Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function. J Thromb Haemost 4:436-442

Department of Medicine, University of California, San Diego, San Diego, California, United States
Journal of Thrombosis and Haemostasis (Impact Factor: 5.72). 03/2006; 4(2):436-42. DOI: 10.1111/j.1538-7836.2006.01744.x
Source: PubMed


The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits alpha(IIb) and beta(3), is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of alpha(IIb)beta(3), thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to alpha(IIb)beta(3) triggers cytoskeletal changes and granule release (outside-in signaling).
Genetic approaches to characterize the molecular pathways involved in alpha(IIb)beta(3) signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs).
alpha(IIb)beta(3) activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIbalpha(+) cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of alpha(IIb)beta(3) outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of alpha(IIb)beta(3) activation.
Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and alpha(IIb)beta(3) signaling in the native cellular environment.

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Available from: Bill Moran, Jan 28, 2015
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    • "In humans, CD41+ megakaryocyte progenitors were generated after co-culturing the ES cells with S17 cells [78]. CD41a+/CD42b+ megakaryocyte lineage cells could be generated from human ES cells after co-cultures with OP9 stromal cells [83]. Platelets were induced from cell populations that expressed CD34, VE-cadherin, CD31, CD41a and CD45 surface antigen markers from both human ES cells and adult fibroblast-derived iPS cells after OP9 co-cultures [78]. "
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    • "OP9 cells are useful feeder cells and support differentiation of both mouse and human ESCs into mature hematopoietic cells in vitro. Gaur’s group observed that after 15–17 days of co-culture in the presence of TPO, ESC-derived MKs began to express specific surface antigens, including two lineage markers, cluster of differentiation (CD) 41a (CD41a; integrin αIIb) and CD42b (GPIbα), but no platelets were detected in this system [26]. We also failed to generate large numbers of platelet-like particles from human ESCs by this basic protocol. "
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    • "The hESC line H9 (NIH designation, WA09), obtained from the University of Wisconsin Alumni Research Foundation, was cultured as described (Gaur et al. 2006). In brief, H9 cells were maintained as undifferentiated colonies by coculture on irradiated CF1 MEFs (Bodnar et al. 2004), plated on 0.1% gelatin-coated 6-well dishes in DMEM high glucose supplemented with 2 mM L-glutamine, 13 penicillin/streptomycin (Gibco BRL), and 10% fetal bovine serum (Hyclone). "
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