We are grateful to Mr. Claus Bus and Ms. Rita Rosendahl at iNANO for their technical assistance. Special thanks to Dr. Daniel Otzen for allowing our frequent use of his ultracentrifuge. This study was supported by the Innovation Fund Denmark (MUSTER project).

NOTE: Mesenchymal stem cell growth medium (MSC media) is prepared beforehand as indicated in the Table of Materials.
1. Cell culture
NOTE: Human mesenchymal stem cells can be obtained from the bone marrow, adipose tissue or other sources11. Alternatively, hMSCs can be bought through a supplier. The BMSCs used in this protocol were derived from the bone marrow of patients and bought from a company.
<ol>
	<li>Thaw 1 x 106 BMSCs into a T175 flask containing 20 mL of MSC media. Incubate the cells at 37 &#176;C with 5% CO2 and replace the media every 2-3 days until 80% confluency.</li>
	<li>Wash the cells with 5 mL of 1x PBS and discard the PBS.</li>
	<li>Detach the cells by adding 5 mL of 0.05% Trypsin-EDTA and incubating the cells for 5 min at 37 &#176;C. Tap the sides of the flask to facilitate detachment.</li>
	<li>Add 15 mL of MSC media to inactivate the trypsin, detach the cells from the surface, and pipette up and down to obtain single cell suspensions.</li>
	<li>Collect the cells in a 50 mL tube and spin down for 5 min at 300 x g to pellet the cells.</li>
	<li>Resuspend the cells in 1 mL of MSC media and count the cells using a hemocytometer. 
	NOTE: Primary human bone marrow MSCs at 80% confluency in a T175 flask is around 2 x 106 cells.</li>
	<li>Plate hMSCs at 2,000 cells/cm2 in fresh MSC media in 5 T175 flasks. Grow the cells at 37 &#176;C with 5% CO2 and replace the media every 2-3 days until 5 flasks of 90% confluent T175 flasks are obtained.</li>
</ol>
2. EVs and RNA-associated biomolecules collection
NOTE: EV collection media is prepared beforehand (Dulbecco&#39;s Modified Eagle&#39;s Medium [DMEM] with 10% fetal bovine serum [FBS] and 1% penicillin/streptomycin [P/S]). EV collection media is normal MSC media, but prepared with commercial EV-depleted FBS (Table of Materials). This is to avoid bovine exRNA contamination from FBS, which normally contains exRNAs associated with EVs, ribonucleoproteins, and lipoproteins. For small RNA library preparation, exRNAs derived from 5 confluent flasks of MSCs are required to enable library construction.
<ol>
	<li>Wash the cells 3x with 20 mL of PBS per T175 flask. Add 20 mL of EV collection media per confluent T175 flask of MSCs and incubate at 37 &#176;C with 5% CO2 for 48 h.</li>
	<li>Collect the media and centrifuge the media for 10 min at 300 x g and 4 &#176;C.</li>
	<li>Collect the supernatant and centrifuge the media for 20 min at 2,000 x g and 4 &#176;C.</li>
	<li>Collect the supernatant and centrifuge the media for 30 min at 15,500 x g and 4 &#176;C. Then collect the supernatant.</li>
	<li>Transfer the media to ultracentrifuge tubes and pellet the exRNAs for 90 min at 100,000 x g and 4 &#176;C. A fixed angle rotor is used here and the pellet is anchored to the side of the tube. Mark the side of the lid and draw a circle on the side of the tube where the pellet is expected.</li>
	<li>Remove the supernatant, dry the inside of the tube by inverting the tube on absorbent paper and use small pieces of absorbent paper to remove the liquid inside the tube without touching the bottom of the tube. Resuspend the pellet in 200 &#956;L of PBS by vortexing for 30 s and pipetting up and down 20x.</li>
	<li>Assess the EVs and biomolecules with nanoparticle tracking analysis (NTA) (Figure 2). 
	NOTE: The EVs and biomolecules can be assessed with nanoparticle tracking analysis (NTA), dynamic light scattering (DLS) or transmission electron microscopy (TEM)12.</li>
	<li>Store the EVs and biomolecules at -80 &#176;C until further downstream experiments. 
	NOTE: If EVs are going to be used for functional studies, 20% glycerol must be added to protect them from rupturing. The cells can be collected using standard procedures if necessary.</li>
</ol>
3. EVs and RNA-associated biomolecules collection of differentiated cells
NOTE: EVs and RNA associated biomolecules can also be collected from the cell culture media while the cells undergo differentiation. The example depicted in the protocol describes osteoblastic differentiation and exRNA collection at day 0 and 7 of differentiation. If no differentiation is required, then skip Section 3 and go to Section 4.
<ol>
	<li>Prepare osteoblastic differentiation media (DMEM with 10% FBS, 1% P/S, 10 mM &#946;-glycerophosphate, 10 nM dexamethasone, 50 &#956;M ascorbate-2-phosphate, and 10 mM 1.25-vitamin-D3) fresh every time.</li>
	<li>Once MSCs are 80% confluent, change the MSC media to osteoblastic differentiation media.</li>
	<li>Replenish the osteoblastic differentiation media after 2-3 days.</li>
	<li>On day 5 of differentiation, remove the media and wash the cells 3x with 20 mL of PBS per T175 flask.</li>
	<li>Add 20 mL of EV collection media containing 10 mM &#946;-glycerophosphate, 10 nM dexamethasone, 50 &#956;M ascorbate-2-phosphate and 10 mM 1.25-vitamin-D3 per confluent T175 flask of MSCs. Incubate the cells at 37 &#176;C with 5% CO2 for 48 h to ensure continued differentiation while collecting the EVs and biomolecules.</li>
	<li>Collect the media on Day 7 and proceed to isolate the EVs and biomolecules as described in steps 2.2-2.6.</li>
	<li>For quality control, seed cells in 96-wells or 6-wells to assess differentiation using an alkaline phosphatase (ALP) activity assay or with quantitative polymerase chain reaction (qPCR), respectively. 
	NOTE: Figure 3 is an example showing osteogenic differentiation of the cells.</li>
</ol>
4. RNA extraction and quality control
<ol>
	<li>Thaw samples from step 2.8 on ice. Extract RNA using an RNA isolation kit (Table of Materials).</li>
	<li>Elute the RNA from the column provided in the RNA isolation kit (Table of Materials) in 100 &#956;L of RNase-free water.</li>
	<li>Concentrate the RNA through ethanol precipitation by adding 1 &#956;L of glycogen, 10 &#956;L of 2 M pH 5.5 sodium acetate and 250 &#956;L of pre-chilled 99% ethanol into 100 &#956;L of purified RNA.</li>
	<li>Incubate the samples at -20 &#176;C overnight to precipitate the RNA. Pellet the RNA by centrifuging for 20 min at 16,000 x g and 4 &#176;C. 
	NOTE: The pellet is white due to co-precipitation with glycogen.</li>
	<li>Remove the supernatant and wash the RNA pellet with 1 mL of 75% ethanol. Pellet the RNA again for 5 min at 16,000 x g and 4 &#176;C.</li>
	<li>Remove the ethanol and leave the lid of the RNA tube open for 5-10 min to air dry the RNA pellet. Resuspend the RNA pellet in 7 &#956;L of RNase-free water.</li>
	<li>Check the RNA quality and concentration using a chip-based capillary electrophoresis machine to detect the RNA according to the manufacturer&#8217;s protocol prior to library construction. 
	NOTE: A representative size distribution of the RNA is shown in Figure 4.</li>
	<li>Extract cellular RNA using a commercial purification kit (Table of Materials) if necessary.</li>
</ol>
5. Library construction and quality control
NOTE: Small RNA libraries are constructed using commercial kits (Table of Materials) with adjustments due to the low RNA input. Library construction is performed on the chilled block.
<ol>
	<li>Chill the heating block for 0.2 mL PCR tubes on ice and pipette 5 &#956;L of the RNA from step 4.6 into 0.2 mL RNase-free PCR tubes on a chilled block.</li>
	<li>Dilute 3&#8217; adaptors (1:10) in RNase-free water in a 0.2 mL RNase-free PCR tube. Add 0.5 &#956;L of diluted adaptor and mix with 5 &#956;L of RNA by pipetting up and down 8x and centrifuge briefly to collect all the liquid at the bottom of the tube.</li>
	<li>Incubate the RNA and 3&#8217; adaptor mix at 70 &#176;C for 2 min in a preheated thermal cycler and then place the sample back on the chilled block.</li>
	<li>Add 1 &#956;L of ligation buffer, 0.5 &#956;L of RNase inhibitor, and 0.5 &#956;L of T4 RNA ligase (deletion mutant) into the RNA and 3&#8217; adaptor mixture. Mix by pipetting up and down 8x and centrifuge briefly.</li>
	<li>Incubate the tube at 28 &#176;C for 1 h in the preheated thermal cycler.</li>
	<li>Add 0.5 &#956;L of stop solution into the sample tube with the tube staying in the thermal cycler, mix by pipetting up and down 8x and continue to incubate at 28 &#176;C for 15 min.</li>
	<li>Dilute 5&#8217; adaptors (1:10) in RNase-free water in a 0.2 mL RNase-free PCR tube. Add 0.5 &#956;L of 5&#8217; adaptor into a separate RNase-free 0.2 mL PCR tube, heat the 5&#8217; adaptor at 70 &#176;C for 2 min in the preheated thermal cycler, and then place the sample on the chilled block.</li>
	<li>Add 0.5 &#956;L of 10 nM ATP, 0.5 &#956;L of T4 RNA ligase to the 5&#8217; adaptor tube, mix by pipetting up and down 8x and centrifuge briefly to collect all the liquids into the bottom. 
	NOTE: Keep the 5&#8217; adaptor on chilled block as much as possible.</li>
	<li>Add 1.5 &#956;L of the 5&#8217; adaptor mixture to the sample from step 5.6, and mix very gently by pipetting 8x slowly. Incubate the sample at 28 &#176;C for 1 h in the preheated thermal cycler.</li>
	<li>Dilute RT primer 1:10 in RNase-free water in an RNase-free 0.2 mL PCR tube. Add 0.5 &#956;L of diluted RT primer into the sample from step 5.9, mix very gently by pipetting up and down 8x slowly and centrifuge briefly.</li>
	<li>Incubate the sample at 70 &#176;C for 2 min in the preheated thermal cycler and then place the sample on a chilled block.</li>
	<li>Add 1 &#956;L of 5x first strand buffer, 0.5 &#956;L of 12.5 mM dNTP mix, 0.5 &#956;L of 100 mM dithiothreitol (DTT), 0.5 &#956;L of RNase inhibitor, and 0.5 &#956;L of reverse transcriptase. Mix very gently by pipetting up and down 8x slowly and centrifuge briefly.</li>
	<li>Incubate the sample at 50 &#176;C for 1 h in the preheated thermal cycler to obtain cDNA.</li>
	<li>Add 4.25 &#956;L of ultrapure water, 12.5 &#956;L of PCR mix, 1 &#956;L of index primer, and 1 &#956;L of universal primer into the cDNA. Mix by pipetting up and down 8x and centrifuge briefly.</li>
	<li>Place the sample in a thermal cycler and set up the cycler as follows: 98 &#176;C for 30 s; 15 cycles of 98 &#176;C for 10 s, 60 &#176;C for 30 s and 72 &#176;C for 15 s; and end the cycle with 72 &#176;C for 10 min. 
	NOTE: The prepared library can be stored at -20 &#176;C for one week.</li>
	<li>Purify the RNA library by separating the library on a DNA gel and cutting the gel (gel extraction) between 140 bp and 160 bp and eluting the library from the gel following the protocol given by the library construction kit. 
	NOTE: In this study, the prepared library from step 5.15 is loaded onto a commercial gel (Table of Materials) and the library between 140 bp and 160 bp is purified out by an automated DNA size fractionator (Table of Materials) according to the manufacturer&#8217;s protocol.</li>
	<li>Concentrate and wash the library from step 5.16 using a column-based PCR purification kit (Table of Materials) and elute the library in 10 &#956;L RNase-free water finally.</li>
	<li>Load 1 &#956;L of the purified library onto a DNA chip (Table of Materials) to check the size of the library following the manufacturer&#8217;s protocol.</li>
	<li>Dilute 1 &#956;L of the purified library (1:1000) in 10 mM pH 8.0 Tris-HCl with 0.05% polysorbate 20 and quantify the library by qPCR using a commercial library quantification kit to quantify the library concentration using the DNA standards in the kit.</li>
	<li>Pool equal amounts of the libraries according to the requirements of the sequencing machine and sequence on a high-throughput sequencing system. 
	NOTE: The library construction of cellular RNA can be done using the same kits by following standard protocol of the kits.</li>
</ol>
6. Bioinformatics pipeline
NOTE: This is an in-house pipeline and the programs used here are listed in the Table of Materials. 
<ol>
	<li>Trim away low-quality reads and remove adaptor sequences from the raw reads.</li>
	<li>Map the clean reads to different kinds of RNAs.
	<ol>
		<li>Annotate tRNAs by allowing two mismatches because the heavily modified tRNA sequences cause frequent base misincorporation by reverse transcriptase.</li>
		<li>Annotate miRNAs by mapping to human miRNAs from miRBase v21 allowing zero mismatches. Since miRNAs are subject to A and U nontemplated 3&#8217;end additions, sequences that do not map are 3&#8217;trimmed of up to three A and/or T nts after which reads are mapped to human miRNAs and other miRNAs from miRBase v21 allowing zero mismatches.</li>
		<li>Annotate to other relevant small RNAs (snRNA, snoRNA, piRNA, and Y RNAs) by allowing one mismatch.</li>
		<li>Map the remaining unmapped reads to long RNA datasets (rRNA, other RNA from Rfam, and mRNA) to assess degradation.</li>
		<li>Annotate the sequences to the human genome.</li>
		<li>Annotate the sequences to bacterial genomes.</li>
		<li>Normalize miRNA expression using the following formula: miRNA counts / the total counts of all mapped miRNAs) x 106.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a protocol for next generation sequencing of exRNAs that enables differential expression analyses from low input samples. Adhering to a specific protocol for EV and exRNA isolation is important because even small alterations (i.e., the ultracentrifugation step or a change in rotor type) can influence the transcriptome and miRNA levels13,14. Thus, regardless of how the exRNA is isolated, it is important to apply the same experimental and bioinfor...

Extracellular and circulating RNAs (exRNAs) are present in various bodily fluids and are resistant towards RNases1,2. Their high abundance, stability and ease of accessibility are attractive for clinical assessment as diagnostic and prognostic markers3. The mode of transport for exRNAs include extracellular vesicles (EVs), association with lipoproteins (such as high-density lipoprotein; HDL) and ribonucleoprotein complexes (such as with Argonaute2 complexes)4.
A subset of exRNAs are microRNAs (miRNAs), which are small non-coding RNAs of about 22 nt that regulate posttranscriptional gene expression. Ex-miRNAs have been implicated in cell-cell communication and regulation of cell homeostasis5. For example, HDL delivers ex-miR-223 to endothelial cells to repress intercellular adhesion molecule 1 (ICAM-1) and inflammation6. Interestingly, miR-223 is also seen transported by extracellular vesicles from leukocytes to lung cancer cells, programming them to take on a more invasive phenotype7. Thus, the transcriptome of ex-miRNAs from various bodily fluids and cell culture medium will greatly improve our understanding of ex-miRNA signaling.
Small RNA sequencing (small RNA seq) is a powerful tool that can be used to understand the transcriptomics of small RNAs. Not only can different samples be compared amongst differentially expressed known RNAs, but novel small RNAs can also be detected and characterized. Consequently, it is also a robust method to identify differentially expressed miRNAs under different conditions. However, one of the hurdles of small RNA seq is the difficulty in generating small RNA seq libraries from low exRNA input fluids like cerebrospinal fluids, saliva, urine, milk, serum and culture media. The TruSeq Small RNA Library Prep protocol from Illumina requires approximately 1 &#181;g of high quality total RNA and the NEBNext Small RNA Library Prep Set protocol from New England Biolabs requires 100 ng-1 &#181;g of RNA8,9. Oftentimes, total RNA from these samples are below detection limit for conventional UV-vis spectrophotometers.
Ex-miRNAs derived from bodily fluids are potentially good prognostic and diagnostic markers. However, in order to study the functional effects or to determine the origin of specific ex-miRNAs, cell culture systems are often used instead. Mesenchymal stem cells (MSCs) have been studied extensively because their EVs have been implicated in alleviating many diseases including myocardial infarction, Alzheimer&#39;s disease and graft versus host disease10. Here, we demonstrate the purification of ex-miRNAs from bone marrow-derived MSCs (BMSCs) and the specific steps used to optimize small RNA library construction, sequencing and data analysis (Figure 1).

<table><tbody><tr><td>Bone Marrow-Derived Mesenchymal Stem Cells</td><td>ATCC</td><td>PCS-500-012</td><td>Cells used in this protocol was bought from ATCC</td></tr><tr><td>MSCGM BulletKit</td><td>Lonza</td><td>PT-3001</td><td>Termed as Mesenchymal Stem Cell Growth Medium (MSC media)</td></tr><tr><td>Exosome-depleted FBS</td><td>Gibco</td><td>A2720801</td><td></td></tr><tr><td>ExRNA collecting media: MSCGM but with the FBS replaced by exosome-depleted FBS</td><td></td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>T175 Flask</td><td>Sarstedt</td><td>833,912</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>806552</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Polycarbonate Bottle with Cap Assembly</td><td>Beckman Coulter</td><td>355618</td><td></td></tr><tr><td>Beckman Coulter Type 60 Ti</td><td></td><td></td><td>Rotor used here</td></tr><tr><td>NucleoCounter</td><td>Chemometec</td><td>NC-3000</td><td>Cell Counter</td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Calbiochem</td><td>35675</td><td>Components of the osteogenic differentiation media</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902-25MG</td><td>Components of the osteogenic differentiation media</td></tr><tr><td>2-Phospho-L-ascorbic&amp;nbsp;acid trisodium salt</td><td>Sigma</td><td>49752-10G</td><td>Components of the osteogenic differentiation media</td></tr><tr><td>1&amp;alpha;,25-Dihydroxyvitamin&amp;nbsp;D3</td><td>Sigma</td><td>D1530-1MG</td><td>Components of the osteogenic differentiation media</td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td>miRNA and total RNA purification kit for step 4.8</td></tr><tr><td>Agilent RNA 6000 Pico Kit</td><td>Agilent Technologies</td><td>5067-1514</td><td>Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis</td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939BA</td><td>Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis</td></tr><tr><td>Agilent High Sensitivity DNA Kit</td><td>Agilent Technologies</td><td>5067-4626</td><td>Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis</td></tr><tr><td>KAPA Library Quantification Kits</td><td>Roche</td><td>KK4824</td><td>Library quantification kit used here</td></tr><tr><td>TruSeq Small RNA Library Prep Kit -Set A (24 rxns) (Set A: indexes 1-12)</td><td>Illumina</td><td>RS-200-0012</td><td>Small RNA library prepartion kit used in this protocol - used in step 5</td></tr><tr><td>Pippin Prep</td><td>Sage Science</td><td></td><td>Automated DNA gel extractor used in this protocol; manual extraction can be done too</td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td>PCR purification in step 5.17</td></tr><tr><td>FASTX_Toolkit</td><td>Cold Spring Harbor Lab</td><td></td><td>Trimming low-quality reads in step 6</td></tr><tr><td>cutadapt</td><td></td><td></td><td>Adaptor removal in step 6</td></tr><tr><td>Bowtie</td><td></td><td></td><td>Mapping of clean reads in step 6</td></tr><tr><td>Samtools</td><td></td><td></td><td>To make the expression profile in step 6</td></tr><tr><td>Bedtools</td><td></td><td></td><td>To make the expression profile in step 6</td></tr></tbody></table>

isolating, sequencing and analyzing extracellular micrornas from human mesenchymal stem cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

The method described in this protocol is optimized to collect exRNA from MSC culture for next generation sequencing. The overall scheme of the workflow is in Figure 1 on the left and the respective quality control checkpoints are on the right.
The morphology of the cells on the day of collection for undifferentiated (Figure 3A) and differentiated (F...

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Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

isolating-sequencing-analyzing-extracellular-micrornas-from-human

Research

JoVE Journal

Genetics

8.1K Views.  Aarhus University. Dit is het eerste papier dat technische problemen aanpakt bij het sequencing van kleine RNAs afgeleid van extracellulaire vikjes uit cellen. NGS-monsters vereisen een strenge voorbereiding en kwaliteitscontrole. Door de aard van EV's wijkt het QC-proces van EV-monsters af van de norm.Het voordeel van dit protocol is de QC stappen daarin voor nieuwe gebruikers. Het aantonen van de procedure zal Junyi Su zijn, een onderzoeksassistent van mijn laboratorium. Om te beginnen, plaat menselijk...

Video: Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Developmental Biology

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Cancer Research

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Bioengineering

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Medicine

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

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

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

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

Biology

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

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