Novel approach for formation of platelet-like particles from mouse embryonic stem cells without using feeder cells.

Megakaryocytes (MKs) and platelet-like particles (PLPs) have generally been obtained by culturing embryonic stem (ES) cells over feeder cells. However, using feeder cells need many labor-consuming processes and the MK and PLP fractions obtained are often contaminated by such cells and their fragments. Here we describe our new culture system for differentiating mouse ES cells to MKs and PLPs without using feeder cells. ES cells are differentiated to cells with MK-like morphology and properties, including proplatelet formation, high ploidy (>8N), and CD41 expression. The culture medium contained PLPs expressing platelet glycoproteins, CD41 and GPIb. Integrin alpha(IIb)beta(3) of PLPs can be activated by thrombin. Addition of the metalloproteinase inhibitor TAPI-2 to the culture increased the surface expression of GPIbalpha and augmented the adhesion of PLPs to immobilized von Willebrand factor through decreasing the shedding of GPIbalpha. Thus our mouse ES cells culture system is a suitable and efficient method for obtaining MKs and functional PLPs that obviates the need for feeder cells.