Figure 4.
Erythromegakaryocytic maturation of G1ME cells. (A) Transmission electron microscopy demonstrating features of megakaryocytic maturation. Control (MIGR1-transduced) cells (top left) are small and lack features of differentiation. GATA-1-rescued cultures contain a population of large cells with megakaryocytic features, including dense chromatin around the periphery of the nuclei, large multilobed nuclei (*), multivesicular bodies (mvb), dense granules (dg), α granules (α), and a developing platelet-membrane demarcation system (dm). Original magnifications are indicated. Photographs were taken by using a microscope (208S transmission electron; Philips) equipped with a Hamamatsu digital camera (Hamamatsu, Japan) and analyzed with visualization software (Advanced Microscope Techniques). (B) RT-PCR analysis showing GATA-1 induction of both erythroid and megakaryocyte genes. Erythroid genes are β major globin and Ahsp. Megakaryocyte genes are platelet factor 4 (Pf4) and glycoprotein (GP) Ibα and Ibβ. PCR cycle numbers for each primer pair are shown at the right side of the panel. (C) GATA-1 induces proliferation arrest as part of terminal maturation. Transduced cells were analyzed by flow cytometry for GFP expression. The fraction of GFP+ cells was normalized to 1.0 at 2 days after retroviral infection and then followed over time. GFP-expressing cells transduced by vector grew normally, whereas GATA-1-expressing cells were outgrown by noninfected cells.