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Updated: Aug 7, 2026
08:56
Isolation of Murine Embryonic Hemogenic Endothelial Cells
Published on: June 17, 2016
Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling
Koji Eto1, Ronan Murphy, Steve W Kerrigan
1Departments of Cell Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Summary
Murine embryonic stem cells generate functional megakaryocytes for studying alphaIIbbeta3 integrin signaling. CalDAG-GEFI enhances agonist-induced fibrinogen binding in these cells.
Area of Science:
- Hematology
- Cell Biology
- Molecular Biology
Background:
- Integrin alphaIIbbeta3 mediates platelet aggregation via fibrinogen binding, regulated by inside-out signaling.
- Megakaryocytes, platelet precursors, also exhibit agonist regulation of alphaIIbbeta3.
- Understanding inside-out signaling in megakaryocytes is crucial for platelet function research.
Purpose of the Study:
- To generate functional megakaryocytes from murine embryonic stem (ES) cells for studying alphaIIbbeta3 integrin signaling.
- To investigate the role of CalDAG-GEFI in agonist-induced alphaIIbbeta3 activation in ES cell-derived megakaryocytes.
Main Methods:
- Coculture of murine ES cells with OP9 stromal cells and specific cytokines (thrombopoietin, IL-6, IL-11) for megakaryocyte differentiation.
- Analysis of megakaryocyte markers and proplatelet formation.
- Functional assays measuring fibrinogen binding to alphaIIbbeta3 in response to agonists.
- Retrovirus-mediated gene expression of green fluorescent protein and CalDAG-GEFI.
Main Results:
- ES cells differentiated into mature, polyploid megakaryocytes expressing alphaIIbbeta3 and producing proplatelets.
- Mature megakaryocytes, but not progenitors, showed agonist-dependent fibrinogen binding via alphaIIbbeta3.
- Retroviral expression of CalDAG-GEFI enhanced agonist-induced Rap1b activation and alphaIIbbeta3-mediated fibrinogen binding.
- Reporter gene expression did not impair megakaryocyte viability or alphaIIbbeta3 function.
Conclusions:
- Murine ES cells provide a valuable source of mature megakaryocytes for research, including integrin signaling studies.
- CalDAG-GEFI plays a significant role in the inside-out signaling pathway regulating alphaIIbbeta3 function.
- This model system facilitates mechanistic studies of integrin regulation and platelet biology.
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