Y-box binding protein 1 in small extracellular vesicles reduces mesenchymal stem cell differentiation to osteoblasts-implications for acute myeloid leukaemia.

Small extracellular vesicles (sEVs) released by acute myeloid leukaemia (AML) cells have been reported to influence the trilineage differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it remains elusive which biological cargo from AML-sEVs is responsible for this effect. In this study, sEVs were isolated from cell-conditioned media and blood plasma using size-exclusion chromatography and ultrafiltration and characterized according to MISEV2018 guidelines. Our results demonstrated that AML-sEVs increased the proliferation of BM-MSCs. Conversely, key proteins that are important for normal haematopoiesis were downregulated in BM-MSCs. Additionally, we revealed that AML-sEVs significantly reduced the differentiation of BM-MSCs to osteoblasts without affecting adipogenic or chondrogenic differentiation. Next, LC-MS/MS proteomics elucidated that various proteins, including Y-box-binding protein 1 (YBX1), were upregulated in both AML-sEVs and BM-MSCs treated with AML-sEVs. Clinically relevant, we found that YBX1 is considerably upregulated in most paediatric AML patient-derived sEVs compared to healthy controls. Interestingly, sEVs isolated after the downregulation of YBX1 in AML cells remarkably rescued the osteoblastic differentiation of BM-MSCs. Altogether, our data demonstrate for the first time that YBX1 containing AML-sEVs is one of the key players that disrupt the normal function of bone marrow microenvironment by reducing the osteogenic differentiation of BM-MSCs.

Investigation of the Presence of DNA in Human Blood Plasma Small Extracellular Vesicles.

Extracellular DNA (ecDNA) is DNA outside of cells, which is a result of various mechanisms. EcDNA is believed to be a cause of various pathogeneses as well as their potential biomarker. EcDNA is believed to also be part of small extracellular vesicles (sEVs) from cell cultures. If ecDNA is present in sEVs in plasma, their membrane may protect it from degradation by deoxyribonucleases. Moreover, sEVs play a role in the intercellular communication, and they can therefore transfer ecDNA between cells. The aim of this study was to investigate the presence of ecDNA in sEVs isolated from fresh human plasma by the ultracentrifugation and density gradient, which serves to exclude the co-isolation of non-sEVs compartments. The novelty of the current study is the investigation of the localization and subcellular origin of the ecDNA associated with sEVs in plasma, as well as the estimation of the approximate concentration. The cup-shaped sEVs were confirmed by transmission electron microscopy. The highest concentration of particles was in the size of 123 nm. The presence of the sEVs markers CD9 and TSG101 was confirmed by western blot. It was found that 60-75% of DNA is on the surface of sEVs, but a part of the DNA is localized inside the sEVs. Moreover, both nuclear and mitochondrial DNA were present in plasma EVs. Further studies should focus on the potential harmful autoimmune effect of DNA carried by plasma EVs or specifically sEVs.