Abstract:
During pregnancy, the human placenta releases extracellular vesicles (EVs) into the maternal circulation which carry diverse cargos, including fetal DNA (fDNA). Also, during pregnancy, fetal microchimerism has been described in mothers which is defined as the permanent translocation of a small number of fetal cells to the maternal body. Studies investigating fetal microchimerism typically utilize short DNA primers/probes against Y-chromosome sequences (e.g. SRY) in mothers who have had male pregnancies. However, it is possible that the identification of fetal cells may, in part, be fDNA (e.g. SRY) delivered into maternal cells by placental EVs. I hypothesized that the placenta releases large quantities of EVs containing fDNA that can transfect maternal cells to create more widespread ‘genetic fetal microchimerism’.
I systematically summarized the biodistribution of EVs in animal models from the literature, including placental EVs, to determine whether their localization aligned to the sites of fetal microchimerism; characterized the fDNA associated with placental EVs; developed methods to load EVs with plasmids; and investigated whether cells can be stably transfected with EV-delivered fDNA or plasmid for sequence-specific transfection.
Placental EVs distributed predominantly to the liver, lungs, and kidneys, which were organs, amongst others, that are reported to be sites of fetal microchimerism. Placental EVs are released in abundance and were associated with copious quantities of fDNA. The fDNA of EV populations mostly uniformly represented the fetal exome and had the capacity to carry large fDNA sizes that could encode intact genes (including SRY), up to 18,008 bp, including the 5’ regulatory region. Placental EVs can be loaded with plasmid DNA by electroporation or directly incubating EVs with the plasmid. Placental EVs are internalized into recipient cells, reaching perinuclear regions and the nucleus. fDNA (including SRY) was delivered into recipient cells by EVs, but stable transfection was not confirmed. Lastly, plasmid carried by placental EVs failed to show successful sequence-specific transfection into cells.
Taken together, my work demonstrates the abundance of placental EVs, their carriage of fDNA, the uptake of EVs into recipient cells, and transient detection of fDNA in recipient cells and this evidence supports the possibility of genetic fetal microchimerism, although this is likely a rare event which is technically challenging to detect, model and validate.