Objectives

1. Epigenetic alteration of Mir-122 and let-7b expression in Adipose Tissue-Derived Mesenchymal Stem Cells by Trichostatin AE. Alizadeh1, N. Zarghami1, M.B. Eslaminejad2, M. Abasi1, A. Barzegar3, S. Jahangir2, S. Hashemzadeh4 1) Department of Medical Biotechnology, School of Advanced Medical Science, Tabriz University of Medical Science, Tabriz, Iran 2) Department of Stem Cell and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 3) Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran4) Department of General Surgery, Faculty of Medicine, Tabriz University of Medical Science, Tabriz, Iran - zarghami@tbzmed.ac.ir Results Objectives Our isolated AMSCs expressed CD44, CD73, and CD90 markers (95-97%). Epithelial-like morphology was observed in AMSCs, surrounded by fibroblastic-cells 20 days after culturing with TSA(Fig 1D). Additionally, among the human miRNAs investigated by the real time PCR, miR-122 was induced and conversely let-7b miRNAs was down-regulated in the TSA-treated group as compared to the control group.(Fig 3) Adipose tissue derived-mesenchymal stem cells (AMSCs) have the potential of differentiation into different lineages. Abundance and non-invasive isolation of AMSCs made them favorite choice for autologus stem cell therapy. MicroRNAs are small, non-coding RNAs with great impact on proliferation and differentiation. Recent study pointed that let-7-family are main microRNAs in exosomes deriving from AMSCs. Also, miR-122 is liver-specific microRNA. Histone-deacetylase inhibitors such as Trichostatin A are epigenetic agents with differentiation-inducing properties. The aim of this study was to investigate the effect of Trichostatin A (TSA) on expression levels of mir-122, let-7a, let-7b, let-7c and let-7d in AMSCs. Figure -2. Analysis of surface markers by flow cytometry Methods Subcutaneous adipose-tissue was obtained with informed consent during surgery from 6 donors in Teaching-Imam Reza Hospital of Tabriz, Iran(Table 1). Isolation of AMSCs was performed using enzyme digestion and established-protocol. AMSCs were stained with combinations of antibodies conjugated with FITC or PE: CD34, MHCII, CD44, CD11b, CD45, and CD90 followed by flow cytometry analysis(Fig 2). Differentiation potential of AMSCs was evaluated by osteogenic and adipogenic induction.(Fig1 A,B) AMSCs were cultured in media containing L-DMEM, EGF, OMS, ITS, bFGF, and various concentrations (0-25µM) of TSA. The colony-forming and MTT assays was performed. The expression of mir-122, let-7a, let-7b, let-7c, and let-7d was investigated by LNA-based Real time PCR in AMSCs, at days 7th, 14th and 21st after TSA treatments. Tabel-1 Characteristics of donors A B Figure-3. Expression levels of miR-122 and Let-7b by Real time PCR References Conclusions D C Our results imply that mir-122 and let-7b might have a possible role in differentiation process. So, these findings may be applicable in production of functional hepatocytes from AMSCs utilizing microRNAs and epigenetic agents. Figure -1. Adipogenic(A), osteogenic(B) differentiation of AMSCs(C), AMSCs after TSA treatments(D)