The Effect of 5-Azacitidine Treatment on the Biologic Characteristics of Bone Marrow Mesenchymal Stem Cells in Patients with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cell characterized by ineffective bone marrow (BM) hematopoiesis and increased risk for leukemic evolution. An abnormal BM microenvironment has been reported to contribute to 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 have previously shown that MSCs from MDS patients display impaired proliferative and clonogenic potential and develop chromosomal alterations during passages.

It has been shown that DNA methylation occurs at a high frequency in MDS and that gene reactivation by hypo-methylating agents may have a positive impact on patients' outcome. Recent evidence also suggests altered global DNA methylation status in BM-MSCs of MDS patients. The methyl transferase inhibitor 5-Azacitidine (ΑΖΑ) is widely used for the treatment of MDS; however the effect of AZA on patients' BM microenvironment and specifically on the MSC compartment has not been extensively studied. In the present study we investigate the functional, molecular and cytogenetic characteristics of ex vivo expanded BM-MSCs from MDS patients receiving AZA, before and after 6-cycle therapy.

MSCs were isolated and expanded from 14 patients with high risk MDS and 10 age- and sex-matched hematologically healthy subjects undergoing surgery for hip replacement, after informed consent. MSCs were expanded and re-seeded for a total of 5 passages (P) and phenotypically characterized by flow cytometry. MSCs were induced to differentiate to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. MSC growth characteristics were assessed by the population doubling time (PDT) and the methyl-triazolyl-tetrazolium (MTT) assay throughout passages. The clonogenic potential of MSCs was evaluated by the colony forming unit fibroblast assay. Conventional cytogenetic and array comparative genomic hybridization (aCGH) analyses were performed in cultured expanded MSCs at P2.

MSCs from MDS patients before and after therapy, displayed normal spindle-shape morphology and immunophenotypic characteristics as shown by the expression of CD90, CD105, CD44, CD29, CD73, and the lack of expression of CD45, CD14 and CD34. MSCs were also able to differentiate into adipocytes and osteoblasts before and after therapy, as evidenced by the Oil Red O and Alizarin Red staining, respectively, and the expression of differentiation specific genes. Cumulative analysis showed that the proliferation potential of patient MSCs, evaluated by the PDT and MTT assays, was statistically significant decreased compared to healthy individuals (P<0.01) but increased significantly following AZA treatment compared to baseline (P<0.05), although not at the level of the healthy controls. A paired analysis of patient samples before and after therapy, also showed that AZA treatment improves significantly the proliferation rate of MDS-MSCs (P<0.05). Similarly, a cumulative and paired analysis showed that the defective (compared to healthy individuals) clonogenic potential of patient MSCs at P2, increased significantly following AZA treatment (P<0.05 and P<0.05, respectively), although not at the level of the healthy individuals. Karyotypic analysis did not reveal any chromosomal abnormalities in MSC samples, before or after AZA treatment. An aCGH analysis of P2 MSCs has - thus far - been performed in three MDS patients before and after AZA therapy. Results have indicated genetic abnormalities (duplications/deletions) in all three samples before treatment that remained unchanged after AZA therapy whereas some new abnormalities also appeared.

The data of this ongoing study on the biological properties of MSCs from MDS patients receiving AZA, show an improvement of the clonogenic and proliferative potential of MSCs following treatment. According to aCGH analysis, the genetic abnormalities of MSCs remain unaffected. The findings of the functional and cytogenetic characteristics of patient MSCs before and after therapy will be individually correlated with the MSC methylation status and with patients' outcome and the results are anticipated to provide novel insights in the role of AZA therapy in the BM microenvironment of MDS patients.