Analysis of in vitro–grown megakaryocytes derived from human CD34⁺cells after RUNX1 suppression. (A) Experimental schema of the studies performed. To mimic FPDMM disease, CD34⁺ cells were infected with shRX- or shNT-lentiviruses on day 2 of differentiation. Infected cells expressing mCherry (mCherry⁺) that were sorted on day 4 of differentiation, were the focus of these studies. From day 5 of differentiation, cells were treated with drugs until day 11 or day 13 to 14 of differentiation. These matured megakaryocytes (Mk) were used for either in vitro or in vivo experiments. (B) Representative flow cytometric data on day 11 of differentiation for agonist-induced surface P-selectin exposure. After stimulation of mCherry⁺ megakaryocytes with indicated doses of thrombin, cells were stained with both anti-hCD41a and hCD62P (P-selectin). (C) The mean ± 1 standard deviation (SD) levels of surface P-selectin were quantified in megakaryocytes stimulated by increasing doses of thrombin as indicated from lighter to darker color. Blue indicates shNT-megakaryocytes; red, shRX-megakaryocytes. In panels D and E, similar studies as in panel C, but shNT- or shRX-megakaryocytes were exposed to TRAP (D) or convulxin (CVX) (E). In panels C-E, N = 3 separate studies, each in duplicate. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, and ∗∗∗∗P ≤ .0001. P values were calculated by 1-way analysis of variance (ANOVA) comparing shRX-megakaryocyte to each shNT-megakaryocyte sample. Also, see supplemental Figure 5 for all 3 agonists on day 14 megakaryocytes showing that all megakaryocytes that were mCherry⁻ for shRX-lentivirus were agonist responsive.