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

描述（由申请人提供）：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：多轮内丝分裂形成多倍体细胞；分界膜系统的研制导致细胞解体的凋亡-死亡程序；并形成称为原血小板的细胞质延伸，血小板从中释放出来。调控Mk承诺、分化、多倍体化和凋亡的机制仍然知之甚少。增加Mk倍性很重要，因为产生的血小板数量随着Mk DNA含量的增加而增加。在生物反应器中体外生成功能性血小板将对输血医学产生重大影响。血小板输注广泛用于血栓性缺陷，每年输注数百万单位。由于捐献血液的收集和处理步骤以及血小板不能冷冻储存，血小板是一种供应有限的昂贵产品。相反，它们在20-240℃下储存3-5天，这增加了细菌污染的风险。血源性病原体也会造成风险，而且接受者的同种异体免疫仍然是一个问题。按照良好生产规范生产的培养源血小板可以为输血治疗提供更安全、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，这些解决方案将使大规模生产用于输血医学的血小板在经济上可行。几个小组已经产生了少量的培养衍生血小板，其功能活性与收获的血小板相似。然而，即使是产生一次输血所需的血小板也是一项重大的技术挑战。大规模的、由培养物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。这将需要改进人类造血和祖细胞（HSPCs）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，因为从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脉管系统中，Mk细胞通过窦壁间隙投射细胞质延伸。Mks也可以进入循环，成熟后在肺血管中产生血小板。mk细胞的破裂与血流和/或细胞变形引起的机械应力有关。因此，Mk的分裂和可能的成熟是一个压力诱导的过程。鉴于Mk成熟和血小板前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含有半合成基质的生物反应器系统，以尽可能地模拟体内条件。在这里，我们关注外源机械（剪切）应力对Mk成熟的影响。我们的体内和离体初步数据表明，肿瘤抑制因子p53在Mk分化开始时被特异性激活。我们的中心假设是p53的作用是通过阻碍细胞周期和促进细胞凋亡来控制多倍体化和从内膜分裂到细胞凋亡的转变。在这个模型中，p53被称为发挥Mk倍性调节因子的作用，从这个意义上说，我们假设这种作用是对内源性（由于多倍体化）和外源性（剪切）应激作出反应。了解p53作为这些压力的传感器的作用，以及作为Mk成熟的可能调节剂，将对血小板生产的可扩展过程的发展具有重要意义。利用平行板流动装置，我们旨在了解剪切应力水平，结合暴露于流动的长度，如何影响培养的人Mk细胞的细胞周期、内膜分裂、凋亡和p53激活（通过特定的乙酰化/去乙酰化事件捕获）。微阵列分析将检查流体作用力对Mk成熟过程中基因表达模式的影响，旨在确定受影响的程序和基因。

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.