This work was funded by University of Helsinki project funding (WBS490302, WBS73714112) Helsinki University Hospital State funding for university-level health research (Y1014SUL05, TYH2016130), Finnish-Norwegian Medical Foundation, and the Selma and Maja-Lisa Selander Fund (Minerva Foundation). We thank Walter Pavicic for providing the modified MSPA protocol and for related technical support. We are grateful to Teemu Masalin (University of Helsinki) for helping us with video production.

This study was approved by the Ethics Committee of Helsinki and Uusimaa Hospital District (ethical approval D. No. 217/13/03/02/2015).
1. Preparation of EV-depleted FBS by ultracentrifugation
<ol>
	<li>Take FBS in (ultra)centrifuge tubes and place them in ultracentrifuge buckets. To ensure that the ultracentrifugation runs smoothly and safely, balance the buckets within 10 mg of each other.</li>
	<li>Load the buckets on a swinging rotor (type SW28, k-factor 246). Place the rotor in the ultracentrifuge and run at 100,000 x g for 19 h at 4 &#176;C.</li>
	<li>Carefully collect the light-colored upper layer of the supernatant (approximately nine-tenths) and transfer to a 50 mL tube. Do not disturb or pipette the dark brown pellet, as it contains EVs from FBS.</li>
	<li>Pass the supernatant through a 0.22 &#181;m filter into a new 50 mL tube.</li>
	<li>Add the filter-sterilized, EV-depleted FBS to cell culture media when growing cells for EV isolation.</li>
</ol>
2. Isolation of osteosarcoma-derived EVs
<ol>
	<li>Plate OS cells (HOS-143B cell line) in a T-175 flask with RPMI 1640 medium, supplemented with 10% normal FBS and 1% antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin). Place the flask in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>When the flask is 70%&#8211;80% confluent, wash the cells with phosphate-buffered saline (PBS) then grow them in media containing 10% EV-depleted FBS (hereafter referred to as EV-depleted media). 
	NOTE: 1 x 106 HOS-143B cells plated in a T175 flask reaches 70% confluency after approximately 60 h.</li>
	<li>During the next 48 h, collect conditioned media from osteosarcoma cells after every 24 h and add fresh EV depleted media.</li>
	<li>Centrifuge the conditioned media at 2500 x g for 20 min at 4 &#176;C to remove cells and cell debris. Transfer the supernatant to a new tube, leaving around 2 mL of media at the bottom. 
	NOTE: If not directly proceeding with EV isolation at this stage, the supernatant can be stored at -80 &#7506;C.</li>
	<li>Pour the supernatant into ultracentrifuge tubes and balance them as earlier. Centrifuge the tubes at 100,000 x g for 2 h at 4 &#176;C.</li>
	<li>Carefully discard the supernatant, leaving around 1 mL at the bottom. Add around 20 mL of PBS (0.1 &#181;m filtered) to the tube and pipette gently to wash and resuspend the EV pellet.</li>
	<li>Balance the tubes and perform another round of ultracentrifugation with the same settings.</li>
	<li>Carefully remove the supernatant and resuspend the EV pellet in 200 &#181;L of PBS by gentle pipetting. Store the EVs in low binding tubes. 
	NOTE: EVs can be used straightaway or else stored in -80 &#176;C until they are needed.</li>
</ol>
3. Characterization of OS-EVs
NOTE: Purified EVs can be characterized by western blotting (WB), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM)16.
<ol>
	<li>Perform WB as per the standard protocol16 with EV markers CD63, TSG101, and Hsp70, and with calnexin as a negative control to indicate purity of the EV sample20.</li>
	<li>For NTA, first dilute the EV sample in 0.1 &#181;m filtered Dulbecco&#39;s PBS to obtain (ideally) 30&#8211;100 particles per frame.
	<ol>
		<li>Take around 500 &#181;L of the sample into a 1 mL syringe and load it into the inlet port of the NTA instrument. Check that there are enough particles per frame, for accurate measurements.</li>
		<li>Open the NTA software and record five videos of 60 s duration, using camera level 13 at ambient temperature. During analysis, use detection threshold = 5 and gain = 10.</li>
	</ol>
	</li>
	<li>Analyze EV samples by TEM as described previously21.</li>
</ol>
4. AT-MSC culture
NOTE: Human adipose tissue for mesenchymal stem cell isolation was provided as liposuction aspirate (Department of Plastic Surgery, Laser Tilkka Ltd., Finland). Written informed consent was taken from the lipoaspirate donors, who were undergoing elective liposuction procedures.
<ol>
	<li>Isolate AT-MSCs from liposuction aspirates using standard mechanical and enzymatic isolation methods22. 
	NOTE: MSCs from other sources (including commercial cell lines) may also be used.</li>
	<li>Culture cells in DMEM/F-12 media supplemented with 10% FBS and 1% antibiotics.</li>
</ol>
5. Treatment of MSCs with OS-EVs
<ol>
	<li>Plate 15,000 AT-MSCs per well in a 24 well plate.</li>
	<li>After 24 h, remove the old media, wash cells with PBS, and change to EV-depleted media.</li>
	<li>Treat cells with OS-EVs (at a particle concentration of 1 x 106 EVs per cell) on Day 1 (24 h after cell adhesion), Day 3 (48 h after Day 1), and Day 5 (96 h after Day 1).
	<ol>
		<li>Stop the OS-EV treatment for timepoint (TP) 0 samples on Day 1, TP 3 samples on Day 3 and TP 7 samples on Day 7 (48 h after Day 5). 
		NOTE: Other timepoint schedules as per the experimental plans can also be followed.</li>
	</ol>
	</li>
	<li>Extract DNA from the MSCs using an appropriate method. Include positive and negative control samples for the data analysis.</li>
</ol>
6. LINE-1 methylation assay
<ol>
	<li>Design the customized LINE-1 probe primers, as done previously by Pavicic et al.23. For methylation probes, select three sequences containing the HhaI restriction site within the promoter region of LINE-1. For control probes, select seven sequences lacking the HhaI restriction site from the rest of the LINE-1 sequence. 
	NOTE: The LINE-1 sequence is available at the GenBank database24 (L1.2, accession no. AH005269.2). Use the MSPA manufacturer&#39;s instructions for designing the probes25.</li>
	<li>Dilute 70 ng of DNA sample in TE buffer to a 5 &#181;L volume.</li>
	<li>Carry out subsequent thermocycling and PCR steps as mentioned in Table 1. Heat the samples for 10 min at 98 &#176;C, then cool to 25 &#176;C.</li>
	<li>Add 3 &#181;L of probe hybridization mix to each sample and run the thermocycler to allow the probes to hybridize to the DNA.</li>
	<li>At room temperature (RT), add 13 &#181;L of post-hybridization mix to each sample. Transfer 10 &#181;L to a second tube.</li>
	<li>Place both sets of tubes in the thermocycler and incubate at 48 &#176;C for at least 1 min.
	<ol>
		<li>While samples are at 48 &#176;C, add 10 &#181;L of the ligation mix to the first set of tubes (undigested series) and 10 &#181;L of the ligation-digestion mix to the second set of tubes (digested series). Run the next thermocycler program.</li>
	</ol>
	</li>
	<li>Spin down the tubes and simultaneously set the thermocycler to 72 &#176;C.</li>
	<li>Add 5 &#181;L of polymerase mix to each tube and place the tubes in the thermocycler. Run the PCR program.</li>
	<li>While the PCR program is running, prepare a solution of 1 mL formamide containing 2.5 &#181;L of size standard. Pipette 10 &#181;L of this solution to each well of an optical 96 well plate (with barcode).</li>
	<li>After PCR, dilute the undigested and digested samples to 1:100 and 1:200 respectively in ultrapure water. Add 2 &#181;L of diluted PCR product to the 96 well plate. Centrifuge the plate at 200 x g for 15&#8211;20 s to remove air bubbles.</li>
	<li>Carry out fragment analysis of samples by capillary electrophoresis. 
	NOTE: The plate should be stored at 4 &#176;C in the dark until analysis.</li>
</ol>
7. Fragment data analysis
<ol>
	<li>Open the capillary electrophoresis results in an electropherogram analysis software.
	<ol>
		<li>Under the Panel column, for one of the samples, choose MLPA from the menu. Click on the Panel header and press Ctrl+D to apply MLPA to all samples.</li>
		<li>In the same way, set the Analysis method to Microsatellite default for all samples.</li>
		<li>Select all samples and click on the Green play button to analyze the samples as per the chosen settings.</li>
		<li>Select all samples and click on the Graph button to visualize the probe peaks.</li>
		<li>Zoom in on the peak region for higher resolution of the individual probe peaks. Make sure that all 10 peaks corresponding to the probes from the LINE-1 probe-mix are labeled. Discard additional peaks (&#60;95 bp and &#62;160 bp).</li>
		<li>In the Genotypes tab, export the results in the comma-separated-values (CSV) format.</li>
	</ol>
	</li>
	<li>Open the CSV file in a data analysis software and sort the data into columns.
	<ol>
		<li>Label the three methylation site peaks based on their approximate sizes (L1-1m at 153 bp, L1-2m at 119 bp, L1-3m at 133 bp). The remaining seven peaks correspond to the control probes. 
		NOTE: Here, values of the L1-2m probe peaks are used, which have a size of 117 bp, since that region has been used in most LINE-1 methylation assays23.</li>
		<li>For each sample (undigested and digested), calculate the sum peak area of all seven control peaks. Divide the peak area of each LINE-1 probe by this sum.</li>
		<li>For each DNA sample, divide the value of the digested sample by that of the undigested sample to obtain the methylation dosage ratio (DM) using the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/60705/60705eq1.jpg" /> 
		Where: DM is methylation dosage ratio, Ax is the area under peak x (e.g., L1-2m peak), and Actrl is the sum peak area of all seven control probes.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This study illustrates how MSPA can be used to detect and quantify the methylation status of a specific genetic element. LINE-1 was the focus here, but the probes can be designed to target a range of genes and sequences. Moreover, there is a growing list of probe mixes available for different applications. MSPA is a simple and robust technique for DNA methylation analysis that does not require bisulfite conversion10. The complete procedure from sample preparation to data analysis takes around 2 days but only involves 4&#8211;5 h of actual hands-on work. It is applicable for small amounts of DNA (as low as 70 ng), as demonstrated here.
The most important part of the methylation analysis protocol is the preparation of custom probe-mixes, such as the LINE-1 probe-mix in this study. Due to their different lengths, LINE-1 probe oligonucleotides were synthesized in the range of 4&#8211;40 nM, so the dissolution step and subsequent dilutions had to be performed carefully, as per the manufacturer&#39;s instructions25. The PCR and several thermocycling programs of the protocol as used here differ from those in the original manufacturer&#39;s version.
There are a few precautions relating to the HhaI enzyme. First, the volume of enzyme used in the ligation-digestion mix depends on the manufacturer, and versions of the enzyme that are resistant to heat-inactivation are not suitable for MSPA. With some enzymes, there may be instances of incomplete digestion, which would result in the formation of faulty PCR products and aberrant peak signals. Lastly, the extent to which the final PCR products are diluted for capillary electrophoresis depend on the probes. Initial runs are likely to involve optimizations, for which it is recommended to test a range of dilutions.
There are certain limitations of MSPA, such as the selective nature of HhaI-mediated DNA digestion. HhaI cleaves DNA only at unmethylated GCGC sequences and disregards other instances of CpG dinucleotides that are not enclosed by a G and C, even if they are located within CpG islands. As a result, the methylation status of such dinucleotides (and those that are located outside the target sequence of the probes) cannot be determined. The LINE-1 probe-mix can include more probes containing additional GCGC sequences from the promoter region24, thereby representing a greater number of methylation sites.
Alternatively, other methylation-specific restriction enzymes, such as SacII and MluI, that have a different restriction site than that of HhaI can be additionally used in MSPA, which may provide a broader representation of global methylation levels26. Secondly, as observed with TP 7 samples, the differences in methylation dosage ratio can be quite subtle. This may be due to the high copy number of LINE-1s in the genome5, which have high levels of global methylation under normal conditions and will largely be unaffected by transient hypomethylation events in only a few CpG sites. Moreover, the MSCs are heterogenous in terms of the stage of differentiation and of the cell cycle, which can affect the baseline methylation value. Lastly, MSPA can only detect relative differences in DNA methylation and requires reference samples for this reason. In such instances, methods that directly detect local methylation may be more appropriate, though they may require the bisulfite conversion step27.
Since this study involved the use of extracellular vesicles, we also demonstrated the preparation of EV-depleted FBS, which is an essential component of cell culture media for EV isolation. Ultracentrifugation is an inexpensive process that can effectively separate EVs from the rest of the FBS. However, the 19 h of centrifugation makes it a more time-consuming method than alternatives, such as ultrafiltration and commercial versions19. Ultracentrifugation also requires careful handling of the sample, such as when balancing the tubes before centrifugation and collecting the supernatant afterwards. Despite these challenges, it is a popular method for separating EVs from biological fluids.

DNA methylation is a major epigenetic modification occurring in human cells. DNA methylation refers to the linkage of methyl groups to cytosine residues in CpG dinucleotides. Such dinucleotides are usually found in clusters (CpG islands) at the 5&#39; region of genes1. In normal cells, most of these dinucleotides exist in an unmethylated state, which allows DNA transcription. Incidentally, many cancers are associated with hypermethylated CpG islands and transcriptomic silencing2, especially in tumor suppressor genes, which in turn contribute to various hallmarks of cancer3.
On the other hand, long interspersed nuclear elements-1 (LINE-1s or L1s) are repetitive, transposable DNA elements that normally have high levels of methylation at CpG islands. Methylation of LINE-1 prevents translocation and helps maintain genome integrity. In several types of cancer, LINE-1 is hypomethylated, resulting in activation and subsequent retrotransposition-mediated chromosomal instability4. LINE-1 accounts for nearly 17% of the human genome5, and its methylation status may serve as an indicator of global genomic methylation levels6. Global LINE-1 hypomethylation is considered to precede the transition of cells to a tumor phenotype7; therefore, it holds promise as a potential marker for early cancer onset.
Currently, there are several methods for methylation analysis, including pyrosequencing, methylation-specific PCR, microarrays, and chromatin immunoprecipitation1. The use of next-generation sequencing has also made it possible to incorporate genome-wide approaches to detection of DNA methylation. Many of these methods rely on bisulfite-treated DNA, in which unmethylated cytosines are converted to uracil and methylated cytosines remain unchanged. However, working with bisulfite-treated DNA has several pitfalls, such as incomplete conversions of unmethylated cytosines to uracil, biased amplification of sequences, and sequencing errors8.
In methylation-specific probe amplification (MSPA), probes composed of two oligonucleotides target DNA sequences containing a restriction site (GCGC) for the methylation-sensitive restriction enzyme HhaI9. After the probes hybridize to DNA, each sample is divided into two sets. Probes in the first set undergo ligation, while probes in the second set undergo ligation followed by HhaI-mediated digestion at unmethylated CGCG sites. Both sets of samples are then amplified by PCR, and the products are separated by capillary electrophoresis. Probes at unmethylated sites are digested by HhaI and are not amplified during PCR, resulting in no peak signals. By contrast, probes at methylated sites are protected from digestion and are therefore amplified during PCR, subsequently generating peak signals10.
MSPA has several advantages over alternative methods. First, it requires a low amount of DNA (50&#8211;100 ng) and is well-suited for analysis of DNA from formalin-fixed paraffin embedded samples10. It does not require bisulfite-treated DNA; in fact, it is unsuitable for DNA that is modified in this way. Many samples can be analyzed at the same time, and MSPA probes can be designed such that they target multiple genes or sequences simultaneously. Additionally, the probes are specific and sensitive for methylated DNA as the HhaI restriction site corresponds to a sequence that is typical of CpG islands10.
This study investigated the effects of osteosarcoma (OS)-derived extracellular vesicles (EVs) on LINE-1 methylation in adipose tissue-derived mesenchymal stem cells (AT-MSCs; Figure 1). EVs are nanoscale, membrane-bound vesicles secreted by most cell types. They carry proteins, lipids, mRNA, microRNA, and additional molecules from parent cells11,12. EVs mediate intercellular communication and play important roles in several pathophysiological conditions13,14. A recent study showed that cancer-derived EVs may transfer active LINE-1 to recipient cells15. It has been reported earlier that EVs from the HOS-143B cell line can alter the methylation status of LINE-1 in MSCs, in addition to other genetic effects16.
When growing cells for EV isolation, it is important to use EV-depleted fetal bovine serum [FBS] in the growth medium, since FBS-derived EVs may interfere with EVs from other sources and hamper the results17,18. Ultracentrifugation is one of the most common methods for depleting EVs from FBS. It is a relatively simple and cost-effective procedure compared to alternatives such as ultrafiltration and commercial EV-depleted FBS19. Here, the protocol also demonstrates how to prepare EV-depleted FBS by ultracentrifugation.
This article presents a detailed protocol for the aforementioned techniques, from isolation of EVs from an OS cell line to the methylation analysis of LINE-1 in OS-EV treated MSCs (Figure 1).

<table><tbody><tr><td>1 mL syringe</td><td>Terumo</td><td>SS+01T1</td><td>for NTA</td></tr><tr><td>24-well plate</td><td>Corning</td><td>3524</td><td>MSC cell culture</td></tr><tr><td>3730xl DNA Analyzer</td><td>Applied Biosystems, ThermoFisher Scientific</td><td>3730XL</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>Beckman Optima LE-80K Ultracentrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>BlueStar Prestained Protein Marker</td><td>Nippon Genetics</td><td>MWP03</td><td>WB: protein marker</td></tr><tr><td>Calnexin (clone C5C9)</td><td>Cell Signaling Technology</td><td>2679</td><td>WB, dilution 1:800</td></tr><tr><td>CD63 (clone H5C6)</td><td>BD Biosciences</td><td>556019</td><td>WB, dilution 1:1000</td></tr><tr><td>Centrifuge 5702 R</td><td>Eppendorf</td><td>5703000010</td><td>For conditioned media and cells</td></tr><tr><td>Centrifuge 5810</td><td>Eppendorf</td><td>5810000010</td><td>For spinning down 96-well plate</td></tr><tr><td>Centrifuge tube (polyallomer, 14x95 mm)</td><td>Beckman</td><td>331374</td><td>Ultracentrifugation</td></tr><tr><td>DMEM/F-12 + GlutaMAX medium</td><td>Gibco, Life Technologies</td><td>31331-028</td><td>For AT-MSC culture</td></tr><tr><td>Fetal bovine serum</td><td>Gibco, Life Technologies</td><td>10270-106</td><td></td></tr><tr><td>GeneScan 500 LIZ size standard</td><td>Applied Biosystems, Life Technologies</td><td>4322682</td><td>for capillary electrophoresis</td></tr><tr><td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td><td>Sigma</td><td>WGA2-10RXN</td><td>for MSPA negative control</td></tr><tr><td>Hi-Di formamide</td><td>Applied Biosystems, Life Technologies</td><td>4311320</td><td>for capillary electrophoresis</td></tr><tr><td>HOS-143B cell line</td><td>ATCC</td><td>CRL-8303</td><td></td></tr><tr><td>Hsp70 (clone 5G10)</td><td>BD Biosciences</td><td>554243</td><td>WB, dilution 1:1000</td></tr><tr><td>IRDye 800CW Goat anti-mouse</td><td>Li-Cor</td><td>926-32210</td><td>WB: secondary</td></tr><tr><td>IRDye 800CW Goat anti-rabbit</td><td>Li-Cor</td><td>926-32211</td><td>WB: secondary</td></tr><tr><td>LINE-1 probe-mix primers</td><td>IDT</td><td></td><td>Sequences in Table 1</td></tr><tr><td>MicroAmp Optical 96-well reaction plate with barcode</td><td>Applied Biosystems, Life Technologies</td><td>4306737</td><td>also requires sealing film</td></tr><tr><td>Micro BCA Protein Assay kit</td><td>ThermoFisher Scientific</td><td>23235</td><td>measure protein concentration</td></tr><tr><td>MiniProtean TGX 10% gels</td><td>Bio-Rad</td><td>456-1034</td><td>WB: gel electrophoresis</td></tr><tr><td>NanoSight LM14C</td><td>Malvern Instruments</td><td></td><td>for NTA</td></tr><tr><td>Nitrocellulose membrane 0.2 &amp;micro;m</td><td>Bio-Rad</td><td>1620112</td><td>WB: protein transfer</td></tr><tr><td>NucleoSpin Tissue XS</td><td>Macherey-Nagel</td><td>740901.50</td><td>for DNA extraction</td></tr><tr><td>Odyssey Blocking Buffer</td><td>Li-Cor</td><td>927-40000</td><td>WB: blocking, antibodies</td></tr><tr><td>PBS, 1X</td><td>Corning</td><td>21-040-CVR</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco, Life Technologies</td><td>DE17-602E</td><td>Antibiotics for culture media</td></tr><tr><td>Protein LoBind tube, 0.5 mL</td><td>Eppendorf</td><td>22431064</td><td>For storing Evs</td></tr><tr><td>REVERT Total Protein Stain and Wash Solution Kit</td><td>Li-Cor</td><td>926-11015</td><td>WB: total protein staining</td></tr><tr><td>RKO cell line</td><td>ATCC</td><td>CRL-2577</td><td>for MSPA positive control</td></tr><tr><td>RPMI medium 1640 + GlutaMAX</td><td>Gibco, Life Technologies</td><td>61870-010</td><td>For HOS-143B cell culture</td></tr><tr><td>SALSA MLPA HhaI enzyme</td><td>MRC-Holland</td><td>SMR50</td><td></td></tr><tr><td>SALSA MLPA reagent kit</td><td>MRC-Holland</td><td>EK1-FAM</td><td></td></tr><tr><td>SALSA MLPA P300 probe-mix</td><td>MRC-Holland</td><td>P300-100R</td><td></td></tr><tr><td>Swinging rotor SW-28</td><td>Beckman Coulter</td><td>342207</td><td>Ultracentrifugation</td></tr><tr><td>Syringe filter, 0.22 &amp;micro;m</td><td>Jet Biofil</td><td>FPE-204-030</td><td>sterile filtering FBS</td></tr><tr><td>Tecnai 12</td><td>FEI Company</td><td></td><td>equipped with Gatan Orius SC&lt;br/&gt; 1000B CCD-camera&lt;br/&gt; (Gatan Inc., USA); for TEM</td></tr><tr><td>TBS, 1X tablets</td><td>Medicago</td><td>09-7500-100</td><td>WB: buffer</td></tr><tr><td>Trans-Blot Turbo</td><td>Bio-Rad</td><td></td><td>WB: transfer</td></tr><tr><td>Thermal cycler</td><td>ThermoFisher Scientific</td><td>TCA0096</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12604-021</td><td>for trypsinization of cells</td></tr><tr><td>TSG101 (clone 4A10)</td><td>Sigma</td><td>SAB2702167</td><td>WB, dilution 1:500</td></tr></tbody></table>

line-1 methylation analysis in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4&#8211;5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

The main goal of this study was to evaluate the epigenetic effects of OS-EVs on MSCs. OS-EVs were isolated from HOS-143B cells using the standard differential centrifugation method. Expression of the typical EV markers CD63, Hsp70, and TSG101 by western blotting confirmed the presence of OS-EVs. (Figure 2A). Absence of calnexin signal indicated purity of the OS-EV isolate. Additional indication of purity was observed with TEM, with intact vesicles of various sizes being present (Figure 2B). The average OS-EV particle concentration was 7.63x1011/mL (Figure 2C). The size distribution of EVs ranged from 50&#8211;500 nm, with around 80% of particles falling within 50&#8211;200 nm range (Figure 2D).
AT-MSCs were treated with OS-EVs and DNA was extracted from the MSCs at different timepoints. LINE-1 methylation was analyzed by MS-MLPA and calculated in terms of methylation dosage ratio. Results from TP 0 were used to determine the baseline methylation levels (dashed line) (Figure 3). At TP 3 (green columns), a decrease in average LINE-1 methylation dosage ratio was observed in MSCs when treated with OS-EVs, falling under baseline methylation levels. This hypomethylating phenomenon was more subtle at TP 7 (blue columns), and the average methylation dosage ratio was slightly higher in both untreated and EV-treated MSCs compared to the baseline.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60705/60705fig1.jpg" /> 
Figure 1: Experimental workflow. EVs were isolated from HOS-143B cells by differential centrifugation and characterized by western blotting, TEM and NTA. AT-MSCs were treated with OS-EVs at different time points (TP 0, 3, an 7). DNA was extracted from MSCs and LINE-1 methylation was analyzed by MSPA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60705/60705fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60705/60705fig2.jpg" /> 
Figure 2: Characterization of EVs. (A) The presence of EV markers CD63, Hsp70 and TSG101 confirmed the presence of EVs by western blotting, while no bands were observed for calnexin, indicating the purity of the EV isolate. 10 &#181;g of protein was loaded for both HOS-143B protein lysate and OS-EVs. (B) TEM confirmed the presence of intact EVs of various sizes in the isolate. (C) Particle concentration and (D) size distribution of OS-EVs were determined by NTA measurements. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60705/60705fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60705/60705fig3a.jpg" /> 
Figure 3: Methylation dosage ratios of LINE-1 from MSCs either untreated or treated with OS-EVs. The dashed grey line represents baseline methylation value, corresponding to TP 0 values. Green columns represent average methylation dosage ratios without and with OS-EV treatment for TP 3 samples, while blue columns represent the same for TP 7 samples. Both TP 3 and TP 7 samples showed a decrease in methylation levels after treatment with OS-EVs. However, the difference was greater in TP 3 samples, where the methylation level after EV treatment was also lower than the baseline value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60705/60705fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img src="/files/ftp_upload/60705/60705table1.jpg" />
Table 1: MSPA reagent mix recipes and thermocycler/PCR programs. The volumes mentioned represent one DNA sample. It should be noted that the polymerase mix should be prepared for 2x as many samples as previous mixes, since there are two sets of tubes at this stage. Details of the subsequent thermocycling or PCR program are provided to the right.

Watch this Scientific Journal Video about LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles at JoVE.com

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

line-1-methylation-analysis-mesenchymal-stem-cells-treated-with

Nanyang Technological University

Research

JoVE Journal

Genetics

5.7K Views.  University of Helsinki. DNA-methylatie is een van de meest bestudeerde epigenetische handtekeningen. Veel kankers worden gekenmerkt door veranderingen in DNA-methylatieniveaus, die worden geassocieerd met afwijkende genexpressie en andere genomische afwijkingen. Lange gestrooide kernelementen zijn repetitieve omslagbare genomische sequenties die normaal gesproken hoge niveaus van methylatie hebben.Echter, ze worden vaak hypermethylated bij kanker, wat resulteert in hun activering en leidt tot chromosomale ins...

Video: LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Cancer Research

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Biochemistry

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

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.

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, ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Bioengineering

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 ...

Medicine

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 ...

Biology

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

Setting a Successful Sorting for Extracellular Vesicle Isolation

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.
First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. 
In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.
Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. ...

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

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