In vitro bioreactor system for platelet formation

摘要 在人类的各种病理中，血小板的产生或功能发生了无数的变化。然而，许多人 这些发病机制和所需的靶向治疗仍不清楚，导致姑息性治疗。在……里面 在体内，巨核细胞与骨髓微血管系统联系在一起，在那里它们延伸前血小板 通过血管内皮细胞突出进入管腔，并释放血小板进入血流。这个 科学界和临床界正在积极寻找产生功能性血小板的新模式 活体，以满足临床需要，以及深入了解机制的基础研究。我们假设 正如我们目前的资助中所展示的那样，设计一个3D骨髓模拟物将推动机械学 了解血小板脱落，并确定未来的治疗方案。测试我们的 假设，在目标1中，我们将使用非血栓形成的丝蛋白生物材料在改良的体外三种 研究巨核细胞释放血小板的骨髓三维组织模型 来自人类诱导多能干细胞(HiPSCs)；与目前使用的资助结果进行比较 脐带血祖细胞来源的巨核细胞。在目标2中，我们将重点介绍生物反应器的使用 研究血小板生成素模拟物对患者来源的人巨核细胞的影响的系统 受遗传性血小板减少症和健康对照的影响。在目标3中，我们将进行研究，以评估 在体外和体内生物反应器系统中释放的血小板的功能。这些措施的结果 研究有望对控制血小板形成的机制有前所未有的洞察力。这些 洞察力将建立在我们体外产生功能性人类血小板的能力上，但具有重要的意义 在这项更新提案中，在细胞来源、疾病洞察和功能评估方面有所改进。这个 根据拟议的科学和技术制定统一的解决方案将澄清 血小板生成素在激活细胞内信号方面对人类巨核细胞行为的模拟， 分化，与细胞外环境的相互作用和血小板的产生，都与主要的 对人类健康的影响。重要的是，该项目的成功结果将为研究人员提供 有了新的专门工具来预测处理巨核细胞的新药的有效性和安全性- 与之相关的疾病。此外，研究结果将为临床相关来源的研究提供重要的下一步。 以及病人治疗所需的功能性人体血小板。

Summary In various human pathologies there are countless alterations in platelet production or function. Yet many of these pathogenesis and the required targeted therapies remain unknown, resulting in palliative treatments. In vivo, megakaryocytes associate with the bone marrow microvasculature where they extend proplatelets that protrude through the vascular endothelium into the lumen and release platelets into the blood stream. The scientific and clinical communities are actively searching for new modes to generate functional platelets ex vivo to address clinical needs as well as for insight into fundamental studies of mechanisms. We hypothesize that engineering a 3D bone marrow mimic, as demonstrated in our current grant, will propel mechanistic understanding of platelet shedding and determine future protocols for therapeutic inquiry. To test our hypothesis, in Aim 1 we will utilize non-thrombogenic silk protein biomaterial in a modified ex vivo three dimensional (3D) tissue model of the bone marrow to study platelet release from megakaryocytes derived from human induced pluripotent stem cells (hiPSCs); to compare with the results in the current grant using megakaryocytes from umbilical cord blood progenitors. In Aim 2 we will focus on the use of the bioreactor systems to study the effects of thrombopoietin mimetics on human megakaryocytes derived from patients affected by inherited thrombocytopenias and healthy controls. In Aim 3 we will conduct studies to assess the functionality of the platelets released in the bioreactor systems in vitro and in vivo. The outcome of these studies is expected to be unprecedented insight into mechanisms that control platelet formation. These insights will build on our ability to generate functional human platelets ex vivo but with significant improvements in cell sources, disease insight and functional assessments in this renewal proposal. The development of a unified solution based on the proposed science and technologies will clarify the impact of thrombopoietin mimetics on human megakaryocyte behaviour in terms of activation of intracellular signaling, differentiation, interaction with the extracellular environment and platelet production, all with major implications for human health. Importantly, the successful outcome of this project will provide researchers with new specialized tools for predicting the efficacy and safety of new drugs to address megakaryocyte-  related diseases. In addition, the results will provide an important next step towards clinically relevant sources and supplies of functional human platelets for patient treatments.