In vitro production of megakaryocytes and platelets from buffy coat derived stem cells

The generation of megakaryocytes (MKs) from hematopoietic stem cells (HSCs) is a critical process in thrombopoiesis, with significant implications for both basic research and clinical applications. This thesis investigates the in vitro culture and differentiation of megakaryocytes from HSCs isolated from human buffy coats, an easily available and rich source of HSCs. The study aims to optimize culture conditions to enhance the efficiency of MK production, providing insights into the molecular mechanisms driving megakaryopoiesis. The MKs produced, are used to investigate the effects of monoclonal antibodies targeting key platelet receptors on MK survival. The study also examines the binding of IgG from immune thrombocytopenia (ITP) patient serum to megakaryocytes and platelets, aiming to better understand the effect of these antibodies on physiological and pathological changes in platelets and megakaryocytes of ITP patients. HSCs were isolated from buffy coats using density gradient centrifugation and immunomagnetic separation, followed by culture in a thrombopoietin-enriched SFEM medium to induce differentiation into megakaryocytes. The produced MKs were characterized by flow cytometry, assessing surface markers such as CD34, CD41, CD42a, and CD61. Monoclonal antibodies against CD41, CD61, CD42a, and CD42b were then applied to the cultured MKs to evaluate their effects on cell survival. In addition, a binding assay was conducted to test the interaction between IgG from ITP patient serum and the cultured MKs, as well as platelets isolated from healthy donors. The extent of IgG binding was measured using flow cytometry, providing insights into the presence of autoantibody against MK and platelets in ITP patients' serum. The results demonstrate a successful protocol for the in vitro culture of megakaryocytes from HSCs derived from buffy coats, with a high yield and functionality of the produced MKs. The study found that monoclonal antibodies targeting CD41, CD61, CD42a, and CD42b significantly affect MK survival. The binding assays revealed enhanced IgG binding to both MKs and platelets in ITP patients, supporting the studies that autoantibodies in ITP contribute to both platelet and MK destruction. This research expands our knowledge of the role of specific receptors in megakaryocyte survival. Moreover, the findings provide a foundation for further investigation into the development of targeted therapies to protect MKs and platelets in patients with immune-mediated platelet disorders.