We have reported a mesencephalic microisland culture method (J Neurosci 1994) and expansion of dopaminergic neurons with microisland culture (SFN Abstr. 1997;23:593). We have also established a protocol for robust expansion and cryopreservation of mesencephalic neural progenitors (MNP) from E14 rat brain. Here, we explored efficient differentiation protocols for the MNPs to dopaminergic neurons. RT-PCR expression analyses of nurr-1, shh, en-1, en-2, smo, ptc, pax-2, pax-8 were carried out in MNP cultures on various extracellular matrix (ECM), i.e. glass, poly-D- lysine (PDL), collagen, laminin, or fibronectin. Nurr-1, an essential transcription factor for dopaminergic neurons, was expressed in MNPs. The strongest expression of nurr-1 was observed in the cells grown on fibronectin. We tested brain derived neurotrophic factor (BDNF), forskolin, dopamine and ascorbic acid in various concentrations and in some combinations. The differentiated MNPs are evaluated at 3, 5, and 7 days in culture by immunostaing fo...