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Identifier 000431319
Title Exosomes derived from Bone Marrow Mesenchymal Stem/Stromal Cells of patients with Myelodysplastic syndromes : Investigation of their role in hematopoiesis.
Alternative Title Εξωσώματα από αρχέγονα μεσεγχυματικά κύτταρα μυελού των οστών σε ασθενείς με μυελοδυσπλαστικά σύνδρομα
Author Βρυώνη, Άντρια
Thesis advisor Ποντίκογλου, Χαράλαμπος
Reviewer Παπαδάκη, Ελένη
Μπερτσιάς, Γεώργιος
Abstract Introduction: Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cell characterized by ineffective bone marrow hematopoiesis and increased risk for leukemic evolution. However, MDS pathogenesis is not restricted to the Hematopoietic Stem Cell (HSC) compartment, an abnormal BM microenvironment has been reported to contribute to BM failure in MDS through defective support of hematopoiesis. Mesenchymal Stem Cells (MSCs) are key components of the Bone Marrow niche. MDS derived MSCs (MDS-MSCs) have been shown to hold intrinsic functional defects and an inability to sustain normal hematopoiesis. 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. 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. Extracellular Vesicles (EVs) are a family of membrane vesicles containing a phospholipid bilayer and are secreted in the extracellular environment by most if not all cells. EVs can be separated into three major classes based on their biogenesis and their size: apoptotic bodies, microvesicles and exosomes. Aim of the study: The aim of the study was the isolation, quantification and verification of the presence of exosomes derived from BM-MSCs from both MDS patients and Heathy donors. Also, another aim was to investigate the effect of AZA treatment on phenotypic, proliferative and differentiation characteristics of BM-MSC derived from High Risk-MDS patients compared with Healthy Donors (HD). Moreover, we wanted to study the incorporation of exosomes derived from MDS-MSCs and HD with CD34+ and observe their effect on clonogenic potentials of Hematopoietic Stem Cells and subsequently hematopoiesis. Materials and Methods: BM-MSCs from MDS patients (n=4), of which 2 were studied also before and after 4 cycles of AZA treatment, and HD (n=3) were ex vivo expanded and re-seeded for a total of 3 passages and phenotypically characterized by flow cytometry. The proliferative potential of BM-MSCs was evaluated via the MTT method, the potential of MSCs to differentiate into adipocytes and osteocytes was evaluated via histochemical stainings (Alizarin Red and Von Kossa for osteocytes, and Oil Red O for adipocytes) and through the expression levels of specific genes (ALP, OSC, DLX5, RUNX-2 for osteocytes and PPARγ and CEBPa for adipocytes). Exosomes from MDA-MB-231 were isolated following consecutive steps of ultracentrifugation and were quantifies using BCA assay. The presence of exosomes was confirmed via Western Blot for two reference proteins Alix and CD9. Also, to examine incorporation of exosomes derived from MDS-MSCs (both before and after AZA treatment) and HD into CD34+ and their effect on their clonogenic potential and subsequently to hematopoiesis. Results: Thus far, our results have shown that cultured MSCs from MDS patients before and after treatment with AZA exhibited similar morphological characteristics compared with MSCs from HD, as both preserve the characteristic spindle-shaped fibroblast-like morphology. The immunophenotypic characteristics of MSCs from MDS patients showed the expression of the typical mesenchymal surface markers, with no differences detected in their expression before and after AZA treatment. In addition, some early results indicated that the proliferation potential of MDS-MSC before and after AZA treatment in vivo appeared to be similar, in contrast with AZA treatment in vitro which reduces the proliferative potential of cultured MDS-MSCs. The differentiation capacity of MDS-MSCs into osteocytes and adipocytes as shown by the stains did not exhibited any morphological difference before and after AZA treatment. However, the gene expression of MDS-MSCs after AZA treatment showed that adipogenesis-related genes were increased, while osteogenesis-related genes were similar with the MDS-MSCs before AZA, but not statistically significant. On the other hand, estimation of the expression levels for the specific genes shown that BM-MSCs from HD excel in their ability to differentiate into adipocytes and osteocytes compared with MDS-MSCs regardless of ΑΖΑ treatment. Moreover, during the study, the isolation of exosomes derived from the MDA-MB-231 cell line using ultracentrifugation was standardized and the isolated exosomes were confirmed by the expression of proteins Alix and CD9. Co-cultures of exosomes derived from random MSCs samples into CD34+ cells indicated that the incorporation of exosomes into CD34+ cells had no important effects on the clonogenic capacity of CD34+ cells comparing with the number of forming colonies of CD34+ control samples (without exosomes). Conclusion: Consequently, all experiments are still ongoing and many more are needed in order to get a valid result, but thus far, it appears that AZA treatment in vivo has no statistically significant effects on MSCs proliferation, it increases the differentiation potential of MSCs into adipocytes and maintains the osteogenic potential of MSCs compared with MDS-MSC before AZA treatment. Further research in MDS-MSCs before and after the treatment with Azacytidine in needed to make the action mechanism of AZA more understandable while providing new data on the pathophysiology of MDS, thus leading to a better therapeutic approach. Moreover, the standardization of exosome isolation, quantification and presence verification was achieved. However, even though EVs have become the focus of great interest, there are still important obstacles to overcome so as to optimize their clinical use.
Language English
Issue date 2020-08-05
Collection   Faculty/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work--Post-graduate theses
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