Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

Sandra Muntión,María-Eugenia Sarasquete,María-Consuelo Del Cañizo,María Diez-Campelo,Marcos González,Fermín Sánchez-Guijo,Beatriz Rosón,Teresa L Ramos,Javier De Las Rivas,Luis Ignacio Sánchez-Abarca,Silvia Preciado,Irena Misiewicz-Krzeminska,Wolfgang Wagner

doi:10.1371/journal.pone.0146722

Abstract

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

Highlights

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematological disorders characterized by the presence of peripheral cytopenias and an increased risk of transformation into acute myeloblastic leukemia (AML)[1, 2]
Immunophenotypic analysis by flow cytometry (FC) showed that all mesenchymal stromal cells (MSC)-derived MVs from healthy donors (HD) and MDS patients displayed a compared pattern
In 6 cases (4 MDS and 2 HD) the presence of MVs in the MSC-derived supernatants was confirmed by transmission electronic microscopy (TEM), whereby these structures were observed to have sizes of around 200nm (Fig 1C)

Summary

Introduction

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematological disorders characterized by the presence of peripheral cytopenias and an increased risk of transformation into acute myeloblastic leukemia (AML)[1, 2]. Our group[5] and others[6,7,8] have shown that MSC exhibit several morphological, functional and genetic alterations in MDS patients In this regard, Raaijmakers et al.[9] have recently demonstrated in a murine model that the deletion of DICER, an RNase III enzyme involved in microRNA biogenesis, in MSC-derived osteoprogenitors affected their differentiation and resulted in peripheral blood cytopenias, myelodysplasia and secondary leukemia, providing evidence that specific molecular alterations in the bone marrow microenvironment could result in clonally impaired hematopoiesis. The vesicles are a mixed population of exosomes and shedding vesicles and both components, despite originating from different cellular structures, participate in the communication between the microenvironment and the HPC In this manuscript we will refer to them as microvesicles (MVs). The hypothesis of our study is that MSC from MDS patients (MDS-MSC) can modify the properties of HPC through release of MVs with a different microRNAs content as compared to MSC from healthy donors (HD-MSC)

Methods

Results

Discussion