The authors would like to thank Dr. Stephen Kingsmore and the Pediatric Genome Medicine Center at Children's Mercy Hospitals and Clinics for the use of their computing clusters for our data analysis, Illumina's field service team (Elizabeth Boyer, Scott Cook and Mark Cook) and technical consultant team for their quick responses and helpful suggestions on the running of the next generation DNA sequencing instrument, HiScanSQ, and data quality analysis. This work was supported in part by National Institutes of Health Grant HL080042 (to S.Q.Y.) and start-up fund and endowment of Children's Mercy Hospitals and Clinics, University of Missouri at Kansas City (to S.Q.Y.).

A flowchart outlining this protocol is displayed in Figure 1.
1. Treatment of Cells with Thrombin, RNA Isolation, Quality Assessment and Quantification of RNA
<ol>
	<li>Culture Human Lung Microvascular Endothelial Cells (HMVEC-LBl) to 90-100% confluence in 6-well plates in EGM-2 medium with 5% FBS, growth factors and antibiotics (Lonza, cat# CC-3202).</li>
	<li>Change media to the starvation media (0% FBS) 30 min prior to treatment with thrombin.</li>
	<li>Treat the cells with 0.05 U/ml thrombin or leave untreated as a control for 6 hr at 37 &deg;C and 5% CO2.</li>
	<li>Isolate total RNA from the treated and control cells using the Ambion mirVana kit according to manufacturer's instructions.</li>
	<li>Assess the quality of the RNA with an Experion StdSense Eukaryotic RNA chip according to the standard protocol on the Experion Automated Electrophoresis Station (<a href="http://www.bio-rad.com" target="_blank">www.bio-rad.com</a>).</li>
	<li>Quantify the RNA using a standard spectrophotometric method.</li>
</ol>
2. Library Construction and Sequencing
<ol>
	<li>Use 1 &mu;g of high quality total RNA per sample as starting material.</li>
	<li>To construct the library, follow the standard procedure from Illumina (protocol # 15008136 Rev. A). In this protocol, two rounds of poly(A) containing mRNA selections are performed to remove rRNA to minimize the rRNA sequencing.</li>
	<li>Assess the quality of the libraries using an Experion DNA 1K chip according to the standard protocol on the Experion Automated Electrophoresis Station (<a href="http://www.bio-rad.com" target="_blank">www.bio-rad.com</a>).</li>
	<li>Quantify the library using qPCR: Use a library that has previously been sequenced as a standard curve and primers specific for the ligated adapters. Use a range of dilutions of the unknown libraries (i.e. 1:100, 1:500 and 1:1,000). Run the qPCR according to the SyberGreen MM protocol and calculate the original stock concentration of each library.</li>
	<li>Dilute the library stocks to 10 nM and store at -20 &deg;C until ready to cluster a flow cell.</li>
	<li>When ready to cluster a flow cell, thaw the cBot reagent plate in a water bath. cBot is an Illumina instrument used to streamline the cluster generation process.</li>
	<li>Wash the cBot instrument.</li>
	<li>Denature the libraries: Combine 13 &mu;l 1x TE and 6 &mu;l 10 &mu;M library and, to the side of the tube, add 1 &mu;l 1 N NaOH (provided by Illumina). Vortex, spin down, incubate at room temperature for 5 min and place the denatured libraries on ice.</li>
	<li>Dilute the libraries: Dilute the denatured libraries with pre-chilled hybridization buffer (HT1, provided by Illumina) by combining 996 &mu;l HT1 and 4 &mu;l denatured library for a final concentration of 12 pM. Place the denatured and diluted libraries on ice.</li>
	<li>Invert each row of tubes of the cBot plate, ensuring that all the reagents are thawed. Spin down the plate, remove/puncture the foil seals and load onto the cBot.</li>
	<li>Aliquot 120 &mu;l of the diluted, denatured libraries to a strip tube, labeled 1-8. Add 1.2 &mu;l diluted, denatured PhiX control library (from Illumina) into each tube as a spike-in control. Vortex and spin down the tubes and load them on the cBot in the correct orientation (tube #1 to the right).</li>
	<li>Load a flow cell and manifold onto the cBot.</li>
	<li>Complete the flow check and begin the clustering run.</li>
	<li>After the run is complete, check reagent delivery across all lanes. Make note of any abnormalities. Either start the sequencing run immediately or store the flow cell in the provided tube at 4 &deg;C.</li>
	<li>Thaw the sequencing-by-synthesis (SBS, Illumina) reagents.</li>
	<li>Load the reagents to the appropriate spots on the reagent trays, making sure not to touch the other reagents after touching the cleavage mix.</li>
	<li>Using a non-sequencing flow cell (i.e. one that was sequenced previously), prime the reagent lines twice.</li>
	<li>Thoroughly clean the sequencing flow cell with 70% EtOH and Kimwipes, followed by 70% EtOH and lens paper. Inspect the flow cell for any streaks. Re-clean it if necessary.</li>
	<li>Load the flow cell onto the sequencer and perform a flow check to ensure that the seal between the manifolds and the flow cell is tight.</li>
	<li>Start the sequencing run.</li>
	<li>Assess the quality metrics (e.g. the cluster density, clusters passing filter, Q30, intensity) as they become available during the run.</li>
	<li>Monitor intensity throughout the run.</li>
	<li>After 101 cycles are completed, perform turnaround chemistry to complete the second read: Thaw the paired end reagents and the second read Incorporation Buffer (ICB, a component of the SBS reagents, Illumina) and load the reagents.</li>
	<li>Continue the sequencing run, assessing 2nd read intensity, Q30 and other quality metrics as the run progresses.</li>
</ol>
3. Data Analysis
<ol>
	<li>Use the latest version of CASAVA (Illumina, currently 1.8.2) to convert the base call files (.bcl) files to .fastq files, setting fastq-cluster-count to 0 to ensure the creation of a single fastq file for each sample. Unzip the fastq files for downstream analysis.</li>
	<li>Perform paired end alignment using the latest versions of TopHat (1.4.1)5, which aligns RNA-seq reads to mammalian-sized genomes using the ultra high-throughput short read aligner(Bowtie,0.12.7)6 and SAMtools (0.1.17)7. SAMtools implements various utilities for post-processing alignments in the SAM format. The reference human transcriptome can be downloaded from iGenomes (<a href="http://www.illumina.com" target="_blank">www.illumina.com</a>). In running TopHat, we used all default parameter settings including the library type option as fr-unstranded (default).</li>
	<li>Using the program CuffDiff, part of the CuffLinks (1.3.0)8 software package, compare the thrombin-treated cells to the controls cells to screen out the differentially expressed gene transcripts in the former based on the human reference transcriptome. This comparison detects the differential expression of known transcripts. Use Microsoft Excel to visualize the result in table form. In running Cufflinks program, we used all default parameter settings. Those gene transcripts with FPKM&lt;0.05 and p&gt;0.05 are filtered out.</li>
	<li>To detect novel isoforms, run Cufflinks without a reference transcriptome. Compare the sample transcript files to the reference genome using Cuffcompare and test the differential expression with Cuffdiff using the combined thrombin transcript files as the reference genome for one analysis and the combined control transcript files as the reference genome for a second analysis. Use Microsoft Excel to visualize the result in tabular format. Again, those gene transcripts with FPKM&lt;0.05 and p&gt;0.05 are filtered out. After this step, investigators may opt to upload a list of newly reported transcripts to the UCSC Genome Browser website (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>) to verify their validity by a manual inspection.</li>
	<li>Submit lists of differentially expressed genes to Ingenuity Pathway Analysis (IPA, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>) for characterization of the genes and pathways affected by the thrombin treatment. In this step, investigators may opt to use CummeRbund (<a href="http://compbio.mit.edu/cummeRbund/" target="_blank">http://compbio.mit.edu/cummeRbund/</a>), an R package that is designed to aid and simplify the task of analyzing Cufflinks RNA-seq output, to help manage, visualize and integrate all of data produced by a Cuffdiff analysis.</li>
</ol>
4. Validation of the RNA-seq Results by Quantitative Real-time-Polymerase Chain Reaction (qRT-PCR)
<ol>
	<li>Perform total RNA isolation from control and thrombin-treated HMVEC-LBl cells, RNA quality assessment and RNA quantification described in Steps 1.4 to 1.6.</li>
	<li>Generate complementary DNA from 1 &mu;g total RNA of each sample with SuperScript III First-Strand Synthesis System RT Kits, following the manufacturer's instructions (Invitrogen, 18080-051).</li>
	<li>Perform qRT-PCR analysis on a Applied Biosystems ViiA 7 Real-Time PCR System using Taqman Assay-on-Demand designed oligonucleotides for the detection of CUGBP, Elav-likefamilymember1(CELF1, Hs00198069_m1), Fanconianemia,complementationgroupD2 (FANDCD2, Hs00276992_m1), TNFreceptor-associated factor 1(TRAF1, Hs01090170_m1),and &beta;-actin (ACTB,Hs99999903_m1). Each sample had a template equivalent to 5 ng of total RNA. Measure quantitation using the DDCt method and normalize to &beta;-actin. Each assay was performed across at least three biological replicates.</li>
</ol>

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10, 57-63 (2009).</li><li>Shendure, J., Ji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+DNA+sequencing.">Next-generation DNA sequencing.</a> Nat. Biotechnol. 26, 1135-1145 (2008).</li><li>Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+an+assessment+of+technical+reproducibility+and+comparison+with+gene+expression+arrays.">RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays.</a> Genome Res. 18, 1509-1517 (2008).</li><li>Zhang, L. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+Reveals+Novel+Transcriptome+of+Genes+and+Their+Isoforms+in+Human+Pulmonary+Microvascular+Endothelial+Cells+Treated+with+Thrombin.">RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin.</a> PLoS One. 7, e31229 (2012).</li><li>Trapnell, C., Pachter, L., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat:+discovering+splice+junctions+with+RNA-Seq.">TopHat: discovering splice junctions with RNA-Seq.</a> Bioinformatics. 25, 1105-1111 (2009).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10, R25 (2009).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat. Biotechnol. 28, 511-515 (2010).</li><li>Khatri, P., Sirota, M., Butte, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+pathway+analysis:+current+approaches+and+outstanding+challenges.">Ten years of pathway analysis: current approaches and outstanding challenges.</a> PLoS Comput. Biol. 8, e1002375 (2012).</li><li>Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+RNA.">Working with RNA.</a> Methods. Mol. Biol. 703, 15-28 (2011).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat. Protoc. 7, 562-578 (2012).</li><li>Robertson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+assembly+and+analysis+of+RNA-seq+data.">De novo assembly and analysis of RNA-seq data.</a> Nat. Methods. 7, 909-912 (2010).</li><li>Garber, M., Grabherr, M. G., Guttman, M., Trapnell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+methods+for+transcriptome+annotation+and+quantification+using+RNA-seq.">Computational methods for transcriptome annotation and quantification using RNA-seq.</a> Nat. Methods. 8, 469-477 (2011).</li><li>Martin, J. A., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+transcriptome+assembly.">Next-generation transcriptome assembly.</a> Nat. Rev. Genet. 12, 671-682 (2011).</li></ol>

No conflicts of interest declared.

Key steps
RNA Handling: RNases will degrade even the most high-quality RNA, therefore care must be taken during the isolation, storage and use of RNA10. Gloves are always worn to prevent contamination by RNases found on human hands. Gloves should be changed often, particularly after touching skin, doorknobs or other common surfaces. A set of pipettes should be dedicated solely to RNA work and all tips and tubes should be RNase-free. RNA isolation and downstream applica...

<table border="1">
 <tbody>
 <tr>
 <td width="183">Reagents or Equipments</td>
 <td width="85">Company</td>
 <td width="185">Catalog number</td>
 <td width="359">Comments</td>
 </tr>
 <tr>
 <td>Human Lung Microvascular Endothelial Cells</td>
 <td>Lonza</td>
 <td>CC-2815</td>
 <td></td>
 </tr>
 <tr>
 <td>Lonza, Bullet Kit</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td>Contains EGM-2, FBS, growth factors and antibiotics</td>
 </tr>
 <tr>
 <td>Thrombin</td>
 <td>Sigma</td>
 <td>T4393</td>
 <td></td>
 </tr>
 <tr>
 <td>Ambion mirVana 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 Preparation Kit</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>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 Control Kit</td>
 <td>Illumina</td>
 <td>FC110-301</td>
 <td></td>
 </tr>
 <tr>
 <td>200 Cycle 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 machine - Viia7</td>
 <td>Life Technologies</td>
 <td>Model #VIIA7 / Equipment #10631261</td>
 <td>Or equivalent </td>
 </tr>
 <tr>
 <td>Experion System </td>
 <td>Bio Rad</td>
 <td>7007001</td>
 <td>Bioanalyzer is an alternative system</td>
 </tr>
 <tr>
 <td>Spectrophotometer </td>
 <td>Bio-Tek</td>
 <td>Epoch Microplate Spectrophotometer</td>
 <td>Or equivalent</td>
 </tr>
 <tr>
 <td>Centrifuge - Sorvall Legend XTR </td>
 <td>Thermo Scientific</td>
 <td>75004521</td>
 <td>Or equivalent</td>
 </tr>
 <tr>
 <td>Magnetic stand</td>
 <td>Life Technologies</td>
 <td>AM10027</td>
 <td></td>
 </tr>
 <tr>
 <td>96-well thermocycler</td>
 <td>General Lab Supplier</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 3. List of Key Reagents and Major Equipment. *, In the video, HiSeq1000 instead of HiScanSQ was demonstrated.</td>
 </tr>
 </tbody>
</table>

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

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

For Step 1: The 28s:18s ratio is traditionally used as an indicator of RNA degradation. Ideally, the 28s peak should have approximately twice the area of the 18s band (a ratio of 2), however this ideal ratio is often not seen in practice. Furthermore, 28s:18s ratios obtained from spectrophotometric methods can underestimate the amount of degradation of the RNA. To more accurately quantify the degradation, and therefore the quality of the RNA sample, the Experion system calculates an RNA Quality I...

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

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

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

Research

JoVE Journal

Biology

21.8K Views.  Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City. This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.