Directed neural differentiation of primate ES cells

University Of Georgia (Uga), Athens GA

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Abstract

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.