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ABSTRACT: The molecular mechanisms that regulate the proliferation and differentiation of induced pluripotent stem (iPS) cells are of great interest. However, no previous studies have examined whether stimulation with nicotine enhances the proliferation and differentiation of iPS cells. In the present study, the western blot analysis revealed that α4-nAchR and α7-nAchR are expressed in mouse iPS cells. Mouse iPS cells were treated with nicotine for 24 h under feeder-free conditions. Induction of mouse iPS cell differentiation into mesodermal progenitor cell was performed on type IV collagen (Col IV)-coated dishes in differentiation medium. On the other hand, induction of mouse iPS cell differentiation into neural progenitor cells was initiated by embryoid body (EB) formation on ultra-low- attachment dishes. After 4 days of formation, ATRA (1 µM) or nicotine (300 nM) was added to the EB cultures and maintained for additional 4 days and plated onto fibronectin-coated plates. The BrdU incorporation assay showed that treatment with 300 nM nicotine significantly increases DNA synthesis of mouse iPS cells or mouse iPS cell-derived mesodermal progenitor cells. This is significantly inhibited by pretreatment with a α4-nAchR antagonist, a α7-nAchR antagonist, or a CaMKП inhibitor. The differentiation potential of mouse iPS cells into mesodermal progenitor cells or neural progenitor cells was not affected by the nicotine treatment. The present study indicates that stimulation of α4-nAchR and α7-nAchR may lead to a significant increase in the proliferation of mouse iPS cells or mouse iPS cell-derived mesodermal progenitor cells through the CaMKП signaling pathway.
Current Medicinal Chemistry 08/2012; · 4.86 Impact Factor
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ABSTRACT: The cyclic AMP/protein kinase A signaling pathway is thought to be involved in neural differentiation of mesenchymal stem cells. In the present study, we examined the involvement of β-adrenoceptor signaling on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells. Mouse iPS cells were cultured on ultra-low-attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 1 μM) and/or the β-adrenoceptor agonist l-isoproterenol (0.3 or 1 μM) were added to the EB cultures for 4 days, then EBs were plated on gelatin-coated plates and cultured for 7 or 14 days. Subtype-specific antibody staining revealed that mouse iPS cells express β(1)-adrenoceptors predominantly. Although treatment with l-isoproterenol alone did not affect the expression of Nestin (a specific marker for neural progenitor cells), l-isoproterenol significantly enhanced ATRA-induced Nestin expression. Pretreatment of EBs with either atenolol (a selective β(1)-adrenoceptor antagonist) or H89 (a protein kinase A inhibitor) significantly inhibited the l-isoproterenol-enhancement of ATRA-induced Nestin expression. In addition, the l-isoproterenol treatment significantly enhanced ATRA-induced expression of NeuN (a neuron-specific nuclear protein). These findings suggest that β(1)-adrenoceptor stimulation enhances ATRA-induced neural differentiation of mouse iPS cells.
Neuroscience Letters 07/2012; 525(1):60-5. · 2.11 Impact Factor
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ABSTRACT: As the clinical use of induced pluripotent stem (iPS) cells may have the potential to overcome current obstacles in stem cell-based therapy, the molecular mechanisms that regulate the proliferation of iPS cells are of great interest. However, to our knowledge, no previous studies have examined whether stimulation with nicotinic acetylcholine receptor (nAchR) enhances the growth of iPS cells. In the present study, we examined the involvement of nAchR in the proliferation of mouse iPS cells.
We performed immunofluorescence staining to determine whether mouse iPS cells could express nAchRs. Mouse iPS cells were treated with nicotine for 24h under feeder-free conditions in the presence of leukemia inhibitory factor (LIF). The DNA synthesis was examined by the BrdU incorporation assay. Intracellular calcium levels were measured using Fluo-4-acetoxymethyl (a cell-permeable calcium indicator). In addition, we examined the involvement of the CaMKП pathway in nicotine-enhanced proliferation of mouse iPS cells.
The fluorescence images revealed that α(4)-nAchR and α(7)-nAchR are expressed on mouse iPS cells. Treatment of the cells with 300nM nicotine significantly increases DNA synthesis. This is significantly inhibited by pretreatment with antagonists of α(4)-nAchR and α(7)-nAchR or a CaMKП inhibitor. In addition, treatment with nicotine increases the intracellular Ca(2+) level dose-dependently in mouse iPS cells. Treatment with nicotine significantly enhances CaMKП phosphorylation.
The present study indicates that stimulation of α(4)-nAchR and α(7)-nAchR may lead to a significant increase in the rate of mouse iPS cell proliferation through enhancement of the CaMKП signaling pathway.
Life sciences 03/2012; 90(17-18):637-48. · 2.56 Impact Factor
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ABSTRACT: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT(1)R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation of mouse iPS cells with AT(1)R may enhance LIF-induced DNA synthesis, by augmenting the generation of superoxide and activating JAK/STAT3, and that AT(1)R stimulation may enhance Col IV-induced differentiation into mesodermal progenitor cells via p38 MAPK activation.
Biochemical and Biophysical Research Communications 03/2012; 420(1):148-55. · 2.48 Impact Factor
