Directed neural differentiation of primate ES cells

University Of Georgia (Uga), Athens GA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] 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.