Fig. 9. More efficient primitive and definitive hematopoiesis is induced by re-plating sorted CD34-positive cells. (A-C) Flow cytometric analysis and cell sorting of cultures on day 10 with human CD34 antibody. A sorting gate in B was defined according to the intensity of staining with an isotype control antibody (A). Reanalysis of the sorted cells confirmed the purity as 95 to 98% (C). (D) Large adherent hematopoietic cell cluster with a cobblestone appearance on day 25. Scale bars: 100 µm. (E-G) May-Giemsa staining (E), human hemoglobin (Hb; FITC) and embryonic hemoglobin (Cy3; F), and Hb (FITC) and fetal hemoglobin (Cy3; G) staining of day 25 erythrocytes grown in the presence of EPO. Nuclei were labeled with Hoechst 33342. Merged images are shown. Scale bars: 10 µm. (H,I) Sequential analysis of the number of erythrocytes (H) and total hematopoietic cells (I), with (black circles) or without (white circles) 10 U/ml EPO. (J) Sequential analysis of the proportion of definitive erythrocytes among total erythrocytes. (K) Sequential analysis of the percentages of enucleated erythrocytes (stippled columns), nucleated erythrocytes (white columns) and myeloid cells (striped columns) in the presence of EPO. Data represent the mean±s.d. of triplicate wells. Representative results from one of three independent experiments are shown.