The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells


Yousef Mortazavi, Ph.D, 1Fatemeh Sheikhsaran, M.Sc, 1Gholamreza Khamisipour Khamisipour, Ph.D, 2,*Masoud Soleimani, Ph.D, 3Ali Teimuri, Ph.D, 4Somayeh Shokri, M.Sc, 1
Department of Molecular Medicine and Genetics, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
Department of Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences, Bushehr, Iran
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Department of Molecular Medicine and Genetics, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
Department of Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences, Bushehr, Iran
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
*Corresponding Address: P.O.Box: 7518759577 Department of Hematology Faculty of Allied Medicine Bushehr University of Medical Sciences Bushehr Iran Email:r.khamisipour@bpums.ac.ir
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Mortazavi Yousef, Sheikhsaran Fatemeh, Khamisipour Gholamreza Khamisipour, Soleimani Masoud, Teimuri Ali, Shokri Somayeh. The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells . Cell J. 2016; 18(2): 189-196.

Abstract

Objective

Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs) as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF) and assessed their neural lineage genes.

Materials and Methods

In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP) gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293) packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR).

Results

We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF), glial fibrillary acidic protein (GFAP) and Microtubule-associated protein 2 (MAP2) genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF) genes reduced.

Conclusion

Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this obstacle may be the utilization of differentiated neural stem cells.