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Differentiation of Monkey Embryonic Stem Cells into Neural Lineages¹

Division of Reproductive Sciences,³ Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon 97006 Department of Obstetrics/Gynecology and Physiology and Pharmacology,⁴ Oregon Health & Science University, Portland, Oregon 97201 Department of Physiology,⁵ Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (4%) or choline acetyltransferase (13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

¹ Supported by NIH grants RR00163, HD18185, and RR15199 (to D.P.W.).

² Correspondence: Don Wolf, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006. FAX: 503-533-2494; wolfd{at}ohsu.edu