COBRE PROJ 10: VSEL AND BRAIN REGENERATION IN A MURINE MODEL OF SLEEP APNEA

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 Magdalena Kucia，Pi 干细胞提供了一种新的潜在来源来替代因慢性缺血而受损的大脑中死亡的神经元和支持组织，例如睡眠呼吸暂停(SA)。一些对动物和短期人类骨髓移植的研究表明，骨髓细胞提供神经元来源，并可以修复脑损伤(例如，在中风期间)。这种功能改善的机制目前是密集研究的重点，因此需要新的研究方法来评估这种策略的有效性。阐明干细胞相关的再生机制对于开发有效的基于干细胞的疗法至关重要，这种疗法可以延长患有可通过组织再生治疗的疾病的患者的寿命。其中一种疾病是睡眠呼吸暂停导致的低氧导致的慢性脑损伤。 基于初步数据，提出了一个新的假说，即CXCR4+表胚源性VSEL池在发育早期沉积在骨髓中。这些细胞随后作为干细胞的备用移动池，可被动员到外周血液中，并在脑再生中发挥重要作用--它们被SDF-1梯度化学吸引。此外，假设这些循环中的VSEL的骨髓池与年龄相关的减少可能导致中枢神经系统(CNS)的老化，导致修复效率降低。为了调查这些问题，提出了四个具体目标。 具体目的1.骨髓源性VSEL的神经分化。我们已经提出的证据表明，骨髓含有VSEL群体，这些外胚层来源的细胞在身体快速生长/扩张的早期发育阶段就沉积在那里。我们将首先优化它们的骨髓分离和神经分化(培养神经球的能力)。接下来，我们将评估与年龄相关的VSEL在骨髓组织中的存在。一旦确定VSEL是否在正常的稳定状态下在外周血液(PB)中以非常低但可检测的水平循环，我们将在睡眠呼吸暂停的小鼠模型中研究它们的动员。 具体目标2.优化外周血中VSEL的动员。由于动员的外周血(MPB)可能是潜在的神经再生的VSEL的来源，我们将优化它们动员到PB中。我们将测试包括选定的生长因子(G-CSF、Flt3-Ligand、VEGF、HGF)和小分子抑制剂(CXCR4拮抗剂T140、C3aR拮抗剂)的各种动员剂对其动员效果的影响。我们还将研究这些细胞在应对缺氧损伤时的动员以及SDF-1CXCR4轴在这一过程中的作用。据推测，CXCR4+VSEL被动员起来，随后以SDF-1依赖的方式被化疗吸引到受损的大脑中。 具体目标3.开发一种扩展VSEL的方法。由于可以从老年人的骨髓和MPB中分离出的VSEL数量相对较少，因此可能需要一个有效的体外扩增系统来获得足够数量的VSEL细胞用于神经再生。体外培养的VSEL也有可能更好地植入和再生脑细胞。我们将使用选定的策略来体外扩增这些细胞，包括选定生长因子的鸡尾酒、骨髓基质支持，以及我们基于从胚胎干细胞(ESMV)分离的膜来源微囊存在的情况下扩增干细胞的新策略。我们观察到，纯化的VSEL细胞能够在与C2C12成肌细胞系共培养的情况下形成球状细胞，其饲养层类似于胚状体。来自这些球体的细胞可以再次(最多传5-7代)长出新的次级球体，或者如果将其移植到促进组织分化的培养物中，则会从所有三个生殖细胞层扩增成细胞。我们将利用这个系统从VSEL衍生的球体中扩增神经细胞。 具体目的4.在睡眠呼吸暂停(SA)小鼠模型中，确定VSEL在体内脑再生中的有效性。VSEL对受损组织功能再生的贡献将在SA的活体小鼠模型中进行测试。我们将比较从增强型绿色免疫荧光蛋白(EGFP+)转基因小鼠分离的同基因VSEL拯救因缺氧而受损的脑细胞的再生潜力以及SDF-1CXCR4轴在这一过程中的作用。我们将采用从骨髓或MPB新鲜分离的VSEL以及体外培养扩增的VSEL。作为大脑再生的读数，我们将使用选定的行为测试。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Magdalena Kucia, PI Stem cells provide a novel potential source to replace dead neurons and supporting tissue in a brain damaged by chronic ischemia as seen for example in sleep apnea (SA). Some investigations of animals and short-term human bone marrow (BM) transplants have demonstrated that bone marrow cells provide a source of neurons and can repair brain damage (e.g., during stroke). The mechanisms of this functional improvement are currently the focus of intense research, creating a need for new study methodologies to assess the effectiveness of such strategies. Elucidation of stem cell-related mechanisms of regeneration is crucial to developing effective stem cell-based therapies that could extend the lifespan of patients with diseases that are treatable by tissue regeneration. One such disorder is chronic brain damage due to hypoxia resulting from sleep apnea. Based on preliminary data, a novel hypothesis is presented that the pool of CXCR4+ epiblast-derived VSELs is deposited in the BM during early development. These cells subsequently serve as a reserve mobile pool of stem cells that may be mobilized into peripheral blood and play an important role in brain regeneration - where they are chemoattracted by an SDF-1 gradient. Furthermore, it is hypothesized that an age-related decrease in the marrow pool of these circulating VSELs may contribute to aging of the central nervous system (CNS), resulting in less effective repair. To investigate these issues, four specific aims were proposed. Specific Aim 1. Neural differentiation of bone marrow-derived VSELs. We have presented evidence that BM contains a population of VSELs and that these epiblast-derived cells are deposited there early in development during rapid body growth/expansion. We will first optimize their isolation from bone marrow and neural differentiation (ability to grow neurospheres). Next we will evaluate the age-related presence of VSELs in bone marrow tissue. Once it is determined whether VSELs circulate under normal steady-state conditions in the peripheral blood (PB) at very low but detectable levels, we will study their mobilization in a murine model of sleep apnea. Specific Aim 2. Optimize mobilization of VSELs into peripheral blood. Since mobilized peripheral blood (mPB) may be a source of VSELs for potential neural regeneration, we will optimize their mobilization into PB. We will test the effect of various mobilizing agents involving selected growth factors (G-CSF, Flt3-ligand, VEGF, HGF) and small-molecule inhibitors (CXCR4-antagonist T140, C3aR antagonist) on the efficacy of their mobilization. We also will investigate mobilization of these cells in response to hypoxia damage and the role of the SDF-1CXCR4 axis in this process. It is hypothesized that CXCR4+ VSELs are mobilized and subsequently chemo-attracted into a damaged brain in an SDF-1-dependent manner. Specific Aim 3. Develop an approach to expand VSELs. Since the number of VSELs that can be isolated from the BM and mPB of older individuals is relatively low, an efficient ex vivo expansion system may be needed to obtain a sufficient number of these cells for neural regeneration. It also is possible that ex vivo culture-derived VSELs will better engraft and regenerate brain cells. We will employ selected strategies to expand these cells ex vivo involving cocktails of selected growth factors, BM stroma support, and our new strategy based on the expansion of stem cells in the presence of membrane-derived microvesicles isolated from embryonic stem cells (ESMV). We observed that purified VSEL cells are able to form spheres in co-cultures with C2C12 myoblastic cell line feeder layers that resemble embryoid bodies. Cells from these spheres may again (up to 5-7 passages) grow new secondary spheres, or if plated into cultures promoting tissue differentiation, expand into cells from all three germ-cell layers. We will employ this system to expand neural cells from VSEL-derived spheres. Specific Aim 4. Determine the efficacy of VSELs in brain regeneration in vivo in a murine model of sleep apnea (SA). The contribution of VSELs to functional regeneration of damaged tissues will be tested in an in vivo mouse model of SA. We will compare the regeneration potential of syngeneic VSELs isolated from enhanced green immunofluorescence protein (EGFP+) transgenic mice to rescue brain cells damaged by hypoxia and the role of the SDF-1CXCR4 axis in this process. We will employ freshly isolated VSELs from BM or mPB as well as VSEL expanded in ex vivo cultures. As a readout of brain regeneration, we will use selected behavioral tests.