Low oxygen tension in combination with FGF2 expansion promotes dopaminergic differentiation of ventral mesencephalic precursor cells
Pia Jensen1,2, Morten Meyer1, Angélique D. Ducray2 and Hans R. Widmer2
1Department of Anatomy and Neurobiology, University of Southern Denmark, Odense, Denmark, 2Department of Neurosurgery, University of Bern, Bern, Switzerland.
Parkinson’s disease (PD) is a neurodegenerative disorder mainly characterized by a progressive loss of dopaminergic neurons in the substantia nigra. Cell replacement therapy in PD is still at an experimental stage and the limited availability of suitable donor tissue presents a major complication for a successful clinical application of this approach. Effective numerical expansion of fetal dopaminergic precursor cells hence might offer a strategy to minimize this problem. In this context the role of oxygen tension may be of critical importance. While physiological oxygen tension in the CNS is in the range of 1-5% traditionally cell culturing is performed at high non-physiological oxygen tension. In line with this notion, positive effects on both neural precursor cell proliferation and dopaminergic differentiation by culturing cells at a more physiological oxygen tension have been reported.
In the present study, we investigated the influence of FGF2 and FGF8 on expansion of precursor cells cultured at high (20%) and low (3%) oxygen tension. For that purpose tissue from embryonic day 12 rat ventral mesencephalon was mechanically dissociated and cultured for 4 days in serum-free medium in the absence (controls) or the presence of FGF2 or FGF8. After mitogen withdrawal and addition of serum, cells were differentiated for 6 days. We first observed that significantly more cells incorporated BrdU in cultures exposed to FGF2 (51 fold) and FGF8 (13 fold) than in control cultures. Notably, expansion at low oxygen tension significantly increased the number of BrdU expressing cells in both FGF2 (1.7 fold) and FGF8 (3.0 fold) exposed cultures as compared to high oxygen tension. After differentiation the number of beta-III-tubulin expressing cells was significantly higher in low as compared to high oxygen tension cultures. Importantly, numbers of tyrosine hydroxylase-immunoreactive (TH-ir) cells was significantly higher in FGF2-expanded and FGF8-expanded cultures as compared to controls. Low oxygen tension in combination with FGF2 expansion resulted in the most pronounced increase in TH-ir cell numbers which was significantly higher as compared to high oxygen tension, while no such effect was detected for HPLC measured dopamine levels released into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation resulted in both a significantly higher number of TH-ir cells and release of dopamine as compared to all other treatment groups. In contrast, such effects were not observed for FGF8-expanded cultures.
Taken together, the combination of FGF2 treatment at low oxygen and exposed to high oxygen tension during differentiation hence provided the most effective numerical expansion of fetal mesencephalic precursor cells. Our findings demonstrate that low oxygen tension serves a critical role for the induction and differentiation of TH expressing neurons in culture.