Inhibition of TPO effects on MK differentiation by extracellular inhibitors of the BMP pathway. Purified CD34⁺ cells were incubated 3 or 7 days in 4GF serum-free medium in the presence or not (−) of 100 ng/mL of TPO or BMP4 alone, or with specific exogenous inhibitors. (A) Cells were incubated with TPO or BMP4 alone (left and right panels, respectively) and in the presence of blocking goat anti–TPO-R antibody and its isotype control goat IgG (20 μg/mL), or blocking monoclonal mouse anti-BMP4 antibody and its isotype control mouse IgG₂b (1 μg/mL). After 3 days of culture, cells were assayed for their CFU-MK content (top panel). Results, expressed as numbers of CFU-MK/1000 seeded cells, represent the mean value plus or minus SEM of 10 experiments. After 7 days, cells were harvested and flow cytometry was used to determine the proportion of CD41⁺ cells in viable cells (gated; bottom panel). Data represent the mean percentage of positive viable cells plus or minus SEM of 9 experiments. The P value of statistical differences between cells treated by simultaneous addition of TPO or BMP4 with isotype control antibodies or with specific blocking antibodies is directly mentioned in the figure. (B) Cells were incubated in the presence of 50 ng/mL of follistatin (Foll) or follistatin-related gene (FLRG). After 3 days of culture, cells were assayed for their CFU-MK content (left panel) and after 7 days for the percentage of CD41⁺ viable cells (gated; right panel). Results, expressed as numbers of CFU-MK/1000 seeded cells or percentage of positive viable cells, represent the mean value plus or minus SEM of 5 experiments. (C) After 7 days, cells were harvested and counted, and platelets and supernatant were collected for PF4 quantification. Results are expressed as the total number of platelets × 10⁶ per 10⁶ initial CD34⁺ and represent the mean of 4 independent experiments. The proportion of CD42b⁺ cells in viable cells (gated) was determined by flow cytometry. The total number of CD42b⁺ cells was calculated and reported to the initial number of CD34⁺ cells. Data represent the mean number of positive CD42b cells/10⁶CD34⁺ plus or minus SEM of 6 experiments. The supernatant was cleared by 10 minutes of centrifugation at 3000g, then placed in 96-well plates for ELISA quantification of PF4 as described in “BMP4 and PF4 quantification.” Results expressed in pg/mL of PF4 represent the mean value plus or minus SEM of 3 experiments. * indicates statistically significant difference from those in nontreated cells (P < .05).