DIFFERENTIATION AND PROLIFERATION POTENTIAL OF NEURAL STEM CELLS IN SUBVENTRICULAR ZONE OF MICE IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGTao ¹ , LIWenjie ² , WANGFeng ¹ , ZHANGYu ¹ , XUAiqiang ¹ ,  SHENYixin ¹

1. Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Suzhou Jiangsu, 215000, P. R. China;
2. Department of Anatomy, Medical College, Soochow University;

Corresponding author：

SHENYixin, Email: 972679925@qq.com

Keywords：

Subventricular zone; Neural stem cells; Differentiation; Proliferation; Mouse

DOI：

10.7507/1002-1892.20150164

Video：

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Objective To establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. Methods NSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used. Results The cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017). Conclusion The NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.

Citation： ZHANGTao, LIWenjie, WANGFeng, ZHANGYu, XUAiqiang, SHENYixin. DIFFERENTIATION AND PROLIFERATION POTENTIAL OF NEURAL STEM CELLS IN SUBVENTRICULAR ZONE OF MICE IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(6): 766-771. doi: 10.7507/1002-1892.20150164 Copy

1.	Shindo T, Matsumoto Y, Wang Q, et al. Differences in the neuronal stem cells survival, neuronal differentiation and neurological improvement after transplantation of neural stem cells between mild and severe experimental traumatic brain injury. J Med Invest, 2006, 53(1-2):42-51.
2.	Guo W, Allan AM, Zong R, et al. Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning. Nat Med, 2011, 17(5):559-565.
3.	Aimone JB, Deng W, Gage FH. Resolving new memories:a critical look at the dentate gyrus, adult neurogenesis, and pattern separation. Neuron, 2011, 70(4):589-596.
4.	Brown J, Cooper-Kuhn CM, Kempermann G, et al. Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur J Neurosci, 2003, 17(10):2042-2046.
5.	Pencea V, Luskin MB. Prenatal development of the rodent rostral migratory stream. J Comp Neurol, 2003, 463(4):402-418.
6.	胡莹莹, 段现花, 陈梅, 等. 新生小鼠海马神经干细胞体外培养条件下的增殖与分化. 中国组织工程研究, 2013, 17(1):79-85.
7.	宋平, 蔡强, 陈谦学, 等. 新生大鼠海马神经干细胞的分离培养、分化与鉴定. 中华实验外科杂志, 2013, 30(4):728-730.
8.	冯世庆, 孔晓红, 孙振辉, 等. 大鼠神经干细胞与小鼠雪旺细胞混合培养的研究. 中华实验外科杂志, 2006, 23(1):81-83.
9.	Müller R, Fischer C, Wilmes T, et al. Phosphoinositide-3-kinases p110α and p110β meditae S phase entry in astroglial cells in the marginal zone of rat neocortex. Front Cell Neurosci, 2013, 7:24.
10.	Yu HC, Hung MH, Chen YL, et al. Erlotinib derivative inhibits hepatocellular carcinoma by targeting CIP2A to reactivate protein phosphatase 2A. Cell Death Dis, 2014, 5:e1359.
11.	Ma DK, Bonaguidi MA, Ming GL, et al. Adult neural stem cells in the mammalian central nervous system. Cell Res, 2009, 19(6):672-682.
12.	Zheng LS, Hitoshi S, Kaneko N, et al. Mechanisms for interferon-α-induced depression and neural stem cell dysfunction. Stem Cell Reports, 2014, 3(1):73-84.
13.	Xu J, Wang H, Liang T, et al. Retinoic acid promotes neural conversion of mouse embryonic. Mol Biol Rep, 2012, 39(2):789-795.
14.	Tsai PC, Bake S, Balaraman S, et al. MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells. Biol Open, 2014, 3(8):741-758.
15.	Liu F, Xuan A, Chen Y, et al. Combined effect of nerve growth factor and brainderived neurotrophic factor on neuronal differentiation of neural stem cells and the potential molecular mechanisms. Mol Med Rep, 2014, 10(4):1739-1745.
16.	Li M, Sun L, Luo Y, et al. High-mobility group box 1 released from astrocytes promotes the proliferation of cultured neural stem/progenitor cells. Int J Mol Med, 2014, 34(3):705-714.
17.	Sun T, Wang XJ, Xie SS, et al. A comparison of proliferative capacity and passaging potential between neural stem and progenitor cells in adherent and neurosphere cultures. Int J Dev Neurosci, 2011, 29(7):723-731.
18.	Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell, 1990, 60(4):585-595.
19.	Suzuki S, Nsmiki J, Shibata S, et al. The neural stem/progenitor cell marker nestin is expressed in proliferative endothelial cells, but not in mature vasculature. J Histochem Cytochem, 2010, 58(8):721-730.
20.	Ferri AL, Cavallaro M, Braida D, et al. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development, 2004, 131(15):3805-3819.
21.	Suh H, Consiglio A, Ray J, et al. In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus. Cell Stem Cell, 2007, 1(15):515-528.
22.	Lee KE, Seo J, Shin J, et al. Positive feedback loop between Sox2 and Sox6 inhibits neuronal differentiation in the developing central nervous system. Proc Natl Acad Sci U S A, 2014, 111(7):2794-2799.
23.	Sarkar A, Hochedlinger K. The sox family of transcription factors:versatile regulators of stem and progenitor cell fate. Cell Stem Cell, 2013, 12(1):15-30.
24.	Graham V, Khudyakov J, Ellis P, et al. SOX2 functions to maintain neural progenitor identity. Neuron, 2003, 39(5):749-765.
25.	Elis P, Fagan BM, Magness ST, et al. SOX2, a persistent marker for multipotential neural stem cells derived embryonic stem cells, the embryonic or stem cells, embryo or the adult. Dev Neurosci, 2004, 26(2-4):148-165.

1. Shindo T, Matsumoto Y, Wang Q, et al. Differences in the neuronal stem cells survival, neuronal differentiation and neurological improvement after transplantation of neural stem cells between mild and severe experimental traumatic brain injury. J Med Invest, 2006, 53(1-2):42-51.
2. Guo W, Allan AM, Zong R, et al. Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning. Nat Med, 2011, 17(5):559-565.
3. Aimone JB, Deng W, Gage FH. Resolving new memories:a critical look at the dentate gyrus, adult neurogenesis, and pattern separation. Neuron, 2011, 70(4):589-596.
4. Brown J, Cooper-Kuhn CM, Kempermann G, et al. Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur J Neurosci, 2003, 17(10):2042-2046.
5. Pencea V, Luskin MB. Prenatal development of the rodent rostral migratory stream. J Comp Neurol, 2003, 463(4):402-418.
6. 胡莹莹, 段现花, 陈梅, 等. 新生小鼠海马神经干细胞体外培养条件下的增殖与分化. 中国组织工程研究, 2013, 17(1):79-85.
7. 宋平, 蔡强, 陈谦学, 等. 新生大鼠海马神经干细胞的分离培养、分化与鉴定. 中华实验外科杂志, 2013, 30(4):728-730.
8. 冯世庆, 孔晓红, 孙振辉, 等. 大鼠神经干细胞与小鼠雪旺细胞混合培养的研究. 中华实验外科杂志, 2006, 23(1):81-83.
9. Müller R, Fischer C, Wilmes T, et al. Phosphoinositide-3-kinases p110α and p110β meditae S phase entry in astroglial cells in the marginal zone of rat neocortex. Front Cell Neurosci, 2013, 7:24.
10. Yu HC, Hung MH, Chen YL, et al. Erlotinib derivative inhibits hepatocellular carcinoma by targeting CIP2A to reactivate protein phosphatase 2A. Cell Death Dis, 2014, 5:e1359.
11. Ma DK, Bonaguidi MA, Ming GL, et al. Adult neural stem cells in the mammalian central nervous system. Cell Res, 2009, 19(6):672-682.
12. Zheng LS, Hitoshi S, Kaneko N, et al. Mechanisms for interferon-α-induced depression and neural stem cell dysfunction. Stem Cell Reports, 2014, 3(1):73-84.
13. Xu J, Wang H, Liang T, et al. Retinoic acid promotes neural conversion of mouse embryonic. Mol Biol Rep, 2012, 39(2):789-795.
14. Tsai PC, Bake S, Balaraman S, et al. MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells. Biol Open, 2014, 3(8):741-758.
15. Liu F, Xuan A, Chen Y, et al. Combined effect of nerve growth factor and brainderived neurotrophic factor on neuronal differentiation of neural stem cells and the potential molecular mechanisms. Mol Med Rep, 2014, 10(4):1739-1745.
16. Li M, Sun L, Luo Y, et al. High-mobility group box 1 released from astrocytes promotes the proliferation of cultured neural stem/progenitor cells. Int J Mol Med, 2014, 34(3):705-714.
17. Sun T, Wang XJ, Xie SS, et al. A comparison of proliferative capacity and passaging potential between neural stem and progenitor cells in adherent and neurosphere cultures. Int J Dev Neurosci, 2011, 29(7):723-731.
18. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell, 1990, 60(4):585-595.
19. Suzuki S, Nsmiki J, Shibata S, et al. The neural stem/progenitor cell marker nestin is expressed in proliferative endothelial cells, but not in mature vasculature. J Histochem Cytochem, 2010, 58(8):721-730.
20. Ferri AL, Cavallaro M, Braida D, et al. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development, 2004, 131(15):3805-3819.
21. Suh H, Consiglio A, Ray J, et al. In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus. Cell Stem Cell, 2007, 1(15):515-528.
22. Lee KE, Seo J, Shin J, et al. Positive feedback loop between Sox2 and Sox6 inhibits neuronal differentiation in the developing central nervous system. Proc Natl Acad Sci U S A, 2014, 111(7):2794-2799.
23. Sarkar A, Hochedlinger K. The sox family of transcription factors:versatile regulators of stem and progenitor cell fate. Cell Stem Cell, 2013, 12(1):15-30.
24. Graham V, Khudyakov J, Ellis P, et al. SOX2 functions to maintain neural progenitor identity. Neuron, 2003, 39(5):749-765.
25. Elis P, Fagan BM, Magness ST, et al. SOX2, a persistent marker for multipotential neural stem cells derived embryonic stem cells, the embryonic or stem cells, embryo or the adult. Dev Neurosci, 2004, 26(2-4):148-165.

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DIFFERENTIATION AND PROLIFERATION POTENTIAL OF NEURAL STEM CELLS IN SUBVENTRICULAR ZONE OF MICE IN VITRO

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