Name: purification of prominin-1+ stem cells from postnatal mouse cerebellum
Uploaded: 2020-04-12T13:00:00
Description: Watch this Scientific Journal Video about Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum at JoVE.com

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

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
<ol><li>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）。</li>
	<li>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...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+paper:+the+cerebellum's+role+in+movement+and+cognition.">Consensus paper: the cerebellum's role in movement and cognition.</a> Cerebellum. 13, 151-177 (2014).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Machold, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+expressed+in+temporally+discrete+pools+of+cerebellar+rhombic-lip+neural+progenitors.">Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors.</a> Neuron. 48, 17-24 (2005).</li><li>Wang, V. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+expression+redefines+the+rhombic+lip+derivatives+and+reveals+novel+lineages+within+the+brainstem+and+cerebellum.">Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum.</a> Neuron. 48, 31-43 (2005).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nature Neurosciences. 16, 1737-1744 (2013).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nature Neurosciences. 8, 723-729 (2005).</li><li>Toren, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133-positive+hematopoietic+stem+cell+"stemness"+genes+contain+many+genes+mutated+or+abnormally+expressed+in+leukemia.">CD133-positive hematopoietic stem cell "stemness" genes contain many genes mutated or abnormally expressed in leukemia.</a> Stem Cells. 23, 1142-1153 (2005).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prominin+1+marks+intestinal+stem+cells+that+are+susceptible+to+neoplastic+transformation.">Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.</a> Nature. 457, 603-607 (2009).</li><li>Man, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Role+for+the+DNA+Sensor+AIM2+in+Stem+Cell+Proliferation+and+Cancer.">Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer.</a> Cell. 162, 45-58 (2015).</li><li>Fleming, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Purkinje+neuron+acts+as+a+central+regulator+of+spatially+and+functionally+distinct+cerebellar+precursors.">The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors.</a> Developmental Cell. 27, 278-292 (2013).</li><li>Panchision, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+flow+cytometric+analysis+of+central+nervous+system+tissue+reveals+novel+functional+relationships+among+cells+expressing+CD133,+CD15,+and+CD24.">Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.</a> Stem Cells. 25, 1560-1570 (2007).</li><li>Beaudoin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+pyramidal+neurons+from+the+early+postnatal+mouse+hippocampus+and+cortex.">Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.</a> Nature Protocols. 7, 1741-1754 (2012).</li><li>Edamakanti, C. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuronal+differentiation+in+human+CNS+stem+cell+progeny+by+leukemia+inhibitory+factor.">Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor.</a> Developmental Neurosciences. 22, 86-95 (2000).</li><li>Silbereis, J., Cheng, E., Ganat, Y. M., Ment, L. R., Vaccarino, F. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP1+is+involved+in+replenishment+of+granule+cell+precursors+following+injury+to+the+neonatal+cerebellum.">YAP1 is involved in replenishment of granule cell precursors following injury to the neonatal cerebellum.</a> Developmental Biology. 1606 (19), 30207 (2019).</li><li>Wang, S. S., Kloth, A. D., Badura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebellum,+sensitive+periods,+and+autism.">The cerebellum, sensitive periods, and autism.</a> Neuron. 83, 518-532 (2014).</li><li>Eberhart, C. 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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?...

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。

<table>
	<tbody>
		<tr>
			<td>0.05%Trypsin</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td>0.05%</td>
		</tr>
		<tr>
			<td>2% B27</td>
			<td>Gibco; Thermo Fisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>2mM EDTA solution</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- Prominin-1 microbeads</td>
			<td>Miltenyi Biote</td>
			<td>130-092-333</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Column MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>culture plates ultra - low attachment</td>
			<td>Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>cysteine</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>D4513-1VL</td>
			<td>250 U/ml</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffer Saline</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution-HBSS</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant Basic Fibroblast Growth Factor</td>
			<td>Promega</td>
			<td>G507A</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Human recombinant Epidermal Growth Factor</td>
			<td>Promega</td>
			<td>G502A</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Sigma</td>
			<td>L5158</td>
			<td></td>
		</tr>
		<tr>
			<td>l-glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td>Lieca TCS SP5 confocal microscopes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-Prominin-1</td>
			<td>Affymetrix eBioscience</td>
			<td>14-1331</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Abcam</td>
			<td>ab27952</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>O4</td>
			<td>Millopore</td>
			<td>MAB345</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td>(100 U/ml)</td>
		</tr>
		<tr>
			<td>Platelet- Derived Growth Factor</td>
			<td>Sigma</td>
			<td>H8291</td>
			<td>10 ng/ml</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-tubulin, b-III</td>
			<td>Sigma</td>
			<td>T2200</td>
			<td>1 in 500</td>
		</tr>
		<tr>
			<td>Rabit anti-GFAP</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td>1 in 500</td>
		</tr>
		<tr>
			<td>Separation columns-MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td>40um</td>
		</tr>
	</tbody>
</table>

purification of prominin-1+ stem cells from postnatal mouse cerebellum

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。

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...

Watch this Scientific Journal Video about Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum at JoVE.com

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

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。

从产后小鼠小脑中纯化Promin-1+干细胞

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...

该协议描述了一种简单且具有成本效益的方法，用于从产后小脑中纯化干细胞。这些干细胞占晚期分子混乱的内皮龙，但也是产后衍生的小脑星细胞。因此，研究这些细胞显然对了解小脑的发展很重要。使用该协议，干细胞的产生与基于 FACS 的策略相当，这种策略通常比其昂贵 10 倍。该协议可以推广，从其他组织，如肠道和骨髓中提取蛋白-1表达干细胞。首先将大脑的盘子放在解剖显微镜下。使用细数五钳从小脑中去除脑膜和大血管，然后用铲子将小脑从脑干中分离。将小脑转移到含有5毫升冰冷HSS溶液的15毫升离心管中。冲洗它，然后将HBSS去掉。再重复洗涤两次。最后一次冲洗后，加入五毫升的木瓜组织分离溶液，这些溶液已预热至37摄氏度。在37摄氏度的水浴中孵育组织15分钟，每三分钟倒置3至5次，慢慢混合管内物。在 Bunsen 燃烧器上进行火抛光，准备宽直径和窄直径玻璃巴斯德移液器。孵育后，用五毫升的HSS溶液洗三次组织，确保避免在脱液时失去组织。上次HBSS洗涤后，将5毫升DPBS与250微升DNAse添加到组织中，用宽直径巴斯德移液器轻轻三叶草10至15次将其分离。然后使用狭窄的巴斯德移液器进一步三化组织浆料10次。如果较大的组织仍然存在，用移液器尖端将它们压在管的底部，然后继续移液，直到达到精细悬浮液。将离心管放在冰上。然后通过40微米细胞过滤器将分离的细胞应变成50毫升的管。在过滤器顶部加10毫升冰冷HSS溶液，确保细胞通过网格。将过滤过的细胞转移到一个新的15毫升离心管中，在4摄氏度下以300倍g离心，10分钟。然后使用真空吸气器完全去除上一液。将细胞颗粒重新悬浮在160微升的冰冷磁柱缓冲液中。在细胞悬浮液中加入40微升抗多明因-1微珠。然后在冰箱中孵育管子15分钟，使抗体与蛋白-1表达细胞结合。用一到两毫升的柱缓冲液X清洗细胞，并在300次g下离心10分钟。吸升液，将颗粒重新悬浮在一毫升柱缓冲液X.将磁柱放在磁支架上，用500微升缓冲液X冲洗一次。用500微升缓冲器X洗三次柱，然后从磁场中取出柱子，放在1.5毫升管中。加入一毫升培养培养剂，将柱塞推入柱中，以冲洗标有蛋白-1珠子的细胞。要通过神经球，将它们与培养培养剂一起转移到无菌的15毫升离心管中。以300次g离心将神经球挖碎5分钟，然后丢弃上经剂。将颗粒与木瓜素或0.05%的三辛辛溶液重新在5毫升分离介质中，并在37摄氏度下孵育细胞10分钟。再次将电池悬架离心。然后将细胞重新在神经球介质的五毫升中重新暂停，然后用巴斯德移液器缓慢上下移液10次，使细胞分离。如文本协议所述，在神经球培养中培养细胞，并在培养7至10天后，如前面所述，用继发性神经球丰富细胞。在血细胞计上计算细胞数，并在未来实验中对多D-lysine涂层板上的最佳细胞数进行镀。柱纯化的表蛋白-1阳性产后小脑干细胞形成神经球的生长因子丰富的神经球介质。这些神经球对普明-1呈阳性，它们用于分离和其他干细胞标记，如巢和GFAP。流中细胞染色阴性为干细胞标记蛋白-1和巢和阳性的神经元标记β-III浴缸。当生长因子的退出和白血病抑制因子或PDGF-AA的存在，普明-1阳性神经球分化成神经元，星形细胞和寡头细胞。在尝试这种技术时，重要的是要确保神经球被推动通过prominin-1抗体，他们可以通过，因为正常细胞也倾向于形成团块，可能看起来像神经球。使用这种隔离方法，可以在健康和疾病条件下描述其衍生血统，如神经元和胶质。这种方法很有价值，为小脑干细胞在小脑发育和疾病状况（如脊柱癌和癌症）中的作用提供了新的见解。

Research

JoVE Journal

Neuroscience

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

科研

神经科学

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Purification of Mouse Brain Vessels

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

从胚胎小鼠小脑分离的神经元的主要培养

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

Isolation and Expansion of Neurospheres from Postnatal P1ÃƒÂ¢Ã‹â€ '3 Mouse Neurogenic Niches

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...