Directed neural differentiation of primate ES cells

灵长类ES细胞的定向神经分化

• 批准号: 6784367
• 负责人: STEVEN L STICE
• 金额: $ 4.7万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-03 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6784367
• 关键词: Directed; neural; differentiation; primate; ES

DESCRIPTION (provided by applicant): Mouse embryonic stem (ES) cells can be directed to differentiate into homogenous populations of neural progenitor cells and then to dopaminergic like cells; unfortunately, human and non-human primate ES cells are very different from mouse ES cells in morphology, cell surface markers, cell culture techniques and differentiation. However, previous data including our own indicates that primate and human ES cells are similar and that new or modified procedures for neural differentiation are required to obtain significant quantities of differentiated neural cells. Primate ES cells are highly unstable and require constant monitoring and laborious techniques are used to maintain semi-uniform undifferentiated cell cultures. The long-term goal of this project is to develop innovative methods of establishing novel and stable primate pluripotent stem cells derived from ES cell lines and to uniformly differentiate these cells down neuronal pathways. Two specific aims address this goal. Specific aim one: To improve the stability and uniformity of starting pluripotent stem cells. We have isolated from primate ES cell cultures, a stable cell type (epithelioid) that is alkaline phosphatase positive but morphologically different than ES cells. Also, mouse studies indicate coculture of ES cells with a human HepG2 cells produces a stable primitive ectoderm like cell (EPL cells). Primate epithelioid and EPL pluripotent cells will be propagated and compared with ES cells for uniformity and state of differentiation using a battery of markers (Oct-4, SSEA and TRA markers, AFP, FGF-5 and brachyury). Proliferation potential and stability of the three cell types will be further tested using LIF and HepG2 conditioned medium (MED11) growth factors. One or both of these novel cell types and ES cells proliferated under optimal culture conditions will be used in specific aim two studies. Specific aim two: To obtain a uniform population of neural stem cells capable of differentiating down neural pathways. Primate ES cells and putative EPL cells will be exposed to our novel neural inducing factor (NIF) and other growth factors. Preliminary studies suggest that NIF does induce primate ES cells to form putative neural-like cell types. Neural differentiation and uniformity will be determined using Sox1, nestin, MAP2, TH, and other markers to monitor uniformity and progression to dopaminergic cell types. The desired outcome is a step-wise and Uniform directed neural differentiation of primate ES cells.

描述(由申请人提供)： 小鼠胚胎干细胞可以定向分化为神经前体细胞，然后分化为多巴胺能细胞；不幸的是，人和非人灵长类ES细胞在形态、细胞表面标志物、细胞培养技术和分化方面与小鼠ES细胞有很大的不同。然而，包括我们自己在内的以前的数据表明，灵长类和人类的ES细胞是相似的，需要新的或修改的神经分化程序才能获得大量的分化神经细胞。灵长类胚胎干细胞高度不稳定，需要持续监测，并使用繁琐的技术来维持半均匀的未分化细胞培养。该项目的长期目标是开发创新的方法，建立来自ES细胞系的新颖而稳定的灵长类多能干细胞，并将这些细胞统一分化为神经元通路。有两个具体目标解决了这一目标。具体目标一：提高启动多能干细胞的稳定性和一致性。我们从灵长类ES细胞培养物中分离出一种稳定的细胞类型(上皮样)，碱性磷酸酶阳性，但形态与ES细胞不同。此外，小鼠研究表明，ES细胞与人类HepG2细胞共培养可产生稳定的原始外胚层样细胞(EPL细胞)。灵长类上皮样细胞和EPL多能细胞将被培养，并使用一系列标记物(OCT-4、SSEA和TRA标记物、AFP、成纤维细胞生长因子-5和短臂)与ES细胞进行一致性和分化状态的比较。使用LIF和HepG2条件培养液(MED11)生长因子将进一步测试三种细胞类型的增殖潜力和稳定性。这些新的细胞类型和在最佳培养条件下增殖的ES细胞中的一种或两种将用于特定目的的两项研究。具体目标二：获得能够向下分化神经通路的统一神经干细胞群体。灵长类ES细胞和可能的EPL细胞将暴露于我们新的神经诱导因子(NIF)和其他生长因子。初步研究表明，NIF确实能诱导灵长类ES细胞形成可能的神经样细胞类型。神经分化和一致性将使用Sox1、Nestin、MAP2、TH和其他标记物来确定，以监测一致性和向多巴胺能细胞类型的进展。预期的结果是灵长类胚胎干细胞逐步和统一的定向神经分化。