Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells
Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells
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DOI：
10.1089/scd.2012.0701
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发表时间：
2013-07-01
影响因子：
4
通讯作者：
Inaba, Toshio
中科院分区：
文献类型：
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作者：
Nishimura, Toshiya;Hatoya, Shingo;Inaba, Toshio
血小板减少症（TTP）是人类和犬常见的血液病。目前，除了输血外，没有有效的治疗方法。在这项研究中，我们报告了从犬胚胎成纤维细胞产生犬诱导多能干细胞（ciPSC），以及从ciPSC产生成熟巨核细胞（MK）和功能性血小板的新方案。使用慢病毒载体产生ciPSC，并在补充有生长因子的0 P9基质细胞上分化成MK和血小板。我们的ciPSC呈现出紧密的圆顶形状，并显示出关键的多能性标志物REX 1的表达和正常的核型。此外，ciPSC通过形成胚状体分化成源自三个胚层的细胞。来源于ciPSC的MK具有超倍性并转化为前血小板。前体血小板在表达特异性MK和血小板标志物CD 41/61的早期释放血小板。有趣的是，当这些血小板被二磷酸腺苷或凝血酶激活时，它们与纤维蛋白原结合。电镜观察显示血小板具有与外周血小板相同的超微结构。因此，我们首次证明了能够在体外分化成MK并释放功能性血小板的ciPSC的产生。我们的用于将ciPSC分化成MK和血小板的系统有望成为犬TTP的关键疗法，并且似乎原则上可扩展以解决人TTP。
Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.