Melatonin effects on Neural-like Phenotype and Melatonin Receptor Expression in human Adipose-derived Mesenchymal Stem Cells

OBJECTIVE: Several studies show that a neural-like differentiation can be induced in Adipose-derived Stem Cells (ASCs) by various strategies. In a previous study, we showed that expression of neural markers was increased when ASCs were cultured in a conditioned medium (CM) from glial cells, such as Olfactory Ensheathing Cells (OEC-CM) or Schwann Cells. As Melatonin (Mel) plays an important role in survival and differentiation of Neural Stem Cells, in the present investigation the effect of this hormone was tested on ASC neural differentiation. The combination of Mel and OEC-CM treatment was also evaluated. MATERIALS AND METHODS: ASCs were isolated from human lipoaspirate. After their stem cell characterization, four groups of cells were cultured: the first served as control, being ASCs cultured in the basal medium; in the second group, Mel was added (1 ?M) to the basal medium; the third group was cultured in OEC-CM; in the last group (OEC-CM-Mel), Mel (1 ?M) was added to the conditioned medium. Each group of cells was processed for immunofluorescence at day 1, 3 and 7 of growth. Antibodies for Neuron Specific Enolase (NSE), Microtubule Associate Protein 2 (MAP2), and for Melatonin Receptor 1 and 2 (MTr1 and MTr2) were tested. RESULTS: A neural-like differentiation was confirmed after OEC-CM treatment. In fact, NSE expression, virtually absent in control cultures, was considerably increased in these conditions. In Mel-treated cultures, NSE increases were also observed, although to a lesser extent. As it was expectable, more evident immunostaining was induced in OEC-CM-Mel group. A similar trend was also found for MAP2 expression. Marked increases were particularly evident for MTr1 immunostaining, whereas MTr2 expression was only weakly increased by any of these treatments. The concentration of Mel at 1 ?M was chosen since it was the lowest to induce effects, without compromising cell growth. CONCLUSION: The present results show that Mel can favor a neural differentiation of ASCs, particularly in association with other induction strategies. Interestingly, it was found that MTr1 expression, already present in native ASCs, was considerably increased after Mel treatment. Since the concentration of Mel used in the present investigation did not affect cell viability and proliferation, it may be considered a suitable tool to be further developed. Future investigations will elucidate the molecular mechanisms underlying the observed effects.