Mesenchymal stromal cells from patients with myelodyplastic syndrome display distinct functional alterations that are modulated by lenalidomide

Abstract

ABSTRACTtypes including MDS associated with single del(5q), and characterized their clonogenicity and differentiation potential. Methods Human samplesTwenty MDS patients were studied; their characteristics are detailed in Online Supplementary Table S1. Our control cohort consisted of one male and five female healthy individuals undergoing orthopedic surgery with a median age of 60 years (range, 56-65). MSC were obtained as previously described. 18 Patients were classified for the study as "lower risk" with and without del(5q) (LR, LR-5q ) (<5% bone marrow blasts and IPSS-low/int-1), and as "higher-risk" (HR) (>5% bone marrow blasts and IPSS-int-2/high). Magnetically sorted HSPC were obtained from peripheral blood of healthy individuals after mobilization. Samples were collected after approval by our Institutional Review Board, and written informed consent was obtained. A detailed description is available in the Online Supplementary Material. Lenalidomide treatmentLEN was kindly provided by Celgene (Munich, Germany) and prepared using 10% dimethyl-sulfoxide as a vehicle. The experiments were performed with 0.1 μM and 1 μM of the drug. Characterization of the mesenchymal stromal cellsClonality, self-renewal, generation of single cell-derived colonies (SCD), expression of cell surface and adhesion molecules with flow cytometry, differentiation potential, proliferation, viability, apoptosis, senescence, and cell cycle were studied. Detailed information about the experiments is provided in the Online Supplementary Material. Co-culture of myelodysplastic syndrome-mesenchymal stromal cells with hematopoietic stem and progenitor cells, clonogenicity and apoptosis assayThe capacity of MSC to support HSPC was tested using a 4-week cobblestone area-forming (CAF-C) assay and a 14-day colony-forming unit granulocyte-erythrocytemonocyte/macrophage (CFU-GEMM) assay as described in the Online Supplementary Material. HSPC or the cell line KG1-α were labeled with carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) prior to co-culture with MSC for 72 h, or left unlabeled and co-cultured for 24 h, with or without the addition of 100 nM recombinant tumor necrosis factor alpha (TNF-α, Peprotech). Apoptosis and proliferation were studied by annexin-V staining and CFSE dilution using flow cytometry. Cytokine measurement and transwell migration assaySupernatant from MSC was obtained after culture with or without LEN and used for enzyme-linked immunoabsorbent assay (ELISA) determinations of SDF-1α, angiopoietin-1 (ANG1), and stem cell factor (SCF) levels. Supernatant was also used for a 4-h transwell migration assay with 1x10 5 HSPC, as described previously. 18 The CXCR4 antagonist AMD3100 (Sigma) was used at a concentration of 10 μM.RNA extraction, complementary DNA synthesis and real time polymerase chain reaction mRNA extracted with Trizol reagent (Invitrogen) was transcribed into complementary-DNA and used for real time polymerase chain reaction (RT-PCR) analysis for fatty acid binding protein 4 (FABP4), SDF-1α , ANG1, and SCF. Ab...

Down-regulation of Dicer1 promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells in patients with myelodysplastic syndrome

“…31,32 Just as predicted, most of the cultured and primary MDS-MSC were larger and irregular and expressed significantly higher amounts of SA-β-gal and the senescence-related molecule, p21. These findings are similar to those of previous studies, 10,14 but the investigators of those studies did not provide a detailed analysis of the different MDS subsets and the functional changes related to senescence in MDS pathology. In our study, not all of the cases exhibited an increase in senescence (22/32, 68.7%).…”

“…In keeping with our findings, some studies demonstrated that MDS-MSC displayed insufficient hematopoietic support capability compared with normal controls. 10,13,14 However, other reports suggested that MDS-MSC may have normal support capacity. 11,12,34 The discrepancies in these results might be attributed to differences between patients, cellular populations and experimental protocols in the various studies.…”

“…10,14 Cellular senescence is usually accompanied by functional changes. Indeed, decreased differentiation has been linked to senescence and aging in MSC.…”

Alteration Analysis of Bone Marrow Mesenchymal Stromal Cells from De Novo Acute Myeloid Leukemia Patients at Diagnosis

“…Such proliferation defects have also been described in MSCs from several other hematopoietic disorders, such as acute lymphoblastic leukemia (ALL) [31,32], chronic lymphocytic leukemia (CLL) [33,34], and especially MDS [16,17,35,36]. A slight decrease of their clonogenic potential (CFU-F) was also observed that was inversely correlated with the PDT values.…”

“…However, it is not yet evident if MSC long-term hematopoietic-supporting activity in AML at diagnosis is intrinsically impaired [12] or not [10]. Otherwise, several recent reports have highlighted the abnormal MSC function in chronic preleukemic diseases such as myelodysplastic syndromes (MDSs) [15][16][17] in contrast to chronic myelogenous leukemia (CML) [18,19].…”