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

描述（由申请人提供）：巨核细胞（Mk）来源于造血干细胞。Mk的分化和成熟经历了几个相互重叠的阶段：多轮核内有丝分裂以形成多倍体细胞;分界膜系统的发展;导致细胞解体的凋亡程序;以及称为前血小板的细胞质延伸的形成，血小板从其中释放。Mk定型、分化、多倍化和凋亡的机制仍知之甚少。增加Mk倍性是重要的，因为产生的血小板数量随着Mk DNA含量增加而增加。在生物反应器中离体生成功能性血小板将对输血医学产生重大影响。血小板输注用于广泛的血栓形成缺陷，每年输注数百万单位。血小板是一种昂贵的产品，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，血小板供应有限。相反，它们在20- 240 ℃下储存3-5天，这增加了细菌污染的风险。血液传播的病原体也构成风险，受体的同种免疫仍然是一个问题。根据良好生产规范生产的培养衍生血小板可以为输血治疗提供更安全和更耐受的供应。改进的理解和控制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用于输血医学的血小板在经济上可行。几个研究小组已经产生了少量的培养衍生的血小板，其功能活性与收获的血小板相似。然而，即使是生产单次输血所需的血小板也是一个重大的技术挑战。大规模的培养衍生的血小板生产需要取得重大进展，才能在经济上具有吸引力。这将需要改进人造血和祖细胞（HSPC）向Mk的扩增，而且还需要改进产生大的多倍体Mk细胞的能力，因为从Mk细胞产生的血小板的数量与细胞的倍性成比例。多倍性Mk细胞的分裂主要发生在BM脉管系统中，由此Mk细胞通过窦壁的间隙投射细胞质延伸。Mks也可以进入循环并在肺血管系统中成熟产生血小板。MK细胞破裂与由于血流和/或细胞变形引起的机械应力相关。因此，Mk分裂和可能的成熟是一个应力诱导的过程。鉴于Mk成熟和前血小板形成也受到与细胞外基质相互作用的影响，血小板生产将需要在流动条件下使用涉及半合成基质的生物反应器系统，以尽可能模拟体内条件。在这里，我们专注于方面的Mk成熟受外源性机械（剪切）应力。我们在体内和体外的初步数据表明，肿瘤抑制基因p53是特异性激活后，启动Mk分化。我们的中心假设是，p53的作用是通过阻碍细胞周期和促进凋亡来控制多倍化和从内有丝分裂到凋亡的转变。在这个模型中，p53被称为Mk倍性调节剂的作用，在这个意义上，我们假设这个作用是响应内源性（由于多倍化）和外源性（剪切）应力。了解p53作为这些压力的转换器的作用，以及作为Mk成熟的可能调节器，将在血小板生产的可扩展过程的发展中非常重要。使用平行板流动装置，我们的目的是了解剪切应力的水平，结合暴露于流的长度，影响细胞周期，内有丝分裂，细胞凋亡和p53激活（捕获的特定乙酰化/脱乙酰化事件）在培养的人Mk细胞。微阵列分析将检查Mk成熟过程中流体力对基因表达模式的影响，旨在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，并且以其非常大的尺寸、高DNA含量和形成脱落血小板（血液凝固所必需的小细胞）的前血小板延伸而区分。在机械应力条件下，前血小板形成发生在骨髓脉管系统中，这对于血小板产生是必不可少的。阐明负责Mk成熟和前血小板形成的机制对于理解导致血小板产生的过程是重要的。这可能导致识别促进许多高倍性Mk细胞产生的因素和培养条件，这些细胞将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理解和模拟体内前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.