Increase in dopaminergic neurons from mouse embryonic stem cell-derived neural progenitor/stem cells is mediated by hypoxia inducible factor-1alpha.

A reliable method to induce neural progenitor/stem cells (NPCs) into dopaminergic (DAergic) neurons has not yet been established. As well, the mechanism involved remains to be elucidated. To induce DAergic differentiation from NPCs, a cytokine mixture (C-Mix) of interleukin (IL)-1beta, IL-11, leukemia-inhibitory factor (LIF), and glial-derived neurotrophic factor or low oxygen (3.5% O(2): L-Oxy) was used to treat embryonic stem (ES) cell-derived NPCs. Treatment with C-Mix increased the number of tyrosine hydroxylase (TH)-positive cells compared with controls (2.20-fold of control). The C-Mix effect was induced by mainly LIF or IL-1beta treatment. Although L-Oxy caused an increase in TH-positive cells (1.34-fold), the combination of L-Oxy with C-Mix did not show an additive effect. Increases in DA in the medium were shown in the presence of C-Mix, LIF, and L-Oxy by high-performance liquid chromatography. Gene expression patterns of neural markers [tryptophan hydroxylase (TPH), GAD67, GluT1, beta-tubulin III, glial fibrillary acidc protein, and TH] were different in C-Mix and L-Oxy treatments. Because increases in hypoxia-inducible factor (HIF)-1alpha protein were found in both treatments, we investigated the effect of HIF-1alpha on differentiation of NPCs to DAergic neurons. Inhibition of HIF-1alpha by the application of antisense oligodeoxynucleotides (ODNs) to NPCs caused a decrease in TH-positive cells induced by LIF treatment. Gene expressions of TH, GAD67, and GluT1 were decreased, and those of TPH, beta-tubulin III, and S-100beta were increased by treatment with just ODNs, indicating the importance of the endogenous effect of HIF-1alpha on neuronal differentiation. These data suggest that enhanced differentiation into DAergic neurons from ES cell-derived NPCs was induced by C-Mix or L-Oxy mediated by HIF-1alpha.