Hematopoietic progenitors within ES-sacs efficiently generate megakaryocytes. (A) Dot plots on flow cytometry showing CD41a (integrin αIib; x-axis) and CD42a (GPIX; y-axis) expression (left panel) or CD41a (x-axis) and CD42b (GPIbα; y-axis) expression (right panel) on day 24. Fifty percent to 60% of the cells appeared to be mature megakaryocytes. Numbers on plots are the percentages of total cells within each quadrant. (B) Floating cells on days 23 to 24 were stained with Hemacolor (Diagnostica Merck, Darmstadt, Germany). (C) Formation of megakaryocytes bearing proplatelets (i-iii) or a megakaryocyte shedding its cytoplasmic membrane for direct releases of platelets (iv). (i) Representative phase contrast photomicrograph. (ii-iv) Immunohistochemical staining demonstrating expression of CD41a (green); nuclei were stained using 4,6-diamino-2-phenylindole (DAPI; blue). Bar represents 20 μm. (D) Top panel: Hematopoietic progenitors within ES-sacs on day 15 were stained for CD34 and CD41a and sorted using the indicated gates (i, ii, iii, and iv). Bottom panel: Percentages of CD41a⁺/CD42b⁺ megakaryocytes among the hematopoietic cells on day 24 that were derived from CD34⁺/CD41a⁻, CD34⁺/CD41a⁺, or CD34⁻/CD41a⁺ hematopoietic progeni-tors within ES-sac on day 15. Error bars represent SD. (E) Undifferentiated hESCs (no. 1), cells sorted on day 15 (no. 2 or no. 3), or mature megakaryocytes on day 24 (no. 4) were collected and prepared as described in “Methods.” Extracted RNAs were used for semiquantitative RT-PCR. The bands in the red squares were obtained with fewer PCR cycles.