Characterization of Ocular Neural Stem Cells

建立细胞多样性对大脑的发育、结构和功能至关重要。 这种多样性的基础是一群具有干细胞特性的神经祖细胞，它们产生区域特异性神经元和神经胶质。因此，了解神经祖细胞的分子和细胞生物学是研究包括视网膜在内的大脑特定区域发育机制的关键。这些发育机制的概括可能为治疗由于特定神经元群体死亡而引起的功能障碍开辟新的途径，如视网膜色素变性和黄斑变性的情况。 考虑到这些目标，我们已经提出了表征神经祖细胞分离的衍生物的眼神经上皮，视网膜和睫状体，其增殖能力，自我更新，维护，并在体内和体外的发展潜力，在以下特定的目标。 首先，眼祖细胞的增殖和分化潜能将在其对有丝分裂原、FGF 2和EGF的反应性的背景下表征。 我们将使用流式细胞术来检查它们的增殖和存活，有限稀释分析来估计它们形成克隆的频率，细胞类型特异性标记的免疫细胞化学和RT-PCR分析来测试它们的多能性，以及克隆密度培养来确定它们的自我更新能力。 此外，我们将确定有丝分裂原受体在眼祖细胞不同亚群中的分布，以了解它们的反应性和关系的基础。其次，将评估Notch信号传导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对Notch信号传导的功能获得和功能丧失扰动的反应。 第三，将在促进分化的条件下体外测定眼祖细胞的分化潜能。我们将使用免疫细胞化学和电生理分析，以评估他们的能力，获得特定的表型，在响应生长因子和神经营养因子，并在共培养条件。 第四，将评估眼祖细胞在体内产生位点特异性细胞的潜力。 我们将使用同种和异位移植和体内激活祖细胞对损伤的反应来实现这一目标。 实现这些目标将提供关于视网膜发育的潜在机制的有价值的信息，并将允许在干细胞治疗中使用眼祖细胞来解决视网膜中的退行性变化，无论是遗传的、年龄相关的还是由于损伤。

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.