Hmga2 promotes megakaryocytic differentiation and enhances megakaryocytic cell proliferation. (A) BM cells (2 × 10⁴) from WT-vector, WT-Hmga2, Jak2^VF/+-vector, and Jak2^VF/+-Hmga2 mice (n = 5-7) were plated in methylcellulose medium supplemented with cytokines. BFU-E and CFU-GM colonies were scored 7 days after plating. (B) CFU-E colonies. BM cells (1 × 10⁵) from WT-vector, WT-Hmga2, Jak2^VF/+-vector, and Jak2^VF/+-Hmga2 mice (n = 6) were plated in methylcellulose medium in the presence of erythropoietin (3 U/mL) or absence of erythropoietin. CFU-E colonies were scored after 2 days. (C) CFU-Mk colonies. BM cells (1 × 10⁵) from WT-vector, WT-Hmga2, Jak2^VF/+-vector, and Jak2^VF/+-Hmga2 mice (n = 5-7) were plated into collagen-based MegaCult medium supplemented with interleukin-3 (IL-3), IL-6, IL-11, and thrombopoietin (TPO). Megakaryocytic (CFU-Mk) colonies were assessed after culturing for 8 days. (D) Expression of Hmga2 significantly increased megakaryocytic cell proliferation in both WT and Jak2^V617F mouse BM. BM cells from the transplanted animals were first cultured in a megakaryocytic culture condition (StemPro-34 medium containing stem cell factor [SCF] and TPO) for 4 days. Flow cytometric analysis confirmed ∼99% cells were CD41⁺ after 4 days of culture. Then equal numbers of megakaryocytic cells were plated, and cell proliferation was assessed by viable cell counts over 6 days. (E) Ex vivo expression of Hmga2 significantly increased CFU-Mk colonies in the BM of both WT and Jak2^V617F mice compared with vector expression (n = 4-6). (F) Ex vivo expression of Hmga2 significantly increased megakaryocytic cell proliferation in both WT and Jak2^V617F mouse BM compared with vector expression. First megakaryocytic culture was established and then cell proliferation was assessed by viable cell counts over 6 days. All data are shown as mean ± SEM. Student t test was used for comparison between 2 groups of mice. *P < .05; **P < .005; ***P < .0005.