Hematopoietic potential of purified BB9⁺ and CD34⁺ hEB populations. Line H1 day 9 hEB cell suspensions (presorted) were FACS-purified into (A) CD34⁺BB9⁺, CD34⁻BB9⁺, and CD34⁻BB9⁻ fractions (postsort analysis of purified positive fractions shown in middle and right panels). (B-D) Sorted cells were cultured onto duplicate fibronectin-coated dishes with SF methylcellulose and hemato-endothelial GFs, as described under “SF clonogenic assays for hematopoietic and hemangioblastic colony-forming cells.” (B,C) single day 9 CD34⁺BB9⁺ and CD34⁻BB9⁺ (but not CD34⁻BB9⁻) hEB cells differentiated into hemangioblastic colonies of mixed primitive erythromyeloid cells and adherent endothelioid cells (MIXED-HE), as well as primitive erythroid (EryP) colonies. Day 14 hEB similarly FACS-purified into these same populations (D), generated more varied CFU including mixed hematoendothelial (MIXED-HE), primitive erythroid (EryP), definitive erythroid (CFU-e-d), and macrophage (M-CFC) colonies. At both days 9 and 14, hematopoietic CFU progenitor activity was greater in CD34-BB9⁺ than in CD34⁺BB9⁺ populations; at Day 14 this represented approximately 6% of CD34⁺BB9⁺ cells, and approximately 10% of CD34⁻BB9⁺ cells. (E,F) Definitive hematopoietic differentiation of these same populations on OP9 stroma with erythromyeloid GFs. Both CD34⁺BB9⁺ and CD34-BB9⁺ (but not CD34⁻BB9⁻) fractions produced robust definitive hematopoietic potential, but CD235a⁺/CD45⁺CD13⁺CD15⁺ definitive-type erythro-myeloid cells had a more robust proliferation in the CD34⁻BB9⁺ fraction.