Molecular Biology Studies with Primary Megakaryocytes
Megakaryocytes are rare hematopoietic cells comprising only about 0.02–0.05% of the bone marrow nucleated cell population. Because of the relative infrequency of megakaryocytes in the bone marrow and their fragility in vitro, studies to characterize expression of platelet-specific genes have mostly been carried out in continuous cell lines originating from leukaemic marrow or blood cells that express a range of megakaryocytic phenotypic properties. HEL cells, which are representative of such cell lines, were instrumental in getting the molecular analysis of megakaryocytes and platelets established, and although these cells are still useful for many studies, it has become clear that they have significant limitations. These limitations include the fact that these lines only approximate megakaryocytes. These lines do not contain α-granules, do not demarcate or release platelets, respond appropriately to thrombopoietin (TPO), or express the high levels of such platelet-specific proteins such as the integrin αIIb and platelet factor 4 (PF4). Other proteins such as the platelet-restricted G protein Gzα has not been detected in any of these cell lines. Further, these cell lines often express a mixture of multiple different lineages and can be easily shifted from one lineage to another.
KeywordsPhenol EDTA Agarose Citrate Chloroform