Embryonic Stem Cell-Derived Platelets as Cellular Therapeutics

描述（由申请人提供）： 目前用于各种临床相关环境的血小板递送具有许多显著的限制，来自储存、污染、多次输血个体中的抑制剂、可变质量和对人类供体的依赖性的问题。我们提出了一种新的策略，从人类胚胎干细胞（hESC）的临床相关数量的血小板的生产，初步数据的支持。这样的血小板可以使我们避免目前的许多限制，并使我们能够开发血小板作为一种机制，用于靶向递送治疗剂到血管损伤部位。该方法将涉及以下步骤，这些步骤将在我们的初步数据的基础上同时进行：1）通过提高hESC对造血中胚层发育的效率并将造血祖细胞定向分化为巨核细胞（Megs）来增强hESC定向分化为Megs。2)通过敲低转录因子GATA 1，将细胞阻滞在巨红系祖细胞（MEP）阶段，以形成自我复制细胞。在重新诱导GATA 1后，这些MEP细胞将完全分化为Megs。还将采取加强MEP以使Megs成熟的战略。3)输注体外生成的血小板或成熟的Megs，以生成新的功能性血小板。4)hESC或自我复制MEP细胞也将被修饰，使得Megs表达储存在其α-颗粒中的目的异位蛋白，用于在血小板活化时释放。基于初步的数据，我们选择在发育中的Megs中表达尿纤溶酶原激活物作为原理验证。我们将证明，由此产生的人血小板允许有针对性地交付这种代理生长血栓，而不引起全身纤维蛋白溶解。 这些研究将与西雅图集群密切合作，涉及3个物种：小鼠、狗和人类。小鼠研究将允许快速进展，然后将在狗作为大型动物模型进行测试，并将我们的进展应用于人类研究。我们相信，在七年的支持下，我们将在从hESC开始生产临床相关数量的功能性血小板方面取得重大进展，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.