Generation of dopaminergic neurons from human fetal mesencephalic progenitors after co-culture with striatal-conditioned media and exposure to lowered oxygen

Mature dopaminergic neurons were generated from human fetal mesencephalic progenitors by co-culture with human fetal striatal-conditioned media (SCM) and exposure to lowered oxygen. Immunofluorescence analysis showed that cells cultured in differentiation media with embryonic SCM had a greater number of TH-positive neurons than those cultured in traditional media without SCM addition. The TH-positive neurons cultured in lowered oxygen (3-5% O-2) had more branches and those branches were longer than those of neurons cultured under 20% O-2 culture conditions. Furthermore, more TH-positive cells with mature morphology were observed in the culture containing combined SCM addition and lowered oxygen. We also observed higher levels of dopamine release under the combined media conditions than observed under any individual media condition when depolarized by high K+ as determined by high-performance liquid chromatography (HPLC). Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA expressions of sonic hedgehog (SHH), glial cell derived neurotrophic factor (GDNF) and tyrosine hydroxylase (TH), which are linked to the generation of dopaminergic neurons, were increased after co-culture with SCM. Lowered O-2 significantly induced mRNA expression of erythropoietin (EPC) and P27, which affect neuronal differentiation, and was accompanied by upregulation of Nurr1, Pitx3 and dopamine transporter (DAT) mRNAs, which are correlated to the maturation of dopaminergic neurons. This study makes an important contribution to understanding the effects of hypoxia and SCM on the differentiation and maturation of TH-positive neurons derived from human fetal mesencephalic progenitors in vitro. (C) 2009 Elsevier Inc. All rights reserved.