从产后小鼠小脑中纯化Promin-1+干细胞
Summary
这里演示的是一种高效且经济高效的方法，用于从产后小鼠小脑中纯化、培养和分化白质干细胞。
Abstract
大多数小脑神经元产生于两个胚胎干细胞利基：一个红唇壁，它产生所有的小脑外皮谷类神经元，和一个心室区利基，它产生抑制性GABAergic Purkinje细胞，这是构成深小脑核和伯格曼胶质的神经元。最近，第三个干细胞利基被描述为从心室区利基的继发生殖区。这种利基的细胞由细胞表面标记素-1定义，并定位为产后小脑的白质。这个利基占晚期出生的分子层GABAergic间神经元以及产后生成的小脑星细胞。除了他们的发育作用，这个利基正在获得翻译的重要性，其参与神经退化和肿瘤发生。由于缺乏有效的纯化技术，这些细胞的生物学一直难以破译。这里演示的是净化、培养和分化这些产后小脑干细胞的有效方法。
Introduction
小脑长期以来一直被认为是协调自愿运动的主要神经元回路1。它接收来自神经轴的宽条输入，包括来自外围的感知信息，从而微调电机输出和协调运动。最近，它还被牵连到通过可能使用类似的信息处理网络22，3，43,4来调节认知和情感。
成人小脑由外小脑皮层和内白质组成。在这些结构内穿插的是深内回核。与神经系统的其他部分类似，小脑的发展是由多能祖细胞（干细胞）的增殖推动的，这些细胞迁移并分化以产生这种组织良好的结构。在早期发育（E10.5_E13.5）中，围绕发育的第四个心室的心室干细胞利基产生GABAergic神经元（即珀金耶细胞、卢加罗细胞、高尔基细胞）以及伯格曼利亚55、6、7、8。6,7,8
在发育后期（产后第一周），红唇中的第二个干细胞利基产生MATH1和内丁表达的祖体，产生兴奋颗粒神经元9,109，10，11，12。,11,12最近，第三个干细胞利基被描述13。这些细胞表达prominin-1（也称为CD133），一种膜覆盖的糖蛋白，定义肠道和造血系统14、15、16的干细胞子集。14,15,16体内命运图显示，这些干细胞在前三个产后周内产生关键的分子层间神经元（即篮子细胞和斯特拉特细胞）以及星细胞。过去，在体外研究这些细胞是很困难的，因为先前的方法需要昂贵且耗时的技术（即荧光激活细胞分拣[FACS]），这些技术依赖于promin-1染色12、13、17。12,13,17该协议描述了一种基于免疫磁性的方法，用于分离这些干细胞，然后可以很容易地培养和分化。
Protocol
所有动物实验都符合NIH的《实验室动物护理和使用指南》（2011年），并经西北大学IACUC（IS00011368议定书）批准。 1. 准备解决方案 制备由无菌酚类红含量Dulbecco的磷缓冲盐水（DPBS）制成的组织分离溶液，包括木瓜素（100 U/ml）、半胱氨酸（0.2毫克/毫升）和DNase（250 U/ml）。 制备DNase溶液稀释100毫克的DNase I（一瓶）的嗜血粉在50 mL的H2O.混合良好，并过滤库…
Representative Results
Prominin-1阳性产后小脑干细胞在神经球介质中形成神经球，富含生长因子（EGF和bFGF）。这些神经球对promin-1染色呈阳性，这是用于分离的标记，也是其他干细胞标记物（如内丁和GFAP13）的污渍（图1）。干细胞标记表达在整个培养过程中保持，最多8个通道20。当退出生长因子时，在LIF和PDGF-AA（支持神经元和胶质分化21，22<su…
Discussion
在产后生命的前3周，Prominin-1表达的小脑干细胞存在于潜在的白质中。它们的增殖受到由Purkinje细胞17支持的声波刺猪通路的严格控制。这些干细胞/祖细胞有助于后来出生的GABAergic间神经元，称为篮子细胞和斯特拉特细胞。这些间神经元存在于分子层中，它们突触到Purkinje细胞上，并通过GABAergic抑制13、17、2317,雕刻PC地形13和功?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢蛋白石实验室成员的建议。这项工作得到了NIH授予1RO1 NS062051和1RO1NS08251（Opal P）的支持
Materials
| 0.05%Trypsin | Thermo Fisher Scientific | 25300054 | 0.05% |
| 2% B27 | Gibco; Thermo Fisher Scientific | 17504001 | |
| 2mM EDTA solution | Corning | 46-034-CI | |
| Anti- Prominin-1 microbeads | Miltenyi Biote | 130-092-333 | |
| bovine serum albumin | Sigma | A9418 | |
| Column MultiStand | Miltenyi Biotec | 130-042-303 | |
| culture plates ultra – low attachment | Corning | 3473 | |
| cysteine | Sigma | C7880 | |
| DNase | Sigma | D4513-1VL | 250 U/ml |
| Dulbecco’s Phosphate Buffer Saline | Thermo Fisher Scientific | 14040141 | |
| Hank's balanced salt solution-HBSS | Gibco | 14025-092 | |
| Human recombinant Basic Fibroblast Growth Factor | Promega | G507A | 20 ng/ml |
| Human recombinant Epidermal Growth Factor | Promega | G502A | 20 ng/ml |
| Leukemia Inhibitory Factor | Sigma | L5158 | |
| l-glutamine | Gibco | 25030081 | |
| Microscopy | Lieca TCS SP5 confocal microscopes | ||
| MiniMACS separator | Miltenyi Biotec | 130-042-102 | |
| mouse anti-Prominin-1 | Affymetrix eBioscience | 14-1331 | 1 in 100 |
| Nestin | Abcam | ab27952 | 1 in 200 |
| Neurobasal medium | Thermo Fisher | 25030081 | |
| O4 | Millopore | MAB345 | |
| Papain | Worthington | LS003126 | (100 U/ml) |
| Platelet- Derived Growth Factor | Sigma | H8291 | 10 ng/ml |
| Poly-D-Lysine | Sigma | P6407 | |
| rabbit anti-tubulin, b-III | Sigma | T2200 | 1 in 500 |
| Rabit anti-GFAP | Dako | Z0334 | 1 in 500 |
| Separation columns-MS columns | Miltenyi Biotec | 130-042-201 | |
| Sterile cell strainer | Fisher Scientific | 22363547 | 40um |
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Citazione di questo articolo
Edamakanti, C. R., Opal, P. Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum. J. Vis. Exp. (158), e60554, doi:10.3791/60554 (2020).