Self-renewal and expansion of hematopoietic progenitors from differentiated hESCs revealed by secondary colony formation. (A) Colonies derived from primary hematopoietic progenitors (1′ CFU) were isolated and placed into secondary colony forming assays (2′ CFU) to assess the self-renewal and expansion capacities of hES-derived hematopoietic progenitors. (B-D) Morphologies of typical secondary colonies, including macrophage (B), granulocyte (C), and erythroid (D) colonies; scale bar measures 100 μm. (E-H) Flow cytometry of pooled secondary CFUs, representing all 2′ CFU subtypes as indicated by staining with CD45, CD33, CD13, and glycophorin A. Markers for positive cells were determined based on isotype controls (inset). (I) The number of 2′ CFUs arising from individual 1′ CFU following 2′ CFU assay (± SEM), indicating the magnitude of progenitor expansion. The inset shows the 2′ CFU formation from primary CB CFU after plating individual or pooled colonies into 2′ CFU assays. Progenitor expansion occurs when the 2′ CFU output is greater than the 1′ CFU input. The magnitude of progenitor expansion was calculated from plating between 1 to 5 individual primary CFUs, corrected to an input of one individual primary CFU.