Impaired Wnt-Pathway Signalling and Reduced Expression of Senescence-Associated Markers in Bone Marrow Mesenchymal Stem Cells of Patients with Myelodysplastic Syndromes

Abstract 272

The pathogenesis of Myelodysplastic Syndromes (MDS) is characterized by the acquisition of multiple consecutive alterations in an early hematopoietic stem cell, leading to a stepwise clonal cell expansion and disease evolution. An abnormal bone marrow (BM) microenvironment, however, might also contribute to the BM failure in MDS through defective support of hematopoiesis. The cellular elements of the BM microenvironment derive from a common progenitor cell, namely the mesenchymal stem cell (MSC). We and others (reviewed in Kastrinaki et al. Curr Stem Cell Res Ther. 2011) have recently reported that MDS-derived BM MSCs show defective proliferative potential, impaired clonogenic capacity and limited cellular expansion during passages. The pathogenetic basis of these abnormalities and their implication in the disease process remain unknown. The aim of this study is to probe the mechanisms underlying the impaired functional properties of BM MSCs in patients with lower risk MDS.

BM MSCs were isolated from 12 patients with lower risk MDS aged 51 to 75 years (median 67.5 years) and 20 healthy volunteers aged 50 to 73 years (median 63.3 years). BM MSCs were expanded and re-seeded for a total of 10 passages (P). To investigate whether the decreased proliferative capacity of MSCs in MDS patients might be related to replicative senescence, genomic DNA was isolated from culture expanded MSCs at P2, P6 and P10 and telomere length was measured by means of real-time PCR using β-globin as control single-copy-gene. Telomere length is proportional to the relative telomere/single-copy-gene ratio (T/S): T/S=2^−δCt(δCt=Ct^telomere-Ct^β-globin). Furthermore, total RNA was extracted from culture-expanded P2, P6 and P10 MSCs and amplified by real-time PCR for the evaluation of the senescence associated genes CDKN1B (p15), CDKN2A (p16), CDKN1A (p21), pRB, TP53 (p53) and PARG1. Relative gene expression was calculated by the δCt method. Given that the Wnt signaling pathway plays a key role in MSC proliferation and differentiation, we next investigated whether the proliferative defect of MSC in MDS might be associated with abnormal expression of genes related to Wnt-mediated signal transduction, using a PCR array. Total RNA was thus isolated from 4 representative MDS and 6 normal MSC cultures at P2. The fold change (FC) for each gene between the group of patients and the group of controls was calculated with the δδCt method (FC=2^−δδCt).

The relative telomere values declined over the entire observation period (P2-P10) in both patient- and control-derived MSCs. T/S values remained constantly higher over passages in MDS MSCs compared to healthy individuals (F=4.362, P<0.05) and the highest difference was obtained in P10 (6545±904 versus 4465±403, respectively, P<0.05). A statistically significant decreased expression of CDKN1B (p15), CDKN1A (p21) and PARG1 was identified in patients compared to the controls time-course (F=6.14, P<0.001; F =4.56, P<0.05 and F=3.716, P<0.05, respectively) implying a decreased senescence rate of MDS-derived MSCs. The expression of CDKN2A (p16) was stable during passages in patients whereas an increase was obtained in healthy subjects time-course but not at statistically significant level. No statistically significant differences were observed in pRB and TP53 (p53) expression between patients and controls time-course. Regarding the Wnt-pathway signaling molecules, we observed that patient MSCs displayed an over-expression of the canonical Wnt pathway inhibitors (αES-FC=3.2, P<0.05; DVL1-FC=3.1, P=0.01; GSK3A-FC=3.9, P<0.01; SENP2-FC=4.6, P<0.01) as well as ligands of the non-canonical Wnt pathway (Wnt5A-FC=3.2, P<0.01; Wnt5B-FC=3.3, P<0.01; Wnt9A-FC=4.14, P<0.01) in comparison to the control group.

MDS-derived MSCs display longer telomeres and decreased expression of senescence-associated genes compared with healthy individuals. The possibility therefore that cellular aging account for the defective proliferative potential of patient MSCs seems rather unlikely On the other hand, activation of the non-canonical Wnt pathway in association with the inhibition of the canonical one can explain, at least in part, the decreased proliferative capacity of MSCs in MDS patients. Collectively our results suggest that aberrant Wnt signaling is actively implicated in the mechanisms underlying the impaired functional properties of BM MSCs in MDS.