Fig. 1. Development of primitive and definitive erythrocytes. Primate ES cells (4x10³ cells/well) were cultured onto OP9 stromal cells for 6 days. The induced cells were harvested and re-cultured at a concentration of 1x10⁵ cells per well onto fresh OP9 stromal cells, with or without 10 U/ml EPO. The floating cells were harvested every other day and analyzed by May-Giemsa staining and immunostaining against human hemoglobin (Hb), fetal hemoglobin (HbF) and embryonic hemoglobin (HbEmb). (A-E) Day 12; (G-J) day 18. (A,F) May-Giemsa staining of erythrocytes. (B,G) Hb (FITC) and HbF (Cy3) staining of erythrocytes. (C-E,H-J) Hb (FITC) and HbEmb (Cy3) staining of erythrocytes. Merged images are shown in B,E,G and J. Nuclei were labeled with Hoechst 33342 in B-E and G-J. Scale bars: 10 µm. (K) Sequential analysis of the number of erythrocytes, with (black circles) or without (white circles) EPO. (L) Sequential analysis of the proportion of definitive erythrocytes (EryD) among total erythrocytes, with (black columns) or without (white columns) EPO. EryD were defined as Hb- and HbF-positive, HbEmb-negative erythrocytes, whereas primitive erythrocytes were Hb-, HbF- and HbEmb-positive. Data represent the mean±s.d. of triplicate wells. Representative results from one of three independent experiments are shown.