In the present study, 60-80% of the ES cell-derived cells from NPCs were positive for the neuronal markers, MAP-2C or NeuN, whereas only a fraction expressed TH (~4%), an anabolic enzyme involved in dopamine production, or ChAT (~13%), which is likewise integral to choline formation. Additional methods to enrich a specific neuronal phenotype in the mouse (i.e., TH-positive neurons) have been reported. For example, high yields of dopaminergic neurons have been obtained after the addition of sonic hedgehog and fibroblast growth factor-8 (FGF8) into ascorbic acid-containing cultures of proliferating ES-derived NPCs. Furthermore, Kawasaki et al. demonstrated that both mouse and monkey ES cells could be directed to differentiate into dopaminergic neurons on coculture with a stromal cell (PA-6 cell) feeder layer; indeed, 35% TH-positive cells were recovered from monkey ES cell cultures. generic Levitra
Additional characterization of functional neuronal phenotypes will help to define the extent of monkey ES cell neuronal differentiation following the current protocol, including gene expression profiles using reverse transcrip-tion-polymerase chain reaction and a determination of physiological behavior. Specifically for dopaminergic neurons from monkey ES cells, this should include a determination of dopamine concentrations in cells and culture medium after induced release by reverse-phase high-performance liquid chromatography and functional capacity using electrophysiological methods. Before use of ES cell-derived dopaminergic neurons in cell-based therapy for Parkinson disease, highly purified cell preparations will be required.
In summary, our results demonstrate that rhesus monkey ES cells are able to generate proliferating NPCs via EB formation and NPC selection and expansion. Furthermore, ES cell-derived NPCs can be stored frozen in highly purified populations before they are subjected to neural differentiation. Enrichment and isolation of monkey ES cell-derived neural stem/progenitor cells, neurons, or glial cells in vitro will facilitate the potential application of stem cell-based therapy in the treatment of neurodegenerative diseases.