Structure of ES-sacs and hematopoietic progenitors within ES-sacs. (A) Immunohistochemical staining of an ES-sac on day 15. The ES-sac expresses UEA-1 lectin-binding activity, CD31, and VEGF-R2, which is indicative of endothelial differentiation (arrows). Many of the cells inside ES-sacs also express CD31 (arrowheads). (B) Parallel sections of an ES-sac stained with hematoxylin-eosin or UEA-I lectin. The structure is composed of multiple cystic regions in which cellular septa express UEA-1 lectin-binding activity (arrowheads, large magnification view). Top image, 40×; bottom 2 images, 200×. (C) Representative flow-cytometry dot plots of surface molecule expression on the cells inside ES-sacs on day 15. The y-axes indicate CD31, VEGF-R2, vascular endothelial (VE)–cadherin, CD41a (integrin αIIb), or CD45; the x-axes indicate CD34 expression. Numbers on plots are the percentages of total cells within each quadrant. (D) Numbers of CD41a⁺ cells per starting 10⁵ hESCs. Using protocol 2, CD41a⁺ cells were obtained more efficiently from cells within ES-sacs on day 15 (adherent cells) than through the use of floating cells on day 7, 10, or 14. Error bars represent SD.