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.

中文翻译：

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小细胞外囊泡中的 Y-box 结合蛋白 1 减少间充质干细胞向成骨细胞的分化——对急性髓系白血病的影响

据报道，急性髓系白血病 (AML) 细胞释放的小细胞外囊泡 (sEV) 会影响骨髓间充质干细胞 (BM-MSC) 的三系分化。然而，目前尚不清楚 AML-sEV 中的哪种生物货物造成了这种效应。在这项研究中，使用尺寸排阻色谱和超滤从细胞条件培养基和血浆中分离出 sEV，并根据 MISEV2018 指南进行表征。我们的结果表明 AML-sEV 增加了 BM-MSC 的增殖。相反，对正常造血重要的关键蛋白在 BM-MSC 中下调。此外，我们发现 AML-sEV 显着降低 BM-MSC 向成骨细胞的分化，而不影响脂肪形成或软骨形成分化。接下来，LC-MS/MS 蛋白质组学阐明了各种蛋白质，包括 Y 盒结合蛋白 1 (YBX1)，在经 AML-sEV 处理的 AML-sEV 和 BM-MSC 中均上调。与临床相关的是，我们发现与健康对照相比，大多数儿科 AML 患者衍生的 sEV 中 YBX1 显着上调。有趣的是，在 AML 细胞中下调 YBX1 后分离的 sEV 显着挽救了 BM-MSC 的成骨细胞分化。总而言之，我们的数据首次证明含有 AML-sEV 的 YBX1 是通过减少 BM-MSC 的成骨分化来破坏骨髓微环境正常功能的关键参与者之一。