Eto Koji, Murphy Ronan, Kerrigan Steve W, Bertoni Alessandra, Stuhlmann Heidi, Nakano Toru, Leavitt Andrew D, Shattil Sanford J
Departments of Cell Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12819-24. doi: 10.1073/pnas.202380099. Epub 2002 Sep 18.
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.
纤维蛋白原与整合素αIIbβ3的结合介导血小板聚集，并且需要激动剂诱导的“由内向外”信号来增加αIIbβ3的亲和力。αIIbβ3的激动剂调节也发生在巨核细胞中，血小板就是由骨髓中的这些细胞产生的。为了便于对由内向外信号传导进行机制研究，我们在此描述了从小鼠胚胎干细胞大量生成巨核细胞的方法。在血小板生成素、白细胞介素-6和白细胞介素-11存在的情况下，将胚胎干细胞与OP9基质细胞共培养8 - 12天，会导致产生前血小板的大型多倍体巨核细胞的发育。这些细胞表达αIIbβ3和血小板糖蛋白Ibalpha，但缺乏造血干细胞、红细胞和白细胞标志物。成熟的巨核细胞而非巨核细胞祖细胞，在血小板激动剂的作用下，通过αIIbβ3特异性结合纤维蛋白原。逆转录病毒介导的报告基因绿色荧光蛋白在胚胎干细胞来源的巨核细胞中的表达不影响细胞活力或αIIbβ3功能。另一方面，通过巨核细胞基因谱分析鉴定为整合素调节剂候选物的Rap1交换因子CalDAG-GEFI的逆转录病毒表达，增强了激动剂诱导的Rap1b激活以及纤维蛋白原与αIIbβ3的结合（P < 0.01）。这些结果表明，胚胎干细胞是用于整合素研究和其他生物学应用的成熟巨核细胞的现成来源，并且提示CalDAG-GEFI参与了αIIbβ3的由内向外信号传导。
