Directed neural differentiation of primate ES cells

描述（由申请人提供）： 小鼠胚胎干细胞（ES细胞）可以定向分化为神经祖细胞的同质群体，然后分化为多巴胺能样细胞;不幸的是，人和非人灵长类ES细胞在形态学、细胞表面标志物、细胞培养技术和分化方面与小鼠ES细胞非常不同。然而，以前的数据，包括我们自己的数据表明，灵长类动物和人类ES细胞是相似的，需要新的或修改的神经分化程序，以获得大量的分化的神经细胞。灵长类动物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。该项目的长期目标是开发创新的方法，建立新的和稳定的灵长类多能干细胞来源于ES细胞系，并均匀分化这些细胞的神经通路。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。此外，小鼠研究表明ES细胞与人HepG 2细胞的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LIF和HepG 2条件培养基（MED 11）生长因子进一步测试三种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：获得能够分化神经通路的均匀神经干细胞群。灵长类ES细胞和假定的EPL细胞将暴露于我们的新的神经诱导因子（NIF）和其他生长因子。初步研究表明，NIF确实诱导灵长类动物ES细胞形成假定的神经样细胞类型。将使用Sox 1、巢蛋白、MAP 2、TH和其他标志物测定神经分化和均匀性，以监测均匀性和向多巴胺能细胞类型的进展。期望的结果是灵长类ES细胞的逐步和均匀的定向神经分化。

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.