Culture, Expansion, and Differentiation of Murine Megakaryocytes from Fetal Liver, Bone Marrow, and Spleen

Megakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the assessment of megakaryocytic potential within hematopoietic precursor cells in the bone marrow by colony-forming assays and expansion and enrichment of MKs from cultured fetal liver or spleen or bone marrow cells. Although these MKs are not synchronized in their maturation, they can be enriched over an albumin step gradient, and one-third to one-half of recovered cells will typically elaborate proplatelets, the immediate precursors of blood platelets. Both protocols require recombinant thrombopoietin (TPO) as a growth factor. Support protocols describe methods for preparing fetal liver cells, identifying mature rodent MKs by staining for acetylcholinesterase activity, and staining (May-Grünwald-Giemsa) mixed populations on cytocentrifuged blood cell preparations.