Megakaryocyte Growth and Development Factor-Induced Proliferation and Differentiation Are Regulated by the Mitogen-Activated Protein Kinase Pathway in Primitive Cord Blood Hematopoietic Progenitors by Serge Fichelson, Jean-Marc Freyssinier, Françoise Picard, Michaela Fontenay-Roupie, Martine Guesnu, Mustapha Cherai, Sylvie Gisselbrecht, and Françoise Porteu Blood Volume 94(5):1601-1613 September 1, 1999 ©1999 by American Society of Hematology

MAPK activation in CB-derived megakaryocytes. MAPK activation in CB-derived megakaryocytes. (A) Kinetic of ERK activation in day 7 and day 10 cells derived from purified CD34+ cells grown in the presence of rhuMGDF. Cells were deprived of growth factor by incubation for 5 hours in cytokine-free medium and stimulated for the indicated times with 100 ng/mL rhuMGDF. (B) Inhibition of rhuMGDF-induced ERK activation by PD98059. Cells were grown for 10 days with rhuMGDF, starved, and stimulated for 60 minutes at 37°C with 100 ng/mL of rhuMGDF in the presence of 6 μmol/L PD98059 (+) or the equivalent amount of DMSO as control (−). ERK activity was detected by immunoblotting whole-cell lysates with antiphospho ERK antibody. The amount of sample loaded in each lane was verified by immunoblotting the same membranes with an antibody recognizing both active and inactive ERK1 and ERK2. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Relative expansion of CD34+ cells in the presence (•, ○) or absence (▪, □) of 6 μmol/L PD98059 induced by MGDF (A) or a mixture of SCF + IL-3 +IL-6 (B). Relative expansion of CD34+ cells in the presence (•, ○) or absence (▪, □) of 6 μmol/L PD98059 induced by MGDF (A) or a mixture of SCF + IL-3 +IL-6 (B). The data have been normalized to the number of purified CD34+ cells seeded on day 0. The results presented are those from a representative experiment of 8 (A) or 3 (B) performed. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Appearance of apoptotic cells in the cultures as measured by annexin V staining. Appearance of apoptotic cells in the cultures as measured by annexin V staining. CD34+ cells were grown with rhuMGDF in the absence or presence of 6 μmol/L PD98059 and stained with annexin V-FITC at the indicated days. The percentage of annexin V–positive cells was determined by FACS analysis. (A) Frequency of apoptotic cells in control cultures (▪) and cultures containing the inhibitor (▨). The results represent mean ± SD from 4 independent experiments. (B) Total number of apoptotic cells in the presence (▪) or absence (•) of PD98059. The results from a representative experiment are shown. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Effect of the MEK inhibitor on DNA synthesis capacity of CD34+ cells grown with rhuMGDF. Effect of the MEK inhibitor on DNA synthesis capacity of CD34+ cells grown with rhuMGDF. Cells from cultures performed in the absence (▪) or presence (▨) of 6 μmol/L PD98059 were harvested at the indicated days and DNA synthesis was assessed by measuring [3H]thymidine incorporation. Results are mean ± SD of triplicate determinations from a representative experiment of 3 performed. The relative increase in [3H]thymidine incorporation in PD98059-treated cells as compared with control DMSO-treated cells is indicated below the bars for each time point. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Effect of the MEK inhibitor on CD34 and CD41 cell surface expression. Effect of the MEK inhibitor on CD34 and CD41 cell surface expression. Representative histograms from 1 of at least 4 experiments performed with blood from different donors are shown for each marker. Cells from control cultures (filled histograms) or cultures containing 6 μmol/L PD98059 (open histograms) were harvested at the indicated days and stained separately with either anti-CD34 antibody coupled to R-PE or anti-CD41 antibody coupled to FITC. In some experiments, labeling was performed with FITC-anti-CD34 and R-PE-anti-CD41 antibodies and similar results were obtained. For each marker, the percentage of positive cells (or the MFI for CD41 at days 10 and 13) are indicated: gray labeling, controls; black labeling, PD98059-treated samples. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Increased frequency of double-stained CD34+CD41+ cells in PD98059-treated cultures. Increased frequency of double-stained CD34+CD41+ cells in PD98059-treated cultures. Cells from cultures containing or not 6 μmol/L PD98059 were harvested at different time intervals and stained simultaneously with R-PE-anti-CD34 and FITC-anti-CD41 (or FITC-anti-CD34 and R-PE-anti-CD41) antibodies. (A) FACS dot plot from a representative experiment on day 7 cells. (B) Percent of CD34+ cells among the CD41+ cell population after 7 and 11 days of culture in the absence (−) or in the presence (+) of PD98059 in 5 independent experiments. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Dose-response analysis of PD98059 effect on the presence of CD34+, CD41+, and CD41+CD34+ cells in MGDF-induced cultures. Dose-response analysis of PD98059 effect on the presence of CD34+, CD41+, and CD41+CD34+ cells in MGDF-induced cultures. CD34+ cells were grown with rhuMGDF in the presence of the indicated concentrations of PD98059 diluted in DMSO or the equivalent volumes of DMSO alone. After 7 days, cells were labeled with FITC-anti-CD41 and R-PE-anti-CD34 antibodies either separately or simultaneously. Results are expressed as a percent of the frequency of positives cells for each marker obtained with control cells. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Evolution of erythroid (Ery), megakaryocytic (MK), and GM clonogenic progenitors in MGDF-induced cultures. Evolution of erythroid (Ery), megakaryocytic (MK), and GM clonogenic progenitors in MGDF-induced cultures. Clonogenic progenitors in cultures performed in the absence or the presence of 6 μmol/L PD98059 were quantified at the indicated days by semisolid culture assays as described in Materials and Methods. (A) Total number of colonies formed with control (▪) or PD98059-treated cultures (▨). The results from a representative experiment, where determinations were done in duplicate, are shown. (B) Percent of increase in relative (▩) or total (□) colony numbers obtained with PD98059-treated cells as compared with control cells. Results are mean ± SD of at least 4 independent experiments for each type of colony. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology

Morphologic analysis of cells from liquid cultures. Morphologic analysis of cells from liquid cultures. MGG staining of cytocentrifugated cells from PD98059-treated (A to C) or control (D to F) cultures, at day 10 (A and D) and day 13 (B, C, E, and F); original magnification × 100. Serge Fichelson et al. Blood 1999;94:1601-1613 ©1999 by American Society of Hematology