Overexpression of GPIbα in ESCs results in the generation of giant platelets in vitro. (A) Schematic diagram of the vector used to generate ESCs that express a human (Hu) GPIbα-EGFP fusion protein. Platelet-specific expression was driven by the human GPIbα promoter. (B) Transfected ESCs were selected with Zeocin and further differentiated into megakaryocytes, proplatelets, and platelets as described in the legends for Figures 2 and 3. Left panel: representative FACS profile of platelets present in the culture media of ESC/EB-derived proplatelet cultures, as defined by forward scatter and expression of GPIbβ. Right panel: expression of huGPIbα in ESC/EB-derived platelets. As shown, most proplatelets lose expression of huGPIbα with time and become GFP-negative (population P1). A minority population (P2, ∼ 10%-20%) remain GFP-positive and continue to express the huGPIbα-EGFP fusion protein. When these 2 populations were back-gated and analyzed for platelet size, GFP-positive platelets were found to be approximately twice the size of GFP-negative platelets, demonstrating that overexpressing GPIbα has the same effect on platelet size as does loss of filamin, and suggesting that the ratio of GPIbα:filamin expression may be an important determinant of platelet size during thrombopoiesis.