Potential effects and molecular mechanisms of melatonin on the dopaminergic neuronal differentiation of human amniotic fluid mesenchymal stem cells

Melatonin, a highly lipophilic molecule secreted by the pineal gland in the brain, plays a role in various biological functions. Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic- and adipogenic-lineage. However, the effect of melatonin in neurogenic differentiation in amniotic fluid (AF)-MSCs remains to be explored, thus we investigated the potential role of melatonin on dopaminergic neuron differentiation in AF-MSCs. The results showed that various concentrations of melatonin did not affect cell viability and proliferative effects of AF-MSCs. Increases in the levels of neuronal protein marker (βIII-tubulin) and dopaminergic neuronal markers (tyrosine hydroxylase, TH and NURR1), but decrease in the level of glial fibrillary acidic protein (GFAP), were observed in melatonin-treated AF-MSCs. Melatonin induced alteration in differential expression patterns of mesenchymal stem cell antigens by reducing CD29, CD45, CD73, CD90 and CD105, but no changing CD34 expressing cells. AF-MSCs were sequentially induced in neurobasal medium containing standard inducing cocktails (ST: bFGF, SHH, FGF8, BDNF), 1 μM melatonin, or a combination of ST and melatonin. The levels of TUJ1, TH, MAP2, NURR1 and dopamine transporter (DAT) were significantly increased in all treated groups when compared with control-untreated cells. Pretreated AF-MSCs with non-selective MT1/MT2 receptors antagonist, luzindole and selective MT2 receptor antagonist, 4-P-PDOT diminished melatonin-induced increase in dopaminergic neuronal markers and phosphorylated ERK but did not diminish increase in phosphorylated CaMKII by melatonin. Pretreatment with mitogen-activated protein kinase (MEK) inhibitor, PD98059 and CaMKII inhibitor, KN-93 were able to abolish increase in the levels of dopaminergic markers in melatonin-treated AF-MSCs. These findings suggest that melatonin promotes dopaminergic neuronal differentiation of AF-MSCs possibly via the induction in ERK and CaMKII pathways through melatonin receptor-dependent and -independent mechanisms, respectively.