Increased maturation but decreased formation of PLPs in human CD34⁺-derived MKs upon knockdown of PDK1. (A) Human CD34⁺ HSPCs cultured for 14 days under MK differentiation conditions transduced with either an shRNA-bearing pGIPZ lentiviral vector (sh_PDK1) or a nonsilencing shRNA control (sh_ctrl). GFP served as a marker for positive transduction. Differentiation potency was quantified by flow cytometry using GPIIb (CD41) and GP1bα (CD42b) as markers for mature MKs. Representative pseudocolor plots of 1 donor with the respective indicated shRNA (left). Quantification of mature CD41/CD42b double-positive MKs assessed in 3 independent experiments with CD34⁺ cells from 2 different donors (right). Wilcoxon matched-pairs signed rank test was used to calculate statistical significance. (B) PLPs of day 14 MKs subgated from CD41-positive events. Representative scatter plot showing clearly defined PLPs (gate adjusted on peripheral blood PLPs) together with histograms indicating the amount of GFP-positive PLPs from 1 exemplarily chosen culture in each condition. Quantification of CD41-subgated GFP-positive PLPs in the PLP gate assessed in 3 independent experiments with CD34⁺ cells from 2 different donors (right). Wilcoxon matched-pairs signed rank test was used to calculate statistical significance. *P < .05 indicates statistically significant difference. SSC, side scatter.