Figure 6.
Figure 6. Globin protein and mRNA expression by ES-derived erythroid cells. (A) Frequencies of ϵ, ζ, γ, or β/δ globin-expressing cells were determined using monospecific antibodies among the erythroid colony-derived cells from dissociated D14EBs, from nonadherent cells (NA cells), or from expanded nonadherent cells (Exp NA cells). (B) RNase protection assays were performed on samples collected from post-SF culture of cells from D7EB, D14EB, nonadherent cells (NA cells), or BFU-E cultures. Erythroid cells derived from human bone marrow (BM) or fetal liver (FL) cultures were used as controls for β- and γ-globin expression. Embryonic blood of a transgenic mouse carrying the human β-globin locus yeast artificial chromosome was used as a control for ϵ- and γ-globin expression. A total of 47 samples were tested, and representative samples are shown. (C) The amount of specific β-locus globin: ϵ, γ, and β mRNA relative to the total amount of β-locus globin mRNA was computed and compared in the groups described in panel B. (D) RNase protection assays were performed on samples collected from post-SF culture of FL, D7EB, or D14EB for the expression of α-locus globin (α and ζ). A total of 10 samples were analyzed, and representative samples are shown. (E) The amount of specific α-locus globin: relative expression of α and ζ mRNA was computed and compared in the groups described in panel D. (F) EBs were generated in the EB medium supplemented with or without fetal bovine serum and with or without VEGF-165 for 7 days. D7EBs then were dissociated and cultured in suspension in the SF medium for 8 days. β-locus mRNA expression was analyzed with RNase protection assays. (G) EBs were generated in the EB medium supplemented with fetal calf serum and VEGF-165. After 7 days, EBs were dissociated and cultured in the SF medium (SF) or SF medium supplemented with 100 ng/mL Flt3-L (SF + Flt3-L) or cocultured with confluent irradiated OP-9 cells (SF + OP9) for 8 days. α-locus mRNA expression was analyzed with RNase protection assays. β-locus mRNA expression also was analyzed, and there were no significant differences among groups (data not shown). (A, C, E, G) Error bars indicate SEM.

Globin protein and mRNA expression by ES-derived erythroid cells. (A) Frequencies of ϵ, ζ, γ, or β/δ globin-expressing cells were determined using monospecific antibodies among the erythroid colony-derived cells from dissociated D14EBs, from nonadherent cells (NA cells), or from expanded nonadherent cells (Exp NA cells). (B) RNase protection assays were performed on samples collected from post-SF culture of cells from D7EB, D14EB, nonadherent cells (NA cells), or BFU-E cultures. Erythroid cells derived from human bone marrow (BM) or fetal liver (FL) cultures were used as controls for β- and γ-globin expression. Embryonic blood of a transgenic mouse carrying the human β-globin locus yeast artificial chromosome was used as a control for ϵ- and γ-globin expression. A total of 47 samples were tested, and representative samples are shown. (C) The amount of specific β-locus globin: ϵ, γ, and β mRNA relative to the total amount of β-locus globin mRNA was computed and compared in the groups described in panel B. (D) RNase protection assays were performed on samples collected from post-SF culture of FL, D7EB, or D14EB for the expression of α-locus globin (α and ζ). A total of 10 samples were analyzed, and representative samples are shown. (E) The amount of specific α-locus globin: relative expression of α and ζ mRNA was computed and compared in the groups described in panel D. (F) EBs were generated in the EB medium supplemented with or without fetal bovine serum and with or without VEGF-165 for 7 days. D7EBs then were dissociated and cultured in suspension in the SF medium for 8 days. β-locus mRNA expression was analyzed with RNase protection assays. (G) EBs were generated in the EB medium supplemented with fetal calf serum and VEGF-165. After 7 days, EBs were dissociated and cultured in the SF medium (SF) or SF medium supplemented with 100 ng/mL Flt3-L (SF + Flt3-L) or cocultured with confluent irradiated OP-9 cells (SF + OP9) for 8 days. α-locus mRNA expression was analyzed with RNase protection assays. β-locus mRNA expression also was analyzed, and there were no significant differences among groups (data not shown). (A, C, E, G) Error bars indicate SEM.

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