Culture of Murine Megakaryocytes and Platelets from Fetal Liver and Bone Marrow

Abstract

Megakaryocytes (MKs) are the largest hematopoietic cells in bone marrow. Since the mid-1990s, recombinant thrombopoietin has been commercially available. Together with the emerging knowledge of mouse models, this has provided an unprecedented contribution to the understanding of external and internal factors that orchestrate the transition as one large MK gives rise to thousands of discoid platelets that are indistinguishable from one another. Taking advantage of the murine fetal liver as a source of MKs allows for analysis at the single cell level and also serves to provide enough cells for classical biochemical analyses. Other sources of MK progenitors are adult bone marrow and spleen, although the yields are normally lower. This chapter describes methods to establish a standard culture of murine MKs from fetal liver and bone marrow along with approaches to purify MKs using bovine serum albumin gradients and magnetic-activated cell sorting. These culture systems allow investigations of MK development (ploidy, surface markers, etc.) and platelet biogenesis, including effects of external factors such as structural proteins or feeder layers.