Gli autori desiderano ringraziare il Dr. Stephen Kingsmore e il Pediatric Medicine Center Genome presso Ospedali Mercy bambini e cliniche per l&#39;uso dei loro cluster di calcolo per l&#39;analisi dei dati, il campo Illumina di assistenza tecnica (Elizabeth Boyer, Scott Cook e Mark Cook) e tecnica team di consulenti per le loro risposte rapide e suggerimenti utili sul funzionamento dello strumento di prossima generazione di sequenziamento del DNA, HiScanSQ, e la qualità dei dati di analisi. Questo lavoro è stato in parte finanziato dal National Institutes of Health di Grant HL080042 (a SQY) e start-up fondo di dotazione e degli Ospedali Mercy bambini e Cliniche, Università del Missouri a Kansas City (a SQY).

<html> Un diagramma di flusso che illustra questo protocollo è mostrato in Figura 1. 1. Il trattamento delle cellule con la trombina, l&#39;RNA Isolation, della Qualità e di quantificazione di RNA <ol><li> La cultura umana del polmone cellule endoteliali microvascolari (HMVEC-LBL) al 90-100% confluenza in piastre da 6 pozzetti in EGM-2 media al 5% di FBS, fattori di crescita e gli antibiotici (Lonza, cat # CC-3202). </li><li> Cambiare supporto ai media fame (0% FBS) 30 minuti prima del trattamento con trombina. </li><li> Trattare le cellule con 0,05 U / ml di trombina o lasciato non trattato come un controllo per 6 ore a 37 ° C e 5% di CO 2. </li><li> Isolare RNA totale dalle cellule trattate e di controllo con il Ambion mir Vana kit secondo le istruzioni del produttore. </li><li> Valutare la qualità del RNA con un eucariote Experion StdSense chip di RNA in base al protocollo standard Automated Stazione elettroforesi Experion (= &quot;_blank&quot;&gt; Www.bio-rad.com). </li><li> Quantificare l&#39;RNA utilizzando un metodo standard spettrofotometrico. </li></ol> 2. Biblioteca Edilizia e Sequencing <ol><li> Usare 1 pg di RNA di alta qualità totale per campione come materiale di partenza. </li><li> Per costruire la biblioteca, seguire la procedura standard da Illumina (protocollo # 15008136 Rev. A). In questo protocollo, due cicli di poly (A) dell&#39;mRNA contenenti selezioni vengono eseguite per rimuovere rRNA per minimizzare il sequenziamento rRNA. </li><li> Valutare la qualità delle biblioteche utilizzando un chip di DNA 1K Experion secondo il protocollo standard sulla Stazione Experion elettroforesi automatica ( <a href="http://www.bio-rad.com" target="_blank">www.bio-rad.com</a> ). </li><li> Quantificare la libreria utilizzando qPCR: Utilizzare una libreria che è già stato sequenziato da una curva standard e primer specifici per gli adattatori legato. Utilizzare una serie di diluizioni delle biblioteche sconosciute (cioè 1:100, 1:500 e 1:1000). Eseguire l&#39;acco qPCRrding al protocollo SyberGreen MM e calcolare la concentrazione originale stock di ogni libreria. </li><li> Diluire le scorte di libreria a 10 nM e conservare a -20 ° C fino al momento di cluster di una cella di flusso. </li><li> Quando si è pronti a raggrupparsi una cella di flusso, scongelare la piastra CBOT reagente in un bagno d&#39;acqua. CBOT è uno strumento Illumina utilizzato per semplificare il processo di generazione di cluster. </li><li> Lavare lo strumento CBOT. </li><li> Denaturare le librerie: Combinare 13 microlitri 1x TE e 6 microlitri 10 mM biblioteca e, al lato del tubo, aggiungere 1 ml di NaOH 1 N (fornito da Illumina). Vortex, spin down, incubare a temperatura ambiente per 5 minuti e posizionare le librerie denaturati su ghiaccio. </li><li> Diluire le librerie: Diluire le librerie denaturato con pre-raffreddata tampone di ibridazione (HT1, fornito da Illumina) combinando 996 microlitri HT1 e 4 microlitri biblioteca denaturato per una concentrazione finale di 12 pM. Posizionare le librerie denaturati e diluito su ghiaccio. </li><li> Capovolgere ogni fila di tubi della piastra CBOT,assicurare che tutti i reagenti vengono scongelate. Centrifugare la piastra, rimuovere / forare le guarnizioni strisce e caricare sul CBOT. </li><li> Aliquotare 120 ml di diluito, le biblioteche denaturati a un cilindro delle strisce, etichetta 1-8. Aggiungere 1,2 microlitri diluito, denaturati biblioteca controllo PhiX (da Illumina) in ciascuna provetta come un picco-in controllo. Vortex e centrifugare le provette e caricarli sul CBOT con l&#39;orientamento corretto (tubo # 1 verso destra). </li><li> Caricare una cella di flusso e collettore sul CBOT. </li><li> Completate il flusso di controllo e iniziare la corsa di clustering. </li><li> Una volta completata l&#39;analisi, controllare la consegna dei reagenti in tutte le corsie. Prendere nota di eventuali anomalie. O iniziare la corsa di sequenziamento immediatamente o conservare la cella di flusso nel tubo fornito a 4 ° C. </li><li> Scongelare il sequenziamento per sintesi (SBS, Illumina) reagenti. </li><li> Caricare i reagenti ai punti giusti sui vassoi reagenti, facendo attenzione a non toccare gli altri reagenti dopo aver toccato il mix scissione. </li><li> Utilizzando un non-sequenziamento cella di flusso (vale a dire quello che è stato sequenziato in precedenza), le linee dei reagenti prime due volte. </li><li> Pulire accuratamente la cella di flusso di sequenza con il 70% EtOH e Kimwipes, seguito dal 70% EtOH e salviette. Ispezionare la cella di flusso per eventuali aloni. Re-pulirlo se necessario. </li><li> Caricare la cella di flusso sul sequencer ed eseguire un flusso di controllare che la tenuta tra il collettore e la cella di flusso è stretto. </li><li> Avviare la corsa di sequenziamento. </li><li> Valutare le metriche di qualità (ad esempio, la densità di cluster, i cluster di passaggio filtro, Q30, di intensità) che vengono resi disponibili durante la corsa. </li><li> Monitorare l&#39;intensità per tutta la corsa. </li><li> Dopo 101 cicli sono completati, effettuare chimica turnaround per completare la seconda lettura: Scongelare i reagenti terminali accoppiati e il secondo tampone Incorporation lettura (ICB, un componente di reagenti SBS, Illumina) e caricare i reagenti. </li><li> Continua la corsa di sequenziamento, valutazione 2 ° letto intensità, Q30 unmetriche di qualità nd altre i progressi di esecuzione. </li></ol> 3. Analisi dei dati <ol><li> Utilizzare la versione più recente di casava (Illumina, attualmente 1.8.2) per convertire i file di base (chiamate. Bcl) file per file. Fastq, impostando fastq-cluster-count a 0 per garantire la creazione di un file fastq unico per ogni campione . Decomprimere i file fastq per l&#39;analisi a valle. </li><li> Eseguire l&#39;allineamento fine accoppiato con le ultime versioni di TopHat (1.4.1) 5, che allinea l&#39;RNA-seq legge di mammiferi di dimensioni genomi utilizzando l&#39;ultra high-throughput allineatore breve lettura (, Bowtie 0.12.7) 6 e SAMtools (0.1. 17) 7. SAMtools implementa varie utilità per la post-elaborazione allineamenti in formato SAM. Il trascrittoma umano di riferimento può essere scaricato dal iGenomes ( <a href="http://www.illumina.com" target="_blank">www.illumina.com</a> ). In esecuzione TopHat, abbiamo usato tutte le impostazioni di default dei parametri tra cui l&#39;opzione di libreria dei tipi come fr-unstranded (default). </li><li> Noizione del programma CuffDiff, parte dei gemelli (1.3.0) 8 pacchetto software, confronta le cellule trattate con trombina alle cellule controlli per escludere dei trascritti genici differenzialmente espressi nella ex basato sul trascrittoma umano di riferimento. Questo confronto rileva l&#39;espressione differenziale dei trascritti noti. Utilizzare Microsoft Excel per visualizzare il risultato sotto forma di tabella. In esecuzione del programma Gemelli, abbiamo usato tutte le impostazioni di default dei parametri. Le trascrizioni del gene con FPKM &lt;0,05 e p&gt; 0,05 sono filtrati. </li><li> Per rilevare isoforme nuovi, eseguire Gemelli senza un trascrittoma di riferimento. Confronta i file di esempio trascrizione al genoma di riferimento usando Cuffcompare e testare l&#39;espressione differenziale con Cuffdiff utilizzando i file combinati trascrizione della trombina come il genoma di riferimento per una analisi e le file di controllo combinati trascrizione come il genoma di riferimento per una seconda analisi. Utilizzare Microsoft Excel per visualizzare il risultato in formato tabulare. Nuovamente, thostrascrizioni elettroniche gene con FPKM &lt;0,05 e p&gt; 0,05 sono filtrati. Dopo questo passo, gli investigatori possono scegliere di caricare un elenco di trascrizioni segnalate di recente al sito UCSC Genome Browser ( <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a> ) per verificare la validità di un controllo manuale. </li><li> Presentazione delle liste di geni differenzialmente espressi Analisi Ingenuity Pathway (IPA, <a href="www.ingenuity.com" target="_blank">www.ingenuity.com</a> ) per la caratterizzazione dei geni e le vie interessate dal trattamento trombina. In questa fase, gli investigatori possono scegliere di utilizzare cummerbund ( <a href="http://compbio.mit.edu/cummeRbund/" target="_blank">http://compbio.mit.edu/cummeRbund/</a> ), un pacchetto di R che è stato progettato per aiutare e semplificare il compito di analizzare Gemelli RNA-seq uscita, per aiutare a gestire, visualizzare e integrare tutti i dati prodotti da una analisi Cuffdiff. </li></ol> 4. Validazione dei RNA-Seq Risultati della quantitativa in tempo reale-Polymerase Chain Reaction (qRT-PCR) <ol><li> Eseguire isolamento dell&#39;RNA totale di controllo e la trombina trattati HMVEC-LBL cellule, RNA qualità di valutazione e quantificazione RNA descritto nei passi 1,4-1,6. </li><li> Generazione di DNA complementare da 1 mcg RNA totale di ciascun campione con SuperScript III First-Strand Synthesis Kit sistema RT, seguendo le istruzioni del produttore (Invitrogen, 18080-051). </li><li> Eseguire qRT-PCR su Applied Biosystems VIIa 7 Real-Time PCR System utilizzando Taqman Assay-on-Demand oligonucleotidi progettati per l&#39;individuazione di CUGBP, Elav-likefamilymember1 (CELF1, Hs00198069_m1), Fanconianemia, complementationgroupD2 (FANDCD2, Hs00276992_m1), TNFreceptor -associated factor 1 (TRAF1, Hs01090170_m1), e β-actina (ACTB, Hs99999903_m1). Ogni campione aveva un modello equivalente a 5 ng di RNA totale. Misurare quantificazione con il metodo DDCT e normalizzare per β-actina. Ogni saggio è stato eseguito in almeno tre repliche biologiche. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

Passaggi chiave Manipolazione di RNA: RNasi peggiora anche i più di alta qualità RNA, pertanto è necessario prestare attenzione durante l&#39;isolamento, lo stoccaggio e l&#39;uso di RNA 10. I guanti sono sempre indossati per prevenire la contaminazione da RNasi presenti sulle mani umane. I guanti devono essere cambiati spesso, in particolare dopo la pelle toccare, maniglie delle porte o altre superfici comuni. Una serie di pipette deve essere dedicato esclusivame...

<table border="1"><tbody><tr><td width="183"> Reagenti o Attrezzature </td><td width="85"> Azienda </td><td width="185"> Numero di catalogo </td><td width="359"> Commenti </td></tr><tr><td> Umani del polmone cellule endoteliali microvascolari </td><td> Lonza </td><td> CC-2815 </td><td></td></tr><tr><td> Lonza, Kit proiettile </td><td> Lonza </td><td> CC-3202 </td><td> Contiene EGM-2, FBS, fattori di crescita e antibiotici </td></tr><tr><td> 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> RNase-Zap </td><td> Life Technologies </td><td> AM9782 </td><td></td></tr><tr><td> Experion StdSens RNA </td><td> Bio-Rad </td><td> 700-7103 </td><td></td></tr><tr><td> TruSeq RNA PreparazioneKit </td><td> Illumina </td><td> FC-122-1001 </td><td></td></tr><tr><td> AMPureXP Perline </td><td> Beckman Coulter </td><td> A63881 </td><td></td></tr><tr><td> Superscript Reverse Transcriptase II </td><td> Life Technologies </td><td> 18064-014 </td><td></td></tr><tr><td> Experion DNA 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 Cluster Generation Kit </td><td> Illumina </td><td> PE-401-3001 </td><td></td></tr><tr><td> PhiX Kit di controllo </td><td> Illumina </td><td> FC110-301 </td><td></td></tr><tr><td> 200 Ciclo di 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> qPCR macchina - Viia7 </td><td> Life Technologies </td><td> Model # VIIA7 / Accessori # 10631261 </td><td> O equivalente </td></tr><tr><td> Experion sistema </td><td> Bio Rad </td><td> 7007001 </td><td> Bioanalyzer è un sistema alternativo </td></tr><tr><td> Spettrofotometro </td><td> Bio-Tek </td><td> Spettrofotometro per micropiastre Epoch </td><td> O equivalente </td></tr><tr><td> Centrifuga - Sorvall Legenda XTR </td><td> Thermo Scientific </td><td> 75004521 </td><td> O equivalente </td></tr><tr><td> Supporto magnetico </td><td> Life Technologies </td><td> AM10027 </td><td></td></tr><tr><td> 96 pozzetti termociclatore </td><td> Generale Lab Fornitore </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 3. Elenco dei reagenti chiave e mi maggiorequipment. *, Nel video, invece di HiSeq1000 HiScanSQ è stata dimostrata. </td></tr></tbody></table>

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

La caratterizzazione di espressione genica in cellule attraverso la misurazione dei livelli di mRNA è uno strumento utile nel determinare come la macchina di trascrizione della cellula è influenzata da segnali esterni (per esempio trattamento di droga), o come cellule differiscono tra uno stato di salute e di uno stato di malattia. Con l&#39;avvento e il perfezionamento continuo della prossima generazione di tecnologia di sequenziamento del DNA, RNA-sequenziamento (RNA-seq) è diventato un metodo sempre più popolare di analisi del trascrittoma di catalogare tutte le specie di trascrizioni, per determinare la struttura della trascrizione di tutti i geni espressi e di quantificare i livelli di espressione di cambiamento del set totale di trascritti in un dato 1,2 cellula, tessuto o organismo. RNA-seq sostituisce progressivamente DNA microarrays come metodo preferito per l&#39;analisi del trascrittoma perché ha i vantaggi di una profilatura transcriptome completa, fornendo un tipo di dato digitale (numero di copie di ogni trascrizione) e non basandosi su qualsiasi sequen genomica notace 3. Qui vi presentiamo un protocollo completo e dettagliato di applicare RNA-seq per trascrittomi profilo in umano cellule endoteliali microvascolari polmonari con o senza trattamento trombina. Questo protocollo si basa sul nostro recente studio pubblicato dal titolo &quot;RNA-Seq rivela trascrittoma Romanzo di geni e loro isoforme umane in cellule endoteliali microvascolari polmonari trattati con trombina,&quot; 4 in cui abbiamo effettuato con successo la prima analisi completa del trascrittoma umane polmonari cellule endoteliali microvascolari trattate con trombina utilizzando RNA-seq. Ha prodotto le risorse senza precedenti per la sperimentazione ulteriore di acquisire conoscenze sui meccanismi molecolari alla base di trombina-mediata disfunzione endoteliale nella patogenesi di condizioni infiammatorie, il cancro, il diabete e le malattie cardiache coronariche, e fornisce nuovi indizi per potenziali bersagli terapeutici per queste malattie. Il testo descrittivo di questo protocolloè diviso in quattro parti. La prima parte descrive il trattamento di polmonari umane cellule endoteliali microvascolari con trombina e isolamento dell&#39;RNA, analisi della qualità e quantificazione. La seconda parte descrive la costruzione della libreria e sequenziamento. La terza parte descrive l&#39;analisi dei dati. La quarta parte descrive una RT-PCR convalida. I risultati rappresentativi di diversi passaggi chiave vengono visualizzati. Consigli utili o precauzioni per aumentare il successo in passaggi chiave sono forniti nella sezione di discussione. Sebbene questo protocollo utilizza polmonari umane cellule endoteliali microvascolari trattate con trombina, può essere generalizzato a trascrittomi profilo sia nelle cellule di mammiferi e non mammiferi e nei tessuti trattati con differenti stimoli o inibitori, o per confrontare trascrittomi in cellule o tessuti tra uno stato di salute e uno stato di malattia.

Per Fase 1: Il 28s: 18s rapporto è tradizionalmente utilizzato come indicatore di degradazione dell&#39;RNA. Idealmente, il picco 28s dovrebbe avere circa due volte l&#39;area della banda 18s (un rapporto di 2), tuttavia questo rapporto ideale spesso non viene osservata nella pratica. Inoltre, 28s: 18s rapporti ottenuti da metodi spettrofotometrici può sottovalutare la quantità di degradazione dell&#39;RNA. Per quantificare più accuratamente la degradazione, e quindi la qualità del campione RNA, il...

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

Questo protocollo presenta una procedura completa e dettagliata di applicare RNA-seq, una potente tecnologia di nuova generazione di sequenziamento del DNA, per trascrittomi profilo in umano cellule endoteliali microvascolari polmonari con o senza trattamento trombina. Questo protocollo è generalizzabile a varie cellule o tessuti colpiti da diversi reagenti o stati di malattia.

RNA-seq Analisi trascrittomi in trombina-trattati e di controllo dell&#39;uomo polmonari cellule endoteliali microvascolari

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

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21.8K Views.  Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City. Questo protocollo presenta una procedura completa e dettagliata di applicare RNA-seq, una potente tecnologia di nuova generazione di sequenziamento del DNA, per trascrittomi profilo in umano cellule endoteliali microvascolari polmonari con o senza trattamento trombina. Questo protocollo è generalizzabile a varie cellule o tessuti colpiti da diversi reagenti o stati di malattia.

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.

Isolamento di cellule umane endoteliali della vena ombelicale (HUVEC)

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

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Biologia

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

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

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

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

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.

Bioingegneria reti umane in topi immunodeficienti microvascolari

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.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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.

Isolamento di microvascolare endoteliale tubi da arterie di resistenza mouse

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.

Analisi dello Stress Ossidativo in embrioni di zebrafish

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.

Isolamento di cellule murine endoteliali Valve

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 Face Detection di ossido nitrico e superossido in strato endoteliale delle arterie Intact

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali 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.

RNA-seq Analisi trascrittomi in trombina-trattati e di controllo dell'uomo polmonari cellule endoteliali microvascolari

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