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

COBRE PROJ 10：睡眠呼吸暂停小鼠模型中的 VSEL 和大脑再生

• 批准号: 7720771
• 负责人: Magdalena J. Kucia
• 金额: $ 22.42万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720771
• 关键词: Aging; Animals; Apnea; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Brain; Brain Injuries; CXCR4 gene; Cell Count; Cell Line; Cell Therapy; Cells; Chronic Brain Damage; Coculture Techniques; Computer Retrieval of Information on Scientific Projects Database; Condition; Data; Deposition; Development; Disease; Effectiveness; Epiblast; Funding; Germ Cells; Grant; Growth; Growth Factor; Human; Hypoxia; Immunofluorescence Immunologic; Individual; Institution; Investigation; Ischemia; Ligands; Longevity; Marrow; Membrane; Methodology; Modeling; Mus; Natural regeneration; Nerve Regeneration; Neuraxis; Neurons; Numbers; Patients; Play; Population; Process; Reading; Research; Research Personnel; Resources; Role; Sleep; Sleep Apnea Syndromes; Source; Stem cells; Stroke; Stromal Cell-Derived Factor 1; System; T140 peptide; Testing; Tissue Differentiation; Tissues; Transgenic Mice; United States National Institutes of Health; Vascular Endothelial Growth Factors; age related; base; behavior test; embryonic stem cell; enhanced green fluorescent protein; functional improvement; in vivo; in vivo regeneration; inhibitor/antagonist; mouse model; novel; peripheral blood; relating to nervous system; repaired; response; small molecule; tissue regeneration

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Magdalena Kucia, PI Stem cells provide a novel potential source to replace dead neurons and supporting tissue in 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 VSEL 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 VSEL may contribute to aging of the central nervous system (CNS), resulting in less effective repair. To investigate these issues, four specific aims are proposed. Specific Aim 1. Neural differentiation of bone marrow-derived VSEL. We have presented evidence that BM contains a population of VSEL and that these epiblast-derived cells are deposited there early in development during rapid body growth/expansion. We will optimize their isolation from bone marrow and neural differentiation (ability to grow neurospheres). Next we will evaluate the age-related presence of VSEL in bone marrow tissue. Once it is determined whether VSEL 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 VSEL into peripheral blood. Since mobilized peripheral blood (mPB) may be a source of VSEL 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+ VSEL are mobilized and subsequently chemoattracted into a damaged brain in an SDF-1-dependent manner. Specific Aim 3. Develop an approach to expand VSEL. Since the number of VSEL 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 VSEL will better engraft and regenerate brain. 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 layer 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 VSEL in brain regeneration in vivo in a murine model of sleep apnea (SA). The contribution of VSEL 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 VSEL isolated from enhanced green immunofluorescence protein (EGFP+) transgenic mice to rescue brain damaged by hypoxia and the role of the SDF-1CXCR4 axis in this process. We will employ freshly isolated VSEL from BM or mPB as well as VSEL expanded in ex vivo cultures. As a read-out of brain regeneration, we will use selected behavioral tests.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 Magdalena Kucia，Pi 干细胞提供了一种新的潜在来源，以替代死亡神经元并支撑慢性缺血破坏的大脑中的组织，例如睡眠呼吸暂停（SA）。对动物和短期人骨髓（BM）移植的一些研究表明，骨髓细胞提供神经元的来源并可以修复脑损伤（例如，在中风期间）。目前，这种功能改进的机制是激烈研究的重点，从而需要新的研究方法来评估此类策略的有效性。阐明与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，这些疗法可以延长可通过组织再生治疗的疾病患者的寿命。这种疾病是由于睡眠呼吸暂停引起的缺氧引起的慢性脑损伤。 基于初步数据，提出了一个新的假设，即在早期开发过程中，CXCR4+ epiblast衍生的VSEL的池沉积在BM中。 这些细胞随后用作可动员到外周血细胞的储备移动池，并在脑再生中起重要作用 - 在这些层被SDF -1梯度介绍。 此外，假设这些循环VSEL的骨髓库的年龄相关减少可能会导致中枢神经系统（CNS）的衰老，从而导致修复效率较低。 为了调查这些问题，提出了四个具体目标。 特定目的1。骨髓来源的VSEL的神经分化。我们提供了证据表明，BM含有VSEL的群体，并且这些细胞衍生的细胞在快速身体生长/扩张期间的发育早期沉积在那里。我们将优化它们从骨髓和神经分化（发展神经球的能力）中的分离。接下来，我们将评估VSER在骨髓组织中的年龄相关的存在。一旦确定了VSEL在非常低但可检测到的水平的正常稳态条件下的稳态条件下循环，我们将在睡眠呼吸暂停的鼠模型中研究它们的动员。 具体目标2。优化将VSEL动员到外周血中。由于动员的外周血（MPB）可能是潜在神经再生的VSEL来源，因此我们将优化它们的动员为Pb。 我们将测试涉及所选生长因子（G-CSF，FLT3-LIGAND，VEGF，HGF）和小分子抑制剂（CXCR4-抗Aantagonist T140，C3AR拮抗剂）的各种动员剂的影响。 我们还将研究这些细胞对缺氧损伤的动员以及SDF-1 CXCR4轴在此过程中的作用。假设CXCR4+ VSEL被动员，然后以SDF-1依赖性方式将其趋化为受损的大脑。 特定目标3。开发一种扩展VSEL的方法。 由于可以从老年人的BM和MPB中分离出的VSEL数量相对较低，因此可能需要有效的离体扩展系统来获得足够数量的这些细胞进行神经再生。 离体培养衍生的VSEL也可能会更好地植入和再生大脑。 我们将采用选定的策略来扩展这些细胞，涉及所选生长因子的鸡尾酒，BM基质支持以及我们的新策略，基于在存在于胚胎干细胞（ESMV）的膜衍生的微泡中的干细胞扩展的情况下。我们观察到，纯化的VSEL细胞能够与类似于胚胎体的C2C12肌细胞细胞系进料器层形成球体。 来自这些球体的细胞可能会再次（多达5-7个通道）生长新的次级球，或者如果将促进组织分化的培养物铺设到培养物中，则会从所有三个细胞层层扩展到细胞中。 我们将利用该系统扩展神经细胞从VSEL衍生的球体扩展。 具体目的4。确定VSER在鼠类睡眠呼吸暂停（SA）的鼠模型中的脑再生中的疗效。 VSEL​​对受损组织功能再生的贡献将在SA的体内小鼠模型中进行测试。我们将比较从增强的绿色免疫荧光蛋白（EGFP+）转基因小鼠中分离出的同性VSEL的再生潜力，以挽救缺氧受损的大脑以及在此过程中SDF-1 CXCR4轴的作用。 我们将使用来自BM或MPB的新鲜隔离VSEL以及在离体文化中扩展的VSEL。 作为脑再生的读出，我们将使用选定的行为测试。