Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro

Hu,Yang; Zhang,Yan; Tian,Kang; Xun,Chong; Wang,Shouyu; Lv,Decheng

doi:10.3892/mmr.2015.4553

Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro

Authors:
- Yang Hu
- Yan Zhang
- Kang Tian
- Chong Xun
- Shouyu Wang
- Decheng Lv
View Affiliations

Affiliations: Department of Orthopedics, The First Hospital Affiliated to Dalian Medical University, Dalian, Liaoning 116011, P.R. China, Institute of Cancer Stem Cells, Cancer Center, Dalian Medical University, Dalian, Liaoning 116044, P.R. China
Published online on: November 11, 2015 https://doi.org/10.3892/mmr.2015.4553
Pages: 49-58
Copyright: © Hu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Recent studies regarding regenerative medicine have focused on bone marrow mesenchymal stem cells (BMSCs), which have the potential to undergo neural differentiation, and may be transfected with specific genes. BMSCs can differentiate into neuron‑like cells in certain neurotropic circumstances in vitro. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are often used to induce neural differentiation in BMSCs in vitro. However, previous studies regarding their combined actions are insufficient. The present study is the first, to the best of our knowledge, to thoroughly assess the enhancement of neural differentiation of BMSCs following transfection with bFGF and NGF. Sprague‑Dawley (SD) rat BMSCs were separated through whole bone marrow adherence, and were then passaged to the third generation. The cells were subsequently divided into five groups: The control group, which consisted of untransfected BMSCs; the plv‑blank‑transfected BMSCs group; the plv‑bFGF‑transfected BMSCs group; the plv‑NGF‑transfected BMSCs group; and the plv‑NGF‑bFGF co‑transfected BMSCs group. Cell neural differentiation was characterized in terms of stem cell molecular expression, and the neuronal morphology and expression of neural‑like molecules was detected in each of the groups. A total of 72 h post‑transfection, the expression levels of neuron‑specific enolase, glial fibrillary acidic protein, and nestin protein, were higher in the co‑transfected group, as compared with the other groups, the expression levels of β‑tubulin III were also increased in the co‑transfected cells, thus suggesting the maturation of differentiated neuron‑like cells. Furthermore, higher neuronal proliferation was observed in the co‑transfected group, as compared with the other groups at passages 2, 4, 6 and 8. Western blotting demonstrated that the transfected groups exhibited a simultaneous increase in phosphorylation of the AKT and extracellular signal‑regulated kinases (ERK) signaling pathway. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of transfected BMSCs.

View Figures

View References

1	Turgeman G, Pittman DD, Müller R, Kurkali BG, Zhou S, Pelled G, Peyser A, Zilberman Y, Moutsatsos IK and Gazit D: Engineered human mesenchymal stem cells: A novel platform for skeletal cell mediated gene therapy. J Gene Med. 3:240–251. 2001. View Article : Google Scholar : PubMed/NCBI
2	Mohammadi R, Azizi S, Delirezh N, Hobbenaghi R, Amini K and Malekkhetabi P: The use of undifferentiated bone marrow stromal cells for sciatic nerve regeneration in rats. Int J Oral Maxillofac Surg. 41:650–656. 2012. View Article : Google Scholar
3	Pereira Lopes FR, Camargo de Moura Campos L, Dias Corrêa J Jr, Balduino A, Lora S, Langone F, Borojevic R and Blanco Martinez AM: Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice. Exp Neurol. 198:457–468. 2006. View Article : Google Scholar : PubMed/NCBI
4	Wu J, Yu W, Chen Y, Su Y, Ding Z, Ren H, Jiang Y and Wang J: Intrastriatal transplantation of GDNF-engineered BMSCs and its neuroprotection in lactacystin-induced Parkinsonian rat model. Neurochem Res. 35:495–502. 2010. View Article : Google Scholar
5	Wen SR, Qi HP, Ren YJ, Liu GJ, Gong FC, Zhong H and Bi S: Expression of δNp73 in hippocampus of APP/PS1 transgenic mice following GFP-BMSCs transplantation. Neurol Res. 33:1109–1114. 2011. View Article : Google Scholar : PubMed/NCBI
6	Ding J, Cheng Y, Gao S and Chen J: Effects of nerve growth factor and Noggin-modified bone marrow stromal cells on stroke in rats. J Neurosci Res. 89:222–230. 2011. View Article : Google Scholar
7	Zhu H1, Yang A, Du J, Li D, Liu M, Ding F, Gu X and Liu Y: Basic fibroblast growth factor is a key factor that induces bone marrow mesenchymal stem cells towards cells with Schwann cell phenotype. Neurosci Lett. 559:82–87. 2014. View Article : Google Scholar
8	Mohammadi R, Azizi S, Delirezh N, Hobbenaghi R and Amini K: Comparison of beneficial effects of undifferentiated cultured bone marrow stromal cells and omental adipose-derived nucleated cell fractions on sciatic nerve regeneration. Muscle Nerve. 43:157–163. 2011. View Article : Google Scholar : PubMed/NCBI
9	Wang S, Yaszemski MJ, Knight AM, Gruetzmacher JA, Windebank AJ and Lu L: Photo-crosslinked poly(epsilon-capro-lactone fumarate) networks for guided peripheral nerve regeneration: Material properties and preliminary biological evaluations. Acta Biomater. 5:1531–1542. 2009. View Article : Google Scholar : PubMed/NCBI
10	Crouzier T, McClendon T, Tosun Z and McFetridge PS: Inverted human umbilical arteries with tunable wall thicknesses for nerve regeneration. J Biomed Mater Res A. 89:818–828. 2009. View Article : Google Scholar
11	Cuevas P, Carceller F, Garcia-Gómez I, Yan M and Dujovny M: Bone marrow stromal cell implantation for peripheral nerve repair. Neurol Res. 26:230–232. 2004. View Article : Google Scholar : PubMed/NCBI
12	Liu WG, Wang ZY and Huang ZS: Bone marrow-derived mesen-chymal stem cells expressing the bFGF transgene promote axon regeneration and functional recovery after spinal cord injury in rats. Neurol Res. 33:686–693. 2011. View Article : Google Scholar : PubMed/NCBI
13	Colafreancesco V and Villoslada P: Targeting NGF pathway for developing neuroprotective therapies for multiple sclerosis and other neurological diseases. Arch Ital Biol. 149:183–192. 2011.
14	Kopen GC, Prockop DJ and Phinney DG: Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA. 96:10711–10716. 1999. View Article : Google Scholar : PubMed/NCBI
15	Li Y, Chen J and Chopp M: Adult bone marrow transplantation after stroke in adult rats. Cell Transplant. 10:31–40. 2001.PubMed/NCBI
16	Li Y, Chopp M, Chen J, Wang L, Gautam SC, XU YX and Zhang Z: Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab. 20:1311–1319. 2000. View Article : Google Scholar : PubMed/NCBI
17	Chopp M, Zhang XH, Li Y, Wang L, Chen J, Lu D, Lu M and Rosenblum M: Spinal cord injury in rat: Treatment with bone marrow stromal cell transplantation. Neuroreport. 11:3001–3005. 2000. View Article : Google Scholar : PubMed/NCBI
18	Maden M: Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat Rev Neurosci. 8:755–765. 2007. View Article : Google Scholar : PubMed/NCBI
19	Bithell A, Finch SE, Hornby MF and Williams BP: Fibroblast growth factor 2 maintains the neurogenic capacity of embryonic neural progenitor cells in vitro but changes their neuronal subtype specification. Stem Cells. 26:1565–1574. 2008. View Article : Google Scholar : PubMed/NCBI
20	Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, Saporta S, Janssen W, Patel N, et al: Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 164:247–256. 2000. View Article : Google Scholar : PubMed/NCBI
21	Mudò G, Bonomo A, Di Liberto V, Frinchi M, Fuxe K and Belluardo N: The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain. J Neural Transm. 116:995–1005. 2009. View Article : Google Scholar : PubMed/NCBI
22	Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, Zhang Y and Mao N: A protocol for isolation and culture of mesen-chymal stem cells from mouse compact bone. Nat Protoc. 5:550–560. 2010. View Article : Google Scholar : PubMed/NCBI
23	Katsetos CD, Legido A, Perentes E and Mörk SJ: Class III beta-tubulin isotype: A key cytoskeletal protein at the crossroads of developmental neurobiology and tumor neuropathology. J Child Neurol. 18:851–867. 2003. View Article : Google Scholar
24	Wang Z, Deng Q, Zhang X and Zhang J: Treatment of injured neurons with bone marrow stem cells cotransfected by hTERT and Ad-BDNF in vitro. J Mol Neurosci. 38:265–272. 2009. View Article : Google Scholar : PubMed/NCBI
25	Fan BS and Lou JY: Enhancement of angiogenic effect of co-transfection human NGF and VEGF genes in rat bone marrow mesenchymal stem cells. Gene. 485:167–171. 2011. View Article : Google Scholar : PubMed/NCBI
26	Tao YX, Xu HW, Zheng QY and FitzGibbon T: Noggin induces human bone marrow-derived mesenchymal stem cells to differentiate into neural and photoreceptor cells. Indian J Exp Biol. 48:444–452. 2010.PubMed/NCBI
27	Delcroix GJ, Curtis KM, Schiller PC and Montero-Menei CN: EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells. Differentiation. 80:213–227. 2010. View Article : Google Scholar : PubMed/NCBI
28	Fan BS and Lou JY: Enhancement of angiogenic effect of co-transfection human NGF and VEGF genes in rat bone marrow mesenchymal stem cells. Gene. 485:167–171. 2011. View Article : Google Scholar : PubMed/NCBI
29	Tobin JE, Xie M, Le TQ, Song SK and Armstrong RC: Reduced axonopathy and enhanced remyelination after chronic demyelination in fibroblast growth factor 2 (Fgf2)-null mice: Differential detection with diffusion tensor imaging. J Neuropathol Exp Neurol. 70:157–165. 2011. View Article : Google Scholar : PubMed/NCBI
30	Fanarraga ML, Avila J and Zabala JC: Expression of unphosphorylated class III beta-tubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci. 11:516–527. 1999. View Article : Google Scholar : PubMed/NCBI
31	Lam HJ, Patel S, Wang A, Chu J and Li S: In vitro regulation of neural differentiation and axon growth by growth factors and bioactive nanofibers. Tissue Eng Part A. 16:2641–2648. 2010. View Article : Google Scholar : PubMed/NCBI