Development and Analysis of Megakaryocytes from Murine Embryonic Stem Cells

With the availability of recombinant thrombopoietin (TPO) and other hematopoietic growth factors, it has been possible to study both megakaryocyte and platelet development in vitro starting with the hematopoietic stem cells of murine or human origin. Importantly, hematopoietic stem cells and the megakaryocyte lineage cells that are derived from them are amenable to genetic manipulation using viral transduction systems. This capability has enabled molecular studies of adhesion and signaling in megakaryocytes in ways not possible with platelets. Megakaryocyte lineage cells can also be derived from murine or human embryonic stem (ES) cells. For many experimental applications, and possibly even for therapeutic ones, the ability to derive large numbers of megakaryocyte progenitors and mature megakaryocytes from ES cells offers a number of potential advantages over methods that rely on the isolation of hematopoietic stem cells from marrow or other sources. This chapter describes detailed methods for deriving megakaryocytes in a large quantity from murine ES cells, for expressing recombinant genes in these megakaryocytes, and for analyzing the functions of integrin αIIbβ3.