Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Academic Article uri icon

Overview

MeSH

  • Animals
  • Cell Differentiation
  • Cell Division
  • Cells, Cultured
  • Fibrinogen
  • Flow Cytometry
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins
  • Mice
  • Peptides
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Retroviridae
  • Signal Transduction
  • Stem Cells
  • Time Factors
  • rap1 GTP-Binding Proteins

MeSH Major

  • Guanine Nucleotide Exchange Factors
  • Integrins
  • Megakaryocytes

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.

publication date

  • October 1, 2002

has subject area

  • Animals
  • Cell Differentiation
  • Cell Division
  • Cells, Cultured
  • Fibrinogen
  • Flow Cytometry
  • Green Fluorescent Proteins
  • Guanine Nucleotide Exchange Factors
  • Humans
  • Integrins
  • Luminescent Proteins
  • Megakaryocytes
  • Mice
  • Peptides
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Retroviridae
  • Signal Transduction
  • Stem Cells
  • Time Factors
  • rap1 GTP-Binding Proteins

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC130543

Digital Object Identifier (DOI)

  • 10.1073/pnas.202380099

PubMed ID

  • 12239348

Additional Document Info

start page

  • 12819

end page

  • 12824

volume

  • 99

number

  • 20