or less. Experimental study exploring the influence of EVs on DNA methylation

Hypothesis: EVs from aged cells can influence the epigenetic landscape of stem cells by inducing DNA methylation changes. Rationale: Epigenetic changes are responsible for maintaining cellular identity and development, and studies have shown that epigenetic plasticity can be influenced by environmental factors. Age-associated changes in the epigenetic landscape are observed in somatic tissues, and this may affect the ability of stem cells to differentiate or maintain pluripotency. EVs from aged cells contain molecules, such as DNA and RNA, which can be taken up by stem cells and modify their epigenetic landscape. It is therefore possible that EVs from aged cells could induce epigenetic changes in stem cells through the delivery of molecules which have been altered by the ageing process. Methodology: To investigate the influence of EVs on DNA methylation, we will isolate EVs from aged cells and stem cells. We will then transfect the stem cells with EVs from the aged cells, and compare the DNA methylation profiles between the transfected cells and the control cells. This will be done using a bisulfite sequencing assay to quantify the methylation levels at specific CpG sites. To further investigate the role of EVs in inducing epigenetic changes, we will knock down the expression of key proteins involved in the uptake of EVs in stem cells and compare the DNA methylation profiles between the transfected cells and the control cells. We will also assess the changes in DNA methylation in the stem cells over time to determine whether EVs can induce persistent epigenetic changes. Quantification: We will quantify the differences in DNA methylation between the control and transfected cells using a bisulfite sequencing assay. We will compare the methylation levels at specific CpG sites between the two groups and calculate the mean methylation levels for each group. Interpretation: We will interpret the data by comparing the mean methylation levels of the control and transfected cells. If the transfected cells show higher levels of methylation at specific CpG sites than the control cells, this will indicate that EVs from aged cells have induced an epigenetic change in the stem cells. We will also compare the changes in DNA methylation in the stem cells over time to determine whether EVs can induce persistent epigenetic changes.