Effects of nicotine on proliferation and differentiation into cardiovascular progenitor cells in human induced pluripotent stem cells

Yasuhiro Watanabe, Toshiaki Ishizuka, Ayako Ozawa, Hazuki Goshima. National Defense Medical College, Department of Pharmacology, Tokorozawa 359-8513, Japan

[Introduction] Systemic treatment with nicotine was found to increase the number of endothelial progenitor cells and angiogenesis in a mouse model of hind-limb ischemia. In addition, it was confirmed that mouse embryonic stem cells (ESCs) express nicotinic acetylcholine receptor (nAchR). Thus, these findings suggested that nAchR stimulation may alter proliferation and differentiation in pluripotent stem cells. The present study examined whether nicotine enhances proliferation or differentiation into cardiovascular progenitor cells in human induced pluripotent stem (iPS) cells. [Materials and Methods] Human iPS cells were cultured under feeder-free conditions. Twenty-four hours after re-plating, the cells were treated with nicotine (10 nM ~ 10 μM) for 24 h. The DNA synthesis of human iPS cells was examined by BrdU incorporation assay. The treatment with 0.6 nM bone morphogenetic protein-4 (BMP-4) and 0.6 nM vascular endothelial growth factor (VEGF) initiated the differentiation of human iPS cells into cardiovascular progenitor cells. Simultaneously, human iPS cells were stimulated with nicotine (10 nM ~ 10 μM) and cultured for 7 days. The differentiation potential into cardiovascular progenitor cells was evaluated by Flk-1 expression using flow cytometry analysis and western blot analysis. The results of comparisons between the means of multiple groups were analyzed by one-way analysis of variance and the Scheffe multiple comparison test. In all tests, differences were considered statistically significant at P<0.05. [Results] The treatment with nicotine (30 nM) significantly enhanced the DNA synthesis of human iPS cells (n=4, P<0.05). The pretreatment with either dihydro-β-erythroidine (30 nM; a α4 nicotinic acetylcholine receptor (nAchR) antagonist), α-Bungarotoxin (300 nM; a α7 nAchR antagonist), or KN93 (500 nM; a calmodulin kinase II (CaMKII) inhibitor) significantly inhibited nicotine-induced DNA synthesis of the cells (Each comparison test was performed in 4 independent experiments. Each P value was lower than 0.05). Western blot analysis showed that CaMKII phosphorylation in human iPS cells was significantly enhanced by the treatment with 30 nM nicotine for 5 min (n=4, P<0.05). Although the treatment with BMP-4 and VEGF significantly induced Flk-1 expression in human differentiated iPS cells (n=4, P<0.05), the stimulation with nicotine did not affect the Flk-1 expression. [Conclusion] These results suggested that the stimulation with nAchR may enhance the DNA synthesis of human iPS cells via augmentation of CaMKII phosphorylation.