Carbachol, along with calcium, indicates new strategy in neural differentiation of human adipose tissue-derived mesenchymal stem cells in vitro

Abstract

Over the past few years, stem cells have represented a promising treatment in neurological disorders due to the well-defined characteristics of their capability to proliferate and differentiate into any cell type, both invitro and invivo. Additionally, previous studies have shown that calcium signaling modulates the proliferation and differentiation of neural progenitor cells. The present study investigated the effect of carbachol (CCh), a cholinergic agonist activating acetylcholine receptors, with and without calcium, on the neural differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) in neural media, including forskolin and 3-isobutyl-1-methyl-xanthine and retinoic acid. For this purpose, first, the MTT assay and acridine orange staining were studied to obtain the optimal concentration of CCh. Next, the differentiation tests, such as cellular calcium assay as well as evaluation of qualitative and quantitative expression of neuronal index markers through immunofluorescence staining and gene expression analysis, respectively, were performed on days 7 and 14 of the differentiation period. According to the results, CCh at 1μM concentration had no cytotoxicity on hADSCs and also induced cell proliferation. Furthermore, CCh with and without calcium increased the expression of neural-specific genes (NSE, MAP2, β-III-tubulin, and MAPK3) and proteins (γ-enolase, MAP2, and β-III-tubulin) as well as the amount of calcium in differentiated hADSCs at 7 and 14 days after induction. In conclusion, the findings suggest that CCh acts as an influential therapeutic factor in the field of neural regenerative medicine and research.