Characterization of MK and EB cultures. (A-B) Megakaryocyte morphology, the MK cultures were predominantly in the megakaryoblast (MB) or promegakaryocyte (pro-MK; horseshoe-shaped nucleus) stage of differentiation, although some large granular MKs (multilobed nucleus) were observed. (C) Erythroblasts (EBs) were in the proerythroblast stage of differentiation. (D) Flow cytometric analysis of MKs and EBs after sorting. After FACS sorting, 99.3% of the cells were positive for CD41. The parallel EB culture (E) was sorted to a purity of 97.2% (based on CD235a expression). (F) Confirmation of differential expression of lineage-associated markers by TaqMan real-time PCR. Data presented as fold difference in expression (corrected for GAPDH) between paired MK and EB cultures. Error bars represent the SEM of biologic (n = 5) and technical (n = 3) replicates combined. Values above the x-axis indicate genes up-regulated in MKs, whereas values below indicate genes up-regulated in EBs. Cells were stained using modified May-Grünwald eosine-methylene blue solution (Merck) and 0.5 mM KH2PO4, pH 7.4. Images were acquired using a Leica DM 1L microscope (Leica, Mannheim, Germany) equipped with a Leica objective C, plan L 40×/0.50 numerical aperture PH2 with a total magnification of 400×. Images were captured using a Leica DC300 digital camera and Leica imaging software (IM500, version 1.20).
