In vitro generation of glucose-responsive insulin producing cells using lentiviral based pdx-1 gene transduction of mouse (C57BL/6) mesenchymal stem cells Saman Rahmati, Najva Alijani, Mehdi Kadivar ⇑ Biochemistry Department, Pasteur Institute of Iran, Tehran, Iran article info Article history: Received 21 June 2013 Available online 4 July 2013 Keywords: Mesenchymal stem cells pdx-1 Gene Insulin-producing cells Lentiviral vectors abstract The objective of this study was to evaluate the potential of this type of recombinant lentivirus to generate glucose-responsive insulin producing cells in vitro. All steps of cloning were confirmed using restriction digests. After the transduction, mesenchymal stem cells gradually began to change their morphology and showed differentiation into islet like structures. RT-PCR results confirmed the expression of insulin1, insulin2 and pdx-1 in differentiated cells. Dithizone staining of mouse MSCs showed the concentration of glucose in islet like structures. ELISA analysis validated the insulin secretion of islet like structures which in the high-glucose medium (25 mmol/l) was 7.44 fold higher than that secreted in the low-glu- cose medium (5 mmol/l). Our results demonstrated that mouse mesenchymal stem cells can be differen- tiated into effective glucose-responsive insulin producing cells through our new recombinant lentiviral transduction of pdx-1 gene in vitro. This new lentiviral vector could be suggested as an effective candi- date for using in gene therapy of type-1 diabetes. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Type1 diabetes is a chronic disease characterized by severe insulin deficiency and hyperglycemia, due to autoimmune destruc- tion of pancreatic islets of Langerhans [1]. Islet cell replacement has been considered as the potential cure for diabetes over the past 30 years. However, this treatment is limited by a shortage of pan- creas donors and immune rejection against islets [2]. In recent years great interests has been generated in MSCs because of their potential use in regenerative medicine. Recent studies demon- strate that Mesenchymal Stem cells (MSCs) have the ability to dif- ferentiate into functional insulin-producing cells (IPCs) could become a promising source of islet cells [3]. It has been demon- strated that mesenchymal stem cells can be differentiated into insulin-producing cells by exposure to environmental inducers or direct delivery of some specified genes [4]. One of the most impor- tant factors which is involved in pancreas development and tran- scription of insulin gene is pancreatic & duodenal homeobox 1 (pdx-1) transcription factor. During embryonic development, a cascade of transcription factors might be activated to initiate the development of the pancreas [5]. The expression of this factor can switch on the differentiation and development of stem cells to pancreatic buds, and induce further differentiation [6]. Among different gene delivery methods, lentiviral vectors have significant advantages over other vector systems. Lentiviral vectors are at the forefront of gene delivery systems for research and clinical applica- tions. These vectors have the ability to efficiently transduce nondi- viding and dividing cells, to insert large genetic segment in the host chromatin, and to sustain stable long-term transgene expression [7]. In this study, in order to generate functional insulin producing cells, we used a lentiviral vector which had been optimized for transduction of mesenchymal stem cells [8] to achieve long time and efficient expression of pdx-1 gene in mouse (C57BL/6) bone marrow derived mesenchymal stem cells. Using this vector system, we show that pdx-1-expressing mouse (C57BL/6) MSCs can differ- entiate into functional and glucose-responsive insulin producing cells. The function of these cells was confirmed by measuring insu- lin production and release upon different doses of glucose. 2. Materials and methods 2.1. Plasmids pcDNA3.1-pdx-1 containing pdx-1 gene had constructed in our lab previously. pSINTREMSEAPHPGKRTTA2S_M2 (We will briefly mention it pSINTREM) was a kind gift from Dr. Isabelle Barde, Uni- versity of UCL in London, UK [8]. pIRES2-EGFP, pMD2.G, psPAX2 and Escherichia coli TOP 10 were purchased from Invitrogen, USA. pTG19-T vector was purchased from Vivantis, Canada. 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.06.092 ⇑ Corresponding author. Address: Department of Biochemistry, Pasteur Institute of Iran, No. 69, Pasteur Ave, Tehran 13164, Iran. Fax: +98 21 66402770. E-mail address: kadivar@pasteur.ac.ir (M. Kadivar). Biochemical and Biophysical Research Communications 437 (2013) 413–419 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc