Abstract Number: PB1608
Meeting: ISTH 2020 Congress
Background: Defects in megakaryopoiesis and platelet production are common causes of inherited thrombocytopenias in children. Megakaryocytes (MKs) are relatively rare in bone marrow comprising 0.5 to 5% of the hematopoietic cells, which makes direct isolation impractical. The published methods of human MK production are complicated and require large blood volumes; thus adapted methodology is needed to allow efficient research on primary MKs physiology in humans.
Aims: To develop a method to differentiate MKs from a low volume of bone marrow aspirates obtained routinely for diagnostic procedures.
Methods: A small volume of bone marrow (0.5-1 mL) was collected from pediatric patients in clinical remission from acute lymphoid leukemia without any treatment (n=5) and undergoing treatment with cytostatic agents (n=5). Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation. The mononuclear band was seeded in media supplemented with cytokines and allowed to differentiate into MKs for 14 days. Flow cytometry analyses were performed to estimate CD41+ and CD42b+ percentage and analyze the ploidy of CD41+ cells. Immunofluorescent confocal microscopy and live-cell imaging were performed to visualize differentiated MKs and proplatelet formation.
Results: Extensive proliferation was observed in the first 3-4 days, with an increase in CD34+ event count from 5-7% to 13-15% on average. Subsequently, an increase of large CD41+CD42b+ events to 30-35% of the live cell population was observed. Half of the analyzed CD41+ events had >4N DNA content. Interestingly, samples from treated patients had no significant differences in MK differentiation. One-step BSA gradient centrifugation on day 14 enriched CD41+ cells up to 70%. Immunofluorescent microscopy revealed relatively large CD41+CD62p+ cells with multi-lobular nuclei. Overnight live-cell imaging showed 3-5% MKs undergoing proplatelet formation.
Conclusions: Our work suggests using a low volume of bone marrow aspirates to generate fully functional MKs is a viable option for studying human MK production ex vivo and in vitro.
To cite this abstract in AMA style:Butov K, Mikhalkin N, Nikolaev R, Volkova A, Osipova E, Machlus K, Panteleev M. Generation of Megakaryocytes from Human Bone Marrow Aspirates as a Simple Diagnostic and Research Tool: Assay Development and Validation [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/generation-of-megakaryocytes-from-human-bone-marrow-aspirates-as-a-simple-diagnostic-and-research-tool-assay-development-and-validation/. Accessed September 27, 2023.
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