Characterization of Ocular Neural Stem Cells

眼神经干细胞的表征

基本信息

批准号：
6944150
负责人：
Iqbal Ahmad
金额：
$ 14.7万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-30 至 2005-03-31
项目状态：
已结题

项目摘要

Establishing cellular diversity is central to the development, structure and function of the brain. Underlying this diversity is a population of neural progenitors with stem cell properties that generates region-specific neurons and glia. Therefore understanding the molecular and cellular biology of neural progenitors holds the key to the mechanism(s) of development of specific regions of the brain including retina. Recapitulation of these developmental mechanisms is likely to open new avenues for treating the impairments of functions that arise due to death of specific neuronal populations as in the case of retinitis pigmentosa and macular degeneration. With these objectives in mind we have proposed to characterize neural progenitors isolated from derivatives of ocular neuroepithelium, the retina and the ciliary body, for their proliferative capacity, self-renewal, maintenance, and developmental potentials in vivo and in vitro under the following specific aims. First, proliferative and differentiation potentials of ocular progenitors will be characterized in the context of their responsiveness to the mitogens, FGF2 and EGF. We will use flow cytometry to examine their proliferation and survival, limiting dilution analysis to estimate their frequency to form clones, immunocytochemical and RT-PCR analyses of cell-type specific markers to test their multipotentiality, and clonal density culture to determine their self-renewal capacity. In addition, we will determine the distribution of mitogen receptors in different sub-populations of ocular progenitors in order to understand the basis of their responsiveness and relationships. Second, the role of Notch signaling in the maintenance of ocular progenitors will be evaluated. We will analyze proliferation and differentiation of these cells in response to gain-of-function and loss-of-function perturbations of Notch signaling. Third, the differentiation potentials of ocular progenitors will be determined in vitro in conditions that promote differentiation. We will use immunocytochemical and electrophysiological analyses to evaluate their ability to acquire specific phenotypes in response to growth factors and neurotrophins, and in co-culture conditions. Fourth, the potential of ocular progenitors to generate site-specific cells in vivo will be evaluated. We will use homotopic and heterotopic transplantation and in vivo activation of progenitors in response to injuries to achieve the aim. Accomplishing these aims will provide valuable information about the underlying mechanism(s) of retinal development and will allow the use of ocular progenitors in stem cell therapy to address degenerative changes in the retina whether inherited, age-related or due to injuries.

建立细胞多样性对于大脑的发育，结构和功能至关重要。这种多样性的基础是具有产生区域特异性神经元和神经胶质的干细胞特性的神经祖细胞的群体。因此，了解神经祖细胞的分子和细胞生物学是包括视网膜在内的大脑特定区域发展机制的关键。这些发育机制的概括很可能会为治疗因特定神经元种群死亡而导致的功能障碍的新途径开放，例如色素性视网膜炎和黄斑变性。考虑到这些目标，我们提出要表征从眼神经上皮衍生物，视网膜和睫状体衍生物中分离出来的神经祖细胞，其增殖能力，自我更新，维持和发育潜力在体内和体外以及以下特定目标下。首先，眼镜祖细胞的增殖和分化潜力将在其对有丝分裂剂FGF2和EGF的反应性的背景下进行表征。我们将使用流式细胞仪检查它们的增殖和存活，限制稀释分析以估算其频率以形成克隆，对细胞类型特异性标志物的免疫细胞化学和RT-PCR分析，以测试其多能性，以及克隆密度培养以确定其自我恢复能力。此外，我们将确定有丝分裂原受体在眼祖细胞不同亚种群中的分布，以了解其反应性和关系的基础。其次，将评估Notch信号在维持眼祖细胞中的作用。我们将分析这些细胞的增殖和分化，以响应功能获得和功能丧失的凹入信号传导。第三，在促进分化的条件下，将在体外确定眼祖细胞的分化潜力。我们将使用免疫细胞化学和电生理分析来评估其对生长因子和神经营养蛋白的响应以及在共培养条件下获取特定表型的能力。第四，将评估眼部祖细胞在体内产生特异性细胞的潜力。我们将使用同型和异位移植以及祖细胞的体内激活，以应对损伤以实现目标。完成这些目标将提供有关视网膜发育的潜在机制的有价值的信息，并允许在干细胞疗法中使用眼祖细胞来解决视网膜的退化变化，无论是遗传，与年龄相关的或受伤所致。