J Korean Neurosurg Soc.  2016 Sep;59(5):437-441. 10.3340/jkns.2016.59.5.437.

Neural Growth Factor Stimulates Proliferation of Spinal Cord Derived-Neural Precursor/Stem Cells

• Han Y
• Kim KT

• KMID: 2351710
• DOI: http://doi.org/10.3340/jkns.2016.59.5.437

Abstract

OBJECTIVE
Recently, regenerative therapies have been used in clinical trials (heart, cartilage, skeletal). We don't make use of these treatments to spinal cord injury (SCI) patients yet, but regenerative therapies are rising interest in recent study about SCI. Neural precursor/stem cell (NPSC) proliferation is a significant event in functional recovery of the central nervous system (CNS). However, brain NPSCs and spinal cord NPSCs (SC-NPSCs) have many differences including gene expression and proliferation. The purpose of this study was to investigate the influence of neural growth factor (NGF) on the proliferation of SC-NPSCs.
METHODS
NPSCs (2×10⁴) were suspended in 100 µL of neurobasal medium containing NGF-7S (Sigma-Aldrich) and cultured in a 96-well plate for 12 days. NPSC proliferation was analyzed five times for either concentration of NGF (0.02 and 2 ng/mL). Sixteen rats after SCI were randomly allocated into two groups. In group 1 (SCI-vehicle group, n=8), animals received 1.0 mL of the saline vehicle solution. In group 2 (SCI-NGF group, n=8), the animals received single doses of NGF (Sigma-Aldrich). A dose of 0.02 ng/mL of NGF or normal saline as a vehicle control was intra-thecally injected daily at 24 hour intervals for 7 days. For Immunohistochemistry analysis, rats were sacrificed after one week and the spinal cords were obtained.
RESULTS
The elevation of cell proliferation with 0.02 ng/mL NGF was significant (p<0.05) but was not significant for 2 ng/mL NGF. The optical density was increased in the NGF 0.02 ng/mL group compared to the control group and NGF 2 ng/mL groups. The density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group (p<0.05). High power microscopy revealed that the density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group.
CONCLUSION
SC-NPSC proliferation is an important pathway in the functional recovery of SCI. NGF enhances SC-NPSC proliferation in vitro and in vivo. NGF may be a useful option for treatment of SCI patients pending further studies to verify the clinical applicability.

Keyword

Neural growth factor; Spinal cord neural precursor/stem cell; Spinal cord injury

MeSH Terms

Animals
Brain
Cartilage
Cell Proliferation
Central Nervous System
Gene Expression
Humans
Immunohistochemistry
In Vitro Techniques
Microscopy
Nerve Growth Factor
Nestin
Rats
Spinal Cord Injuries
Spinal Cord*
Nerve Growth Factor

Figure

• 

  Fig. 1 Optical density ratio in the lower concentration of NGF 0.02 group (*) was 108.3 (average of five independent determinations; p=0.0007). NPSC proliferation was significantly increased in the NGF 0.02 group (*) compared to the control group. NGF : nerve growth factor, NPSC : neural precursor/stem cells.

• 

  Fig. 2 The optical density of cell proliferation was increased in the NGF 0.02 ng/mL group (A) compared to the control group (B) and the NGF 2 ng/mL group (C) (original magnification, ×10). NGF : nerve growth factor.

• 

  Fig. 3 The expression of nestin at the cord ventral portion in the SCI-NGF group (*) was significantly increased when compared to the SCI-vehicle group. SCI : spinal cord injury, NGF : nerve growth factor.

• 

  Fig. 4 High power microscopy revealed that the density of nestin in the SCI-NGF group (A) was significantly increased compared to the SCI-vehicle group (B). The black bars denote 50 um. SCI : spinal cord injury, NGF : nerve growth factor.

Reference

1. Ahmed S, Reynolds BA, Weiss S. BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci. 1995; 15:5765–5778. PMID: 7643217.

2. Al-Daccak R, Charron D. Allogenic benefit in stem cell therapy : cardiac repair and regeneration. Tissue Antigens. 2015; 86:155–162. PMID: 26206374.

3. Anderson DK, Hall ED. Pathophysiology of spinal cord trauma. Ann Emerg Med. 1993; 22:987–992. PMID: 8503537.

4. Beattie MS. Inflammation and apoptosis : linked therapeutic targets in spinal cord injury. Trends Mol Med. 2004; 10:580–583. PMID: 15567326.

5. Chen SQ, Cai Q, Shen YY, Cai XY, Lei HY. Combined use of NGF/BDNF/bFGF promotes proliferation and differentiation of neural stem cells in vitro. Int J Dev Neurosci. 2014; 38:74–78. PMID: 25124373.

6. Choi KC, Yoo DS, Cho KS, Huh PW, Kim DS, Park CK. Effect of single growth factor and growth factor combinations on differentiation of neural stem cells. J Korean Neurosurg Soc. 2008; 44:375–381. PMID: 19137082.

7. Duggal N, Schmidt-Kastner R, Hakim AM. Nestin expression in reactive astrocytes following focal cerebral ischemia in rats. Brain Res. 1997; 768:1–9. PMID: 9369294.

8. Ghosh A, Greenberg ME. Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis. Neuron. 1995; 15:89–103. PMID: 7619533.

9. Holmin S, Almqvist P, Lendahl U, Mathiesen T. Adult nestin-expressing subependymal cells differentiate to astrocytes in response to brain injury. Eur J Neurosci. 1997; 9:65–75. PMID: 9042570.

10. Iwai H, Nori S, Nishimura S, Yasuda A, Takano M, Tsuji O, et al. Transplantation of neural stem/progenitor cells at different locations in mice with spinal cord injury. Cell Transplant. 2014; 23:1451–1464. PMID: 23998989.

11. Joannides AJ, Webber DJ, Raineteau O, Kelly C, Irvine KA, Watts C, et al. Environmental signals regulate lineage choice and temporal maturation of neural stem cells from human embryonic stem cells. Brain. 2007; 130(Pt 5):1263–1275. PMID: 17472984.

12. Jordan PM, Ojeda LD, Thonhoff JR, Gao J, Boehning D, Yu Y, et al. Generation of spinal motor neurons from human fetal brain-derived neural stem cells : role of basic fibroblast growth factor. J Neurosci Res. 2009; 87:318–332. PMID: 18803285.

13. Kim KT, Kim HJ, Cho DC, Bae JS, Park SW. Substance P stimulates proliferation of spinal neural stem cells in spinal cord injury via the mitogen-activated protein kinase signaling pathway. Spine J. 2015; 15:2055–2065. PMID: 25921821.

14. Kuo YC, Shih KH, Yang JT. Capillary electrophoresis of bone marrow stromal cells with uptake of heparin-functionalized poly(lactide-co-glycolide) nanoparticles during differentiation towards neurons. Electrophoresis. 2010; 31:315–323. PMID: 20084630.

15. Kwon BK, Fisher CG, Dvorak MF, Tetzlaff W. Strategies to promote neural repair and regeneration after spinal cord injury. Spine (Phila Pa 1976). 2005; 30(17 Suppl):S3–S13.

16. Kwon BK, Tetzlaff W, Grauer JN, Beiner J, Vaccaro AR. Pathophysiology and pharmacologic treatment of acute spinal cord injury. Spine J. 2004; 4:451–464. PMID: 15246307.

17. Li J, Lepski G. Cell transplantation for spinal cord injury : a systematic review. Biomed Res Int. 2013; 2013:786475. PMID: 23484157.

18. Liu F, Xuan A, Chen Y, Zhang J, Xu L, Yan Q, et al. Combined effect of nerve growth factor and brain derived neurotrophic factor on neuronal differentiation of neural stem cells and the potential molecular mechanisms. Mol Med Rep. 2014; 10:1739–1745. PMID: 25051506.

19. Liu RH, Morassutti DJ, Whittemore SR, Sosnowski JS, Magnuson DS. Electrophysiological properties of mitogen-expanded adult rat spinal cord and subventricular zone neural precursor cells. Exp Neurol. 1999; 158:143–154. PMID: 10448426.

20. Lo V, Esquenazi Y, Han MK, Lee K. Critical care management of patients with acute spinal cord injury. J Neurosurg Sci. 2013; 57:281–292. PMID: 24091430.

21. Milligan ED, Hinde JL, Mehmert KK, Maier SF, Watkins LR. A method for increasing the viability of the external portion of lumbar catheters placed in the spinal subarachnoid space of rats. J Neurosci Methods. 1999; 90:81–86. PMID: 10517276.

22. Nakamura M, Okano H. Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells. Cell Res. 2013; 23:70–80. PMID: 23229514.

23. Olson L. NGF and the treatment of Alzheimer's disease. Exp Neurol. 1993; 124:5–15. PMID: 8282080.

24. Olson L, Nordberg A, von Holst H, Bäckman L, Ebendal T, Alafuzoff I, et al. Nerve growth factor affects 11C-nicotine binding, blood flow, EEG, and verbal episodic memory in an Alzheimer patient (case report). J Neural Transm Park Dis Dement Sect. 1992; 4:79–95. PMID: 1540306.

25. Pfenninger CV, Steinhoff C, Hertwig F, Nuber UA. Prospectively isolated CD133/CD24-positive ependymal cells from the adult spinal cord and lateral ventricle wall differ in their long-term in vitro self-renewal and in vivo gene expression. Glia. 2011; 59:68–81. PMID: 21046556.

26. Piao MS, Lee JK, Jang JW, Kim SH, Kim HS. A mouse model of photochemically induced spinal cord injury. J Korean Neurosurg Soc. 2009; 46:479–483. PMID: 20041059.

27. Rosenberg MB, Friedmann T, Robertson RC, Tuszynski M, Wolff JA, Breakefield XO, et al. Grafting genetically modified cells to the damaged brain : restorative effects of NGF expression. Science. 1988; 242:1575–1578. PMID: 3201248.

28. Rowland JW, Hawryluk GW, Kwon B, Fehlings MG. Current status of acute spinal cord injury pathophysiology and emerging therapies : promise on the horizon. Neurosurg Focus. 2008; 25:E2. PMID: 18980476.

29. Saeed H, Ahsan M, Saleem Z, Iqtedar M, Islam M, Danish Z, et al. Mesenchymal stem cells (MSCs) as skeletal therapeutics - an update. J Biomed Sci. 2016; 23:41. PMID: 27084089.

30. Sahin Kaya S, Mahmood A, Li Y, Yavuz E, Chopp M. Expression of nestin after traumatic brain injury in rat brain. Brain Res. 1999; 840:153–157. PMID: 10517963.

31. Tator CH. Update on the pathophysiology and pathology of acute spinal cord injury. Brain Pathol. 1995; 5:407–413. PMID: 8974623.

32. Tederko P, Krasuski M, Kiwerski J, Nyka I, Bialoszewski D. Strategies for neuroprotection following spinal cord injury. Ortop Traumatol Rehabil. 2009; 11:103–110. PMID: 19502667.

33. Tsuji O, Miura K, Fujiyoshi K, Momoshima S, Nakamura M, Okano H. Cell therapy for spinal cord injury by neural stem/progenitor cells derived from iPS/ES cells. Neurotherapeutics. 2011; 8:668–676. PMID: 21909829.

34. Tuszynski MH. Intraparenchymal NGF infusions rescue degenerating cholinergic neurons. Cell Transplant. 2000; 9:629–636. PMID: 11144960.

35. von Euler M, Seiger A, Sundström E. Clip compression injury in the spinal cord : a correlative study of neurological and morphological alterations. Exp Neurol. 1997; 145(2 Pt 1):502–510. PMID: 9217086.

36. Vonk LA, de Windt TS, Slaper-Cortenbach IC, Saris DB. Autologous, allogeneic, induced pluripotent stem cell or a combination stem cell therapy? Where are we headed in cartilage repair and why: a concise review. Stem Cell Res Ther. 2015; 6:94. PMID: 25976213.

37. Wang Q, Matsumoto Y, Shindo T, Miyake K, Shindo A, Kawanishi M, et al. Neural stem cells transplantation in cortex in a mouse model of Alzheimer's disease. J Med Invest. 2006; 53:61–69. PMID: 16537997.

38. Williams LR. Hypophagia is induced by intracerebroventricular administration of nerve growth factor. Exp Neurol. 1991; 113:31–37. PMID: 2044677.

39. Winkler J, Ramirez GA, Kuhn HG, Peterson DA, Day-Lollini PA, Stewart GR, et al. Reversible Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites after intraventricular administration of nerve growth factor. Ann Neurol. 1997; 41:82–93. PMID: 9005869.

40. Zhang L, Jiang H, Hu Z. Concentration-dependent effect of nerve growth factor on cell fate determination of neural progenitors. Stem Cells Dev. 2011; 20:1723–1731. PMID: 21219132.