Los autores desean agradecer al Dr. Stephen Kingsmore y el Centro de Medicina del Genoma pediátrica en hospitales Mercy infantiles y clínicas para el uso de clusters sus computacionales para el análisis de datos, el equipo de iluminación de campo de servicio (Elizabeth Boyer, Cook Scott y Cook, Mark) y técnicos consultor equipo por sus respuestas rápidas y sugerencias útiles sobre el funcionamiento del instrumento generación siguiente secuenciación de ADN, HiScanSQ y análisis de datos de calidad. Este trabajo fue apoyado en parte por los Institutos Nacionales de Salud Grant HL080042 (a SQY) y la puesta en marcha del fondo y la dotación de los hospitales Mercy niños y las Clínicas de la Universidad de Missouri en Kansas City (a SQY).

<html> Un diagrama de flujo delinear este protocolo se muestra en la figura 1. 1. El tratamiento de células con trombina, Aislamiento de ARN, Evaluación de la Calidad y cuantificación de ARN <ol><li> Cultivar células endoteliales microvasculares de pulmón (HMVEC-LBL) a 90-100% de confluencia en placas de 6 pocillos en medio EGM-2 con 5% de SFB, factores de crecimiento y antibióticos (Lonza, cat # CC-3202). </li><li> Cambiar el medio a los medios de inanición (0% FBS) 30 min antes del tratamiento con trombina. </li><li> Se tratan las células con 0,05 U / ml de trombina o dejar sin tratar como control durante 6 horas a 37 ° C y 5% CO 2. </li><li> Aislar el ARN total de las células tratadas y de control utilizando el Ambion mir Vana kit de acuerdo con las instrucciones del fabricante. </li><li> Evaluar la calidad del ARN con un chip de Experion Eukaryotic StdSense ARN de acuerdo con el protocolo estándar en la estación de Electroforesis Experion automatizada (= &quot;_blank&quot;&gt; Www.bio-rad.com). </li><li> Cuantificar el ARN utilizando un método espectrofotométrico estándar. </li></ol> 2. Biblioteca de la construcción y secuenciación <ol><li> Usar 1 mg de ARN de alta calidad total por muestra como material de partida. </li><li> Para construir la biblioteca, siga el procedimiento estándar de Illumina (protocolo # 15008136 Rev. A). En este protocolo, dos rondas de poli (A) que contienen selecciones de ARNm se realiza para eliminar rRNA para minimizar la secuenciación de ARNr. </li><li> Evaluar la calidad de las bibliotecas utilizando un chip de ADN Experion 1K de acuerdo con el protocolo estándar en la estación de Electroforesis Experion automatizada ( <a href="http://www.bio-rad.com" target="_blank">www.bio-rad.com</a> ). </li><li> Cuantificar la biblioteca mediante qPCR: utilizar una biblioteca que ha sido previamente secuenciados como una curva estándar y cebadores específicos para los adaptadores ligados. Utilizar una serie de diluciones de las bibliotecas desconocidos (es decir, 1:100, 1:500 y 1:1000). Ejecute el acco qPCRrding con el protocolo SyberGreen MM y calcular la concentración de la población original de cada biblioteca. </li><li> Diluir los fondos de las bibliotecas a 10 nM y se almacena a -20 ° C hasta el momento de clúster de una célula de flujo. </li><li> Cuando esté listo para clúster de una celda de flujo, descongele la placa reactivo CBOT en un baño de agua. CBOT es un instrumento utilizado Illumina para agilizar el proceso de generación de cluster. </li><li> Lave el instrumento CBOT. </li><li> Desnaturalizar las bibliotecas: Combinar 13 l TE 1x y 6 l 10 mM biblioteca y, al lado del tubo, añadir 1 l 1 N NaOH (proporcionado por Illumina). Vortex, girar hacia abajo, se incuba a temperatura ambiente durante 5 min y colocar las librerías desnaturalizadas en hielo. </li><li> Diluir las bibliotecas: Diluir las bibliotecas desnaturalizadas con tampón de hibridación pre-enfriada (HT1, proporcionado por Illumina) mediante la combinación de 996 l HT1 y 4 desnaturalizado biblioteca l para una concentración final de 12 pM. Coloque las bibliotecas desnaturalizados y diluido en hielo. </li><li> Invertir cada fila de tubos de la placa de CBOT,asegurar que todos los reactivos se descongelan. Girar hacia abajo la placa, elimine / perforar los sellos de aluminio y cargar en el CBOT. </li><li> Alícuota de 120 l de las bibliotecas diluidos, desnaturalizadas a un tubo de tira, etiquetados 1-8. Añadir 1,2 l biblioteca diluida, desnaturalizado control de PhiX (de iluminación) en cada tubo como una espiga en los de control. Vortex y centrifugar los tubos y los carga en el CBOT en la orientación correcta (tubo n º 1 a la derecha). </li><li> Cargue una celda de flujo y colector en el CBOT. </li><li> Completar el flujo de comprobar y comenzar el largo agrupación. </li><li> Después de la ejecución haya finalizado, compruebe la entrega de reactivos en todos los carriles. Tome nota de cualquier anomalía. Cualquiera de iniciar la ejecución de la secuencia inmediatamente o almacenar la celda de flujo en el tubo provisto a 4 ° C. </li><li> Descongelar la secuenciación por síntesis (SBS, Illumina) reactivos. </li><li> Cargue los reactivos a los lugares adecuados en las bandejas de reactivos, asegurándose de no tocar los otros reactivos después de tocar la mezcla de escisión. </li><li> El uso de un non-secuenciación de flujo de células (es decir, que fue secuenciado previamente), las líneas principales de reactivos en dos ocasiones. </li><li> Limpiar a fondo la celda de flujo de secuenciación con EtOH al 70% y Kimwipes, seguido por 70% de EtOH y papel para lentes. Inspeccione la celda de flujo para las rayas. Vuelva a limpiar si es necesario. </li><li> Cargar la celda de flujo en el secuenciador y realizar un flujo de comprobar para asegurarse de que el sello entre los colectores y la celda de flujo es ajustado. </li><li> Inicie la carrera de secuenciación. </li><li> Evaluar los indicadores de calidad (por ejemplo, la densidad de cluster, racimos pasan filtro, Q30, intensidad) a medida que estén disponibles durante el funcionamiento. </li><li> Monitorear intensidad a lo largo de la carrera. </li><li> Después de 101 ciclos se han completado, realizar la química de respuesta para completar la segunda lectura: Descongelar los reactivos extremos apareados y el búfer de lectura Incorporación segundo (ICB, un componente de los reactivos de SBS, Illumina) y cargar los reactivos. </li><li> Continúa la carrera de secuenciación, la evaluación 2 ª lectura intensidad, Q30 unand métricas de calidad de otros como los avances de ejecución. </li></ol> 3. Análisis de Datos <ol><li> Utilice la última versión de yuca (Illumina, actualmente 1.8.2) para convertir los archivos de llamadas bases (. Bcl) archivos para archivos. Fastq, estableciendo fastq-cluster-conteo a 0 para garantizar la creación de un archivo fastq único para cada muestra . Descomprimir los archivos fastq para el análisis de aguas abajo. </li><li> Realice la alineación final emparejado con las últimas versiones de TopHat (1.4.1) 5, que se alinea RNA-seq lee a los mamíferos del tamaño de los genomas utilizando el ultra alto rendimiento a corto alineador de lectura (Bowtie, 0.12.7) 6 y SAMtools (0,1. 17) 7. SAMtools implementa varias utilidades para las alineaciones de post-procesamiento en el formato SAM. El transcriptoma humano de referencia se pueden descargar desde iGenomes ( <a href="http://www.illumina.com" target="_blank">www.illumina.com</a> ). Al correr TopHat, hemos utilizado todos los parámetros por defecto, incluyendo la opción de tipo de biblioteca como fr-unstranded (por defecto). </li><li> NosotrosCIÓN DEL CuffDiff programa, parte de los gemelos (1.3.0) 8 paquete de software, comparar las células tratadas con trombina en las células controles para detectar a las transcripciones de genes expresados ​​diferencialmente en la antigua base de referencia el transcriptoma humano. Esta comparación detecta la expresión diferencial de las transcripciones conocidas. Utilice Microsoft Excel para visualizar el resultado en forma de tabla. Al correr el programa Gemelos, hemos utilizado todos los parámetros por defecto. Las transcripciones de genes con FPKM &lt;0,05 yp &lt;0,05 se filtran. </li><li> Para detectar isoformas novela, ejecute Gemelos sin transcriptoma de referencia. Comparar los archivos de transcripción de ejemplo en el genoma de referencia utilizando Cuffcompare y probar la expresión diferencial con Cuffdiff utilizando los archivos de trombina combinados de transcripción como el genoma de referencia para un análisis, y los archivos de control combinados de transcripción como el genoma de referencia para un segundo análisis. Utilice Microsoft Excel para visualizar el resultado en formato tabular. Una vez más, thostranscripciones gen E con FPKM &lt;0,05 y p&gt; 0,05 se filtran. Después de este paso, los investigadores pueden optar por subir una lista de las transcripciones recientemente denunciados a la página web del genoma UCSC ( <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a> ) para verificar su validez mediante una inspección manual. </li><li> Enviar las listas de genes expresados ​​diferencialmente a Ingenuity Pathway Analysis (IPA, <a href="www.ingenuity.com" target="_blank">www.ingenuity.com</a> ) para la caracterización de los genes y las vías afectadas por el tratamiento de la trombina. En este paso, los investigadores pueden optar por usar faja ( <a href="http://compbio.mit.edu/cummeRbund/" target="_blank">http://compbio.mit.edu/cummeRbund/</a> ), un paquete de R que está diseñado para ayudar y simplificar la tarea de analizar Gemelos RNA-seq salida, para ayudar a administrar, visualizar e integrar todos los datos producidos por un análisis Cuffdiff. </li></ol> 4. La validación de los resultados de la RNA-seq por cuantitativa en tiempo real de Po-lymerase reacción en cadena (QRT-PCR) <ol><li> Realice aislamiento de ARN total de control y tratados con trombina HMVEC-LBL células, el ARN de calidad de evaluación y cuantificación del ARN se describe en los pasos 1.4 a 1.6. </li><li> Generar ADN complementario a partir del ARN 1 microgramos totales de cada muestra con SuperScript III First-Strand Synthesis Kits sistema RT, siguiendo las instrucciones del fabricante (Invitrogen, 18080-051). </li><li> Realizar análisis de QRT-PCR en un Applied Biosystems VIIa 7 Real-Time PCR System usando TaqMan ensayo-on-Demand oligonucleótidos diseñados para la detección de CUGBP, elav-likefamilymember1 (CELF1, Hs00198069_m1), Fanconianemia, complementationgroupD2 (FANDCD2, Hs00276992_m1), TNFreceptor -factor asociado 1 (TRAF1, Hs01090170_m1), y β actina-(ACTB, Hs99999903_m1). Cada muestra tenía una plantilla equivalente a 5 ng de ARN total. Medir utilizando el método de cuantificación DDCT y normalizar para β-actina. Cada ensayo se realizó a través de al menos tres repeticiones biológica. </li></ol>

<ol>
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No hay conflictos de interés declarado.

Los pasos clave Manejo de ARN: RNasas se degradará hasta la más alta calidad del ARN, por lo tanto se debe tener cuidado durante el aislamiento, el almacenamiento y el uso de RNA 10. Los guantes se usan siempre para prevenir la contaminación por RNasas se encuentran en manos humanas. Los guantes deben ser cambiados con frecuencia, especialmente después de tocar la piel, pomos de las puertas u otras superficies comunes. Un conjunto de las pipetas deben estar dedic...

<table border="1"><tbody><tr><td width="183"> Los reactivos o equipos </td><td width="85"> Empresa </td><td width="185"> El número de catálogo </td><td width="359"> Comentarios </td></tr><tr><td> Las células pulmonares humanas endoteliales microvasculares </td><td> Lonza </td><td> CC-2815 </td><td></td></tr><tr><td> Lonza, Kit de bala </td><td> Lonza </td><td> CC-3202 </td><td> Contiene EGM-2, FBS, factores de crecimiento y antibióticos </td></tr><tr><td> La trombina </td><td> Sigma </td><td> T4393 </td><td></td></tr><tr><td> Ambion mir Vana Kit </td><td> Life Technologies </td><td> AM 1560 </td><td></td></tr><tr><td> RNasa-Zap </td><td> Life Technologies </td><td> AM9782 </td><td></td></tr><tr><td> Experion StdSens ARN </td><td> Bio-Rad </td><td> 700-7103 </td><td></td></tr><tr><td> TruSeq ARN PreparaciónEquipo </td><td> Illumina </td><td> FC-122-1001 </td><td></td></tr><tr><td> AMPureXP Beads </td><td> Beckman Coulter </td><td> A63881 </td><td></td></tr><tr><td> Superíndice II Reverse Transcriptase </td><td> Life Technologies </td><td> 18064-014 </td><td></td></tr><tr><td> Experion ADN 1K </td><td> Bio-Rad </td><td> 700-7107 </td><td></td></tr><tr><td> QuantiTect SyberGreen </td><td> Qiagen </td><td> 204163 </td><td></td></tr><tr><td> PE Generación Cluster Kit </td><td> Illumina </td><td> PE-401-3001 </td><td></td></tr><tr><td> PhiX Kit de control </td><td> Illumina </td><td> FC110-301 </td><td></td></tr><tr><td> 200 Ciclo SBS Kit </td><td> Illumina </td><td> FC-401-3001 </td><td></td></tr><tr><td> HiScanSQ * </td><td> Illumina </td><td> SY-103-2001 </td><td></td></tr><tr><td> CBOT </td><td> Illumina </td><td> SY-301-2002 </td><td></td></tr><tr><td> máquina qPCR - Viia7 </td><td> Life Technologies </td><td> Modelo # VIIA7 / Equipo # 10631261 </td><td> O el equivalente </td></tr><tr><td> Experion Sistema </td><td> Bio Rad </td><td> 7007001 </td><td> Bioanalyzer es un sistema alternativo </td></tr><tr><td> Espectrofotómetro </td><td> Bio-Tek </td><td> Espectrofotómetro para Microplacas Epoch </td><td> O el equivalente </td></tr><tr><td> Centrífuga - Sorvall Legend XTR </td><td> Thermo Scientific </td><td> 75004521 </td><td> O el equivalente </td></tr><tr><td> Soporte magnético </td><td> Life Technologies </td><td> AM10027 </td><td></td></tr><tr><td> De 96 pocillos del termociclador </td><td> Laboratorio General de Proveedor </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 3. Lista de los reactivos clave y E Majorquipment. * En el video, en lugar de HiSeq1000 HiScanSQ se demostró. </td></tr></tbody></table>

rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells

La caracterización de la expresión génica en las células a través de la medición de los niveles de mRNA es una herramienta útil en la determinación de cómo la maquinaria transcripcional de la célula se ve afectado por señales externas (por ejemplo, tratamiento de drogas), o cómo difieren entre las células un estado saludable y un estado de enfermedad. Con el advenimiento y refinamiento continuo de la tecnología de ADN secuenciación de próxima generación, ARN-secuenciación (RNA-seq) se ha convertido en un método cada vez más popular de análisis del transcriptoma para catalogar todas las especies de transcripciones, para determinar la estructura de la transcripción de todos los genes expresados ​​y cuantificar los cambios en los niveles de expresión de la serie total de transcripciones en un 1,2 dado célula, tejido u organismo. RNA-seq está reemplazando gradualmente microarrays de ADN como un método preferido para el análisis de transcriptoma porque tiene las ventajas de un perfil de un transcriptoma completa, proporcionando un dato de tipo digital (número de copias de cualquier transcripción) y no depender de cualquier secuencial genómico conocidoce 3. A continuación, presentamos un protocolo completo y detallado para aplicar RNA-seq para transcriptomes perfil pulmonares humanas en células endoteliales microvasculares con o sin tratamiento con trombina. Este protocolo se basa en el estudio recientemente publicado titulado &quot;RNA-seq revela Transcriptome novela de genes y sus isoformas en humanos células endoteliales microvasculares pulmonares tratadas con trombina,&quot; 4 en el que realizó con éxito el primer análisis completo de transcriptoma humano pulmonar de células endoteliales microvasculares tratados con trombina utilizando ARN-seq. Cedió recursos sin precedentes para nuevos experimentos para comprender mejor los mecanismos moleculares que subyacen a la trombina mediada por disfunción endotelial en la patogénesis de las enfermedades inflamatorias, cáncer, diabetes y enfermedad cardiaca coronaria, y proporciona nuevas pistas para posibles dianas terapéuticas para estas enfermedades. El texto descriptivo de este protocolose divide en cuatro partes. La primera parte se describe el tratamiento de humanos pulmonar de células endoteliales microvasculares con trombina y el aislamiento de ARN, análisis de la calidad y la cuantificación. La segunda parte describe la construcción de bibliotecas y la secuenciación. La tercera parte se describe el análisis de datos. La cuarta describe un ensayo de validación de RT-PCR. Los resultados representativos de varios pasos claves se muestran. Consejos útiles y precauciones para impulsar el éxito en los pasos claves se proporcionan en la sección de debate. Aunque este protocolo utiliza humanos pulmonar de células endoteliales microvasculares tratados con trombina, que se pueden generalizar a transcriptomes perfil tanto en células de mamíferos y no mamíferos y en los tejidos tratados con diferentes estímulos o inhibidores, o para comparar transcriptomes en células o tejidos entre un estado saludable y un estado de enfermedad.

Para el Paso 1: El 28s: relación de 18 años se utiliza tradicionalmente como un indicador de la degradación del ARN. Idealmente, el pico 28s debería tener aproximadamente dos veces el área de la banda de 18 años (una proporción de 2), sin embargo esta relación ideal no se ve a menudo en la práctica. Además, 28s: 18s coeficientes obtenidos a partir de métodos espectrofotométricos se puede subestimar la cantidad de degradación del ARN. Para cuantificar con mayor precisión la degradación, y ...

Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

Este protocolo presenta un procedimiento completo y detallado de aplicar RNA-seq, una potente tecnología de próxima generación de secuenciación de ADN, a transcriptomes perfil en humanos células endoteliales microvasculares pulmonares con o sin tratamiento con trombina. Este protocolo es generalizable a diversas células o tejidos afectados por diferentes reactivos o estados de enfermedad.

RNA-seq Análisis de transcriptomes en trombina tratamiento y de control pulmonar humana células endoteliales microvasculares

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

rna-seq-analysis-transcriptomes-thrombin-treated-control-human

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21.8K vistas.  Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City. Este protocolo presenta un procedimiento completo y detallado de aplicar RNA-seq, una potente tecnología de próxima generación de secuenciación de ADN, a transcriptomes perfil en humanos células endoteliales microvasculares pulmonares con o sin tratamiento con trombina. Este protocolo es generalizable a diversas células o tejidos afectados por diferentes reactivos o estados de enfermedad.

Video: RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

El aislamiento de las células endoteliales de vena umbilical (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

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 ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingeniería Humanos redes microvasculares en ratones inmunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Aislamiento de microvascular endotelial Tubos de arterias de resistencia Ratón

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análisis de estrés oxidativo en embriones de pez cebra

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Aislamiento de las células murinas de la válvula endoteliales

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Detección de la cara del óxido nítrico y superóxido en la capa endotelial de las arterias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.