Fig. 5. Development of hematopoietic colonies. Methylcellulose colony-forming assays were performed on day 8. Using adherent cells dissociated with trypsin treatment, only a few GM colonies were observed (A,B). When the medium was replaced with methylcellulose-containing medium, erythroid colonies were generated on the OP9 stromal cell layer (C-F). (A,C) Morphology of a GM colony (A) and a primitive erythroid colony (C). (B,D) May-Giemsa staining of a GM colony (B) and a primitive erythroid colony (D). (E,F) Human hemoglobin (Hb; FITC) and fetal hemoglobin (Cy3; E), and Hb (FITC) and embryonic hemoglobin (Cy3; F) staining of erythrocytes in a primitive erythroid colony. Nuclei were labeled with Hoechst 33342. Merged images are shown. Scale bars: 100 µm in A,C; 10 µm in B,D-F. (G) RT-PCR analysis of globin gene expression in erythroid colonies. M, size marker; BM, adult cynomolgus monkey bone marrow cells; K, human erythroblastic cell line K562; O, OP9 stromal cells; EC1-EC3, primitive erythroid colonies. Seven other individual erythroid colonies showed similar globin gene expression patterns.