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.

A esquemática del protocolo de generación BOEC se muestra en la Figura 1. 1. Extracción de Sangre y gradiente de densidad de centrifugación <ol><li> Para cada donante, añadir 3 ml de citrato de sodio a cada uno de dos tubos de centrífuga de 50 ml cónicos. Recoger 60 ml de sangre por punción venosa y añadir 30 ml a cada tubo. Invierta suavemente 2-3 veces para mezclar. Utilice tan sólo 40 ml de sangre en el protocolo, con volúmenes de citrato ajustados en consecuencia. NOTA: Es esencial que las muestras de sangre se procesan dentro de 2 horas de la recolección. Retraso en el procesamiento de los resultados de sangre en una marcada reducción en el rendimiento colonia consecuencia. </li><li> Preparar nuevos tubos cónicos que contienen medio de centrifugación en gradiente de densidad por primera invirtiendo el frasco varias veces para mezclar. En una campana estéril, añadir 15 ml de medio de centrifugación en gradiente de densidad por cada 50 ml tubo cónico (1 tubo por cada 10 ml de sangre, 6 tubos por donante). </li><li> Diluir la sangre 1: 1 con fosfato de DulbeccoSaline -Buffered (DPBS). Incline el tubo que contiene el medio de centrifugación en gradiente de densidad a un ángulo de 20 ° con la superficie de trabajo. El uso de una ayuda de la pipeta y una pipeta serológica de 25 ml, la capa lentamente 21 ml de sangre diluida en la parte superior del medio mediante la adición gradual de sangre por la pared del tubo inclinado. NOTA: Si lo hace, minimizar la mezcla de la sangre con la capa de gradiente de densidad y producirá una capa leucocitaria más apretado, así como un rendimiento mejorado. </li><li> Centrifugar las muestras a 400 xg durante 35 min a TA con el acelerador y el freno. </li><li> Durante este período de centrifugación, comenzará el recubrimiento de colágeno se detalla en los pasos 2.1 - 2.4. Asegúrese de que estos pasos se han completado antes de proceder al paso 3.1. </li></ol> 2. El colágeno Recubrimiento de T-75 Frasco y Preparación del medio de cultivo NOTA: Realice los siguientes pasos en una campana de cultivo celular. <ol><li> Preparar una solución de colágeno / ml 50 mg diluyendo Stock TyPE I colágeno en 10 ml de ácido acético 0,02 M Ejemplo:. para el colágeno de stock a una concentración de 4,05 mg / ml, añadir 123,5 l de colágeno a 10 ml de ácido acético 0,02 M. Filtro estéril suspensión de colágeno antes de su uso. </li><li> Utilizando una pipeta serológica, añadir 7,5 ml de solución de colágeno a un matraz de cultivo de células T-75. Este volumen da 5 mg de colágeno / cm2 (o 375 mg / frasco). Escudo matraz durante 1 hora a RT. </li><li> Preparar el medio de crecimiento endotelial utilizado para la generación BOEC completándolo medio basal endotelial con los suplementos de factores de crecimiento proporcionadas por el fabricante. Añadir todos los suplementos, pero no agregue suero. Para preparar 15 ml de medio de generación BOEC, añadir 2,5 ml de suero bovino fetal definido (FBS) a 12,5 ml de medio de crecimiento endotelial que contiene los suplementos de factores de crecimiento. </li><li> Continúe con los pasos descritos en la sección 3. Después del período de recubrimiento 1 hr, aspirar la solución de colágeno y lava el ácido acético residual mediante pipeteoen 10 ml de DPBS. Aspirar el DPBS y repita este paso de lavado una vez más. Si la suspensión celular obtenida durante el paso 3.3 no está listo para el recubrimiento en este momento, añadir 5 ml de medio de generación BOEC al matraz para mantener el revestimiento de colágeno a partir de secado-out. </li></ol> 3. Recogida y Chapado de PBMNCs <ol><li> Después de la centrifugación en gradiente de densidad se indica en el paso 1.4, recoger cuidadosamente la capa leucocitaria usando una pipeta de transferencia de plástico estéril. Colección de la capa leucocitaria debe producir aproximadamente 20 ml de suspensión de células y plasma de cada tubo. Evitar la transferencia de el medio de gradiente de densidad. </li><li> Diluir la suspensión de células mononucleares 1: 1 en DPBS e invierta para mezclar. Centrifugar a TA durante 20 min a 300 xg con freno y acelerador al máximo. </li><li> Después de la centrifugación, aspirar las células sobrenadante y resuspender mediante la adición de 1 ml de medio de generación BOEC a cada pellet y pipeteando arriba y abajo varias veces. Celular piscina suspensiones und reponer volumen total de 10 ml con el medio restante. </li><li> Para obtener una estimación del número total de células, muestra de 10 l de suspensión celular y se diluye 50x con 490 l de solución de Turk, que se lisar células rojas de la sangre. Contar una muestra de 10 l de la suspensión celular diluida usando un hemocitómetro. NOTA: 60 ml de sangre debe producir aproximadamente 100 a 150 x 10 6 células blancas de la sangre para la siembra. </li><li> Placa suspensión celular entera en una sola, T-75 frasco de colágeno recubierto, top-up volumen medio a 15 ml / frasco y la cultura a 37 ° C en un ambiente humidificado contiene 5% de CO 2. Las células chapados en este momento representan el paso 0. NOTA: Es posible congelar las PBMNCs aislados antes del aislamiento BOEC con el fin de derivar las BOECs en un momento posterior o con el fin de transportar los PBMNCs a un laboratorio diferente, donde los BOECs se pueden generar. Sin embargo, es importante señalar que PBMNCs congelación podrían reducir la eficiencia de aislamiento BOEC. See sección 8 a continuación para el método. </li></ol> Cultivo Celular 4. A largo plazo <ol><li> Cambio de medio cada 2 días mediante la adición de 15 ml de medio de generación BOEC fresco (5: 1 mezcla de medio de crecimiento de células endoteliales y definido FBS). Para fines de semana, cambios de medio pueden retrasarse a cada 3 días. NOTA: colonias consecuencia deben aparecer entre los 7 y 14 días de cultivo. </li><li> Supervisar el frasco de cultivo BOEC en los días 7-14 de la aparición de colonias excrecencia. Identificar las colonias como grupos circulares de células que presentan un clásico morfología de adoquines endotelial. </li><li> Una vez que una colonia o múltiples colonias se identifican en el matraz, continúe con cambios de medio y permitir que las colonias crezcan hasta aproximadamente 1.000 a 2.000 células por colonia antes de pases. Determinar el número de células por colonia a través de una estimación visual aproximada. NOTA: Como guía, se acostumbra a paso colonias excrecencia iniciales Una semana después de la identificación de la primera colonia consecuencia. </li> &lt;li&gt; células paso por aclarado matraces dos veces con 10 ml de DPBS. Añadir 5 ml de 1X tripsina-EDTA y se incuban en una incubadora a 37 ° C durante 5 min. Después de 5 min, neutralizar la tripsina con 10 ml de medio (que contiene FBS) y llevar las células en suspensión con pipeteo repetido. </li><li> Suspensión Centrifugar a 300 xg durante 5 min, se resuspende en 15 ml de medio fresco y generación BOEC placa entera suspensión celular a un nuevo matraz T-75 (que representa el paso 1, P1). No se requiere ningún recubrimiento de colágeno. </li><li> Continuar cambios de medio como se describe más arriba hasta que las células son confluentes (aproximadamente 3-5 x 10 6 células por frasco). Una vez confluentes, las células de paso como se ha descrito en el paso 4.3. NOTA: Desde el paso 2 en adelante, las células ya no requieren la generación de medio BOEC y puede ser cultivado en un medio de crecimiento de células endoteliales y 10% estándar FBS inactivado por calor. Revestimiento de colágeno puede ser utilizado como un paso opcional en el futuro, ya que no hay evidencia que sugiere que la presencia de colágeno puede mejorar prolifer BOEClas tasas de inflación. </li><li> Para pases continuación, la placa no menos de 750.000 células por matraz T-75 como bajas densidades celulares pueden causar los BOECs para detener la proliferación. </li></ol> 5. La congelación y descongelación Culturas BOEC <ol><li> Trypsinize células como se describe en la sección 4.3 y se centrifuga a 300 g durante 5 min. Aspirar el sobrenadante y resuspender en 10 ml de medio. Esto no requiere un medio de crecimiento de células endoteliales; DMEM con 10% FBS estándar será suficiente. Recoger una muestra de 10 l de la suspensión de células para llevar a cabo un recuento de células manual utilizando un hemocitómetro. </li><li> Centrifugar de nuevo y resuspender en 2x10 6 células / ml en medio de criopreservación enfriado con hielo (40% de DMEM con 50% de FBS y 10% de DMSO). Añadir 0,5 ml (10 6 células) a cada vial, lugar viales en un hielo isopropanol frío recipiente de crioconservación y colocar en un congelador a -80ºC durante al menos 2 horas antes de transferir a nitrógeno líquido. No deje las células a -80 ° C durante más de 24 horas. </li&gt; <li> Para descongelar las células, añadir 10 ml de medio precalentado a un tubo de centrífuga cónico de 15 ml. NOTA: Al descongelar a cabo el paso 1 células, descongelar en un medio de crecimiento de células endoteliales suplementado con 20% FBS definido por los primeros 2 días después de la descongelación. Esto conduce a un aislamiento de células más estable en el futuro. </li><li> Extraer células del nitrógeno líquido y descongelación en un 37 ° C baño de agua con agitación suave hasta que sólo un pequeño cristal de hielo se deja en el vial. Añadir el contenido del vial gota a gota al tubo de centrífuga cónico y centrifugar a 300 xg durante 5 min. </li><li> Aspirar el sobrenadante y resuspender el sedimento celular en 10 ml de EGM-2MV + 10% de FBS. Añadir a T-75 frasco y medio superior hasta 15 ml. </li></ol> 6. Caracterización de BOECs por Citometría de Flujo <ol><li> Una vez que las colonias BOEC descritos en la sección 4 se les ha permitido ampliar, trypsinize células como se describe en la sección 4.4 y resuspender en una concentración de 10 6 células por ml en tampón de tinción (DPBS ingenioh 2% de FBS y EDTA 2 mM). </li><li> Combine 10 5 células con anticuerpos conjugados con fluorocromo dirigidos contra CD45, CD14, CD34, CD31 (uso todos a la dilución 1:20) o VEGFR2 (diluir 1:10) (ver Tabla 1 para más detalles). Incubar durante 30 minutos a 4 ° C en la oscuridad. </li><li> Lavar las células con 1 ml de tampón de tinción y se centrifuga a 300 xg durante 5 min. Sobrenadante Aspirar y resuspender en 400 l de tampón de tinción para el análisis. Mantenga las células a 4 ° C y protegido de la luz antes del análisis. </li><li> Realizar análisis de citometría de un citómetro equipada en detectar los anticuerpos conjugados con fluorescencia utilizados en el ensayo. </li><li> Utilice una muestra BOEC sin teñir para identificar la población de células en el citómetro mediante el ajuste hacia delante y voltajes de dispersión lateral, así como los voltajes para los canales de FITC y APC. </li><li> Determinar gating umbrales utilizando células isotipo manchado para cada fluorocromo. Evaluar positividad para cada marcador de superficie celular basado en la presencestá analizando e de un cambio máximo en la intensidad de fluorescencia frente al control de isotipo para el fluorocromo. </li></ol> 7. Caracterización de BOECs por Microscopía de inmunofluorescencia <ol><li> BOECs placa en una pocillos 24, placa de cultivo tisular de fondo plano sin cubreobjetos a una densidad de 5 x 10 4 células en 500 l de medio de crecimiento de células endoteliales + 10% de FBS por pocillo y se dejó a adherirse y crecer a 37 inactivado por calor ° C, 5% de CO 2 hasta que las células cubrir al menos el 70-80% de la superficie de cada pocillo (estimación de confluencia mediante inspección visual bajo un Microscopio de luz). </li><li> Lavar las células en cada pocillo durante 5 min con 1 ml de DPBS. </li><li> Fijar las células O / N a 4 ° C con 500 l de solución de paraformaldehído al 4% en DPBS. </li><li> Lavar las células de 3 x 5 min con 1 ml de DPBS por pocillo. Añadir y eliminar los DPBS utilizando una pipeta y un aspirador, respectivamente. </li><li> Permeabilizar las células para 3 x 10 min con 0,2% de polisorbato 20 en DPBS. </li&gt; <li> Se incuban las células durante 1 hora a RT en tampón de bloqueo que contiene 10% de FBS en DPBS. </li><li> Células de la mancha a 4 ° CO / N en 300 l de tampón de dilución de anticuerpo (0,1% de albúmina de suero bovino en DPBS) que contiene anticuerpos primarios dirigidos contra CD34 (10 mg / ml), CD144 (1: 300) o vWF (1: 250) . Ver Tabla 1 para más detalles. </li><li> Lavar anticuerpos primarios no consolidados para 5 x 10 min con DPBS. </li><li> Se incuban las células durante 1 hora a RT con el anticuerpo secundario marcado con fluorescencia apropiada, como se detalla en la Tabla 1 (todos a 1: 200). </li><li> Lavar secundaria de anticuerpos no unidos para 5 x 10 min con DPBS. </li><li> Se incuban las células con 1 mg / ml 4 &#39;, 6-diamidino-2-fenilindol (DAPI) en DPBS durante 10 min a RT. </li><li> Lavar las células con DPBS durante otros 3 x 5 min. </li><li> Celdas de imagen a 100 aumentos utilizando un microscopio invertido equipado con una fuente de luz externa para la excitación de fluorescencia, y capturar imágenes utilizando un adjuntarcámara digital ed para su posterior análisis fuera de línea. </li></ol> 8. congelación y descongelación PBMNCs <ol><li> Al final de la etapa 3.2, después de la centrifugación, aspirar el medio y alterar manualmente el sedimento celular con repetido pipeteado arriba y hacia abajo utilizando una punta de pipeta P1000. Añadir 1 ml de medio de congelación, 90% de suero y 10% de DMSO, asegurándose de mezclar suavemente para producir una suspensión celular. Transferir la suspensión celular a un criovial y congelar y descongelar como se describe en la Sección 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.

Hasta hace poco, se creía que la generación post-natal de nuevos vasos sanguíneos que se produzca exclusivamente a través de un proceso conocido como angiogénesis, definido como el surgimiento de nuevos vasos a partir de las células endoteliales de los vasos preexistentes. 1 Este proceso contrasta de la vasculogénesis, o la formación de novo de vasos sanguíneos a partir de progenitores endoteliales, que se cree que se producen exclusivamente durante la embriogénesis. 2 Sin embargo, estudios más recientes han identificado y aislado células progenitoras endoteliales circulantes (EPC) en la sangre periférica de adultos. Estas células poseen la capacidad de diferenciarse en células endoteliales maduras en cultivo y se cree que participar en la vasculogénesis postnatal. 3,4 Los protocolos para el aislamiento y expansión de estas EPCs típicamente involucran el cultivo de células mononucleares de sangre periférica (PBMNCs) en medios que contienen factores de crecimiento endoteliales, incluyendo vasculfactor de crecimiento endotelial ar (VEGF) y las culturas EPC factor de crecimiento fibroblástico-2 5-8. producen una variedad de diferentes tipos de células de manera espectacular. Culturas iniciales (&lt;7 días) están dominados por un tipo celular de monocitos, conocida en la literatura como &quot;primeros&quot; EPC. A pesar de su nombre, estas células expresan el marcador CD14 de monocitos, son negativos para el marcador CD34 progenitor y expresan niveles sólo mínimas de la clásica marcadores endoteliales CD31 y VEGF receptor 2 (VEGFR2). 5 cultura Continuación da lugar a una población secundaria de las células, conocido como EPC finales de excrecencia o células endoteliales excrecencia de sangre (BOECs), que aparecen como colonias discretas de células similares a las endoteliales. A diferencia de los principios de EPCs monocíticas, BOECs, que también han sido llamados formadoras de colonias (células endoteliales ECFCs), células endoteliales o células endoteliales excrecencia late-excrecencia, exhiben la morfología de adoquines que es típico de monocapas de células endoteliales y son muy similares en marcador de superficie5 y la expresión génica 9 a madurar las células endoteliales. La generación de células similares a las endoteliales de sangre periférica ofrece varias ventajas, en particular para el estudio de la disfunción de la célula endotelial asociada con trastornos vasculares tales como la hipertensión arterial pulmonar (HAP) 10 o von Willebrand enfermedad. 11 Antes de la disponibilidad de BOECs, endotelial células sólo se podrían derivar de órganos explantados en el momento de la muerte o el trasplante de órganos, o aislada de la vena umbilical en el nacimiento. Esta disponibilidad reducida representado una seria limitación para la comprensión de la biología de las células endoteliales de pacientes con trastornos cardiovasculares, así como las interacciones entre las células endoteliales y, o bien células de la sangre o células murales. Además, el aislamiento y el cultivo de una población pura de células endoteliales a partir de estas fuentes es técnicamente difícil y las células obtenidas por estos métodos exhiben solamente un limited capacidad proliferativa. Por lo tanto, BOECs ofrecen un sustituto valiosa para el aislamiento y cultivo de células endoteliales primarias derivadas del paciente. Además de sus aplicaciones in vitro, BOECs también son potencialmente útiles en terapias de trasplante de células autólogas. Estas aplicaciones incluyen tanto el trasplante de células endoteliales para promover la neovascularización (véase 12 y referencias en él), así como la generación de células madre pluripotentes inducidas (CMPI). 13 iPSCs derivados de BOEC se pueden utilizar para el modelado de la enfermedad y ofrecen un potencial inmenso como el de partida material para las terapias con células autólogas. BOECs reprogramar más rápido y con una mayor eficiencia que los fibroblastos de la piel. Además, BOECs también permite la generación de células iPS que están libres de anomalías del cariotipo, que es una característica esencial de cualquier tecnología que será adecuado para aplicaciones de traslación. La capacidad de generar iPSCs de una muestra de sangre de un pacientelso elimina la necesidad de una biopsia de piel y la generación de fibroblastos de la piel, facilitando de este modo la generación de células de pacientes con trastornos de la cicatrización de heridas, o los muy jóvenes. El protocolo se detalla a continuación, aprobado por y realizadas de conformidad con las directrices del Comité de Servicio de Ética de la Investigación Nacional (este de Inglaterra), proporciona un método sencillo y fiable para la generación de BOECs con una eficiencia superior al 90% a partir de un volumen relativamente pequeño (60 ml) de sangre periférica. Estas células son altamente proliferativa y pueden ser pasados ​​repetidamente, lo que permite la generación de cientos de millones de células de una única muestra de sangre.

<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.

Aislamiento de células mononucleares de la capa leucocitaria de 60 ml de sangre normalmente produce 100-150x10 6 células blancas de la sangre totales. Cuando chapada en un solo matraz T-75, el gran número de células en la suspensión celular no lisadas hace que sea difícil de resolver células adherentes mediante microscopía de campo claro individuales. Cambios de medio repetidas dan lugar a la liquidación de las células no adherentes y permiten la visualización de la población adherente de monocitos &quot;primeros&quot; EPC. Al día 7, el T-75 contendrá aproximadamente 7x10 6 de estas células adherentes alargados. Desde el día 7 al 14, las colonias de BOECs deben aparecer dentro del matraz (Figura 2A). Colonias primero aparecen como 3 a 5 células adyacentes. Número de colonias excrecencia puede variar de 1 a 10 colonias por 60 ml de sangre. En general, los donantes más jóvenes (es decir, 20-25 años de edad) tienden a producir un mayor número de colonias excrecencia, que también aparecen antes en la cultura.BOECs proliferan de este grupo central de las células para formar colonias circulares que consisten en varios cientos de células. Las células dentro de estas colonias presentan una clásica morfología de adoquines endotelial. Es preferible paso colonias iniciales cuando contienen aproximadamente 1.000 células por colonia. Colonias consecuencia suelen alcanzar este tamaño de 7 días después de su aspecto original en la cultura. Una vez que las colonias originales se pasan a un nuevo matraz T-75, deberían proliferar para formar una monocapa confluente (3-5x10 6 células por matraz) dentro de los 5 días o menos (Figura 2B). En un pequeño porcentaje de aislamientos (&lt;10%), las colonias excrecencia no aparecen o no proliferan suficientemente tras este paso inicial pases. BOECs que exhiben baja proliferación después de su primer pases rara vez van a convertirse en aislamientos BOEC estables ya menudo detener la proliferación en dos o tres pasajes. El monocíticaEPC principios contenidos en las culturas BOEC son no proliferativa y típicamente se borran de las culturas dentro de 1-2 pasajes. Liquidación de estas células tempranas se debe a la muerte celular, la insuficiencia de las células tempranas de volver a adherirse después de pases y la dilución de los principios de EPCs no proliferativas con pases repetidos. Células Passage 1 o bien se pueden pases más en la cultura o congelaron para su uso posterior. Una vez que se genera un aislamiento estable, las células pueden ser pasados ​​a una velocidad de 1 matraz confluente a 3-5 nuevos matraces T-75 hasta el paso 8 o 9 antes de convertirse en reposo. Una vez más, la densidad celular es crítica a través de la cultura. En nuestra experiencia, no menos de 750.000 células deben ser chapada en un matraz T-75, como densidades celulares más bajos puede causar la detención del crecimiento. A pesar del alto potencial proliferativo de BOECs, las células deben tampoco se les permitirá convertirse overconfluent (es decir.,&gt; 5x10 6 células por frasco) ya que esto puede causar convers<html> ion de BOECs a un fenotipo no proliferativa. Proponemos que cualquier célula está etiquetado como un BOEC debe mostrar superficie celular apropiado y tinción intracelular de marcadores endoteliales. Caracterización de BOECs se puede lograr mediante citometría de flujo (Figura 3) o microscopía de fluorescencia (Figura 4), ​​después de la tinción para los marcadores típicos de células endoteliales. BOECs son positivas para el CD31 marcadores de superficie endotelial y VEGFR2 y son negativos para el marcador CD14 de monocitos y el marcador CD45 pan-leucocitario. BOECs también posess cuerpos de Weibel-Palade y así expresar el factor de Von Willebrand (vWF) como tinción citoplasmática discreta, punteada. A diferencia de otros tipos de células endoteliales maduras, tales como la arteria pulmonar o células endoteliales aórticas, BOECs también expresan el progenitor y la activación marcador CD34 en su superficie. ftp_upload / 53384 / 53384fig1.jpg &quot;/&gt; Figura 1. Diagrama esquemático del protocolo de generación BOEC. Células mononucleares de sangre periférica son aisladas de sangre venosa mediante centrifugación en gradiente de densidad y se cultivaron en placas recubiertas con colágeno en medio de crecimiento endotelial con adición de FBS definidos. Colonias BOEC aparecen dentro de 7-14 días de cultivo. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig1large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53384/53384fig2.jpg" /> Figura 2. Imágenes campo claro de culturas BOEC representativos. (A) colonias consecuencia aparece en culturas entre los días 7 y 14. Las colonias se presentan como colecciones de células de tipo endotelial, que se disponen en una sola capa de adoquines y proliferan radialmente hacia fuera desde un punto central. Alrededor de las colonias excrecencia son el mono adherentecélulas cytic que componen la gran mayoría de las células en las primeras culturas. Estas células, previamente descritas como &quot;principios&quot; células progenitoras endoteliales, tienen una morfología de tipo huso y expresan el marcador CD14 monocitos. (B) A raíz de los pases, los BOECs altamente proliferativas hacerse cargo de las culturas, ya que las células monocíticas no proliferativas o bien mueren o dejan de volver a conectar después de pases. Barra de escala 250 micras. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="figura 3" src="/files/ftp_upload/53384/53384fig3.jpg" /> Figura 3. Caracterización de BOECs por citometría de flujo. BOECs se tripsinizaron y se tiñeron con anticuerpos de control de isotipo conjugados con fluorescencia (gris pico de llenado) o anticuerpos dirigidos contra marcadores de superficie específicos (línea roja). Marcadores de superficie para la caracterización de citometríaincluyen los marcadores hematopoyéticos CD45 y CD14, los marcadores endoteliales CD31 y VEGFR2 y la progentior y marcador de activación endotelial CD34. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53384/53384fig4.jpg" /> Figura 4. La tinción de inmunofluorescencia de BOECs para marcadores de células endoteliales. Inmunofluorescencia imágenes representativas de BOECs immunostained con anticuerpos dirigidos contra endotelial marcador de superficie celular CD144 (VE-cadherina, panel superior derecho) y la glicoproteína de sangre Factor von Willebrand (vWF, panel inferior derecho) . Paneles correspondientes que muestran tinción DAPI nuclear se muestran a la izquierda. Barra de escala 50 micras. para CD144. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig4large.jpg" target="_blank">Haga clic aquí para ver una mayor</a>versión de esta 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.

Generación y Cultura de Sangre Consecuencia endoteliales Las células de la sangre periférica humana

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

generation-culture-blood-outgrowth-endothelial-cells-from-human

Research

JoVE Journal

Biology

18.8K vistas.  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.

Video: 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.

Enriquecimiento de las células NK a partir de sangre humana con el Kit de RosetteSep de Tecnologías de la StemCell

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

Investigación

Biología

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.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

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.

Caracterización fenotípica y funcional de las células endoteliales Formadoras de Colonias derivados de la sangre del cordón umbilical

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.

Generación de las células madre pluripotentes inducidas a partir de sangre periférica mediante el STEMCCA vector lentiviral

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.

Purificación y microRNA perfiles de exosomas derivados de la sangre y Cultura Medios

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 iPS Generación Celular de sangre usando episomas y HDAC inhibidores

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.

De dos fotones de imágenes de Ca intracelular<sup&gt; 2 +</sup&gt; Manejo y producción de óxido nítrico en el endotelio y las células del músculo liso de la aorta de rata aislada

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).

Las proteínas Análisis de inmunofluorescencia de endógenos y exógenos Centrómero-cinetocoro

"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.

Aislamiento y caracterización de microvesículas de la sangre periférica

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.

Un ensayo de cizalla uniforme para los receptores humanos de plaquetas y de superficie celular a través de Viscometry de placa cónica

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