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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Magdalena Kucia，PI 干细胞提供了一个新的潜在来源，以取代死亡的神经元和支持组织在脑损伤的慢性缺血，如在睡眠呼吸暂停（SA）。对动物和短期人类骨髓（BM）移植的一些研究已经证明，骨髓细胞提供神经元的来源并且可以修复脑损伤（例如，在中风期间）。这种功能改善的机制目前是密集研究的焦点，需要新的研究方法来评估这些策略的有效性。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，这些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。一种这样的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，提出了一种新的假设，即CXCR 4+外胚层衍生的VSEL池在早期发育期间沉积在BM中。 这些细胞随后充当干细胞的储备移动的库，其可被动员到外周血中并在脑再生中发挥重要作用-其中它们被SDF-1梯度化学吸引。 此外，假设这些循环VSEL的骨髓池中与年龄相关的减少可能有助于中枢神经系统（CNS）的老化，导致较不有效的修复。 为了研究这些问题，提出了四个具体目标。 具体目标1。骨髓源性VSEL的神经分化。我们已经提出了证据，BM包含一个人口的VSEL和这些外胚层来源的细胞沉积在那里的早期发展过程中快速的身体生长/扩张。我们将优化它们从骨髓和神经分化（生长神经球的能力）中的分离。接下来，我们将评估骨髓组织中与年龄相关的VSEL的存在。一旦确定VSEL是否在正常稳态条件下以非常低但可检测的水平在外周血（PB）中循环，我们将在睡眠呼吸暂停的小鼠模型中研究其动员。 具体目标2。优化VSEL进入外周血的动员。由于动员的外周血（mPB）可能是潜在的神经再生的VSEL的来源，我们将优化其动员到PB。 我们将测试涉及选定的生长因子（G-CSF、Flt 3-配体、VEGF、HGF）和小分子抑制剂（CXCR 4拮抗剂T140、C3 aR拮抗剂）的各种动员剂对其动员功效的影响。 我们还将研究这些细胞对缺氧损伤的反应以及SDF-1的作用CXCR 4轴在此过程中。假设CXCR 4 + VSEL被动员，随后以SDF-1依赖性方式被化学吸引到受损的大脑中。 具体目标3。开发一种扩展VSEL的方法。 由于可以从老年个体的BM和mPB分离的VSEL的数量相对较低，因此可能需要有效的离体扩增系统来获得足够数量的这些细胞用于神经再生。 离体培养衍生的VSEL也可能更好地植入和再生脑。 我们将采用选定的策略来体外扩增这些细胞，涉及选定的生长因子的鸡尾酒，BM基质支持，以及我们的新策略，该策略基于在从胚胎干细胞（ESMV）分离的膜衍生微泡存在下干细胞的扩增。我们观察到纯化的VSEL细胞能够在与C2 C12成肌细胞系饲养层的共培养物中形成类似于胚状体的球体。 来自这些球的细胞可以再次（最多5-7代）生长新的次级球，或者如果接种到促进组织分化的培养物中，则扩增成来自所有三个生殖细胞层的细胞。 我们将使用该系统从VSEL衍生的球体中扩增神经细胞。 具体目标4。在睡眠呼吸暂停（SA）的鼠模型中确定VSEL在体内脑再生中的功效。 将在SA的体内小鼠模型中测试VSEL对受损组织的功能性再生的贡献。我们将比较从增强型绿色免疫荧光蛋白（EGFP+）转基因小鼠中分离的同基因VSEL的再生潜力，以挽救缺氧所致的脑损伤，并比较SDF-1的作用。CXCR 4轴在此过程中。 我们将采用从BM或mPB新鲜分离的VSEL以及在离体培养物中扩增的VSEL。 作为大脑再生的一个读数，我们将使用选定的行为测试。