Cultivation, identification and differentiation of neural stem cells

doi:10.3969/j.issn.2095-4344.2017.17.014

Abstract

Abstract:

BACKGROUND: Neural stem cell transplantation is an emerging therapeutic option in the recovery of neural lesions and neurodegenerative diseases. Neural stem cell culture and differentiation lay a foundation for the further study.
OBJECTIVE: To improve the techniques for the isolation, cultivation, differentiation and identification of neural stem cells, and to explore the biological characteristics of cells.
METHODS: The neural stem cells from C57BL/6 fetal rats were isolated and cultured in vitro using neurophere culture method followed by morphological and ultrastucture examination. The growth curve and cell cycle of passage 3 cells were drawn and analyzed. Nestin expression was tested by immunofluorescence. Neural stem cells induced in 1% and 10% fetal bovine serum were identified using anti-GFAP, anti-βIII-tubulin and anti-MBP by immunofluorescence.
RESULTS AND CONCLUSION: The neurospheres exhibited strong cell proliferation ability. Under transmission electron microscope, there was a high nuclear/cytoplasmic ratio in the neural stem cells, indicating a low differentiation degree. Immunofluorescence analysis revealed that neural stem cells were positive for Nestin. The induced cells were positive for GFAP, βIII-tubulin, and MBP, indicating these cells were induced to differentiate into astrocytes, neurons and oligodendrocytes, and there were more neurons in 1% fetal bovine serum than those in 10% fetal bovine serum. In conclusion, we could successfully isolate neural stem cells in C57BL/6 mice, and low concentration of fetal bovine serum contributes to more neurons differentiated from neural stem cells.

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, Cells, Cultured, Serum, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Zhu Qiong, Hao Yue-juan, Gao Shun-ji, Chen Zhong, Liu Zheng, Xu Ya-li. Cultivation, identification and differentiation of neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2708-2713.

Figures/Tables 2

2.1 神经干细胞的形态在解剖显微镜下剥离脑膜及血管，得到大脑半球。采用机械分离和酶消化法得到大量单细胞。原代培养第1天光镜下见部分细胞贴壁，部分细胞悬浮呈单细胞，折光性好，第2天悬浮细胞部分聚集呈细胞团，大小不均，较大者约有十几个细胞，克隆球直径逐渐增大，呈桑葚状，随着直径的增大，部分克隆球中心变暗。将克隆球消化传代后，克隆球大小更加均匀，形态更加规整，未见明显突起，折光性好(图2)。 2.2 神经干细胞的增殖能力由细胞生长曲线(图3)可见，前2 d细胞生长缓慢，之后细胞增殖较快，进入对数生长期，从第6天开始，细胞增殖缓慢，进入平台期。细胞周期结果显示(图4)，53.23%的神经干细胞处于G0+G1期，G2期占12.58%，S期占34.19%，G2/G1=1.99。根据增殖公式计算得增殖指数为87.86%，说明绝大部分细胞处于增殖状态。 2.3 神经干细胞的超微结构透射电镜观察神经干细胞体积较小，胞核大且明显，核内染色质丰富，以常染色质为主，核仁较大，胞浆少，核浆比大，胞浆中含有大量的核糖体和线粒体，其余细胞器很少，表明该细胞分化程度低。大量的核糖体和线粒体表明细胞合成代谢能力旺盛，生命活动力强。细胞间可见细胞连接(图5)。 2.4 神经干细胞标志蛋白Nestin的表达免疫荧光结果显示(图6)，无论是接种的克隆球，还是单个细胞，细胞核均较小，被DAPI染成蓝色，胞浆呈绿色，说明Nestin呈阳性表达。克隆球向外呈放射状，越靠近中心，细胞密度越大，细胞越紧密，不易被染色，故中心未见染色细胞。 2.5 神经干细胞的诱导分化免疫荧光结果显示，大多数细胞GFAP反应阳性，胞浆丰富呈绿色，有多个短突起，贴壁牢固，说明大部分神经干细胞分化为星形胶质细胞(图7A)；少量细胞MBP反应阳性，呈绿色，说明少数神经干细胞分化为少突胶质细胞(图7B)；部分细胞βtubulin Ⅲ反应阳性，呈红色，突起较少但较长，说明部分神经干细胞分化为神经元(图7C，D)。其中，含体积分数为1%胎牛血清的培养基能诱导分化生成更多的神经元(图7D)，而含体积分数为10%胎牛血清的培养基能诱导分化生成更多的星形胶质细胞，说明低体积分数的血清有助于神经干细胞向神经元细胞分化，而高体积分数血清促使神经干细胞向胶质细胞分化。该分化结果进一步证实了分离培养得到的细胞为神经干细胞，且该细胞具有多向分化能力。"

References

[1] Gage FH. Mammalian neural stem cells. Science. 2000;287 (5457):14331438.

[2] Ma W, Fitzgerald W, Liu QY, et al. CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels. Exp Neurol. 2004;190(2): 276-288.

[3] Swistowski A, Peng J, Liu Q, et al. Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions. Stem Cells. 2010;28(10):1893-1904.

[4] Li Z, Zeng Y, Chen X, et al. Neural stem cells transplanted to the subretinal space of rd1 mice delay retinal degeneration by suppressing microglia activation. Cytotherapy. 2016;18(6): 771-784.

[5] Haus DL, López-Velázquez L, Gold EM, et al. Transplantation of human neural stem cells restores cognition in an immunodeficient rodent model of traumatic brain injury. Exp Neurol. 2016;281:1-16.

[6] Curtis E, Gabel BC, Marsala M, et al. 172 A Phase I, Open-Label, Single-Site, Safety Study of Human Spinal Cord-Derived Neural Stem Cell Transplantation for the Treatment of Chronic Spinal Cord Injury. Neurosurgery. 2016;63 Suppl 1:168-169.

[7] Mazzini L, Gelati M, Profico DC, et al. Human neural stem cell transplantation in ALS: initial results from a phase I trial. J Transl Med. 2015;13:17.

[8] Zhang W, Wang PJ, Sha HY, et al. Neural stem cell transplants improve cognitive function without altering amyloid pathology in an APP/PS1 double transgenic model of Alzheimer's disease. Mol Neurobiol. 2014;50(2):423-437.

[9] George PM, Steinberg GK. Novel Stroke Therapeutics: Unraveling Stroke Pathophysiology and Its Impact on Clinical Treatments. Neuron. 2015;87(2):297-309.

[10] Merkle FT, Alvarez-Buylla A. Neural stem cells in mammalian development. 2006;18(6):704-709.

[11] Gritti A, Parati EA, Cova L, et al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci. 1996;16(3): 1091-1100.

[12] Gage FH. Neurogenesis in the adult brain. J Neurosci. 2002; 22(3):612-613.

[13] Arvidsson A, Collin T, Kirik D, et al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002;8(9):963-970.

[14] Yamashita T, Ninomiya M, Hernández Acosta P, et al. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. J Neurosci. 2006;26(24):6627-6636.

[15] Qu T, Brannen CL, Kim HM, et al. Human neural stem cells improve cognitive function of aged brain. Neuroreport. 2001; 12(6):1127-1132.

[16] Zhao R, Xuan Y, Li X, et al. Age-related changes of germline stem cell activity, niche signaling activity and egg production in Drosophila. Aging Cell. 2008;7(3):344-354.

[17] Kirik OV, Vlasov TD, Korzhevski? DÉ. Nestin and Musashi1 as the markers of neural stem cells in rat telencephalon following transitory focal ischemia. Morfologiia. 2012;142(4):19-24.

[18] Eng LF. Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate filaments in differentiated astrocytes. J Neuroimmunol. 1985;8(4-6):203-214.

[19] Sun L, Lee J, Fine HA. Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury. J Clin Invest. 2004;113(9):1364-1374.

[20] Warrington AE, Barbarese E, Pfeiffer SE. Stage specific, (O4+GalC-) isolated oligodendrocyte progenitors produce MBP+ myelin in vivo. Dev Neurosci. 1992;14(2):93-97.

[21] Ciccolini F. Identification of two distinct types of multipotent neural precursors that appear sequentially during CNS development. Mol Cell Neurosci. 2001;17(5):895-907.

[22] Han J, Calvo CF, Kang TH, et al. Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans. Cell Rep. 2015;10(7):1158-1172.

[23] Yang JW, Ma W, Luo T, et al. BDNF promotes human neural stem cell growth via GSK-3β-mediated crosstalk with the wnt/β-catenin signaling pathway. Growth Factors. 2016; 34(1-2):19-32.

[24] Huat TJ, Khan AA, Pati S, et al. IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells BMC Neurosci. 2014;15:91.

[25] Xiong LL, Chen ZW, Wang TH. Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews. Neural Regen Res. 2016;11(4):591-596.

[26] Zhao H, Steiger A, Nohner M, et al. Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards βIII-Tubulin+ Neurons. PLoS One. 2015;10(6):e0129625.

[27] Chen X, Liu Y, Zhang Z, et al. Hypoxia stimulates the proliferation of rat neural stem cells by regulating the expression of metabotropic glutamate receptors: an in vitro study. Cell Mol Biol (Noisy-le-grand). 2016;62(3):105-114.

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

[29] Keirstead HS. Stem cell transplantation into the central nervous system and the control of differentiation. J Neurosci Res. 2001;63(3):233-236.

[30] Hung CH, Young TH. Differences in the effect on neural stem cells of fetal bovine serum in substrate-coated and soluble form. Biomaterials. 2006;27(35):5901-5908.

[31] Chen WW, Blurton-Jones M. Concise review: Can stem cells be used to treat or model Alzheimer's disease. Stem Cells. 2012;30(12):2612-2618.

[32] Obernier K, Tong CK, Alvarez-Buylla A. Restricted nature of adult neural stem cells: re-evaluation of their potential for brain repair. Front Neurosci. 2014;8:162.

[33] Crawford DC, Jiang X, Taylor A, et al. Astrocyte-derived thrombospondins mediate the development of hippocampal presynaptic plasticity in vitro. J Neurosci. 2012;32(38): 13100-13110.

[34] Bernardinelli Y, Muller D, Nikonenko I. Astrocyte-synapse structural plasticity. Neural Plast. 2014;2014:232105.

[35] Blanco-Suárez E, Caldwell AL, Allen NJ. Role of astrocyte-synapse interactions in CNS disorders. J Physiol. 2016 Jul 5. [Epub ahead of print]

[36] Hynes SR, Rauch MF, Bertram JP, et al. A library of tunable poly(ethylene glycol)/poly(L-lysine) hydrogels to investigate the material cues that influence neural stem cell differentiation. J Biomed Mater Res A. 2009;89(2):499-509.

[37] Lee IC, Wu YC, Cheng EM, et al. Biomimetic niche for neural stem cell differentiation using poly-L-lysine/hyaluronic acid multilayer films. J Biomater Appl. 2015;29(10):1418-1427.