Fig. 2.
Fig. 2. BCR-ABL expression at the onset of hematopoiesis expands multipotent and myeloid progenitors with suppression of differentiation toward erythroblasts. / (A) Morphology of day 7 ES-differentiated hematopoietic progenitors appeared unaltered on BCR-ABL induction. Cytospin preparations were stained with May-Grünwald/Giemsa-Wright. Slides were photographed at ×1000. (B) BCR-ABL expression does not skew hematopoietic differentiation toward multipotent and myeloid progenitors. The absolute percentages of multipotent and myeloid progenitors on day 7 of ES in vitro differentiation in the presence and absence of BCR-ABL induction were determined by flow cytometry using EGFP as a surrogate marker for BCR-ABL expression. Multipotent progenitors were defined by coexpression of cell surface markers C-KIT and CD34 and lack of CD11b expression or lack of lineage panel TER-119, B220, CD4, CD8, CD11b, Gr-1 expression. Myeloid progenitors were defined with cell surface markers C-KIT, CD34, and CD11b. The ratio of multipotent to myeloid progenitors was calculated from the absolute percentages in the presence and absence of BCR-ABL induction. (C) BCR-ABL tyrosine kinase activity is required to expand ES-derived hematopoietic progenitors. The effect of the BCR-ABL tyrosine kinase inhibitor imatinib mesylate on progenitor expansion was analyzed by counting the total number of ES day 7 cells after imatinib mesylate addition on ES day 5 differentiation, in the presence and absence of BCR-ABL induction. BCR-ABL–induced cultures led to BCR-ABL expression in 54% of the cells by EGFP levels, and cell counts were normalized to 100% BCR-ABL expression. Imatinib mesylate was added at both 1 μM and 10 μM concentrations with similar results. Results represent the mean ± SEM of an experiment conducted in triplicate. Similar results were obtained in 3 independent experiments. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON total cell population, multipotent progenitors, and myeloid progenitors was P < .01. Statistical analysis (paired ttest) of BCR-ABL OFF versus BCR-ABL ON and BCR-ABL ON plus imatinib mesylate versus BCR-ABL ON ES day 7 cultures wereP < .01. (D) Selective expansion of multipotent and myeloid progenitors but not erythroblasts on BCR-ABL induction. Total cell counts of day 7 ES-derived hematopoietic progenitors combined with percentages of multipotent progenitors (panel B), myeloid progenitors (panel B), and erythroblasts (defined by expression of TER-119) were used to obtain cell numbers of specific populations. BCR-ABL–induced cultures led to BCR-ABL expression in 54% of the cells by EGFP levels and cell counts were normalized to 100% BCR-ABL expression. Results represent the mean ± SEM of an experiment conducted in triplicate. Similar results were obtained in 3 independent experiments. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON multipotent and myeloid progenitors wasP < .01. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON erythroblasts wasP = .18, thereby representing an insignificant difference. (E) The tyrosine kinase activity of BCR-ABL is required to suppress hematopoietic differentiation toward erythroblasts. The effect of the BCR-ABL tyrosine kinase inhibitor imatinib mesylate on erythroblast differentiation was analyzed by examining the percentage of TER-119+ erythroblasts generated after addition of imatinib mesylate on ES day 5 differentiation, in the presence and absence of BCR-ABL induction. BCR-ABL–induced cultures led to its expression in 67% of the cells by EGFP levels and TER-119 percentages were normalized to 100% BCR-ABL expression. Imatinib mesylate was added at both 1 μM and 10 μM concentrations with similar results. Representative result of an experiment conducted in triplicate wells. Similar results were obtained in another 2 independent experiments. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON TER-119+ erythroblasts was P < .01. X indicates fold.

BCR-ABL expression at the onset of hematopoiesis expands multipotent and myeloid progenitors with suppression of differentiation toward erythroblasts.

(A) Morphology of day 7 ES-differentiated hematopoietic progenitors appeared unaltered on BCR-ABL induction. Cytospin preparations were stained with May-Grünwald/Giemsa-Wright. Slides were photographed at ×1000. (B) BCR-ABL expression does not skew hematopoietic differentiation toward multipotent and myeloid progenitors. The absolute percentages of multipotent and myeloid progenitors on day 7 of ES in vitro differentiation in the presence and absence of BCR-ABL induction were determined by flow cytometry using EGFP as a surrogate marker for BCR-ABL expression. Multipotent progenitors were defined by coexpression of cell surface markers C-KIT and CD34 and lack of CD11b expression or lack of lineage panel TER-119, B220, CD4, CD8, CD11b, Gr-1 expression. Myeloid progenitors were defined with cell surface markers C-KIT, CD34, and CD11b. The ratio of multipotent to myeloid progenitors was calculated from the absolute percentages in the presence and absence of BCR-ABL induction. (C) BCR-ABL tyrosine kinase activity is required to expand ES-derived hematopoietic progenitors. The effect of the BCR-ABL tyrosine kinase inhibitor imatinib mesylate on progenitor expansion was analyzed by counting the total number of ES day 7 cells after imatinib mesylate addition on ES day 5 differentiation, in the presence and absence of BCR-ABL induction. BCR-ABL–induced cultures led to BCR-ABL expression in 54% of the cells by EGFP levels, and cell counts were normalized to 100% BCR-ABL expression. Imatinib mesylate was added at both 1 μM and 10 μM concentrations with similar results. Results represent the mean ± SEM of an experiment conducted in triplicate. Similar results were obtained in 3 independent experiments. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON total cell population, multipotent progenitors, and myeloid progenitors was P < .01. Statistical analysis (paired ttest) of BCR-ABL OFF versus BCR-ABL ON and BCR-ABL ON plus imatinib mesylate versus BCR-ABL ON ES day 7 cultures wereP < .01. (D) Selective expansion of multipotent and myeloid progenitors but not erythroblasts on BCR-ABL induction. Total cell counts of day 7 ES-derived hematopoietic progenitors combined with percentages of multipotent progenitors (panel B), myeloid progenitors (panel B), and erythroblasts (defined by expression of TER-119) were used to obtain cell numbers of specific populations. BCR-ABL–induced cultures led to BCR-ABL expression in 54% of the cells by EGFP levels and cell counts were normalized to 100% BCR-ABL expression. Results represent the mean ± SEM of an experiment conducted in triplicate. Similar results were obtained in 3 independent experiments. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON multipotent and myeloid progenitors wasP < .01. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON erythroblasts wasP = .18, thereby representing an insignificant difference. (E) The tyrosine kinase activity of BCR-ABL is required to suppress hematopoietic differentiation toward erythroblasts. The effect of the BCR-ABL tyrosine kinase inhibitor imatinib mesylate on erythroblast differentiation was analyzed by examining the percentage of TER-119+ erythroblasts generated after addition of imatinib mesylate on ES day 5 differentiation, in the presence and absence of BCR-ABL induction. BCR-ABL–induced cultures led to its expression in 67% of the cells by EGFP levels and TER-119 percentages were normalized to 100% BCR-ABL expression. Imatinib mesylate was added at both 1 μM and 10 μM concentrations with similar results. Representative result of an experiment conducted in triplicate wells. Similar results were obtained in another 2 independent experiments. Statistical analysis (paired t test) of BCR-ABL OFF versus BCR-ABL ON TER-119+ erythroblasts was P < .01. X indicates fold.

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