Generation of mouse megakaryocytes. (A) Embryonic day 13.5 fetal liver was disaggregated, depleted of Ter119⁺-committed erythroblasts by immunomagnetic bead negative selection, and cultured in thrombopoietin for 3-5 days. (B) Surface staining for the megakaryocyte antigen CD41 after 5 days of culture (blue). The red line shows the unstained control. (C) Cytocentrifuge preparations stained with May-Grunwald-Giemsa showing large cells with megakaryocyte morphology. The left panel shows cells after 3 days of culture. The large megkaryocytes were enriched by sedimentation on a discontinuous bovine serum albumin (BSA) gradient (middle panel). The right panel shows megakaryocyte morphology after 5 days of culture. Original magnification of 200× (left and middle panels) and 400× (right panel). Photographs were obtained on a Carl Zeiss Axioskop 2 microscope equipped with a color digital camera (Axiocam, Carl Zeiss). (D) Ploidy, as measured by DNA content on day 3 megakaryocyte cultures. (E-F) Messenger RNA expression in megakaryocyte cultures as measured by quantitative reverse-transcription (Q-RT)–PCR. Values were normalized to Gapdh and are plotted relative to expression levels in the input Ter119^– cells at day 0, which are assigned an arbitrary value of 1. Panel E shows induction of marker genes in megakaryocyte (MEG) cultures. Panel F shows down-regulation of Klf1 mRNA–encoding erythroid transcription factor erythroid Krüppel-like factor and induction of Fli1 mRNA, which encodes a megakaryocyte-enriched transcription factor.