Directed neural differentiation of primate ES cells

描述（由申请人提供）：小鼠胚胎干（ES）细胞可分化为神经祖细胞的同质群体;不幸的是，人和灵长类动物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细胞有很大不同。灵长类动物和人类ES细胞是高度不稳定的，需要不断监测和费力的技术来维持半均匀的未分化细胞培养。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细胞系分离的新型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。有两个具体目标涉及这一目标。具体目标一：提高多能干细胞起始的稳定性和均一性。我们已经从灵长类动物ES细胞培养物中分离出一种稳定的细胞类型（上皮样），它是碱性磷酸酶阳性的，但在形态上不同于ES细胞。小鼠研究表明，ES细胞与人HepG 2细胞共培养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，并使用一系列标志物（Oct-4、SSEA和TRA标志物、AFP、FGF-5和brachyury）与ES细胞比较均匀性和分化状态。将使用不含饲养层的LIF和HepG 2条件培养基（MEDII）生长因子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2研究。具体目标二：获得均匀的神经干细胞群。灵长类ES细胞和EPL细胞将暴露于一种新的神经诱导因子（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确定使用Sox 1，巢蛋白，HuC/D，MAP 2，NF 200和NeuN标记的神经分化的均匀性。期望的结果是灵长类ES细胞的逐步和统一的定向神经分化，以更好地理解胚胎和胎儿中的神经干细胞形成，并作为有效和安全的神经细胞治疗的模型系统。

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.