Summary
生动的图像是研究在实时的细胞行为的有力工具。在这里,我们描述延时视频-显微镜的脑皮层细胞,允许颁布在沿袭前进过程中主要的神经干细胞分化的神经元和神经胶质的阶段详细的审查的协议。
Abstract
大脑皮质发育过程中祖细胞经过几轮的对称和非对称细胞分裂生成新的祖细胞或分裂后神经元。后来,一些祖细胞切换到 gliogenic 的命运,将添加到的星形胶质细胞和少突胶质细胞的人口。使用延时视频显微镜的脑皮层细胞培养,有可能控制模式的细胞分裂和细胞周期参数的祖细胞的细胞和分子机制的研究。同样,命运的分裂后的细胞可以被审查,使用细胞特异性荧光记者蛋白质或后成像免疫细胞化学。更重要的是,所有这些功能,可以在单个细胞水平,允许致力于生成的特定类型的细胞的祖细胞的鉴定分析了。也可以使用病毒介导的基因转染,允许细胞自主和非细胞自主的现象的研究执行操纵基因的表达。最后,融合荧光蛋白的使用允许选定蛋白质过程划分与对比与女儿细胞命运的对称和非对称分布的研究。在这里,我们描述的延时视频显微镜方法图像几天到脑皮层小鼠细胞和细胞分裂、 细胞周期长度和新生成的细胞命运的模式进行了分析。我们还描述祖细胞,可以用于操纵基因的兴趣或简单标签与记者蛋白细胞转染的简便方法。
Introduction
神经干细胞 (NSC) 生成大脑皮质发育过程中神经元和星形细胞。在早期的-corticogenesis,神经干细胞经过几轮的对称的细胞分裂,并展开祖池。然后,神经干细胞发生不对称分裂生成神经元直接或间接通过中间体1。只有在中期-向晚-corticogenesis,祖细胞切换来生成星形胶质细胞和少突胶质细胞2,,34。然而,控制细胞增殖和分化,以及命运限制给代的独特类型的神经元或星形细胞祖细胞的贡献的完整机制仍然是激烈辩论4,,56。个人皮层神经干细胞生成神经元、 星形胶质细胞和少突胶质细胞的潜力已经广泛研究体外和体内使用大量的技术如: 生活成像在单个单元格文化7,8,9,10,11,,1213;生活影像在高密度文化文化3,,1415;生活影像在切片文化16,,1718;无性系分析使用病毒载体介导遗传标记在高密度文化14,15,19,,2021;无性系分析体内使用逆转录病毒22,23,24,25,26,27,28,29,30,31,,3233;与此同时,并用是克隆分析体内转基因动物34。
每个这些技术提出了利与弊。例如,在体内沿袭跟踪易发生混为一谈、 劈裂错误3,导致矛盾的结论,对潜在的个别皮质祖细胞。此外,在体外和体内研究基于标签祖细胞在早期的时间点和后的细胞谱系分析可能受到细胞死亡在沿袭进展35期间未检测到发生。因此,适当的系统,以分析单一神经干细胞的潜力必须允许鉴定所有细胞生成,以及细胞的命运,在世系的适当表征。原代细胞和实时成像的结合提供此设置。使用单一细胞培养和时间推移视频显微镜,寺庙等人有开关示宗族的个人大脑皮质祖细胞从神经到 gliogenesis11。后来,他们用于同一系统显示从单皮质祖12生成不同类型的神经元。然而,这一制度提出了一个重要的警告: 只有 1%的皮质祖细胞在早期 corticogenesis 孤立产生无性系 4 个或更多的后代9。后增加了 FGF2,细胞生成 4 或更多单元格的频率增加到 8-109。不过,这个数字是太小了,考虑到几乎所有皮质祖细胞增殖在这一阶段。此外,FGF2 对命运规范的潜在影响,也不能排除36。为了克服这些局限性,我们使用支持的两个心室扩散 (Pax6 表示) 的高密度细胞培养和室管膜下 (Tbr2 表示) 皮质祖细胞15。此外,这些文化的实时观察表明,NSC 血统进展的几个特征转载这些条件,如细胞分裂、 细胞周期延长、 潜力的单个细胞生成神经元和神经胶质,除其他外的模式下3,15。最近,我们有也用于此系统表明,CREB 信号影响小鼠37未成熟脑皮质神经元的细胞存活。因此,我们认为该延时视频显微镜的小鼠脑皮层细胞生长在高密度是强大和方便的工具,研究细胞和分子机制的细胞周期、 细胞分裂、 细胞存活和细胞命运规范模式。后者可以完成用转基因动物,允许特定细胞的命运,在真正的时间38,,3940鉴定或后影像学免疫细胞化学3,38,,4142使用。
在这里,我们提供一步一步的协议,准备支持的神经干细胞的增殖和对下一代的神经元和星形细胞的脑皮层细胞培养。我们还讨论了利用逆转录病毒介导基因转染来操纵基因表达的个体细胞,它可以在单个细胞水平使用时间推移视频显微镜跟踪和标识。这个协议可以用于研究脑皮层的细胞从一开始到结束了在啮齿类动物,corticogenesis 但须根据阶段14的几个调整。孤立的从其他来源的神经干细胞研究也可以使用时间推移视频显微镜的 2D 的文化,但适当的培养体系应由比较细胞行为在体外和体内38,43。
Subscription Required. Please recommend JoVE to your librarian.
Discussion
脑皮层细胞的实时观察允许分析细胞的增殖、 细胞分裂、 细胞周期长度、 细胞分化和细胞生存3,14,,1537的模式。更重要的是,它允许单个细胞谱系,导致颁布了神经干细胞向神经元3过程的中间阶段的鉴定研究。最后,这个文化体系与生物工程工具来操纵基因表达组合是强大的技术,研究细胞自主作用的选定的目标5。这里介绍的方法可以修改,以研究大脑皮质祖细胞在不同发育阶段14,孤立,以及从其他来源38,42分离的神经干细胞系。类似的方法也用于研究发展的视网膜41的细胞谱系。
在这里,我们显示一个简单的实验,使用逆转录病毒介导的基因转染到记者荧光蛋白标签祖细胞。然而,可以使用其他病毒载体、 化工/电转染或表达神经的细胞类型特异性启动子38,39控制下的荧光蛋白的转基因动物实现类似的目标。所有这些控制基因表达的方法也可以用于诱导或抑制基因表达的兴趣,使分子机制的研究涉及神经干细胞谱系进展15,,1839。我们也预见到这个系统可以用于评价如何不同表达的特定蛋白的水平调节行为和神经元/星形分化,类似于做在造血系统40。此外,它可能有助于阐明单祖细胞生成单独神经元的血统的潜力。
这里介绍的方法与其他技术旨在成像哺乳动物神经干细胞在活的动物或切片文化相比,有一些重要的优势。首先,低成本是方法的一个重要的优势。简单的倒置的显微镜配备传输和荧光灯光和相机控制的基于计算机的软件可以用来获得图像的二维文化为多几个星期。其次,用于为这些实验的动物数量是明显小于其他方法。第三,该系统允许的环境条件,从而使细胞自主和非细胞自主影响的不同操作的分析进行精确的控制。最后,单个单元格可以毫不含糊地观察到长达 15 天,允许精确重构的大的谱系树,这是目前不可能都在大脑皮层切片文化或在体内。另一方面,系统可能会显示与组织的损失相关联的缺点。因此,我们建议在这些二维文化中观察到的细胞行为应理想证实其他实验的体内。
以前的数据,使用此系统表明,细胞周期延长体内皮质祖48是转载体外15。神经源性和 gliogenic 潜力的祖细胞也模仿二维培养系统中的个体大脑皮质这里描述3。最后,细胞增殖和细胞周期出口比率观察体内可以也模仿使用此单元格文化系统15,18。因此,我们认为那生活-成像的脑皮层细胞培养在本议定书中所述的条件是强大和方便的方法,研究细胞和分子机制控制前体细胞的增殖、 神经元和神经胶质细胞分化和细胞命运规范。
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
作者没有透露。
Acknowledgments
这项工作由 CNPq (委员会全国德日托 Científico 电子技术学校),披风 (也 de Aperfeiçoamento de Pessoal de Nível 高级) 和 FAPERN (该片 de Amparo 共同做格兰德北里)。
Materials
Name | Company | Catalog Number | Comments |
Hank's Balanced Salt Solution (HBSS) | Invitrogen Life Technologies | 14175129 | |
HEPES | Sigma-Aldrich | H3375-25G | |
Penicillin/streptomycin | Gibco | 15140122 | |
Dulbecco Modified Eagle's Medium (DMEM) | Gibco | 12400-024 | |
Fetal Calf Serum (FCS) | Gibco | 10437028 | |
Glucose | Gibco | A2494001 | |
B27 | Gibco | 17504044 | |
trypsin-EDTA (0.05%) | Gibco | 25300054 | |
Paraformaldehyde | Sigma | 16005 | |
Goat serum | Sigma-Aldrich | 69023 | |
Triton X-100 | VWR International Ltd. | 306324N | |
Isoflurane | Sigma | 792632 | |
anti-MAP2, mouse | Sigma | M4403 | |
anti-GFP chicken | Aves | 0511FP12 | |
DAPI | Sigma | D9542 | |
Goat anti-mouse alexa 594 | Invitrogen | A11005 | |
Goat anti-chicken alexa 488 | Invitrogen | A11039 | |
ImageJ | NIH | ||
tTt | ETH Zurich | ||
Cell observer microscope | Zeiss | ||
Pasteur pipette | |||
PBS | |||
The Tracking Tool (tTt) software | https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html | download link |
References
- Kriegstein, A., Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci. 32, 149-184 (2009).
- Miller, F. D., Gauthier, A. S. Timing is everything: making neurons versus glia in the developing cortex. Neuron. 54 (3), 357-369 (2007).
- Costa, M. R., Bucholz, O., Schroeder, T., Götz, M. Late Origin of Glia-Restricted Progenitors in the Developing Mouse Cerebral Cortex. Cereb Cortex. 19 (Suppl 1), i135-i143 (2009).
- Knoblich, J. A. Mechanisms of asymmetric stem cell division. Cell. 132 (4), 583-597 (2008).
- Costa, M. R., Müller, U. Specification of excitatory neurons in the developing cerebral cortex: progenitor diversity and environmental influences. Front Cell Neurosci. 8, 449 (2015).
- Schitine, C., Nogaroli, L., Costa, M. R., Hedin-Pereira, C. Astrocyte heterogeneity in the brain: from development to disease. Front Cell Neurosci. 9, 76 (2015).
- Temple, S. Division and differentiation of isolated CNS blast cells in microculture. Nature. 340 (6233), 471-473 (1989).
- Davis, A. A., Temple, S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature. 372 (6503), 263-266 (1994).
- Qian, X., Davis, A. A., Goderie, S. K., Temple, S. FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells. Neuron. 18 (1), 81-93 (1997).
- Qian, X., Goderie, S. K., Shen, Q., Stern, J. H., Temple, S. Intrinsic programs of patterned cell lineages in isolated vertebrate CNS ventricular zone cells. Development. 125 (16), 3143-3152 (1998).
- Qian, X., et al. Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron. 28 (1), 69-80 (2000).
- Shen, Q., et al. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat Neurosci. 9 (6), 743-751 (2006).
- Ravin, R., et al. Potency and fate specification in CNS stem cell populations in vitro. Cell Stem Cell. 3 (6), 670-680 (2008).
- Costa, M. R., Kessaris, N., Richardson, W. D., Götz, M., Hedin-Pereira, C. The marginal zone/layer I as a novel niche for neurogenesis and gliogenesis in developing cerebral cortex. J Neurosci. 27 (42), 11376-11388 (2007).
- Costa, M. R., Wen, G., Lepier, A., Schroeder, T., Götz, M. Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex. Development. 135 (1), 11-22 (2008).
- Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., Kriegstein, A. R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci. 7 (2), 136-144 (2004).
- Noctor, S. C., Martínez-Cerdeño, V., Kriegstein, A. R. Distinct behaviors of neural stem and progenitor cells underlie cortical neurogenesis. J Comp Neurol. 508 (1), 28-44 (2008).
- Bultje, R. S., Castaneda-Castellanos, D. R., Jan, L. Y., Jan, Y. N., Kriegstein, A. R., Shi, S. H. Mammalian Par3 regulates progenitor cell asymmetric division via notch signaling in the developing neocortex. Neuron. 63 (2), 189-202 (2009).
- Williams, B. P., Read, J., Price, J. The generation of neurons and oligodendrocytes from a common precursor cell. Neuron. 7 (4), 685-693 (1991).
- Williams, B. P., Read, J., Price, J. Evidence for multiple precursor cell types in the embryonic rat cerebral cortex. Neuron. 14 (6), 1181-1188 (1995).
- Heins, N., et al. Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci. 5 (4), 308-315 (2002).
- Luskin, M. B., Pearlman, A. L., Sanes, J. R. Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus. Neuron. 1 (8), 635-647 (1988).
- Luskin, M. B., Parnavelas, J. G., Barfield, J. A. Neurons, astrocytes, and oligodendrocytes of the rat cerebral cortex originate from separate progenitor cells: an ultrastructural analysis of clonally related cells. J Neurosci. 13 (4), 1730-1750 (1993).
- Price, J., Thurlow, L. Cell lineage in the rat cerebral cortex: a study using retroviral-mediated gene transfer. Development. 104 (3), 473-482 (1988).
- Walsh, C., Cepko, C. L. Clonally related cortical cells show several migration patterns. Science. 241 (4871), 1342-1345 (1988).
- Walsh, C., Cepko, C. L. Widespread dispersion of neuronal clones across functional regions of the cerebral cortex. Science. 255 (5043), 434-440 (1992).
- Parnavelas, J. G., Barfield, J. A., Franke, E., Luskin, M. B. Separate progenitor cells give rise to pyramidal and nonpyramidal neurons in the rat telencephalon. Cereb Cortex. 1 (6), 463-468 (1991).
- Grove, E. A., Williams, B. P., Li, D. Q., Hajihosseini, M., Friedrich, A., Price, J. Multiple restricted lineages in the embryonic rat cerebral cortex. Development. 117 (2), 553-561 (1993).
- Mione, M. C., Danevic, C., Boardman, P., Harris, B., Parnavelas, J. G. Lineage analysis reveals neurotransmitter (GABA or glutamate) but not calcium-binding protein homogeneity in clonally related cortical neurons. J Neurosci. 14 (1), 107-123 (1994).
- Mione, M. C., Cavanagh, J. F., Harris, B., Parnavelas, J. G. Cell fate specification and symmetrical/asymmetrical divisions in the developing cerebral cortex. J Neurosci. 17 (6), 2018-2029 (1997).
- Reid, C. B., Liang, I., Walsh, C. Systematic widespread clonal organization in cerebral cortex. Neuron. 15 (2), 299-310 (1995).
- McCarthy, M., Turnbull, D. H., Walsh, C. A., Fishell, G. Telencephalic neural progenitors appear to be restricted to regional and glial fates before the onset of neurogenesis. J Neurosci. 21 (17), 6772-6781 (2001).
- Reid, C. B., Walsh, C. A. Evidence of common progenitors and patterns of dispersion in rat striatum and cerebral cortex. J Neurosci. 22 (10), 4002-4014 (2002).
- Gao, P., et al. Deterministic progenitor behavior and unitary production of neurons in the neocortex. Cell. 159 (4), 775-788 (2014).
- Schroeder, T.
Imaging stem-cell-driven regeneration in mammals. Nature. 453 (7193), 345-351 (2008). - Morrow, T., Song, M. R., Ghosh, A. Sequential specification of neurons and glia by developmentally regulated extracellular factors. Development. 128 (18), 3585-3594 (2001).
- Landeira, B. S., et al. Activity-Independent Effects of CREB on Neuronal Survival and Differentiation during Mouse Cerebral Cortex Development. Cereb Cortex. , 1-11 (2016).
- Costa, M. R., et al. Continuous live imaging of adult neural stem cell division and lineage progression in vitro. Development. 138 (6), 1057-1068 (2011).
- Pilaz, L. J., et al. Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain. Neuron. 89 (1), 83-99 (2016).
- Hoppe, P. S., et al. Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios. Nature. 535 (7611), 299-302 (2016).
- Gomes, F. L., et al. Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions. Development. 138 (2), 227-235 (2011).
- Ortega, F., Berninger, B., Costa, M. R. Primary culture and live imaging of adult neural stem cells and their progeny. Methods Mol Biol. 1052, 1-11 (2013).
- Ponti, G., Obernier, K., Guinto, C., Jose, L., Bonfanti, L., Alvarez-Buylla, A. Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice. Proc Natl Acad Sci U S A. 110 (11), E1045-E1054 (2013).
- Jagasia, R., et al. GABA-cAMP response element-binding protein signaling regulates maturation and survival of newly generated neurons in the adult hippocampus. J Neurosci. 29 (25), 7966-7977 (2009).
- Costa, M. R., Jagasia, R., Berninger, B. Directed Neuronal Differentiation of Embryonic and Adult-Derived Neurosphere Cells. Protocols for Neural Cell Culture. Doering, L. C. , Humana Press. New York. 29-49 (2009).
- Hilsenbeck, O., et al. Software tools for single-cell tracking and quantification of cellular and molecular properties. Nat Biotechnol. 34 (7), 703-706 (2016).
- Ortega, F., et al. Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level. Nat Protoc. 6 (12), 1847-1859 (2011).
- Takahashi, T., Nowakowski, R. S., Caviness, V. S. Jr The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J Neurosci. 15 (9), 6046-6057 (1995).