Isolation and manipulation of rostral mesencephalic tegmental progenitor cells from rat

Abstract A technique for isolating mitotic progenitor cells from the embryonic rostral mesencephalon is described. Culture of the progenitor cells in complete media with subsequent staining for neuron specific enolase (NSE) revealed that only 0.6% of the cells were NSE immunoreactive. Coculturing the progenitor cells with established striatal cultures did not result in conversion of any of the cells to the dopamine neuron phenotype (tyrosine hydroxylase immunoreactive (THir) neurons). In contrast, co-culture of progenitor cells with established mesencephalic cultures produced a statistically significant, and in some cases (three of twelve), dramatic increase in the number of THir cells. The THir cells that were present had more pronounced process extension than those observed in mesencephalic mono-cultures. Culturing progenitor cells in transwell baskets that were continuously exposed to media but physically separated from established mesencephalic cultures growing underneath the baskets led to the conversion of only a few progenitor cells to THir neurons in four of twelve transwell studies suggesting that cell-cell contact between progenitor cells and mesencephalic cells is required for the conversion. This co-culture technique also increased the number of THir neurons in the mesencephalic cultures although the increase was not profound enough to explain the increase observed in traditional co-culture. These data suggest that mitotic progenitor cells can be isolated from fetal rat tissue and successfully converted to the dopamine neuron phenotype. Progenitormesencephalic co-culture appears to increase the number of THir cells in both tissue sources mediated in part by soluble factor(s) although cell-cell contact and presumably extracellular matrix proteins play a more substantial role. These progenitor cells may prove useful as a tissue source for transplantation procedures in Parkinson's disease.