Dll4 represses terminal MK differentiation in vitro. Human cord blood CD34⁺ cells were plated in the presence of huTPO and huSCF. (A) Notch1 and 2 expression throughout megakaryocytic differentiation. Analysis by quantitative RT-PCR of the expression levels of Notch1 and Notch2 in sorted cell populations at day 5 throughout MK differentiation. mRNA expression of each gene was normalized to that of HPRT mRNA and calibrated to the gene/HPRT ratio in HEL cells. Results were obtained from 3 independent experiments. Sequences of the PCR primers are described in supplemental Table 1. Human cord-blood CD34⁺ cells were plated onto control or Dll4Fc-coated wells in the presence of huTPO and huSCF. (B-C) Flow cytometric analysis of MK differentiation from human cord blood CD34⁺ cells plated onto control or Dll4Fc-coated wells in the presence of huTPO and huSCF. Flow cytometric analyses were performed at days 5, 7, and 10 (1 representative experiment). (C) Histogram representation of the % of CD41a⁺CD42b⁺ cells at day 10 of the culture. Mean ± SEM from 6 independent experiments. (D-E) At day 10, sorted CD41a⁺CD42b⁺ cells were plated onto Dll4Fc-coated wells in the presence of huTPO, and proplatelet-forming cells were evaluated 3-4 days later. (D) Phase-contrast microscopy of days 13-14 in control and Dll4Fc cultures. Original magnification was ×100. (E) Histogram representation of phase contrast microscopy analyses of platelet-forming cells are presented in (E). Mean ± SEM from 7 independent experiments.