title: Embryonic stem cell differentiation system for evaluating gene functions involved in physiological megakaryocytic differentiation.
abstract: Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP(+) cells among CD41(+) cells). Insertion of full-length or small core β-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (∼60% GFP(+) cells among CD41(+) cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Japan. okadabos@phs.osaka-u.ac.jp
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Okada
authors.forename: Masaaki
authors.initials: M
authors.lastname: Yonekura
authors.forename: Miho
authors.initials: M
authors.lastname: Watanabe
authors.forename: Tomoko
authors.initials: T
authors.lastname: Nakai
authors.forename: Atsuko
authors.initials: A
authors.lastname: Wakimura
authors.forename: Mikiko
authors.initials: M
authors.lastname: Shimizu
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kamikawa
authors.forename: Mie
authors.initials: M
authors.lastname: Kitayama
authors.forename: Kenji
authors.initials: K
authors.lastname: Kitajima
authors.forename: William C
authors.initials: WC
authors.lastname: Aird
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D005796:Genes / Q000502:physiology*; D056426:Genetic Loci; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D038101:Insulator Elements; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D019076:Transgenes; D055544:beta-Globins / Q000235:genetics
chemical_list: D055544:beta-Globins; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase