Hematopoietic potential of ACE⁺ blast colonies. (A) Secondary colony replating efficiencies of individually picked blast colonies. Blast colonies from hESC Line H1 day 9-10 hEB cells were generated, as described in “Methods,” in the presence of 50 ng/mL each of SCF, BMP4, FGF2/heparin + VEGF (SBFH + V), 50 ng/mL each of SCF, BMP4, FGF2/heparin + TPO + VEGF (SBFH + T + V), or 50 ng/mL each of SCF, BMP4, FGF2/heparin + TPO + VEGF + IL-6 (SBFH + T + V + IL-6). The percentage of individually picked blast colonies that formed secondary hematopoietic-only (H), or hematopoietic and endothelial (HE) colonies, for each condition is shown. The addition of IL-6 did not increase the frequency of blast colony generation (data not shown) but greatly enhanced their secondary replating potential to aproximately 80%. Shown is a representative experiment (done at least twice), with the number of single blast colonies evaluated per condition (n = 11). Day 5-9 hEB-derived blast colonies were individually plucked, and secondarily recultured (5-6 days after single hEB cell plating) into single wells on either (B) fibronectin-coated dishes containing SF methylcellulose medium and GFs (SCF, BMP4, Flt3L, TPO, IGF-1, GCSF, GMCSF, IL-3, IL-6, EPO, VEGF), or (C) OP9 bone marrow stromal layers containing the same GFs, for definitive hematopoietic differentiation. Day 5 hEB blast colonies secondarily recultured into SF conditions consistently resulted in simultaneous differentiation into primitive, YS-like CD235a/CD45- erythro-myelopoietic (B, left panel and far right panels), and adherent endothelial cells that take up acetylated Dil-LDL (B, middle panel). Nuclei are stained blue with DAPI. These erythroblasts express only ε and γ, but not β hemoglobin chains (C; “SF,” middle panel). In contrast, single blast colonies recultured onto OP9 layers with human lymphohematopoietic growth factors (EPO, SCF, Flt3L, TPO, IL-3, IL-6, G-CSF, GM-CSF, IL-2, IL-7, IL-15) produced definitive-type CD235a/CD45⁺ erythromyelopoietic differentiation (C, top left panel), with enucleating β hemoglobin-expressing erythrocytes (C, bottom left panel), definitive-type myeloid cells (C, top right panel, CD13 + CD15 + CD45+), and also NK-lymphoid (CD56 + CD45+) but scarce amounts of phenotypic B-lymphoid cells (CD19+ CD45+; C, bottom right panel).