Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Bābul (Aug 2017)
Creatine and retinoic acid effects on the induction of autophagy and differentiation of adipose tissue-derived stem cells into GABAergic-like neurons
Abstract
BACKGROUND AND OBJECTIVE: Deficit of inhibitory GABAergic neurons as a part of central nervous system (CNS) pathogenesis was reported in neurodegenerative disorders; and adipose-derived stem cells (ADSCs) were shown to be a feasible option for cell transdifferentiation in neuronal disorders therapy. In this study, the role of autophagy in differentiation was considered by evaluating the expression of the autologous genes of LC3, P62 and GABARAP in fatty stem cells and after the pre-induction stage. METHODS: In this experimental study, under sterile conditions ADSCs were obtained from pararenal fat of two male adult rats. The cells were divided into three groups of fatty stem cells, pre-induction and induction. Following third passages of cell culture, ADSCs were preinduced to neural-like cells (NLCs) using 1mM β-mercaptoethanol (βME) and 10µM retinoic acid (RA), and then NLCs were induced by creatine(Cr) in 1, 5, 10, 20 millimolar for 5 days. In induction stage, the effects of creatine on differentiation were studied by anti nestin and GABA antibody immunostainig. The roles of GABARAP, LC3 and p62 autophagy genes in transdifferentiation were assessed by RT-PCR. FINDINGS: Immunocytochemical studies on ADSCs using CD49d indicated that cultured cells were ADSCs. In the immunochemical studies of the induction stage, at a dose of 10 mM creatinine for 5 days, the expression of the GABA neurons and the nestin-like neuronal cell marker were 58±2% and 56±5%, respectively which had a significant difference with other doses and control group (p<0.05). RT-PCR results indicated that in pre-induced cells autophagy genes of GABARAP, LC3 and p62 were expressed but only P62 gene was expressed in fatty stem cells. CONCLUSION: This study demonstrated that fatty stem cells after induction are able to express nestin and GABA neuronal markers. GABARAP, LC3 and p62 autophagy genes were expressed in pre-induced cells, which indicates the potential role of autophagy in the differentiation of fatty stem cells into nerve-like cells.
