Role of Mesenchymal Stromal Cells and their Extracellular Vesicles microRNAs in JAK2 Myeloproliferative Neoplasms

Abstract

Compared to HD, BM-MSC from MPN patients showed similar morphology and differentiation capacity, with an increased proliferation rate with less apoptotic cells. BM-MSC from MPN expressed comparable levels of CD73, CD44, CD90 and CD166, whereas they were negative for haematopoietic markers. The median expression of CD105 was lower in BM-MSC from MPN patients (p <0.05) when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD. RT-PCR was performed in two genes to confirm these results, demonstrating that HDAC8 and MYADM genes were upregulated. Next, we observed a significant increase in the number of CFU-GM when MPN-HPC were co-cultured with MPN-MSC compared to HD-MSC. MPN-MSC also showed the ability to support healthy HPC in LTBMC. However, MPN-MSC showed alterations in the expression of genes associated to the maintenance of haematopoiesis. The inhibition of HDAC8 in BM-MSC from MPN was confirmed by RT-PCR and WB assays, when the cells were treated with PCI34051. HDAC8-selective inhibition also induced a cell cycle arrest in the MPN BM-MSC, with an increase of the percentage of apoptotic cells. Co-cultures of BM-MNC from MPN patients with neoplastic stroma previously treated with HDAC8 inhibitor induced a decrease in MNC cell viability (p=0.028), CFU-GM (p=0.018) and an increase of TP53 expression. Regarding the EV studies, we showed that the characterisation by TEM and NanoSight revealed that EV-MSC from both groups exhibited a size and morphology characteristic of EV, and were positive for CD63 (WB), the characteristic marker of EV. By MFC, EV released from BM-MSC of both groups (HD and JAK2) were defined as particles less than 1µM of diameter, positive for CD90, CD44 and CD73 and for EV markers. At the same time they were negative for CD34 and CD45, demonstrating the specificity of monoclonal labelling. When the content of microRNA was analysed (8 HD-MSC and 11 MPN-MSC), we observed an overall increase in the microRNA expression in the EV from patients, yet without reaching statistical significance. Using RT-PCR, we observed a significant overexpression (p=0.032) of miR-155 in the EV derived from MPN-MSC. We also observed an increase of CD34+ cell viability, after the incorporation of EV from both groups (HD and JAK2). A significant increase (p=0.04) of miR-155 expression was observed in the HD CD34+ cells, after incorporating MPN-MSC-derived EV. In addition, an increase of CFU-GM was observed when neoplastic CD34+ cells incorporated the EV derived from MSC from MPN patients (p=0.056). Conclusions These results suggest that MPN-MSC display different proliferative rate, immunophenotypic markers, gene expression profile and HDAC8 overexpression compared to HD-MSC. The inhibition of HDAC8 expression by its specific inhibitor decreases the capacity of the stroma to support haematopoietic cells from MPN patients, suggesting that HDAC8 may be a potential therapeutic target. Furthermore, we suggest that EV rel...