The aim of this study was to produce dopaminergic neurons in vitro from human embryonic stem (hES) cells following treatment of various neurotrophic factors. MB03 hES cells were induced by retinoic acid (RA) or basic fibroblast growth factor (bFGF), which were further treated with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)-alpha in each induction method during neuron differentiation days. At the final differentiation stage (21 days), all treatment groups revealed very similar levels (bFGF, 76-78%; RA, 70-74%) of mature neurons (anti-NF-200) in two induction methods irrespective of the addition of BDNF or TGF-alpha. In addition, immunostaining and HPLC analyses revealed higher levels of tyrosine hydroxylase (20+/-2.3%) and dopamine (265.5+/-62.8 pg/ml) in the bFGF- and TGF-alpha-treated hES cells than in RA- or BDNF-treated hES cells. These data are one of the first reports on the generation of dopaminergic neurons of hES cells in vitro. Also, our results indicate that TGF-alpha may be successfully used in the bFGF induction protocol and yield higher numbers of dopaminergic neurons from hES cells.
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