Coculture of CD34⁺ cells with MSCs promotes proplatelet and platelet production. (A) MK differentiation protocol. Peripheral blood CD34⁺ cells were cultured in the absence (Ctrl) or presence of a monolayer of MSCs in a serum-free medium containing a CC220 cytokine cocktail from days 0 to 7 and with TPO (50 ng/mL) from days 7 to 14. (B) Level of proliferation. Viable cells were counted on days 7 and 10 of culture using an automatic cell counter, and the fold increase over the input of CD34⁺ cells at day 0 was calculated (mean ± standard error of the mean [SEM] in 3 experiments; Student t test, P > .05, not significant [n.s.]). (C) Representative differential interference contrast (DIC) microscopy photographs of culture well at day 7. Zoomed images of the boxed region show extensive proplatelet development in the presence of MSCs. Scale bar, 50 μm. (D) Proportion of MK extending proplatelets at day 14 (22.1 ± 1.5% with MSCs vs 9.5 ± 1.1% for the control; mean ± SEM in 3 experiments; Student t test, ***P < .001). (E) Amount of culture-derived platelets. The cell suspension was subjected to multiple pipetting on day 14 of culture, and platelet-like elements were detected and counted by flow cytometry (mean ± SEM in 3 experiments; Student t test, **P < .01).