Directed neural differentiation of primate ES cells

灵长类ES细胞的定向神经分化

• 批准号: 6620006
• 负责人: STEVEN L STICE
• 金额: $ 15.81万
• 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/6620006
• 关键词: Primates; alkaline phosphatase; cell differentiation; cell line; cell morphology; cell proliferation; ectoderm; embryonic stem cell; immunocytochemistry; method development; nerve stem cell; tissue /cell culture

DESCRIPTION (provided by applicant): Mouse embryonic stem (ES) cells can differentiate into homogenous populations of neural progenitor cells; unfortunately, human and primate ES cells are very different from mouse ES cells in morphology, cell surface markers, cell culture techniques and differentiation. Primate and human ES cells are highly unstable and require constant monitoring and laborious techniques are used to maintain semi-uniform undifferentiated cell cultures. This instability leads to uncontrolled differentiation. The long-term goal of this project is to develop innovative methods of establishing novel and stable primate primitive ectoderm divided from ES cell lines and to uniformly differentiate these to neural stem cells equivalent to neural tube cells. 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. 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 cell types will be further tested using LIF and HepG2 conditioned medium (MEDII) growth factors without feeder layers. 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. Primate ES cells and EPL cells will be exposed to a novel neural inducing factor (NIF) and other neural growth factors. Preliminary studies suggest that NIF does induce primate ES cells to form putative neural-like cell types. Rigorous spatial and temporal experiments will determine uniformity of neural differentiation using Sox1, nestin, HuC/D, MAP2, NF200 and NeuN markers. The desired outcome is a step-wise and uniform directed neural differentiation of primate ES cells to better understand neural stem cell formation in the embryo and fetus and to serve as a model system for effective and safe neural cell therapies.

描述（申请人提供）：小鼠胚胎干（ES）细胞可以分化成同质的神经祖细胞群；不幸的是，人类和灵长类动物 ES 细胞在形态、细胞表面标记、细胞培养技术和分化方面与小鼠 ES 细胞有很大不同。灵长类动物和人类 ES 细胞高度不稳定，需要持续监测，并且需要使用费力的技术来维持半均匀的未分化细胞培养物。这种不稳定性导致不受控制的分化。该项目的长期目标是开发创新方法，建立从ES细胞系中分离出来的新型且稳定的灵长类原始外胚层，并将其均匀分化为相当于神经管细胞的神经干细胞。有两个具体目标可以实现这一目标。具体目标一：提高起始多能干细胞的稳定性和均一性。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（上皮样细胞），呈碱性磷酸酶阳性，但形态与 ES 细胞不同。小鼠研究表明，ES 细胞与人 HepG2 细胞共培养可产生稳定的原始外胚层样细胞（EPL 细胞）。将繁殖灵长类上皮样细胞和 EPL 多能细胞，并使用一系列标记物（Oct-4、SSEA 和 TRA 标记物、AFP、FGF-5 和 brachyury）与 ES 细胞进行比较，以了解分化的均匀性和状态。将使用 LIF 和 HepG2 条件培养基 (MEDII) 生长因子（无饲养层）进一步测试细胞类型的增殖潜力和稳定性。这些新型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标的两项研究。具体目标二：获得均匀的神经干细胞群。灵长类动物 ES 细胞和 EPL 细胞将接触新型神经诱导因子 (NIF) 和其他神经生长因子。初步研究表明，NIF 确实诱导灵长类动物 ES 细胞形成假定的神经样细胞类型。严格的空间和时间实验将使用 Sox1、nestin、HuC/D、MAP2、NF200 和 NeuN 标记来确定神经分化的均匀性。期望的结果是灵长类动物 ES 细胞的逐步、统一的定向神经分化，以更好地了解胚胎和胎儿中神经干细胞的形成，并作为有效和安全的神经细胞治疗的模型系统。