INTR10

Authors:

Jensen P 1,2; Meyer M 1; Widmer HR 2

Institutions:

1 Department of Anatomy and Neurobiology, University of Southern Denmark, Odense, Denmark
2 Department of Neurosurgery, University of Bern, Bern, Switzerland.

Title of abstract : Influence of oxygen on expansion and dopaminergic differentiation of ventral mesencephalic precursor cells

Abstract text:

In vitro expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson’s disease, but generating dopaminergic neurons from precursors has proven difficult. Here we investigated influence of FGF2 and FGF8 on expansion and differentiation of such precursors cultured at high (20%) and low (3%) oxygen tension.
Tissue from embryonic day 12 rat ventral mesencephalon was mechanically dissociated and cultured for 4 or 8 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.
Expansion at low oxygen tension significantly increased the number of cells incorporating BrdU in both FGF2 (1.7 fold; p<0.01) and FGF8 (3.0 fold; p<0.001) exposed cultures as compared to high oxygen tension. Notably, only FGF2-expanded cultures contained significantly more tyrosine hydroxylase-immunoreactive (TH-ir) neurons (2.2 fold; p<0.001) following differentiation at low as compared to high oxygen tension. Cultures both expanded and differentiated at low oxygen tension contained more TH-ir neurons than cultures only expanded or differentiated at low oxygen. Interestingly, the low oxygen culturing resulted in reduced levels of released dopamine per TH-ir cell as detected in the culture medium by HPLC analysis. This effect, however, could be reversed following two days at high oxygen tension. Thus, low oxygen tension likely affected synthesis and/or release of dopamine and the maturity of the TH-ir neurons. We conclude that the procedures employed in this study constitute an efficient method for expansion of dopaminergic precursors.

Supported by Danish Center for Stem Cell Research, the Danish Parkinson Association, the Swiss National Science Foundation and as part of the European Science Foundation EUROCORES Programme EuroSTELLS, by funds from the European Commision Sixth Framework Programme.

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