If the plating density after passaging is too low, the BOECs will not proliferate. Conversely, if the colonies are allowed to become overconfluent before passaging, the cells will also cease to proliferate and have the tendency to convert into an elongated, mesenchymal cell phenotype. If few colonies appear from days 7 to 14, or if the colonies are small in size, troubleshooting can include increasing cell density by passaging P0 colonies into a T-25 flask instead of a T-75.
Once the technique is mastered, the resultant BOECs can be used in several applications, including in vitro studies of endothelial cell biology, disease modeling and drug screening, as well as in vivo cell transplantation therapies. An important consideration for the development of any cell therapy process is to use cells that are free from pathogenic mutations. We have previously shown that BOECs isolated using our protocol possess genomes that are free from copy number variations and are thus representative of the individual from which they were collected. In addition, we have also demonstrated that the majority of BOEC-derived iPSC lines are free from copy number variations.13 This contrasts with previous reports of copy number variation in fibroblast-derived iPSCs. To date, these cells remain the only iPSCs for which this degree of genomic fidelity has been reported. This feature is important for the field of iPSC biology and the use of iPSCs in disease modeling, drug screening and future cell transplantation therapies.

Até recentemente, acreditava a geração pós-natal de novos vasos sanguíneos a ocorrer exclusivamente através de um processo conhecido como angiogênese, definida como o surgimento de novos vasos a partir das células endoteliais dos vasos pré-existentes. 1 Este processo contrasta da vasculogênese, ou a formação de novo de vasos sanguíneos a partir de células progenitoras endoteliais, o que foi pensado para ocorrer exclusivamente durante a embriogénese. 2 No entanto, estudos mais recentes foram identificados e isolados circulantes células progenitoras endoteliais (CPE) no sangue periférico de adultos. Estas células possuem a capacidade de se diferenciarem em células endoteliais maduras em cultura e são acreditados para participar na vasculogénese pós-natal. 3,4 Os protocolos para o isolamento e expansão destas EPCs envolvem tipicamente a cultura de células mononucleares de sangue periférico (PBMNCs) em meio contendo factores de crescimento endoteliais, incluindo Vasculfactor de crescimento endotelial AR (VEGF) e factor de crescimento de fibroblastos-2. 5-8 culturas EPC produzir uma variedade de tipos de células diferentes dramaticamente. Culturas iniciais (&lt;7 dias) são dominadas por um tipo de célula monocítica, conhecido na literatura como &quot;precoces&quot; EPCs. Apesar do seu nome, estas células expressam o CD14 marcador de monócitos, são negativas para CD34 marcador de células progenitoras e expressar apenas níveis mínimos dos marcadores CD31 endotelial clássica e VEGF receptor 2 (VEGFR2). 5 cultura contínua dá origem a uma população secundária de células, conhecido como EPCs final excrescência ou células endoteliais conseqüência sangue (BOECs), que aparecem como colónias discretas de células endoteliais-like. Ao contrário das primeiras EPCs monocíticas, BOECs, que também tem sido chamado de formação de colónias de células endoteliais (ECFCs), células endoteliais ou células endoteliais excrescência de fim de crescimento, exibem a morfologia paralelepípedos que é típico de monocamadas de células endoteliais e são altamente semelhantes em marcador de superfície5 e 9, a expressão do gene para amadurecer as células endoteliais. A geração de células endoteliais semelhantes a partir de sangue periférico oferece várias vantagens, particularmente para o estudo da disfunção das células endoteliais associadas com desordens vasculares, tais como hipertensão arterial pulmonar (HAP) 10 ou de von Willebrand doença. 11 Antes da disponibilidade de BOECs, endotelial células só pode ser derivada a partir de órgãos explantados no momento da morte ou transplante de órgãos, ou isolado a partir da veia umbilical ao nascimento. Esta disponibilidade reduzida representada uma limitação séria para a compreensão da biologia das células endoteliais a partir de pacientes com doenças cardiovasculares, assim como as interacções entre as células endoteliais e as células do sangue ou quer células murais. Além disso, o isolamento e a cultura de uma população pura de células endoteliais a partir destas fontes é tecnicamente exigente e as células obtidas por estes métodos apresentam apenas uma LIMITEd capacidade proliferativa. BOECs portanto oferecer um substituto valioso para o isolamento e cultura de células endoteliais primárias derivadas do paciente. Para além das suas aplicações in vitro, BOECs também são potencialmente úteis em terapias de transplante de células autólogas. Estas aplicações incluem tanto o transplante de células endoteliais para promover neovascularização (ver 12 e referências ai), bem como a geração de células estaminais pluripotentes induzidas (iPSCs). 13 iPSCs BOEC-derivados pode ser usada para modelar doenças e oferecer imenso potencial como o ponto de partida material para terapias com células autólogas. BOECs reprogramar mais rápido e com uma maior eficiência do que os fibroblastos da pele. Além disso, também permitem que BOECs para a geração de iPSCs que estão livres de anomalias karyotypic, o que é uma característica essencial de qualquer tecnologia que será adequado para aplicações de translação. A capacidade para gerar iPSCs de uma amostra de sangue de um pacientelso elimina a necessidade de uma biópsia da pele e a geração de fibroblastos da pele, facilitando assim a geração de células de pacientes com distúrbios cicatrização de feridas, ou os muito jovens. O protocolo detalhado abaixo, aprovado pelo e conduzida de acordo com as orientações do Comitê de Serviço de Ética em Pesquisa Nacional (leste da Inglaterra), fornece um método simples e confiável para a geração de BOECs com rendimento superior a 90% de um volume relativamente pequeno (60 ml) de sangue periférico. Estas células são altamente proliferativas e podem ser passadas várias vezes, permitindo a geração de centenas de milhões de células a partir de uma única amostra de sangue.

<table>
	<tbody>
		<tr>
			<td colspan="4">For blood collection</td>
		</tr>
		<tr>
			<td>60 mL syringe with luer-lok tip</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>19G Surflo Winged Infusion Set</td>
			<td>Terumo</td>
			<td>SV-19BL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>StarLab</td>
			<td>E1450</td>
			<td>2 per donor</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Martindale Pharmaceuticals</td>
			<td>270541</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For buffy coat isolation</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile wrapped plastic transfer pipettes</td>
			<td>Appleton Woods</td>
			<td>KC231</td>
			<td></td>
		</tr>
		<tr>
			<td>Turk&#8217;s Solution</td>
			<td>Millipore</td>
			<td>1.093E+09</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For cell culture, passaging and freezing cells</td>
		</tr>
		<tr>
			<td>Type 1 Collagen (derived from rat tail)</td>
			<td>BD Biosciences</td>
			<td>35-4236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.02M Acetic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>prepared in reagent grade water</td>
		</tr>
		<tr>
			<td>Endothelial Growth Medium-2MV 
			(containing Bullet Kit, but not serum)</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Note: It is essential that the medium does not contain heparin.&#160;&#160; 
			Do not use EGM-2.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (U.S.), Defined</td>
			<td>Hyclone</td>
			<td>SH30070</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Trypsin EDTA</td>
			<td>Gibco</td>
			<td>T4174</td>
			<td>Dilute to 1x in PBS prior to use</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>C1562</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For flow cytometric characterization</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD14</td>
			<td>BD Biosciences</td>
			<td>555397</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD31</td>
			<td>BD Biosciences</td>
			<td>555445</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-human CD34</td>
			<td>BD Biosciences</td>
			<td>555824</td>
			<td>Mouse IgG1k, Clone: 581/CD34 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>560976</td>
			<td>Mouse IgG1k, Clone: HI30 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>&#160;APC-conjugated mouse anti-human VEGFR2</td>
			<td>R&#38;D Systems</td>
			<td>FAB357A</td>
			<td>Mouse IgG1, Clone: 89106 
			Dilution: 1:10</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555751</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>R&#38;D Systems</td>
			<td>IC002A 
			Dilution: 1:10</td>
			<td>Clone: 11711</td>
		</tr>
		<tr>
			<td>EDTA, 0.5M solution</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For immunofluorescent microscopy&#160;</td>
		</tr>
		<tr>
			<td>Corning Costar 24-well tissue culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD34 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB72271</td>
			<td>Clone 756510, IgG1, use at 10 &#956;g/ml</td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-human VE-cadherin (CD144)</td>
			<td>R&#38;D Systems</td>
			<td>AF938</td>
			<td>Antigen affinity- purified IgG, use at 1:300</td>
		</tr>
		<tr>
			<td>Monoclonal rabbit anti-human Von Willebrand Factor (vWF)</td>
			<td>Abcam</td>
			<td>ab154193</td>
			<td>Clone EPSISR15, use at 1:250</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
			<td>Polyclonal, 2 mg/ml, use at 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11055</td>
			<td>Polyclonal, 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 568 conjugate</td>
			<td>Life Technologies</td>
			<td>A-10042</td>
			<td>Polyclonal,&#160; 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use at 1 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

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.

Isolamento de buffy coat células mononucleares a partir de 60 ml de sangue normalmente produz 100-150x10 6 glóbulos brancos totais. Quando plaqueadas em um único frasco T-75, o grande número de células na suspensão de células não lisadas torna difícil de resolver as células aderentes utilizando microscopia de campo claro individuais. Alterações médias repetidas resultar na depuração de células não-aderentes e permitir a visualização da população aderente de monocítica &quot;precoces&quot; EPCs. No dia 7, o T-75 conterá aproximadamente 7x10 6 destas células aderentes alongados. A partir do dia 7 e 14, as colónias de BOECs deve aparecer dentro do frasco (Figura 2A). Colônias primeiro aparecem como 3 a 5 células adjacentes. Número de colónias excrescência pode variar de 1 a 10 colónias por 60 ml de sangue. Em geral, os doadores mais jovens (isto é, 20-25 anos de idade) tendem a produzir um maior número de colónias de crescimento, que também aparecem anteriormente na cultura.BOECs proliferam a partir deste grupo central de células para formar colónias circulares consistindo de várias centenas de células. As células dentro dessas colônias apresentam uma morfologia clássico cobblestone endotelial. É preferível a passagem colónias iniciais quando eles contêm aproximadamente 1000 células por colónia. Colónias excrescência tipicamente atingir este tamanho de 7 dias após a sua aparência original na cultura. Uma vez que as colónias originais são passadas para um novo frasco T-75, eles devem proliferam para formar uma monocamada confluente (3-5x10 6 células por frasco) em menos de 5 dias ou menos (Figura 2B). Em uma pequena porcentagem de isolamentos (&lt;10%), colônias conseqüência não aparecem, ou não proliferam suficientemente seguindo este passo inicial passaging. BOECs que apresentam baixo proliferação após a sua primeira passaging raramente passam a se tornar isolamentos BOEC estáveis ​​e muitas vezes param de proliferar dentro de dois a três passagens. O monocyticEPCs primeiros contidos dentro das culturas BOEC são não-proliferativa e são tipicamente apuradas a partir das culturas num período de 1-2 passagens. Liquidação destas células precoces é devido à morte das células, a insuficiência das células iniciais para re-aderir depois passaging e diluição dos primeiros EPCs não-proliferativas com passagens repetidas. A passagem 1 células podem ser passadas mais em cultura congelada ou para baixo para uso posterior. Uma vez que o isolamento estável é gerado, as células podem ser passadas a uma taxa de 1 frasco confluente de 3-5 novos frascos T-75 até passagem 8 ou 9, antes de se tornar quiescente. Mais uma vez, a densidade celular é crítica ao longo de cultura. Em nossa experiência, nada menos do que 750.000 células devem ser colocadas em um frasco T-75, como densidades celulares mais baixas podem causar a paragem do crescimento. Apesar do alto potencial proliferativo de BOECs, as células também não deve ser permitido tornar-se overconfluent (ie.,&gt; 5x10 6 células por frasco), pois isso pode causar convers<html> ião de BOECs para um fenótipo não-proliferativa. Propomos que qualquer célula que está sendo rotulado como um BOEC deve exibir superfície celular apropriada e coloração intracelular para marcadores endoteliais. Caracterização de BOECs pode ser conseguido por citometria de fluxo (Figura 3) ou microscopia de fluorescência (Figura 4), ​​após coloração para marcadores de células endoteliais típicos. BOECs são positivos para o CD31 marcadores de superfície endotelial e VEGFR2 e são negativos para o marcador CD14 monócitos e pan-leucócitos CD45 marcador. BOECs também posess corpos de Weibel-Palade e, assim, expressar fator de von Willebrand (vWF) como, coloração citoplasmática pontuada discreta. Ao contrário de outros tipos de células endoteliais maduras, tais como a artéria pulmonar ou células endoteliais aórticas, BOECs também expressam o marcador de células progenitoras e de activação CD34 na sua superfície. ftp_upload / 53384 / 53384fig1.jpg &quot;/&gt; Figura 1. Diagrama esquemático do protocolo de geração BOEC. As células mononucleares de sangue periférico são isolados a partir de sangue venoso por centrifugação com gradiente de densidade e cultivadas em placas revestidas com colagénio em meio de crescimento endotelial contendo FBS definido. Colónias BOEC aparecer dentro de 7-14 dias de cultura. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53384/53384fig2.jpg" /> Figura 2. Imagens de campo claro de culturas BOEC representativas. (A) colônias conseqüência aparecer nas culturas entre os dias 7 e 14. As colónias apresentam como colecções de células endoteliais-like, que são dispostos em uma monocamada de paralelepípedos e proliferam radialmente para fora a partir de um ponto central. Ao redor das colônias conseqüência são os mono aderentecytic células que constituem a grande maioria das células em culturas iniciais. Estas células, previamente descritos como &quot;precoce&quot; células progenitoras endoteliais, têm uma morfologia fusiforme e expressar o marcador CD14 monocytic. (B) Na sequência passaging, os BOECs altamente proliferativas assumir culturas, como as células monocíticas não-proliferativa quer morrer ou deixar de voltar a ligar depois passaging. Scale bar 250 um. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53384/53384fig3.jpg" /> Figura 3. Caracterização de BOECs por citometria de fluxo. BOECs foram tripsinizadas e marcadas com anticorpos de controlo de isotipo conjugados com fluorescência (Gray pico a cheio) ou de anticorpos dirigidos contra marcadores de superfície específicos (linha vermelha). Marcadores de superfície para a caracterização de citometriaincluem os marcadores hematopoiéticas CD45 e CD14, os marcadores endoteliais CD31 e VEGFR2 eo progentior e marcador de ativação endotelial CD34. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53384/53384fig4.jpg" /> Figura 4. Immunofluorescent coloração de BOECs para marcadores de células endoteliais. Imagens de imunofluorescência representativos de BOECs imunocoradas com anticorpos dirigidos contra marcador da superfície das células endoteliais CD144 (VE-caderina, o painel superior direito) e da glicoproteína sangue Fator de von Willebrand (vWF, painel inferior direito) . Painéis correspondentes que mostram a coloração DAPI nuclear estão apresentados à esquerda. Scale bar 50 um. para CD144. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig4large.jpg" target="_blank">Por favor clique aqui para ver um maior</a>versão desta figura.

Watch this Scientific Journal Video about Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood at JoVE.com

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

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 ...

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18.8K visualizações.  University of Cambridge. 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.

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

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Enriquecimento das células NK do sangue humano com o Kit RosetteSep de StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Biologia

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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, ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Purificação e microRNA Profiling de Exosomes derivados do sangue e Meios de Cultura

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Eficiente Geração células iPS a partir de sangue, utilizando epissomas e HDAC Inibidores

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Dois fótons de imagem de cálcio intracelular<sup&gt; 2+</sup&gt; Manuseio e produção de óxido nítrico no endotélio e músculo liso células de uma aorta de rato isolada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Proteínas Análise de imunofluorescência de endógenos e exógenos Centromere-cinetócoro

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

Isolamento e caracterização de microvesículas a partir de sangue periférico

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Um ensaio de cisalhamento uniforme para receptores de superfície de células e plaquetas humanas através de Viscometry de cone-Plate

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...