Defect in proplatelet formation by MK cells derived from FPD/AML iPSC lines. (A) Schema of protocol used for MK differentiation. iPSC lines were seeded on OP9 cells with vascular endothelial growth factor (VEGF) and thrombopoietin (TPO) at day 0. After 7 days, stem cell factor (SCF) was added to the culture medium. MK (CD41⁺CD42⁺) cells were sorted at day 14 and further cultured in the presence of TPO and SCF. Analysis of proplatelet formation and qRT-PCR were performed at day 19. (B) Representative microscopic images representing control and patient proplatelet-forming MKs. (C) The percentage of proplatelet-forming MKs was estimated by counting MKs exhibiting 1 or more cytoplasmic processes with areas of constriction. A total of 200 cells per well was counted. The histograms show 1 representative experiment of 5, each in triplicate. Error bars represent ± SD of triplicate. (D) qRT-PCR analysis of MYL9 and MYH10 expression level in MKs. The histograms show 1 representative experiment of 4 for MYL9 and 5 for MYH10, each in triplicate. Data are normalized to PPIA and L32 transcript level, and expression is compared with control C3. Similar results were obtained but only results normalized to PPIA are shown. Error bars represent ± SD of triplicate. **P < .01; ***P < .001; Student t test.