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Directed neural differentiation of primate ES cells

灵长类ES细胞的定向神经分化

基本信息

批准号：
6711089
负责人：
STEVEN L STICE
金额：
$ 15.88万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-03 至 2006-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6711089
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

项目摘要

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（由申请人提供）：小鼠胚胎干细胞（ES）可以分化为神经祖细胞的同质群体；不幸的是，人类和灵长类动物胚胎干细胞在形态、细胞表面标记、细胞培养技术和分化方面与小鼠胚胎干细胞有很大不同。灵长类动物和人类胚胎干细胞高度不稳定，需要持续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的方法，从胚胎干细胞系中分离出新的、稳定的灵长类动物原始外胚层，并将其统一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。具体目的一：提高起始多能干细胞的稳定性和均匀性。我们已经从灵长类胚胎干细胞培养中分离出一种稳定的细胞类型（上皮样细胞），碱性磷酸酶阳性，但在形态上与胚胎干细胞不同。小鼠研究表明，胚胎干细胞与人HepG2细胞共培养可产生稳定的原始外胚层样细胞（EPL细胞）。将灵长类上皮样细胞和EPL多能细胞进行繁殖，并使用一系列标记（Oct-4、SSEA和TRA标记、AFP、FGF-5和brachyury）与胚胎干细胞进行均匀性和分化状态的比较。使用LIF和HepG2条件培养基（MEDII）生长因子，不加饲喂层，进一步测试细胞类型的增殖潜力和稳定性。这些新型细胞类型中的一种或两种以及在最佳培养条件下增殖的胚胎干细胞将用于特定的两项研究。具体目标二：获得统一的神经干细胞群体。灵长类胚胎干细胞和EPL细胞将暴露于一种新型神经诱导因子（NIF）和其他神经生长因子。初步研究表明，NIF确实能诱导灵长类胚胎干细胞形成假定的神经样细胞类型。使用Sox1、nestin、HuC/D、MAP2、NF200和NeuN标记物进行严格的时空实验，确定神经分化的均匀性。期望的结果是灵长类胚胎干细胞的逐步和统一的定向神经分化，以更好地了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。