title: Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling.
abstract: Fibrinogen binding to integrin alphaIIbbeta3 mediates platelet aggregation and requires agonist-induced "inside-out" signals that increase alphaIIbbeta3 affinity. Agonist regulation of alphaIIbbeta3 also takes place in megakaryocytes, the bone marrow cells from which platelets are derived. To facilitate mechanistic studies of inside-out signaling, we describe here the generation of megakaryocytes in quantity from murine embryonic stem (ES) cells. Coculture of ES cells for 8-12 days with OP9 stromal cells in the presence of thrombopoietin, IL-6, and IL-11 resulted in the development of large, polyploid megakaryocytes that produced proplatelets. These cells expressed alphaIIbbeta3 and platelet glycoprotein Ibalpha but were devoid of hematopoietic stem cell, erythrocyte, and leukocyte markers. Mature megakaryocytes, but not megakaryocyte progenitors, specifically bound fibrinogen by way of alphaIIbbeta3 in response to platelet agonists. Retrovirus-mediated expression of the reporter gene, green fluorescent protein, in ES cell-derived megakaryocytes did not affect viability or alphaIIbbeta3 function. On the other hand, retroviral expression of CalDAG-GEFI, a Rap1 exchange factor identified by megakaryocyte gene profiling as a candidate integrin regulator, enhanced agonist-induced activation of Rap1b and fibrinogen binding to alphaIIbbeta3 (P < 0.01). These results establish that ES cells are a ready source of mature megakaryocytes for integrin studies and other biological applications, and they implicate CalDAG-GEFI in inside-out signaling to alphaIIbbeta3.
authors.affiliation: Departments of Cell Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Koji
authors.initials: K
authors.lastname: Eto
authors.forename: Ronan
authors.initials: R
authors.lastname: Murphy
authors.forename: Steve W
authors.initials: SW
authors.lastname: Kerrigan
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bertoni
authors.forename: Heidi
authors.initials: H
authors.lastname: Stuhlmann
authors.forename: Toru
authors.initials: T
authors.lastname: Nakano
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Leavitt
authors.forename: Sanford J
authors.initials: SJ
authors.lastname: Shattil
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D010455:Peptides / Q000737:chemistry; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D016023:Integrins; D008164:Luminescent Proteins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C415483:RASGRP2 protein, human; C485927:Rasgrp2 protein, mouse; D049452:Green Fluorescent Proteins; D005340:Fibrinogen; D020827:rap1 GTP-Binding Proteins