Proportion and difference of neural stem cells and neurons from different embryonic mouse brain tissues

doi:10.3969/j.issn.2095-4344.2017.21.014

Abstract

Abstract:

BACKGROUND: At present, mouse embryonic neural stem cells (NSCs) culture has been skillfully operated by many labs, but there are differences existing about which part are dissociated to get NSCs. Embryonic 14 days (E14) mouse brain tissues are widely used for culturing NSCs, but there are less studies about the detailed percentage and difference of NSCs separated from different brain tissues.

OBJECTIVE: To test the proportion and difference of NSCs and neurons percentage from E14 mouse whole brain, cortex and forebrain, providing quantized data for optimizing the isolation of high-purity NSCs.

METHODS: E14 C57BL/6 mouse whole brain, cortex and forebrain tissues were separated and dissociated into single cells that were adherently cultured for 3.0-4.0 hours and labeled by DAPI. Then the cells were immunostained with NSCs specific marker, Nestin, and neuron specific marker, Tuj1, to identify NSCs and neurons percentage by calculating Nestin⁺/DAPI and Tuj1⁺/DAPI. In addition, real-time PCR assay was used to test Nestin and Tuj1 mRNA expression in the E14 mouse whole brain, cortex and forebrain.
RESULTS AND CONCLUSION: (1) Immunocytochemical results showed that there were a large amount of Nestin⁺ and Tuj1⁺ cells in the whole brain, cortex and forebrain of E14 mice. NSCs percentage in the forebrain was obviously higher than that in the whole brain (P < 0.01) and cortex (P < 0.05), while the percentage of neurons in the forebrain was significantly lower than that in the whole brain (P < 0.05) and in the cortex (P < 0.001). (2) Real-time PCR results showed that the Nestin mRNA expression in the forebrain was significantly higher than that in the whole brain (P < 0.05) and slightly higher than that in the cortex (P > 0.05); the Tuj1 mRNA expression in the forebrain was significantly lower than that in the whole brain (P < 0.05) and in the cortex (P < 0.05). These findings indicated that the forebrain had the most NSCs and the least neurons compared with the whole brain and the cortex. In summary, E14 mouse forebrain has the highest percentage of NSCs compared with the whole brain and cortex, which is a better source to obtain NSCs for the following cell culture experiments.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neural Stem Cells, Brain, Cerebral Cortex, Prosencephalon, Tissue Engineering

CLC Number:

R394.2

Zhang Feng-lan, Yang Lu-jun, Xiao Zhi-cheng. Proportion and difference of neural stem cells and neurons from different embryonic mouse brain tissues [J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3364-3369.

Figures/Tables 1

2.1 E14小鼠不同脑组织Nestin免疫荧光染色和分析观察E14小鼠全脑、大脑皮质和前脑组织分离培养细胞的Nestin免疫荧光染色结果，发现：Nestin阳性细胞的细胞质为绿色荧光，胞核不着色，因培养时间较短(3.0-4.0 h)，细胞形态多数为圆形或椭圆形，边缘清晰，只有极少数细胞伸出细长分枝，DAPI阳性细胞核呈蓝色；3种脑组织经消化分离后，几乎全为单个散在细胞，未见明显细胞团块，且几乎没有碎裂细胞残片，整体染色背景较干净；前脑组织中Nestin阳性细胞比例略高于全脑和大脑皮质。见图1A。将细胞免疫荧光数据进行统计分析，发现：在分离培养的E14小鼠前脑组织中Nestin阳性神经干细胞所占比例最高，为(37.48±0.52)%；大脑皮质组织中比例最低，为(33.07±0.62)%；全脑组织中比例介于二者之间，为(33.92±0.81)%。前脑组织中Nestin阳性神经干细胞的比例显著高于全脑(P < 0.05)和大脑皮质(P < 0.01)。见图1B。该结果表明前脑组织中Nestin阳性神经干细胞比例明显高于全脑和大脑皮质。 2.2 E14小鼠不同脑组织中Nestin mRNA的表达水平通过Real time PCR实验检测E14小鼠全脑、大脑皮质和前脑组织中Nestin mRNA的表达水平，结果显示：E14小鼠前脑组织中Nestin mRNA的表达水平(1.58±0.23)明显高于全脑组织(设定为1，P < 0.05)，略高于大脑皮质(1.15±0.04，P > 0.05)，见图2。该结果表明前脑组织中Nestin mRNA的表达水平较高。 2.3 E14小鼠不同脑组织Tuj1免疫荧光染色及分析观察E14小鼠全脑、大脑皮质和前脑组织中分离培养细胞的Tuj1免疫荧光染色结果，发现：Tuj1阳性神经元的细胞质为红色荧光，胞核不着色，也因培养时间较短(3.0-4.0 h)，大部分阳性细胞为圆形或椭圆形，只有少数细胞具有细长的突起，DAPI阳性细胞核呈蓝色；Tuj1免疫荧光染色整体背景较干净，未见非特异性着色；前脑组织中Tuj1阳性细胞比例略低于全脑和大脑皮质，见图3A。将细胞免疫荧光数据进行统计分析，发现：在分离培养的E14小鼠前脑组织中Tuj1阳性细胞所占比例最低(74.03±0.70)%；全脑组织中比例略低(76.97±0.81)%；大脑皮质组织中比例最高(80.81±0.29)%；前脑组织中Tuj1阳性细胞比例明显低于全脑(P < 0.05)和大脑皮质(P < 0.001)；另外，全脑组织中的Tuj1阳性细胞比例低于大脑皮质，且差异有显著性意义(P < 0.01，未标注)，见图3B。该结果表明前脑组织中Tuj1阳性神经元比例明显低于全脑和大脑皮质。 2.4 E14小鼠不同脑组织中Tuj1 mRNA的表达水平通过Real time PCR实验检测E14小鼠全脑、大脑皮质和前脑组织中Tuj1 mRNA的表达水平，结果显示：E14小鼠前脑组织中Tuj1 mRNA的表达水平(0.71±0.08)明显低于全脑组织中的表达水平(设定为1，P < 0.05)，也低于大脑皮质组织(1.04±0.04，P < 0.05)，但皮质组织与全脑组织之间差异无显著性意义(P > 0.05)，见图4。该结果表明前脑组织中Tuj1 mRNA的表达水平较低。"

References

[1] Yamaguchi M, Seki T, Imayoshi I, et al. Neural stem cells and neuro/gliogenesis in the central nervous system: understanding the structural and functional plasticity of the developing, mature, and diseased brain. J Physiol Sci. 2016;66(3):197-206.

[2] Conti L, Cattaneo E. Neural stem cell systems: physiological players or in vitro entities. Nat Rev Neurosci. 2010;11(3): 176-187.

[3] Akerblom M, Sachdeva R, Jakobsson J. Functional Studies of microRNAs in Neural Stem Cells: Problems and Perspectives. Front Neurosci. 2012;6:14.

[4] Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci. 1992;12(11):4565- 4574.

[5] English D, Sharma NK, Sharma K, et al. Neural stem cells- trends and advances. J Cell Biochem. 2013;114(4): 764-772.

[6] Gaspard N, Vanderhaeghen P. From stem cells to neural networks: recent advances and perspectives for neurodevelopmental disorders. Dev Med Child Neurol. 2011;53(1):13-17.

[7] Walker T, Huang J, Young K. Neural Stem and Progenitor Cells in Nervous System Function and Therapy. Stem Cells Int. 2016;2016:1890568.

[8] Duncan T, Valenzuela M. Alzheimer's disease, dementia, and stem cell therapy. Stem Cell Res Ther. 2017;8(1):111.

[9] Ottoboni L, Merlini A, Martino G. Neural Stem Cell Plasticity: Advantages in Therapy for the Injured Central Nervous System. Front Cell Dev Biol. 2017;5:52.

[10] Azari H, Sharififar S, Rahman M, et al. Establishing embryonic mouse neural stem cell culture using the neurosphere assay. J Vis Exp. 2011;(47): 2457.

[11] Torrado EF, Gomes C, Santos G, et al. Directing mouse embryonic neurosphere differentiation toward an enriched neuronal population. Int J Dev Neurosci. 2014;37:94-99.

[12] Ma QH, Futagawa T, Yang WL, et al. A TAG1-APP signalling pathway through Fe65 negatively modulates neurogenesis. Nat Cell Biol. 2008;10(3):283-294.

[13] 张涛,李文杰,王峰,等. 小鼠室管膜下区神经干细胞增殖及分化研究[J]. 中国修复重建外科杂志, 2015,29(6):766-771.

[14] 许汉鹏,苟琳,杨浩. 中枢神经系统不同部位来源的神经干细胞在体外生长特性的比较[J].解剖学报,2004,35(4): 358-362.

[15] 白连琴,刘娜,李腾腾,等. 新生鼠和胎鼠海马神经干细胞分离和培养方法的比较[J].神经解剖学杂志,2015,31(4):481-486.

[16] 张蓬勃,李卫松,高明,等. 小鼠胚胎神经干细胞的体外培养与鉴定[J].中国当代儿科杂志,2011, 13(3):244-247.

[17] Cao F, Hata R, Zhu P, et al. Conditional deletion of Stat3 promotes neurogenesis and inhibits astrogliogenesis in neural stem cells. Biochem Biophys Res Commun. 2010;394(3): 843-847.

[18] Ao X, Liu Y, Qin M, et al. Expression of Dbn1 during mouse brain development and neural stem cell differentiation. Biochem Biophys Res Commun. 2014;449(1):81-87.

[19] 杨蓬勃,张军峰,张建水,等. 人胎脑额叶和海马中星形胶质细胞的发育性变化[J].西安交通大学学报,2014,35(5):576-580.

[20] Vishwakarma SK, Bardia A, Tiwari SK, et al. Current concept in neural regeneration research: NSCs isolation, characterization and transplantation in various neurodegenerative diseases and stroke: A review. J Adv Res. 2014;5(3):277-294.

[21] 闵娜,胡强夫,李晓培,等. 异氟醚对新生大鼠海马神经干细胞增殖及分化的影响[J].中国组织工程研究,2016,20(1):118-122.

[22] Telias M, Ben-Yosef D.Neural stem cell replacement: a possible therapy for neurodevelopmental disorders?Neural Regen Res. 2015;10(2): 180-182.

[23] 石海杉,吴文,徐建兰.内源性神经干细胞的研究进展[J].实用医学杂志,2013,29(19): 3252-3254.

[24] 孙鼐,李春伟,赵伟新,等. 异氟醚抑制海马神经干细胞增殖并可促其向神经元分化[J].中国组织工程研究,2016,20(10): 1488-1493.

[25] Jiang XF,Yng K,Yang XQ,et al.Elastic modulus affects the growth and differentiation of neural stem cells. Neural Regen Res. 2015;10(9): 1523-1527.

[26] Louis SA, Mak CK, Reynolds BA. Methods to culture, differentiate, and characterize neural stem cells from the adult and embryonic mouse central nervous system. Methods Mol Biol. 2013;946:479-506.

[27] De Waele J, Reekmans K, Daans J, et al. 3D culture of murine neural stem cells on decellularized mouse brain sections. Biomaterials. 2015;41:122-131.

[28] Lee DY, Yeh TH, Emnett RJ, et al. Neurofibromatosis-1 regulates neuroglial progenitor proliferation and glial differentiation in a brain region-specific manner. Genes Dev. 2010;24(20):2317-2329.

[29] 张在金,董艳,卢亦成. 大鼠胎脑不同部位神经干细胞比较性培养的研究[J]. 中华神经外科疾病研究杂志, 2008,7(4):325-327.

[30] 马浚宁,高俊玮,候博儒,等, 新生小鼠海马、嗅球及皮质神经干细胞的分离培养及鉴定[J]. 中国组织工程研究,2014,18 (45): 7266-7272.

[31] Seaberg RM, Smukler SR, van der Kooy D. Intrinsic differences distinguish transiently neurogenic progenitors from neural stem cells in the early postnatal brain. Dev Biol.2005;278(1):71-85.

[32] Maslov AY, Barone TA, Plunkett RJ, et al. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci. 2004;24(7): 1726-1733.

[33] Guo W, Patzlaff NE, Jobe EM, et al. Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse. Nat Protoc. 2012;7(11):2005-2012.

[34] Bonaventura G, Chamayou S, Liprino A, et al. Different Tissue-Derived Stem Cells: A Comparison of Neural Differentiation Capability. PLoS One. 2015;10(10):e0140790.

[35] Xu R, Wu C, Tao Y, et al. Description of distributed features of the nestin-containing cells in brains of adult mice: a potential source of neural precursor cells. J Neurosci Res. 2010;88(5): 945-956.

[36] Lee R, Kim IS, Han N, et al. Real-time discrimination between proliferation and neuronal and astroglial differentiation of human neural stem cells. Sci Rep. 2014;4:6319.

[37] Zhang J, Jiao J. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis. Biomed Res Int. 2015;2015:727542.

[38] Farzanehfar P, Lu SS, Dey A, et al. Evidence of functional duplicity of Nestin expression in the adult mouse midbrain. Stem Cell Res. 2017;19:82-93.

[39] 刘吉星,候博儒,杨文桢,等.神经干细胞体外培养鉴定及诱导分化的表型特征[J].中国组织工程研究,2015,19(19):3054-3060.

[40] Mine Y, Tatarishvili J, Oki K, et al. Grafted human neural stem cells enhance several steps of endogenous neurogenesis and improve behavioral recovery after middle cerebral artery occlusion in rats. Neurobiol Dis. 2013;52:191-203.

[41] Lucchinetti E, Zeisberger SM, Baruscotti I, et al. Stem cell-like human endothelial progenitors show enhanced colony-forming capacity after brief sevoflurane exposure: preconditioning of angiogenic cells by volatile anesthetics. Anesth Analg. 2009;109(4):1117-1126.

[42] 刘罡,吴毅,贾杰,等.神经干细胞体外分化抗原表达的研究[J].中国运动医学杂志,2009,28(1):55-59.