miR-145 and Fli-1 regulate megakaryocyte and erythroid differentiation in vivo. (A) Relative to an empty vector, miR-145 overexpression reduces peripheral blood platelet counts, whereas an miR-145 sponge increases platelet counts 5 to 6 weeks after bone marrow transplantation into BALB/C recipient mice. Platelet counts were assessed from peripheral blood of mice transplanted with hematopoietic progenitors infected with an empty vector (empty), a vector overexpressing miR-145 (miR-145), or a vector expressing a sponge targeting miR-145 (miR-145 sponge). (B) Relative to an empty vector (empty), overexpression of Fli-1 promotes total megakaryocyte differentiation 5 to 6 weeks after bone marrow transplantation into C57BL/6 animals. The percentage of GFP-positive mononucleated cells from the megakaryocyte lineage was assessed by flow cytometry with antibodies against CD41 and CD61. Values are mean ± SEM (n = 3). (C) H&E and von Willebrand factor staining of bone marrow from the sternum of C57BL/6 mice transplanted with HSCs infected with control virus (empty), virus overexpressing miR-145 (miR-145), and virus overexpressing Fli-1 (Fli-1). Arrowheads indicate normal megakaryocytes; and arrows, smaller megakaryocytes with hypolobated nuclei. Note the increased numbers of megakaryocytes in Fli-1-overexpressing HSCs seen both by hematoxylin and eosin and von Willebrand factor staining. Original magnifications: H&E ×1000; and von Willebrand factor ×200. The samples were analyzed using an Olympus BX41 microscrope with the objective lens of 100×/0.75 Olympus UPlanFL or 20×/0.75 Olympus UPlanFL (Olympus). The pictures were taken using Olympus QColor5 and analyzed with acquisition software QCapture Pro Version 6.0 (QImaging) and Adobe Photoshop CS3 (Adobe). (D) Quantitation of hypolobated micromegakarycoytes was performed by pathologic analysis of an independent set of transplantations into BALB/C animals. (E) Bone marrow cells from empty, miR-145, or Fli-1–overexpressing C57BL/6 recipient animals were plated in megakaryocyte differentiation media. Megakaryocyte colonies were quantitated from 1.1 × 106 sorted cells. These experiments were performed in triplicate and replicated twice. (F) Bone marrow cells from empty, miR-145, or Fli-1–overexpressing C57BL/6 recipient animals were plated in methylcellulose, and 1 × 105 transduced bone marrow cells were assayed for erythroid blast colonies. Values are mean ± SEM (n = 3) with propagated error. These experiments were performed in triplicate and replicated twice. *P < .05.
