Embryonic Stem Cell-Derived Platelets as Cellular Therapeutics

描述(由申请人提供)： 目前各种临床相关环境下的血小板输送有许多明显的限制，从储存、污染、多次输血的个体中的抑制剂、质量可变和对人类捐赠者的依赖等问题。我们提出了一种从人类胚胎干细胞(HESCs)中生产临床相关数量的血小板的新策略，并得到了初步数据的支持。这样的血小板可以让我们避免目前的许多限制，并允许我们开发血小板作为一种机制，将治疗剂定向输送到血管损伤部位。该方法将包括以下步骤，这些步骤将在我们初步数据的基础上同时进行：1)通过提高hESC向造血中胚层发育的效率并引导造血祖细胞进入MEG谱系，促进hESCs定向分化为巨核细胞(MEG)。2)通过抑制转录因子GATA1，阻止处于MEP阶段的细胞形成自我复制的细胞。在GATA1重新诱导后，这些MEP细胞将完成向MEGS的分化。还将采取战略，将MEP提升到成熟的Megs。3)注入体外产生的血小板或成熟的MEGs，以产生一波活跃的新的、有功能的血小板。4)hESCs或自我复制的MEP细胞也将被修饰，以便MEG表达一种储存在其α-颗粒中的异位蛋白，以便在血小板激活时释放。根据初步数据，我们选择在发育中的MEGS中表达尿纤溶酶原激活物作为原则证明。我们将证明，由此产生的人类血小板允许这种药物靶向输送到不断增长的血栓中，而不会导致全身纤溶。 这些研究将与西雅图星系团密切合作，涉及3个物种：老鼠、狗和人类。老鼠的研究将允许快速发展，然后将作为大型动物模型在狗身上进行测试，并将我们的进步应用于人类研究。我们相信，在七年的支持下，我们将在从人类胚胎干细胞开始的临床相关数量的功能性血小板的生产方面取得重大进展，并为大规模验证性研究和临床应用做好准备。

DESCRIPTION (provided by applicant): Present day delivery of platelets for various clinically relevant settings has a number of significant limitations, from issues of storage, contamination, inhibitors in multiply transfused individuals, variable quality and dependency on human donors. We propose a novel strategy for production of clinically relevant numbers of platelets from human embryonic stem cells (hESCs), supported by preliminary data. Such platelets may allow us to avoid many of the present limitations and allow us to develop platelets as a mechanism for the targeted delivery of therapeutic agents to sites of vascular injury. The approach will involve the following steps which will be pursued simultaneously building on our preliminary data: 1) Enhance directed differentiation of hESCs into megakaryocytes (Megs) by improving the efficiency of hESC to hematopoietic mesoderm development and directing hematopoietic progenitors into the Meg lineage. 2) Arrest cells at the Meg-erythroid progenitor (MEP) stage to form self-replicating cells by knockdown of the transcription factor GATA1. Upon re-induction of GATA1, these MEP cells will complete differentiation into Megs. Strategies to enhance MEP to mature Megs will also be pursued. 3) Infuse either ex vivo-generated platelets or mature Megs to generate a vigorous wave of new, functional platelets. 4) Either hESCs or the self-replicating MEP cells will also be modified so that the Megs express an ectopic protein of interest stored in their a-granules for release upon platelet activation. Based on preliminary data, we chose as proof-of-principle to express urinary plasminogen activator in the developing Megs. We will demonstrate that the resulting human platelets allow targeted delivery of this agent to growing thrombi without causing systemic fibrinolysis. These studies will be done in close collaboration with the Seattle Cluster and will involve 3 species: mice, dogs and humans. Mice studies will allow rapid advances that will then be tested in dogs as a large animal model as well as apply our advances to human studies. We believe that over the seven years of support that we will have achieved significant advances in the production of clinically relevant numbers of functional platelets beginning with hESCs and be ready for large-scale confirmatory studies and clinical application.