Exosome signaling pathway Diabetes Mellitus Type 2 sera increased Exosome Secretion pathway Activity in Human Marrow Mesenchemal Stem Cell in Vitro Jafar Rezaie,*1 Mahdi mazhar Goshchi, 2 Milad Pezeshki, 3 elhameh shokrollahie,1 Malek soleimani Mehranjani, 3 Mohammad Ali Shariatzadeh, 3 Reza Rahbarghazi 1 1.Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2.Student Research Committee, Urmia University of Medical Science, Urmia, Iran. 3.Department of Biology, Faculty of Science, Arak University, Arak, Iran. *Correspondence to: Jafar Rezaie;Stem Cell Research Center,Tabriz University of Medical Sciences, Tabriz, Iran. E-mail: J.rezaie88@gmail.com Background: Exosomes, nano-sized cell-derived membrane vesicles (30-120 nm), secreted by many cell types. Exosomes participate in physiological and pathological processes. studies described adverse effects of diabetes mellitus type 2 (DM2) on stem cell function and biology. In the present study we aimed to study the effect of serum from type 2 diabetic mellitus patients on the exosome secretion pathway in human mesenchymal stem cells in vitro. Exosome signaling pathway Exosome biogenesis Methods: For in vitro assays, hMSCs were classified into two groups as follows; Control: cells ‎received DMEM/LG and 10% sera from healthy subjects; Diabetic groups treated with ‎DMEM/LG and 10% diabetic sera over a period of 7 days. To investigate expression of Alix, ‎CD63, Rab27a, Rab27b, and Rab8b genes, we performed real -time PCR (Rotor-Gene 3000, ‎Corbett Robotics) using SYBR Green PCR Master Mix (Cat no: YT2551, Iran). To further ‎confirmation of CD63 level, protein level of CD63 was examined using western blotting method ‎and densitometry analysis was performed with ImageJ software ver.1.44p (NIH). Furthermore, ‎exosome secretion quantified by acetylcholinesterase (ACE) assay using kit (Cat No: BXC0801; ‎Biorexfars). Choline esterase activity was calculated by following formula; Activity (U/l) = ‎‎∆Abs/min × 65800. Data are expressed as mean±SD. Student’s t-test was used to calculate the ‎significance of differences between groups. Values of P<0.05 were considered statistically ‎significant.‎ Result: Compared to control group, the mRNA level of Alix, CD63, Rab27a, and Rab8b genes in ‎diabetic group was significantly increased (P<0.01). Furthermore, mRNA level of ‎Rab27b was enhanced as compared to control group (P<0.05). Data showed ‎that the protein level of CD63 was significantly enhanced in diabetic group (P<0.01). Acetylcholinesterase activity was significantly increased in diabetic group (P<0.001).‎ Conclusion: The present study provides the evidence of a specific mechanism that stem cell ‎biology was altered through exosome signaling pathway in diabetic mellitus. As a result, we ‎consider diabetic hMSC release more exosomes through upregulation of genes involved in ‎exosome secretion pathway.‎ Reference: 1.‎Rezaie J, Ajezi S, Avci ÇB, Karimipour M, Geranmayeh MH, Nourazarian A, et al. Exosomes and ‎their Application in Biomedical Field: Difficulties and Advantages. Molecular Neurobiology.1-22.‎ 2.‎Record M, Subra C, Silvente-Poirot S, Poirot M. Exosomes as intercellular signalosomes and ‎pharmacological effectors. Biochemical pharmacology. 2011;81(10):1171-82.‎ 3. Yan J, Tie G, Wang S, Messina KE, DiDato S, Guo S, et al. Type 2 diabetes restricts multipotency of mesenchymal stem cells and impairs their capacity to augment postischemic neovascularization in db/db mice. Journal of the American Heart Association. 2012;1(6):e002238 ‎