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

DNA methylation is one of the most studied epigenetic signatures. Many cancers are characterized by changes in DNA methylation levels, which are associated with aberrant gene expression and other genomic abnormalities. Long interspersed nuclear elements are repetitive transposable genomic sequences that normally have high levels of methylation.

However, they often become hypermethylated in cancer, which result in their activation and leads to chromosomal instability. In this study, we treated mesenchymal stem cells with osteosarcoma-derived extracellular vesicles to see whether cancer EVs can affect LINE-1 methylation in MECs. Fetal bovine serum, which is an integral component of mammalian cell culture media, contains a large number of bovine-derived EVs.

These Evs will interfere with analysis of Evs from cells of our interest, therefore, when growing cells for EV isolation, it is important to use EV-depleted FBS. Take FBS in ultracentrifuge tubes and place them in ultracentrifuge buckets. Balance the buckets to within 10 milligrams of each other prior to loading them on a swinging rotor.

Place the rotor in the ultracentrifuge and run at 100, 000 G for 19 hours at four degrees Celsius. After the run, carefully collect the light colored upper layer of the supernatant and transfer it to a 50 milliliter tube. Do not disturb or pipet the dark brown pellet as it contains EVs from FBS.

Pass the supernatant through a 0.22 micron filter into a new 50 milliliter tube. This filter-sterilized, EV-depleted FBS can now be added to cell culture media when growing cells for EV isolation. Collect conditioned media from osteosarcoma cells grown in EV-depleted media.

To remove cells and cell debris, centrifuge the conditioned media at 2500 G for 20 minutes at four degrees Celsius. Transfer the supernatant to ultracentrifuge tubes and balance them as earlier. Centrifuge the tubes at 100, 000 G for two hours at four degrees Celsius.

Carefully discard the supernatant leaving around one milliliter at the bottom. Add around 20 milliliters of PBS to the tube and pipet gently in order to wash and re-suspend the EV pellet. Balance the tubes and carry out another round of ultracentrifugation with the same settings.

Carefully remove the supernatant and re-suspend the EV pellet in 200 microliters of PBS by gentle pipetting. Store the EVs in low-binding tubes. Purified EVs are characterized by western blotting, nanoparticle tracking analysis, and transmission electron microscopy.

Plate 15, 000 MSCs per well in a 24 well plate. After 24 hours, remove old media, wash the cells with PBS, and change to EV-depleted media. Treat cells with OS-EVs at the scheduled time points.

After stopping the EV treatment, extract DNA from the cells using an appropriate method. For methylation analysis, we used a custom-made methylation-specific probe amplification method. Design the customized LINE-1 probes, as done previously, by Pavicic and others.

For methylation probes, select three sequences containing the HhaI restriction site within the promoter region. For control probes, select seven sequences lacking the HhaI restriction site from the rest of the LINE-1 sequence. Dilute 70 nanograms of DNA sample in TE buffer to five microliters.

Heat the samples for 10 minutes at 98 degrees Celsius then cool to 25 degrees Celsius. Add three microliters of probe hybridization mix to each sample and run the thermocycler to allow the probes to hybridize to DNA. At room temperature, add 30 microliters of post-hybridization mix to each sample.

Transfer 10 microliters to a second tube. Place both sets of tubes in the thermocycler and incubate at 48 degrees Celsius for at least one minute. While the samples are at 48 degrees Celsius, add 10 microliters of ligation mix to the first set of tubes and 10 microliters of ligation-digestion mix to the second set of tubes.

Run the next thermocylcer program. Then, spin down the tubes and simultaneously set the thermocycler to 72 degrees Celsius. Add five microliters of polymerase mix to each tube and place the tubes in the thermocycler.

Run the PCR program. While the PCR program is running, prepare a solution of one milliliter formamide containing 2.5 microliters of size-standard. Pipet 10 microliters of this solution to each row of an optical 96 well plate.

After PCR, dilute the undigested and the digested samples to one to one hundred and one to two hundred, respectively in ultra pure water. Add two microliters of diluted PCR product to the 96 well plate. Centrifuge the plate at 200 G for 15 to 20 seconds to remove air bubbles.

Carry out fragment analysis of samples by capillary electrophoresis. Open the capillary electrophoresis results in an electropherogram analysis software. Select a sample and set the panel to MLPA from the menu.

Click on the panel and press control D to apply this setting to all samples. In a similar way, set the analysis method to microsatellite default for all samples. Select all samples and click on the green play button to run analysis, then select all samples and click on the graph button to visualize the probe peaks.

Zoom in on the peak region for a higher resolution of the individual peaks. Make sure that all 10 peaks corresponding to the probes from the LINE-1 probe mix are labeled. In the genotypes tab, export the results in the CSV format.

Open the CSV file in a data analysis software and sort the data into columns. Label the three methylation site peaks based on their sizes. The remaining seven peaks correspond to the control probes.

Here we are using the LINE-1 2M probe peaks which have a size of roughly 117 base pairs. For each sample, calculate the sum peak area of all seven control peaks. Divide the peak area of each LINE-1 probe by this sum.

For each sample, divide the value of the digested sample by that of the undigested sample to obtain the methylation dosage ratio. Western blotting analysis confirmed the presence of OS-EVs with signals observed for TSG101, HSP70, and CD63. Absence of calnexin signal indicated that the EV sample was pure.

Additional indication of purity was observed with TEM with intact vesicles of various sizes present in the OS-EV sample. The OS-EV particle concentration and size distribution was measured by NTA. 80%of the EVs were in the size range of 50 to 200 nanometers.

Here we can see the methylation dosage ratios of LINE-1 from MSCs at different time points of the EV treatment. The gray line represents baseline methylation level from time point zero. At time point three, there is a decrease in the average methylation dosage ratio after administration of EVs, thereby indicating hypermethylation.

This hypermethylating effect of the EVs is less pronounced at time point seven. Here we have shown a simple, highly sensitive, and robust technique for methylation analysis. It requires a low amount of DNA with no bisulfite conversion involved and it is also suitable for DNA isolated from paraffin-embedded samples.

The whole method, with the experimental and analytical parts, takes around two days. Due to a high copy number of LINE-1 in the genome, the baseline methylation of LINE-1 is high in normal samples. Therefore, the differences in methylation levels of samples, as observed with this method, may be subtle.