DIFFERENTIATION OF C17.2 NEURAL STEM CELLS INTO NEURAL CELLS INDUCED BY SERUM-FREE CONDITIONED MEDIUM OF OLFACTORY ENSHEATHING CELLS AND CELL VIABILITY DETECTION OF DIFFERENTIATED CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGLei ^1,2 , DUANDa ¹ , ZHAOZhenyu ¹ , TENGXiaohua ¹ , LIUBo ¹ , GELite ^1,2 ,  LUMing ^1,2

1. Department of Neurosurgery, the 163rd Hospital of Chinese PLA, the Second Affiliated Hospital of Hunan Normal University, Changsha Hunan, 410003, P. R. China;
2. Medical College of Hunan Normal University;

Corresponding author：

LUMing, Email: lumingcs163@163.com

Keywords：

Neural stem cells; Olfactory ensheathing cells; Induced differentiation; Cell viability; Mice

DOI：

10.7507/1002-1892.20140140

Video：

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Objective To study the possibility of the C17.2 neural stem cells (NSCs) differentiating into neural cells induced by serum-free condition medium of olfactory ensheathing cells (OECs) and to detect the cell viability of the differentiated cells. Methods OECs were isloated and cultured from the olfactory bulbs of 3-day-old postnatal mouse to prepare serum-free condition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 15% FBS and the cell fusion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group, and non-induced C17.2 NSCs served as the blank control group. The growth condition of cells was observed with inverted microscope. After 5 days, the immunofluorescence staining[microtubule-associated protein 2 (MAP-2) and β-tubulin-Ⅲ] and Western blot (Nestin, β-tubulin-Ⅲ, and MAP-2) were carried out to identify the neural cells derived from NSCs. The cell viabilities were measured by MTT assay and the quantity of lactate dehydrogenase (LDH) release in the medium. Results In the experimental group, the C17.2 NSCs bodies began to contract at 24 hours after induction, and the differentiated cells increased obviously with long synapse at 3 days after induction; in the control group, the cell morphology showed no obvious change at 24 hours, cell body shrinkage, condensation of nuclear chromatin, and lysis were observed at 3 days. The immunofluorescence staining showed that β-tubulin-Ⅲ and MAP-2 of C17.2 NSCs were positive at 5 days after induction, and Western blot suggested that the expression of Nestin protein declined significantly and the expressions of β-tubulin-Ⅲ and MAP-2 protein were increased in the experimental group, showing significant differences when compared with those in the control group and blank control group (P<0.05). The LDH release and the cell viability were 130.60%±6.86% and 62.20%±3.82% in the experimental group, and were 178.20%±5.44% and 18.00%±3.83% in the control group respectively, showing significant differences between 2 groups (P<0.05). The LDH release and the cell viability of experimental group and control group were significantly lower than those of blank control group (100%) (P<0.05). Conclusion Neurotrophic factors from OECs play an important role in inducing C17.2 NSCs differentiation into neural cells and keeping the viability of differentiated cells after induction.

Citation： WANGLei, DUANDa, ZHAOZhenyu, TENGXiaohua, LIUBo, GELite, LUMing. DIFFERENTIATION OF C17.2 NEURAL STEM CELLS INTO NEURAL CELLS INDUCED BY SERUM-FREE CONDITIONED MEDIUM OF OLFACTORY ENSHEATHING CELLS AND CELL VIABILITY DETECTION OF DIFFERENTIATED CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(5): 633-638. doi: 10.7507/1002-1892.20140140 Copy

1.	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.
2.	Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature, 2002, 417(6884):39-44.
3.	Chen BY, Wang X, Wang ZY, et al. Brain-derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells, possibly by triggering the Wnt/β-catenin signaling pathway. J Neurosci Res, 2013, 91(1):30-41.
4.	Plane JM, Andjelkovic AV, Keep RF, et al. Intact and injured endothelial cells differentially modulate postnatal murine forebrain neural stem cells. Neurobiol Dis, 2010, 37(1):218-227.
5.	阳运康, 蒋电明, 阳明明. 角质细胞生长因子促进人胚神经干细胞增殖与分化的实验研究. 中国修复重建外科杂志, 2013, 27(10):1246-1251.
6.	汪雷, 宋跃明, 袁海峰, 等. NEP1-40基因修饰对神经干细胞移植后存活和分化的影响. 中国修复重建外科杂志, 2013, 27(11):1368-1374.
7.	Rubio MP, Muñoz-Quiles C, Ramón-Cueto A. Adult olfactory bulbs from primates provide reliable ensheathing glia for cell therapy. Glia, 2008, 56(5):539-551.
8.	Deumens R, Koopmans GC, Lemmens M, et al. Neurite outgrowth promoting effects of enriched and mixed OEC/ONF cultures. Neurosci Lett, 2006, 397(1-2):20-24.
9.	Tan CW, Ng MH, Ohnmar H, et al. Sciatic nerve repair with tissue engineered nerve:Olfactory ensheathing cells seeded poly (lactic-co-glygolic acid) conduit in an animal model. Indian J Orthop, 2013, 47(6):547-552.
10.	Luo Y, Zou Y, Yang L, et al. Transplantation of NSCs with OECs alleviates neuropathic pain associated with NGF downregulation in rats following spinal cord injury. Neuroscience Letters, 2013, 549:103-108.
11.	陶晓宇, 曾瑜, 荣明强, 等. 嗅鞘细胞诱导神经干细胞分化后神经元电生理特性的研究. 中国微侵袭神经外科杂志, 2010, 15(8):361-365.
12.	陶晓宇, 曾瑜, 段答, 等. OECs无血清上清液体外诱导UB-MSCs向神经细胞分化. 湖南师范大学学报:医学版, 2010, 7(4):1-5.
13.	Snyder EY, Deiteher DL, Walsh C, et al. Multipotent neural cell line can engraft and participate in development of mouse cerebellum. Cell, 1992, 68(1):33-51.
14.	Einstein O, Ben-Hur T. The changing face of neural stem cell therapy in neurologic diseases. Arch Neurol, 2008, 65(4):452-456.
15.	Schwartz PH, Brick DJ, Stover AE, et al. Differentiation of neural lineage cells from human pluripotent stem cells. Methods, 2008, 45(2):142-158.
16.	Choi KC, Yoo DS, Cho KS, et al. Effect of single growth factor and growth factor combinations on differentiation of neural stem cells. J Korean Neurosurg Soc, 2008, 44(6):375-381.
17.	Lu H, Li M, Song T, et al. Retrovirus delivered neurotrophin-3 promotes survival, proliferation and neuronal differentiation of human fetal neural stem cells in vitro. Brain Research Bulletin, 2008, 77(4):158-164.
18.	Pahnke J, Mix E, Knoblich R, et al. Over expression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells. ExP Cell Res, 2004, 297(2):484-494.
19.	Tang ZP, Xie XW, Shi YH, et al. Combined transplantation of neural stem cells and olfactory ensheathing cells improves the motor function of rats with intracerebral hemorrhage. Biomed Environ Sci, 2010, 23(1):62-67.
20.	Ao Q, Wang AJ, Chen GQ, et al. Combined transplantation of neural stem cells and olfactory ensheathing cells for the repair of spinal cord injuries. Med Hypotheses, 2007, 69(6):1234-1237.
21.	吴丹萍, 柳林, 曹莉. 嗅鞘细胞与睫状神经营养因子协同促进视神经损伤大鼠视神经修复的研究. 中华眼科杂志, 2013, 49(11):1020-1028.
22.	Zhao RR, Fawcett JW. Combination treatment with chondroitinase ABC in spinal cord injury-breaking the barrier. Neurosci Bull, 2013, 29(4):477-483.
23.	Liu Y, Teng X, Yang X, et al. Shotgun proteomics and network analysis between plasma membrane and extracellular matrix proteins from rat olfactory ensheathing cells. Cell Transplant, 2010, 19(2):133-146.
24.	Lundqvist J, Andaloussi-Lilja J, Svensson C, et al. Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests. Toxicology in Vitro, 2013, 27(5):1565-1569.
25.	Cromer Berman SM, Kshitiz, Wang CJ, et al. Cell motility of neural stem cells is reduced after SPIO-labeling, which is mitigated after exocytosis. Mag Reson Med, 2013, 69(1):255-262.

1. 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.
2. Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature, 2002, 417(6884):39-44.
3. Chen BY, Wang X, Wang ZY, et al. Brain-derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells, possibly by triggering the Wnt/β-catenin signaling pathway. J Neurosci Res, 2013, 91(1):30-41.
4. Plane JM, Andjelkovic AV, Keep RF, et al. Intact and injured endothelial cells differentially modulate postnatal murine forebrain neural stem cells. Neurobiol Dis, 2010, 37(1):218-227.
5. 阳运康, 蒋电明, 阳明明. 角质细胞生长因子促进人胚神经干细胞增殖与分化的实验研究. 中国修复重建外科杂志, 2013, 27(10):1246-1251.
6. 汪雷, 宋跃明, 袁海峰, 等. NEP1-40基因修饰对神经干细胞移植后存活和分化的影响. 中国修复重建外科杂志, 2013, 27(11):1368-1374.
7. Rubio MP, Muñoz-Quiles C, Ramón-Cueto A. Adult olfactory bulbs from primates provide reliable ensheathing glia for cell therapy. Glia, 2008, 56(5):539-551.
8. Deumens R, Koopmans GC, Lemmens M, et al. Neurite outgrowth promoting effects of enriched and mixed OEC/ONF cultures. Neurosci Lett, 2006, 397(1-2):20-24.
9. Tan CW, Ng MH, Ohnmar H, et al. Sciatic nerve repair with tissue engineered nerve:Olfactory ensheathing cells seeded poly (lactic-co-glygolic acid) conduit in an animal model. Indian J Orthop, 2013, 47(6):547-552.
10. Luo Y, Zou Y, Yang L, et al. Transplantation of NSCs with OECs alleviates neuropathic pain associated with NGF downregulation in rats following spinal cord injury. Neuroscience Letters, 2013, 549:103-108.
11. 陶晓宇, 曾瑜, 荣明强, 等. 嗅鞘细胞诱导神经干细胞分化后神经元电生理特性的研究. 中国微侵袭神经外科杂志, 2010, 15(8):361-365.
12. 陶晓宇, 曾瑜, 段答, 等. OECs无血清上清液体外诱导UB-MSCs向神经细胞分化. 湖南师范大学学报:医学版, 2010, 7(4):1-5.
13. Snyder EY, Deiteher DL, Walsh C, et al. Multipotent neural cell line can engraft and participate in development of mouse cerebellum. Cell, 1992, 68(1):33-51.
14. Einstein O, Ben-Hur T. The changing face of neural stem cell therapy in neurologic diseases. Arch Neurol, 2008, 65(4):452-456.
15. Schwartz PH, Brick DJ, Stover AE, et al. Differentiation of neural lineage cells from human pluripotent stem cells. Methods, 2008, 45(2):142-158.
16. Choi KC, Yoo DS, Cho KS, et al. Effect of single growth factor and growth factor combinations on differentiation of neural stem cells. J Korean Neurosurg Soc, 2008, 44(6):375-381.
17. Lu H, Li M, Song T, et al. Retrovirus delivered neurotrophin-3 promotes survival, proliferation and neuronal differentiation of human fetal neural stem cells in vitro. Brain Research Bulletin, 2008, 77(4):158-164.
18. Pahnke J, Mix E, Knoblich R, et al. Over expression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells. ExP Cell Res, 2004, 297(2):484-494.
19. Tang ZP, Xie XW, Shi YH, et al. Combined transplantation of neural stem cells and olfactory ensheathing cells improves the motor function of rats with intracerebral hemorrhage. Biomed Environ Sci, 2010, 23(1):62-67.
20. Ao Q, Wang AJ, Chen GQ, et al. Combined transplantation of neural stem cells and olfactory ensheathing cells for the repair of spinal cord injuries. Med Hypotheses, 2007, 69(6):1234-1237.
21. 吴丹萍, 柳林, 曹莉. 嗅鞘细胞与睫状神经营养因子协同促进视神经损伤大鼠视神经修复的研究. 中华眼科杂志, 2013, 49(11):1020-1028.
22. Zhao RR, Fawcett JW. Combination treatment with chondroitinase ABC in spinal cord injury-breaking the barrier. Neurosci Bull, 2013, 29(4):477-483.
23. Liu Y, Teng X, Yang X, et al. Shotgun proteomics and network analysis between plasma membrane and extracellular matrix proteins from rat olfactory ensheathing cells. Cell Transplant, 2010, 19(2):133-146.
24. Lundqvist J, Andaloussi-Lilja J, Svensson C, et al. Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests. Toxicology in Vitro, 2013, 27(5):1565-1569.
25. Cromer Berman SM, Kshitiz, Wang CJ, et al. Cell motility of neural stem cells is reduced after SPIO-labeling, which is mitigated after exocytosis. Mag Reson Med, 2013, 69(1):255-262.

Chinese Journal of Reparative and Reconstructive Surgery

DIFFERENTIATION OF C17.2 NEURAL STEM CELLS INTO NEURAL CELLS INDUCED BY SERUM-FREE CONDITIONED MEDIUM OF OLFACTORY ENSHEATHING CELLS AND CELL VIABILITY DETECTION OF DIFFERENTIATED CELLS

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