Treatment with deferoxamine increases neurons from neural stem/progenitor cells

Abstract

Neural transplantation is a promising approach for treating neurodegenerative disease. Neural stem/progenitor cells (NPCs) are self-renewing and multipotent and thus are good candidates for donor cells when they have been clearly defined to differentiate into neurons. As neuronal differentiation follows cell cycle exit, we investigated whether neuron production from NPCs is increased by treatment with cell cycle blockers. NPCs from E12.5 rat ventral mesencephalon were cultured as neurospheres in DMEM/F12 medium containing N2 supplements and bFGF. Treatment of NPCs with deferoxamine, a G1/S phase blocker, increased the number of β-tubulin III-positive cells after differentiation, concomitant with increases of MAP2 mRNA and protein, and a decrease of GFAP protein. Further, an increase in β-tubulin III/BrdU double-positive cells and a decrease in GFAP/BrdU double-positive cells were confirmed. In real-time PCR, the expressions of p21 cip1 , p27 kip1 and p57 kip2 mRNAs remained unaltered for 8 h after treatment with deferoxamine but were significantly elevated after 1 day. Deferoxamine specifically enhanced the elevation of p27 kip1 mRNA at 1–2 days and the accumulation of p27 kip1 protein at 3 days, along with the activation of neuroD promoter and the elevation of neuroD mRNA. Transfection of p27 kip1 into NPCs induced activation of neuroD promoter and increase of number of β-tubulin III-positive cells. These data suggest that pretreatment with deferoxamine increases the number of neurons from NPCs related to prolonged p27 kip1 elevation and activation of the neuroD signaling pathway. In this way, regulation of the cell cycle should be a useful first step in engineering NPCs for neural transplantation.