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

  1. Lopes Ramos, Teresa
Supervised by:
  1. Consuelo del Cañizo Fernández-Roldán Director
  2. Luis Ignacio Sánchez-Abarca Bernal Co-director
  3. Fermín Sánchez-Guijo Martín Co-director

Defence university: Universidad de Salamanca

Fecha de defensa: 05 September 2016

Committee:
  1. Jesús María Hernández Rivas Chair
  2. J. R. González Porras Secretary
  3. Antonio Medina Almeida Committee member
Department:
  1. MEDICINA

Type: Thesis

Abstract

Background Bone marrow-derived mesenchymal stromal cells (BM-MSC) are crucial for haematopoietic niche maintenance. There is evidence of continuous cross-talk between the neoplastic cells and BM-MSC, inducing the modulation of both populations, which may favour the emergence and progression of myeloproliferative neoplastic (MPN) disease. Extracellular vesicles (EV) have emerged as new play-ers in cell-to-cell communication. These structures are associated with the bidirectional transfer of proteins, mRNA, microRNA between cancer cells and stromal cells, inducing functional and genetic alterations in neighbouring cells. Objectives and methodology - To characterise and compare BM-MSC from MPN patients (JAK2V617F) with BM-MSC from HD and CML patients, population doubling, multilineage differentiation, immunophenotype, apoptosis and cell cycle assays were performed. - To study the presence of genomic alterations in the BM-MSC from ET and PV patients, and com-pare to control MSC (HD), a global analysis of gene expression profile from the different experimental groups was performed, using Affymetrix Oligoarrays (Human Gene 1.0 ST arrays). RT-PCR and Western blot (WB) were performed to confirm the results. - To evaluate if the alteration in the BM-MSC from JAK-2 MPN patients modifies the capacity to sup-port normal and MPN haematopoietic progenitor cells, clonogenic (CFU-GM) and long-term BM cul-ture (LTBMC) assays were performed. - To identify altered genes and pathways in the MPN-MSC which could potentially be used as thera-peutic targets, we selected from the previous experiments histone deacetylase 8 (HDAC8). Then we treated BM-MSC from HD and MPN patients with a specific inhibitor PCI34051 at concentration of 25µM during 48h. The effects of HDAC8i on the BM-MSC was evaluated by annexing V (apoptosis), cell cycle, RT-PCR and WB. To assess the impact of this inhibition on the capacity of MPN-MSC to support haematopoiesis, BM mononuclear cells (BM-MNC) were co-cultured in transwell for 48h, with PCI34051-treated and non-treated BM-MSC. After co-culture, cell viability, CFU-GM assays and TP53 expression were analysed. - To compare the microRNA content in the MSC-derived extracellular vesicles (EV) from HD and MPN patients, and to study the functional alterations that may be induced when incorporated into haematopoietic progenitor cells. Firstly, EV were isolated from BM-MSC of MPN patients and HD, and were purified by ultracentrifugation. For EV characterisation, transmission electron microscopy (TEM), NanoSight, flow cytometry (MFC) and WB for CD63 were performed. To evaluate microRNA content into EV-MSC from both groups (patients and controls), expression of miRNA was analysed using 384-well microfluidic cards (TaqMan® MicroRNA Array A). EV incorporation was demonstrated by fluorescence microscopy and MFC, where HPC (CD34+cells obtained by immunomagnetic selection) were co-cultured with EV previously labelled with Vybrant Dil. To study HPC modification induced by the incorporation of EV, apoptosis and clonogenic assays were performed. Results 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 up-regulated. 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 associ-ated 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 ob-served 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 released from MPN-MSC represent a mechanism of intercellular communication between malignant stromal and haematopoietic cells, through the transfer of genetic information that may be relevant in the pathophysiology of these diseases.