Blast colonies derived from day EBs contain primitive erythroid and myeloid cells. (A-D) After 8 days of methylcellulose culture, day 4 MIXl1⁺PDGFRα⁺ cells gave rise to hematopoietic colonies representing different stages of blast colony maturation. (A) Early stage colonies often contained a dense central core (white arrowhead) with a morphology distinct from the surrounding hematopoietic cells. (B,C) In more mature colonies, this feature was lost as cells within the colony underwent hemoglobinization. (D) Some colonies also contained adherent cells (black arrowheads). (E-G) Colonies arising from the day 4 MIXL1⁺PDGFRα⁺ fraction after 11 days of methylcellulose culture displayed phenotypes indicative of erythroid, myeloid, and bipotential progenitors. (H-M) Cytocentrifuge preparations of day 4 colonies after 13 days of methylcellulose confirmed the presence of nucleated primitive erythroid and myeloid cells. Enucleated erythroid cells were also observed (* in panel H) as well as cells with the morphologic appearance of neutrophils (n), megakaryocytes (mk), macrophages (mø), and mast cells (m). Panels K-M are derived from a cytocentrifuge preparation of a single erythroid colony similar to that shown in E. (N-P) Flow cytometric analysis of 15-day methylcellulose cultures showing that the majority of cells express the erythroid marker glycophorin A (GLYA) or CD45. Approximately 90% of cells also express the pan-hematopoietic marker CD43 and, of these, approximately 20% also express CD34.