| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
We have established a protocol for robust expansion and cryopreservation of mesencephalic neural progenitors (MNP) from E12 and E14 rat brain.
Keton study: D-β-hydroxybutyrate (keton) protects mesencephalic dopaminergic neurons (MDNs) in an N-methyl-4-phenylpiridinium (MPP^+) model Parkinson's disease. (Proc Natl Acad Sci USA, 2000)
GAPDH analysis of MPP+ induced apoptosis: We demonstrated MPP^+ cause selective death of MDNs bearing apoptotic morphological changes. We found overexpression and nuclear oligonucleotide for GAPDH (Neuroreport, 2001)
Growth and differentiation of MNPs: effect of serum and extra cellular matrix: MNPs were prepared from 1mm^3 tissue blocks isolating from E14 rat ventral mesencephalon. Dispersed cells were cultured in flasks using an expansion medium with/without serum priming (SP: 24 hours - 2% FCS). Expanded MNPs were collected at 14 days in cultures and were frozen for later experiments. Striatal neural progenitors (SNPs) were also prepared by the same procedu
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res. Thawed MNPs and SNPs were cultured 7 days in EM with/without SP. Growth rates of MNPs were 33 folds in SP cultures and 123 folds in serum-free cultures. On the contrary, those of SNPs were 111 folds and 13.2 folds, respectively. These results indicated that the effects of serum to neural progenitors are different between sources of the progenitors. 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 found a combination of BDNF, dopamine, and forskolin increased the efficiency of differentiation of MNPs to dopaminergic neurons in serum-free cultures on fibronectin as ECM. This culture technique will provide new experimental model of dopaminergic neurons relevant to Parkinson's disease. Less
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