Lentiviral Mediated Overexpression of NGF in Adipose-derived Stem Cells
|Akram Alizadeh1, Mansoureh Soleimani2, Jafar Ai1, Ali Fallah3, Seyed Jafar Hashemian4, Hajar Estiri5, Mohammad Reza Nourani6 and Seyed Nasser Ostad7*|
|1Department of Tissue Engineering, Faculty of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran|
|2Cellular and Molecular Research Center and Department of Anatomical Sciences, Iran University of Medical Sciences, Tehran, Iran|
|3Mede Bioeconomy Company, Tehran, Iran|
|4Diabetes Research Center, Endocrinology and metabolism clinical science institute, Tehran University of Medical Sciences, Tehran, Iran|
|5Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran|
|6Tissue Engineering division, Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran|
|7Department of Tissue Engineering, Faculty of Advanced Technologies in Medicine and Department of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran|
|Corresponding Author :||Seyed Nasser Ostad
Department of Tissue Engineering
Faculty of Advanced Technologies in Medicine and Department of Toxicology & Pharmacology
Faculty of Pharmacy, Tehran University of Medical Sciences
Tel: +98 21 66959105
E-mail: [email protected]
|Received July 24, 2015; Accepted August 25, 2015; Published August 27, 2015|
|Citation:Alizadeh A, Soleimani M, Ai J, Fallah A, Hashemian SJ, et al. (2015) Lentiviral Mediated Overexpression of NGF in Adipose-derived Stem Cells. Clon Transgen 4:142. doi:10.4172/2168-9849.1000142|
|Copyright: ©2015 Alizadeh A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.|
Introduction: Human adipose-derived stem cells (ADSCs) are multipotent stem cells that can self-renew and differentiate into various types of cells such as adipocytes, osteocytes, and neural cells. These stem cells can be isolated by minimally invasive technique in large amounts. ADSCs are a useful resource for cell therapy and regenerative medicine. Nerve growth factor (NGF) is the first neurotrophin factor discovered and characterized for its anti-apoptotic role in neural development. NGF can promote neuronal survival and neurite outgrowth and it also promotes neuron differentiation and migration. Moreover, research showed that NGF could protect axons from inflammatory damage, improve cognitive function in damaged brain models, and function in the prevention and treatment of neurological diseases like Alzheimer’s disease. In this study we use Lentiviral vector-mediated gene transfer technique to deliver NGF gene to ADSCs and overexpress this factor in ADSCs. Method and Materials: ADSCs extracted from human adipose tissue after lipoaspiration by digestion method. ADSCs characterized with Flowcytometry and differentiation assay in adipogenic and osteogenic differential media. The NGF gene was cloned in pCDH-513B-1 (System Bioscience, Mountain View, CA, United States) under a cytomegalovirus (CMV) promoter. Recombinant lentiviruses were produced according to the Prof. Trono lab protocol with some modifications in HEK 293T cells. The spinfection method was used to transduce ADSCs. NGF expression was assayed using fluorescent microscope to trace green fluorescent protein (GFP) marker, RT-PCR and western blotting.
Results: Extracted ADSCs had mesenchymal morphology and differentiated into adipocytes and osteocytes in differentiating media. HEK293T easily transfected with pCDH-513B-1 and over 99% of them expressed GFP so we gathered pseudoviruses from the supernatant. ADSCs transduced with these pseudoviruses transferred NGF and after transduction expressed GFP, as seen under fluorescent microscope. RT-PCR and western blotting verified NGF overexpression in them.
Conclusion: ADSCs can be transduced with pseudo lentiviruses transferring NGF leading to overexpression of NGF.