Role of shear forces in proplatelet production from stem cells in bioreactors

描述(申请人提供)：巨核细胞(MKs)来源于造血干细胞。MK的分化和成熟经历了几个重叠的阶段：多轮内减数分裂形成多倍体细胞；分界膜系统的形成；导致细胞解体的凋亡-死亡程序；以及称为原血小板的细胞质延伸的形成，从中释放血小板。Mk的定位、分化、多倍化和细胞凋亡的机制目前还不清楚。增加Mk倍体很重要，因为产生的血小板数量随着Mk DNA含量的增加而增加。功能血小板在生物反应器中的体外生成将对输血医学产生重大影响。血小板输注被广泛用于治疗血栓缺乏症，每年有数百万个单位被输注。由于从捐献的血液中收集和处理步骤，以及血小板不能冷冻储存的事实，血小板是一种有限供应的昂贵产品。相反，它们在20-240摄氏度的温度下储存3-5天，这会增加细菌污染的风险。血液传播的病原体也构成风险，受者的同种异体免疫仍然是一个问题。培养衍生的血小板，在使用良好的制造规范下生产，可以为输血疗法提供更安全和更耐受的供应。更好的理解和控制MK成熟的能力对于提供组织工程解决方案至关重要，这些解决方案使大规模生产血小板用于输血医学在经济上是可行的。有几个小组已经产生了少量的培养衍生的血小板，其功能活性与采集的血小板相似。然而，即使生产一次输血所需的血小板也是一个重大的技术挑战。大规模、培养衍生的血小板生产要想在经济上变得有吸引力，还需要取得重大进展。这将需要改进人类造血和祖细胞(HSPC)向MK细胞的扩增，但也需要提高产生大型多倍体MK细胞的能力，因为MK细胞产生的血小板数量与细胞的倍性成正比。多倍体Mk细胞的分裂主要发生在BM的血管系统中，Mk细胞通过窦壁的缝隙投射细胞质延伸。巨噬细胞集落刺激因子也可以进入循环并成熟，在肺血管系统中产生血小板。MK细胞破裂与血流和/或细胞变形引起的机械应力有关。因此，MK的断裂和可能的成熟是一个应激诱导的过程。鉴于MK的成熟和原血小板的形成也受到与细胞外基质相互作用的影响，血小板的产生将需要在流动条件下使用包含半合成基质的生物反应器系统来尽可能地模拟体内的条件。在这里，我们集中在外源机械(剪切)应力对Mk成熟的影响方面。我们的体内和体外初步数据表明，肿瘤抑制基因P53在MK分化启动时被特异性激活。我们的中心假设是，P53的S的作用是通过阻止细胞周期和促进细胞凋亡来控制多倍体和从内丝分裂到凋亡的转变。在这个模型中，P53被称为Mk倍体调节因子，在这个意义上，我们假设这个作用是对内源(由于多倍化)和外源(剪切)压力的响应。了解P53作为这些压力的转导，以及作为MK成熟的可能调节因子的作用，对于开发可扩展的血小板生产过程将是重要的。利用平行板流动装置，我们旨在了解切应力水平和流动暴露时间如何影响培养的人Mk细胞的细胞周期、内丝分裂、凋亡和P53激活(通过特定的乙酰化/去乙酰化事件捕获)。微阵列分析将检测MK成熟过程中流体作用力对基因表达模式的影响，目的是识别受影响的程序和基因。 公共卫生相关性：巨核细胞(MK)来源于造血(血液)干细胞，其特点是体积非常大，DNA含量高，并形成能释放血小板的原血小板延伸，而血小板是凝血所必需的小细胞。在机械应力条件下，原血小板的形成发生在骨髓血管系统中，这对血小板的产生是必不可少的。阐明MK成熟和原血小板形成的机制对于理解导致血小板产生的过程是很重要的。这可能导致确定促进产生许多高倍体的Mk细胞的因素和培养条件，使其能够在生物反应器中生产大量用于医疗输血的血小板。为实现这一目标而设计的大规模过程要求我们尽可能地了解和模拟体内原血小板形成的复杂过程，这是本项目的一个重要目标。

DESCRIPTION (provided by applicant): Megakaryocytes (Mks) are derived from hematopoietic stem cells. Mk differentiation and maturation progresses through several overlapping stages: multiple rounds of endomitosis to form polyploid cells; development of a demarcation membrane system; an apoptotic-death program, which leads to cell disintegration; and formation of cytoplasmic extensions called proplatelets, from which platelets are released. The mechanisms governing Mk commitment, differentiation, polyploidization, and apoptosis remain poorly understood. Increasing Mk ploidy is important because the number of platelets produced increases with Mk DNA content. Ex vivo generation in bioreactors of functional platelets would have a major impact in transfusion medicine. Platelet transfusions are used for a wide range of thrombotic deficiencies and several million units are transfused each year. Platelets are an expensive product in limited supply due to the collection and processing steps from donated blood and the fact that platelets cannot be stored frozen. Rather they are stored for 3-5 days at 20-240C, which increases the risk of bacterial contamination. Blood-borne pathogens also pose a risk, and alloimmunization of recipients remains a problem. Culture-derived platelets, produced under using Good Manufacturing Practices, could provide a safer and more tolerated supply for transfusion therapies. Improved understanding and the ability to control Mk maturation will be critical for providing tissue engineering solutions that make it economically feasible to produce platelets in large scale for transfusion medicine. Several groups have generated small quantities of culture-derived platelets with functional activity similar to that of harvested platelets. However, producing even the platelets required for a single transfusion presents a major technological challenge. Major advances are needed for large-scale, culture-derived platelet production to become economically attractive. This will require improvements in the expansion of human hematopoietic and progenitor (HSPCs) cells into Mks, but also the ability to produce large, polyploid Mk cells, since the number of platelets produced from an Mk cell is proportional to the cell's ploidy. Breakup of polyploidy Mk cells takes place largely in the BM vasculature whereby Mk cells project cytoplasmic extensions through the gaps of sinus walls. Mks can also go into circulation and mature to produce platelets in the lung vasculature. Mk-cell breakup is associated with mechanical stresses due to blood flow and/or cell deformation. Thus, Mk breakup and possibly maturation is a stress-induced process. In view of the fact that Mk maturation and proplatelet formation are also affected by interactions with extracellular matrix, platelet production will need to engage bioreactor systems involving semi-synthetic matrices under flow conditions to simulate, to the extent possible, the in vivo conditions. Here we focus on the aspects of Mk maturation affected by exogenous mechanical (shear) stress. Our in vivo and ex vivo preliminary data suggest that the tumor suppressor p53 is specifically activated upon initiation of Mk differentiation. Our central hypothesis is that p53's role is to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. In this model, p53 is called to play the role of Mk ploidy regulator, and in this sense we hypothesize that this role is to respond to endogenous (due to polyploidization) and exogenous (shear) stress. Understanding the role of p53 as a transducer of these stresses, but also as a possible regulator of Mk maturation will be important in the development of scalable processes for platelet production. Using a parallel-plate flow apparatus, we aim to understand how the level of shear stress, in combination with the length of exposure to flow, affect cell cycling, endomitosis, apoptosis and p53 activation (as captured by specific acetylation/deacetylation events) in cultured human Mk cells. Microarray analysis will examine the impact of fluid forces on the gene expression patterns during Mk maturation aiming to identify the programs and genes affected. PUBLIC HEALTH RELEVANCE: Megakaryocytes (Mks) are derived from hematopoietic (blood) stem cells, and are distinguished by their very large size, high DNA content, and the formation of proplatelet extensions which shed platelets, the small cells necessary for blood coagulation. Proplatelet formation takes place in the bone marrow vasculature under mechanical stress conditions, which are essential for the platelet production. Elucidating the mechanisms responsible for Mk maturation and proplatelet formation is important for understanding the process that leads to platelet production. This could lead to identification of factors and culture conditions that promote the generation of many, high-ploidy Mk cells that would enable the production, in bioreactors, of large numbers of platelets for medical transfusions. Design of large-scale processes for achieving this goal requires that we understand and mimic to the extent possible the complex process of in vivo proplatelet formation, and this is an important goal of this project.