Hamzic, Edita (2012) Characterisation of mesenchymal stroma and discovery of biomarkers in aplastic anemia. (PhD thesis), Kingston University, .

Abstract

Haemopoietic activity in Aplastic anemia (AA) is significantly reduced and is generally attributed to failure of Haemopoietic stem cells (HSC) within the bone marrow. The extent to which Mesenchymal stromal cells (MSC) are involved in the functional restriction of HSC is largely unknown. To understand this, the physical and functional properties of AA MSC were studied in vitro. MSC were characterised by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross¬over culture assays. Results show several alterations in the physiological and functional status of MSC in AA. Although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was significantly reduced; furthermore, their proliferative and clonogenic capacity was markedly decreased and ability to sustain haemopoiesis was also reduced as assessed by total cell proliferation and clonogenic ability of HSC. It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability significantly weaker. The second aim of this study was to identify serum proteins that change expression in untreated AA patients compared to Anti-thymocyte globulin treated patients and healthy controls that might be useful in understanding and monitoring of AA. Serum was profiled using SELDI¬TOFIMS, detecting a number of differentially expressed protein peaks. A combined strategy of chromatographic fractionation, ID gel electrophoresis, passive elution, trypsin digestion and MALDI-TOFIMS or LC-MS/MS analysis, formally identified six discriminatory peaks, further validated with Western blot and ELISA. They include acute phase response proteins: Haptoglobin, Transthyretin, Alpha-l-aeid glycoprotein and Apolipoprotein Al as well as Apolipoprotein Cl and C3 and represent novel disease- and therapy- associated AA biomarkers. Inflammatory biomarkers in AA serum are consistent with immune-mediated pathology of AA and suppressive action of Anti-thymocyte globulin, and can potentially be used for disease detection, diagnosis and monitoring.

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