Production of functional platelets by differentiated embryonic stem (ES) cells in vitro

• Published in: Blood 2003-12, Vol.102 (12), p.4044-4051
• Main Authors: Fujimoto, Tetsuro-Takahiro, Kohata, Satoshi, Suzuki, Hidenori, Miyazaki, Hiroshi, Fujimura, Kingo
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blood Platelets - cytology; Blood Platelets - metabolism; Cell Culture Techniques - methods; Cell Differentiation; Cell differentiation, maturation, development, hematopoiesis; Cell physiology; Cell Size; Coculture Techniques; Fundamental and applied biological sciences. Psychology; Megakaryocytes - cytology; Mice; Microscopy, Electron; Molecular and cellular biology; Platelet Function Tests; Stem Cells - cytology; Stromal Cells - cytology; Thrombopoiesis; Time Factors; Transfection
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2003-06-1773

Megakaryocytes and functional platelets were generated in vitro from murine embryonic stem (ES) cells with the use of a coculture system with stromal cells. Two morphologically distinctive megakaryocytes were observed sequentially. Small megakaryocytes rapidly produced proplatelets on day 8 of the differentiation, and large hyperploid megakaryocytes developed after day 12, suggesting primitive and definitive megakaryopoiesis. Two waves of platelet production were consistently observed in the culture medium. A larger number of platelets was produced in the second wave; 104 ES cells produced up to 108 platelets. By transmission electron microscopy, platelets from the first wave were relatively rounder with a limited number of granules, but platelets from the second wave were discoid shaped with well-developed granules that were indistinguishable from peripheral blood platelets. ES-derived platelets were functional since they bound fibrinogen, formed aggregates, expressed P-selectin upon stimulation, and fully spread on immobilized fibrinogen. These results show the potential utility of ES-derived platelets for clinical applications. Furthermore, production of gene-transferred platelets was achieved by differentiating ES cells that were transfected with genes of interest. Overexpression of the cytoplasmic domain of integrin β3 in the ES-derived platelets prevented the activation of αIIbβ3, demonstrating that this system will facilitate functional platelet studies. (Blood. 2003;102:4044-4051)