As a result of lacking of effective treatment tools for PD, symptomatic relief and delay in the progression of the disease remain the choice for treatment by using pharmaceutical medications which become not effective in the advanced stages of the disease. So, the effective generation of DAergic neurons in vitro from MSCs will be one of the promising therapeutic approach in treatment of this disease by replacing the lost neurons with healthy and functional ones (3, 23).
In the present study, we isolated MSCs from adipose tissue due to their high proliferation rate that can be differentiated into different cell types such as osteoblasts, chondrocyte, adipocytes, myocytes and neuronal cells as DAergic cells. Also, they could be obtained using minimally invasive methods in comparison with other sources of MSCs, have less ethical issues, provide more homogenous stem cells with less variation in morphological features, and be ideal for the assessment of environmental changes (24). MSCs were enzymatically (collagenase) isolated and cultured from rat adipose tissues. After 5–6 days, the morphology of cells was changed from round into spindle shaped and MSCs with fibroblast shape were demonstrated from other cells by their ability to adhere to the polystyrene tissue culture flask. The cells were sub cultured into several passages at 70–80% confluence. This cell morphology has been confirmed by many other studies which isolated MSCs from adipose tissue of different animal species as rat (20), rabbit (25), dog (26), horse (27–28), and human (29).
After that, AD-MSCs were confirmed by flow cytometry. The results showed that these cells were positive for MSCs surface markers as CD90 and CD105 with 92.5%% and 87.2% respectively and negative for hematopoietic stem cells (HSCs) surface markers as CD 45 and CD34 with 4.9% and 1% respectively. This is in agreement with Hasani et al., (20) who also isolated MSCs from rat adipose tissue. Several studies confirmed that the AD-MSCs showed high positivity for MSC markers as CD73, CD90, and CD105 and integrin markers as CD29 and CD44 but showed negativity for HSCs as CD45 or an endothelial cell marker as CD31 (30–31). There are some confirmed properties for MSCs that are suggested by the international society for cellular therapy including: plastic adherence, a specific set of cell surface markers (CD73, CD90, CD105) and absence of CD14, CD34, CD45, and human leucocyte antigen-DR (32). The isolated cells are agreed with these criteria so these cells are AD-MSCs.
Different studies reported the ability of MSCs derived from different sources to differentiate into DAergic neurons. Khademizadeh et al., (33) who proved that the human AD-MSCs were able to differentiate into DAergic neurons in presence of cocktail of differentiating factors (FGF-2, FGF-8, and Sonic HedgeHog (SHH)) in neurobasal medium and B27 supplement. Also, Urrutia and his colleagues who compared the MSCs capacity from different sources to the neural differentiation in the presence of differentiation factor and retinoic acid in alpha-MEM, they documented that the most applicable source of producing DAergic neurons was adipose tissue (34). Moreover, Thangnipon et al. (35) proved that amniotic fluid MSCs were differentiated into cholinergic neurons by using BMP-9 and N-benzylcinamide.
However, the potential role of melatonin in inducing neuronal differentiation in MSCs has not been reported. Several studies have shown that melatonin is a neuroprotective agent that induces neurogenesis in embryos. Melatonin combined with physical exercise potentiates adult rat hippocampus neurogenesis by enhancing cell survival (36), and administration of melatonin before and during sleep deprivation increases the number of neural precursor cells in adult rats (37). It has been reported that melatonin can induce neuronal differentiation in PC12 cells and induced pluripotent stem cells (38).
Our differentiation results showed that the ability of melatonin to differentiate the AD-MSCs into cells that appeared to be more elongated and with neural-like processes interacting with each other in MSCs + M group. Also, the DN + M group showed completely differentiated DAergic cells after 12 days from induction with melatonin and neurobasal media with essential growth factors. On the other hand, there was no completely differentiation in DN group, which proved that melatonin can induce AD-MSCs into DAergic neurons in presence of neurobasal media with essential growth factors.
These results are also proved by qPCR which indicated that in DN + M group, there was a marked expression of MAP-2 which is a specific marker for neurons and implicated in microtubule synthesis and stabilization by crosslinking with intermediate filaments which involved in stabilizing the dendritic shape during neuron development (39) while, in other treated groups, the expression level of this genes were reduced. Moreover, dopamine ELISA results were in the same line of these results where there was a significant increase in dopamine concentration in DN + M group but it was declined in other groups. All above results are indicated that there was complete differentiated occurred in DN + M group in contrast to other treated groups. Our results are in agree with Phonchai et al., (21) who documented that the melatonin has proliferative effects and differentiation abilities to induce the DAergic neuronal differentiation of human amniotic fluid MSCs.
Autophagy is shown to be crucial in many processes engaged in stem cells differentiation like digestion of redundant organelles and ubiquitinated proteins, protecting cells from DNA damages, preservation of ATP levels by recycling the metabolites of which their biosynthesis is highly energy consuming (40), surviving in nutritional stress conditions and providing nutrients required in activation of stem cells (41).
Different studies documented that the autophagy process is activated during differentiation of embryonic stem cells (ESCs), adult stem cells (ASCs), hematopoietic stem cells (HSCs), MSCs, and neuronal stem cells (NSCs). In 2018, Hasani, and his teamwork documented that there was downregulation for m-TOR gene, the main negatively regulator of autophagy process, during DAergic differentiation derived from human AD-MSCs. Also, other studies showed that there was a decline in mTOR during differentiation of MSCs using beta mercaptoethanol (42) and osteogenic differentiation (43). Currently, there was an evidence for the ability of melatonin to activate the autophagy during neural differentiation through reducing the expression of m-TOR level (44). These findings are consistent with our results which indicated that there was a significantly reduction in m-TOR gene expression level in DN + M group compared to other groups. This mean that the melatonin can activate the autophagy process during differentiation of AD-MSCs into DAergic cells.
Moreover, Autophagy is a highly conserved intracellular catabolic progress, which eliminates damaged organelles and long-lived proteins by isolating these cytoplasmic components in autophagosomes and delivering them to lysosomes for degradation. The results of transmission electron microscope which used to observe the autophagosome formation and analyze both the qualitative and quantitative change of autophagy, showed that there was a markedly increase in formation of autophagosome in DN + M group in contrast to other treated groups. These results are agreed with Liu et al., (45).
The accumulation of misfolded α-Syn may be addressed as the most critical cause of cellular toxicity in PD pathology that finally led to the cell apoptosis (46). It has been recognized that malfunction of the natural destructive process, such as autophagy, may lead to the accumulation of toxic proteins (47). Hence, increasing autophagy may be pursued as a potential and attractive therapeutic approach to reduce PD complications by intracellular degradation of α-Syn (6). As a result of the ability of melatonin to increase the autophagy during neural differentiation (44), it also can reduce the secretion of α-Syn to remove its aggregation. based on gene expression and ELISA results in our study, there was a marked reduction in α-Syn level in DN + M group when compared with other groups.
In conclusion, our study showed that the effective role of melatonin on differentiation of MSCs derived from adipose tissue (AD-MSCs) into DAergic cells and also its effect on how it could modulate or enhance the autophagy process of the differentiated cells in parallel with decreasing the secretion of α-Syn protein from the differentiated cells which may be an alternative way of cell replacement therapy to provide a perfect treatment for Parkinson's disease.