Effect of monocyte- and osteoclast-derived extracellular vesicles on adipose tissue-derived mesenchymal stem/stromal cells. Homo sapiens

Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from monocytes and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human monocytes and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. Monocyte- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated monocytespromoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. Monocyte-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that monocytes facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. This article is protected by copyright. All rights reserved. Overall design: Microarray analysis was performed on total RNA isolated from adipose tissue-derived mesenchymal stem/stromal cells (AT-MSC) obtained from water-assisted lipotransfer liposuction aspirates from 5 female donors. AT-MSCs were cultured in maintenance medium (MM) containing 10% fetal bovine serum depleted of extracellular vesicles (EVs), supplemented with: 1) EVs isolated from the conditioned medium of CD14+ monocytes obtained from 2 human buffy coats, activated with 10 ng/ml lipolysaccharide (MC-EVs) 2) EVs isolated from the conditioned medium of osteoclasts cultured on tissue culture polystyrene, differentiated from the CD14+ monocytes by M-CSF/RANK-L stimulation (OC TCPS-EVs) 3) EVs isolated from the conditioned medium of osteoclasts, activated for mineral resorption by culture on hydroxyapatite coatings, differentiated from the CD14+ monocytes by M-CSF/RANK-L stimulation (OC HA-EVs) 4) 50 µM L-Ascorbic acid 2-phosphate, 10 µM beta-glycerophosphate disodium salt hydrate, 100 nM dexamethasone (osteogenic differentiation medium, ODM) EV-depleted MM without supplementation was used as the reference condition. The RNA was pooled into 3 distinct pools per condition, assembled from 5 donor replicates. The starting amount of total RNA was 100 ng. Complementary DNA (cDNA) was generated using GeneChip®WT Plus Reagent Kit (Affymetrix). Micorarray analysis was performed using Clariom D Human Arrays (Affymetrix).