In vitro bioreactor system for platelet formation

总结 在各种人类病理中，血小板产生或功能存在无数的改变。然而许多 这些发病机制和所需的靶向治疗仍然未知，导致姑息治疗。在 在体内，巨核细胞与骨髓微血管系统结合，在那里它们延伸前血小板， 通过血管内皮突出到管腔中并将血小板释放到血流中。的 科学界和临床界正在积极寻找新的模式来产生功能性血小板， 体内，以满足临床需求，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所展示的那样，设计一个3D骨髓模拟物将推动机械 了解血小板脱落并确定未来的治疗方案。来测试我们 假设，在目的1中，我们将在改良的离体三个细胞中利用非血栓形成丝蛋白生物材料。 骨髓的三维（3D）组织模型，以研究血小板从巨核细胞中的释放， 从人类诱导多能干细胞（hiPSC）;与目前的研究结果相比， 来自脐带血祖细胞的巨核细胞。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来自患者的人巨核细胞的作用的系统 受遗传性血小板减少症影响的人和健康对照组。在目标3中，我们将进行研究， 在体外和体内生物反应器系统中释放的血小板的功能性。成果的 研究预计将是前所未有的深入了解机制，控制血小板形成。这些 这些见解将建立在我们离体产生功能性人类血小板的能力上， 在此更新提案中，细胞来源、疾病洞察和功能评估的改进。的 根据拟议的科学和技术制定统一的解决方案，将澄清 血小板生成素模拟物对人巨核细胞在细胞内信号传导激活方面的行为的影响， 分化，与细胞外环境的相互作用和血小板的产生，所有这些都与主要的 对人类健康的影响。重要的是，该项目的成功成果将为研究人员提供 用新的专门工具来预测新药的疗效和安全性，以解决巨核细胞- 相关疾病。此外，该结果将为临床相关来源提供重要的下一步 以及用于患者治疗的功能性人血小板的供应。

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.