Clonogenic potential of ES-derived cells during differentiation and morphology of BFU-E-derived erythroblasts. (A) Single cell suspension was prepared from D7EB, D14EB, nonadherent cells (NA cells), or expanded nonadherent cells (Exp NAcells) and cultured to detect the clonogenic potential of hematopoietic progenitors present. The D7EB data were from 3 experiments. The D14EB data were pooled from 6 experiments. The nonadherent and expanded nonadherent cells data were pooled from 2 experiments. (B) Most of the colonies from expanded nonadherent cells were of erythroid lineage. (C) A single BFU-E colony from the CFU-C culture of expanded nonadherent cells. (D) Smears prepared from picked BFU-E colonies of expanded nonadherent cells were stained with benzidine and counterstained with hematoxylin to reveal the nuclei. Most of the cells in the BFU-E colonies stained positive for hemoglobin with the presence of some macrophages (arrow). (A,B,D) Error bars indicate SEM. (E) While most of the hemoglobin-positive erythroblasts had the morphology of fetallike definitive erythroblasts, few erythroblasts (< 2%) resembled embryonic erythroblasts, with large cytoplasm-to-nucleus ratio (arrow). Panels B and C were taken under a Leica MZ6 dissecting microscope (Heidelberg, Germany) with 1.6 × and 4 × original magnification using a Nikon coolpix995 camera (Melville, NY). The picture in panel C has been enlarged 20 ×. Panel D was taken under an Olympus BH2 microscope (Melville, NY) with a 40 ×/0.70 NA Olympus objective lens using a Nikon coolpix995 camera. Panel E was taken under a Leica DMLB microscope with a 40 ×/0.70 NA PL Fluotar objective lens using an RT Slider Spot camera using spot version 4.0.9 (Diagnostic Instruments, Sterling Heights, MI). The picture has been enlarged 94 ×.