Abstract
Megakaryocytes (MKs) are specialised cells which function to produce platelets and release them into the blood stream. MKs descend from hematopoietic stem cells (HSCs), via a stepwise differentiation model. A reduction in platelet number or function is described as thrombocytopenia and is typically accompanied by bleeding abnormalities. Surprisingly, a mutation in CYCS, the gene encoding cytochrome c, was identified in patients who had thrombocytopenia but no other deficiencies. This prompted the need for investigation into the energy demands of MKs as there is currently no understood role for cytochrome c in thrombopoiesis. To do so, a MK cell model was developed using K562 cells and phorbol 12- myristate 13-acetate (PMA). To elucidate a megakaryocytic specific function for cytochrome c, the mitochondrial respiratory capacity of the cell models was explored. Cytochrome c shuttles electrons via redox reactions in the electron transport chain (ETC); therefore, it was hypothesised that MKs may have specific energy demands that lead to thrombocytopenia in patients with CYCS mutations. Furthermore, this study aimed to test whether inhibition of mitochondrial ROS impacted on the megakaryocytic differentiation capability of K562 cells.
It was determined that treatment with 0.5 nM PMA for three and six days represented a partially and fully differentiated megakaryocyte respectively. This cell model was used in further experiments looking at the energy demands of megakaryocytes. Using the Seahorse Cell Mito Stress test, the mitochondrial respiratory capacity was compared between wild type (WT) K562 cells and the differentiating MK cell models. Although no significant differences in the energy demands of MKs throughout differentiation were noted, this study suggests that partially differentiated MKs had increased maximal and spare respiratory capacity. K562 cells treated with mitochondrial inhibitors and PMA did not show any significant results when comparing their differentiation capability to cells with PMA alone. There did appear to
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be a positive correlation between CD61 mean fluorescent intensity (MFI) and oxygen consumption rate (OCR).
These results suggest that there is variation in the energy demands of MKs as they differentiate, however, the findings are inconclusive. Further study should be done to confirm the observations made of the increased maximal and spare capacity of partially differentiated megakaryocytes. It would be beneficial to re-define a partially and fully differentiated MK cell model using different cell markers.