Name: zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis
Uploaded: 2018-02-05T14:30:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

这项工作得到了来自邓恩基金会和德克萨斯大学医学分会 (p.w) 人类感染和免疫研究所的资助。

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作者没有利益冲突声明。

培养和操纵 hNSCs 的能力提供了一个关键的工具, 可用于各种目的从建模人类疾病到高通量药物筛选10,11,12,14, 15、16、17。许多问题仍有待解决, 如人胎脑神经干细胞或其子代如何易受 ZIKV 感染, 不同的 ZIKV 菌株是否会以同样的效率感染干细胞, 以及神经系统发育过程中的感染如何导致小头。我们使用此 hNSC 区域性来调查 Zika 病毒关联的神经病11,14。通过使用三个个体捐献者的三株细胞, 我们也能够比较个体差异与 ZIKV 感染后观察到的神经性缺损的敏感性11。这项技术的一个局限是 hNSC 样品的可及性, 以及对适当的文化至关重要的条件培养基。为了绕过这一障碍, 请与相应的作者联系, 安排一个物质转移, 因为在这个协议中使用的单元不是商业可用的。
体外使用 hNSCs 的研究不仅提供了对基本干细胞行为的独到见解, 还为进行临床前期研究提供了良好的平台。然而, 培养 hNSCs 的技术挑战可以作为一个障碍, 使用它们作为有效和可重复的模型。在本议定书中, 我们概述了一些关键步骤和方法, 可用于减少培养方法的变异性, 并产生健康和稳定的 hNSC 干细胞培养。使用 hNSC 干细胞文化的缺点是, 细胞对任何刺激都非常敏感。即使有了上述详细的协议, 重要的是要密切注意 hNSCs 的烧瓶或菜肴的处理, 因为微妙的变化, 在协议可以导致变化的干细胞行为。
本方法最关键的部分是生长因子的鸡尾酒和不同培养基的制备。如果 hNSC 文化的加倍时间是非常低的, 或许多细胞 adherie 到文化船 (当他们应该是不附着的), 第一步是保证使用的成长因素是适当的集中, 并且不过期。本议定书的所有生长因子都有一个非常短的保质期, 一旦解冻 (5-6 天)。此外, 我们还利用了来自多家公司的增长因素, 并注意到维持文化所需的质量和集中的差异。因此, 我们强烈建议使用材料表中详细描述的精确增长因子。传代步骤 (步骤 3.1-3.22) 也是常见的错误来源。如果有许多死细胞在离解过程之后, 减少移的次数向上和向下离解细胞, 因为太多的物理离解可能导致细胞死亡。或者, 如果有许多细胞群在离解步之后, 增加移的活力或次数向上和向下, 因为这些星团将很快成为大球体, 将无法保持在文化中的可行性。如果文化得到适当的维护, 感染与 ZIKV 是相对简单的。
虽然 ZIKV 感染是本议定书中详述的治疗方法, 但可以使用这种 hNSC 文化系统进行广泛的研究。该模型系统可用于筛选药物, 检查个体易感性, 并调查各种疾病和发育障碍的基本机制14。这一系统也是理想的基因组研究, 因为培养细胞没有被基因操纵和显示很少到没有染色体异常, 甚至多达80通道10,11。我们相信这种培养体系是理想的模拟体外如何环境刺激, 如感染, 药物, 酒精, 毒素等, 可以影响神经系统的发展。

Zika 病毒 (ZIKV) 是一种黄传播的性行为, 或由蚊子媒介的伊蚊蚊和白纹伊蚊蚊子。ZIKV 最近被认为是一个严重的公共卫生威胁, 由于其易于传播和附属神经系统症状1。其中一个最有关的神经效应是发育的小头在胎儿出生的孕妇感染的母亲2,3。小头是一种神经发育障碍, 在胎儿发育和出生时, 其头部小于典型的大小, 周长低于平均4的2标准偏差。较小的头围通常伴有各种并存, 如发育迟缓、癫痫、视力和听力丧失以及喂养困难。
最近的研究使用动物模型或诱导多能干细胞来研究 ZIKV 感染对神经的影响5,6,7,8。虽然这些研究有助于我们对 ZIKV 的认识, 但使用不同物种或基因修饰的细胞可能会耗费时间和/或增加可能混淆 ZIKV 对开发神经细胞的影响的其他变量5,6,7,8. 然而, hNSC 文化的困难, 特别是本协议中描述的非粘附干细胞文化, 是文化对用于进行区域性9的方法非常敏感。培养基成分的任何变化, 甚至培养容器的物理处理, 都足以引起细胞9的反应。为了解决这些问题, 我们开发了一个体外人胎儿脑源性神经干细胞 (hNSCs) 培养机械询问 ZIKV 对胎儿神经干细胞的影响。使用我们的方法, hNSCs 保持超过80通道没有明显的表型变化10。此外, 染色体的改变, 如体是无或最小的11。这种 hNSC 文化成长为一种非附着的干细胞文化。球的一个优点是, 球体在文化中创造了一个独特的环境生态位, 比起二维区域性9更能反映在体内的利基。该协议的另一个优点是可以从 hNSC 文化中获得多种细胞类型, 从而使调查人员能够观察给定变量对 hNSC 生存和分化的影响。本议定书适用于寻求回答有关中枢神经系统发育或功能障碍的机械问题的个人。下面的协议描述了如何扩大 hNSC 的文化感染与 ZIKV, 并随后区分 hNSCs, 以观察感染的影响, 分化过程。它还包括存储 hNSCs 用于长期使用的方法, 并将 hNSCs 分化成各种类型的神经元, 以便进一步调查 ZIKV 引起的大脑畸形的缺陷11。我们相信, 这项议定书也有兴趣, 调查人员试图了解任何环境刺激, 如感染或毒素对神经干细胞的生存和分化的影响。

<table><tbody><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>F12</td><td>Gibco</td><td>11765-054</td><td></td></tr><tr><td>Glucose (10%)</td><td>Sigma</td><td>G8644</td><td></td></tr><tr><td>HEPES (1M)</td><td>Corning/cellgro</td><td>25-060-c1</td><td></td></tr><tr><td>Pen Strep (100x)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I4011</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T2036</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Heparin Sigma</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>basic fibroblast growth factor</td><td>R&amp;amp;D system</td><td>233-FB</td><td></td></tr><tr><td>epidermal growth factor</td><td>R&amp;amp;D system</td><td>236-EG</td><td></td></tr><tr><td>leukemia inhibitory factor</td><td>R&amp;amp;D system</td><td>7734-LF</td><td></td></tr><tr><td>Laminin</td><td>Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>2.5% trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>Sigma</td><td>T6522</td><td></td></tr><tr><td>Poly-D-Lysine hydrobromide(PDL)</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>B-27 supplement</td><td>Gibco/Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650-100ml</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning/cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A4378-25G</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson ImmunoResearch Lab</td><td>005-000-121</td><td></td></tr><tr><td>Triton X-100</td><td>FisherBiotech</td><td>BP151-500</td><td></td></tr><tr><td>Nestin antibody</td><td>BD Transduction Laboratories</td><td>611659</td><td>1:200 dilution</td></tr><tr><td>Class III beta-tubulin antibody (TuJ1)</td><td>Covnce</td><td>MMS-435p</td><td>1:2000 dulution</td></tr><tr><td>GFAP antibody</td><td>Millipore</td><td>AB5804</td><td>1:1000 dilution</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>1:2000 dilution</td></tr><tr><td>ZIKV antibody</td><td>World Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical Branch</td><td></td><td>1:2000 dilution</td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Forma</td><td>Model# 3110</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo fisher Scientific</td><td>75004221</td><td></td></tr><tr><td>Biological safety cabinet</td><td>Forma scientific Claas II A/B3</td><td>Model# 1284</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Forma scientific,Inc</td><td>model# 8516</td><td></td></tr><tr><td>Microscope(phase contrast image)</td><td>Hp 2230 workstation</td><td>Product#NOE25us#ABA</td><td></td></tr><tr><td>Microscope (epifluorescent image)</td><td>Nikon Eclipse 80i</td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon TE2000-E microscope with C1si confocal system</td><td></td><td></td></tr></tbody></table>

zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis

人胎脑神经干细胞是一种独特的非基因修饰的模型系统, 研究各种刺激对人类发育神经生物学的影响。而不是使用动物模型或基因修饰的诱导多能细胞, 人类神经干细胞提供了一个有效的体外系统, 以检查治疗效果, 筛选药物, 或检查个体差异。在这里, 我们提供了详细的协议, 用于扩展人类胎儿脑神经干细胞在无血清培养基中的培养, 通过不同的启动程序将它们分化成各种神经元亚型和星形胶质细胞, 并进行冷冻和恢复这些细胞。此外, 我们描述了一个使用人胎脑神经干细胞研究 Zika 病毒感染的过程。

培养的 hNSCs 在他们的增殖阶段将增长作为非黏附球 (图 1)。紧接在 hNSC 通道之后, 将会有许多单独的单元格, 它们将在接下来的几天内聚集并开始形成球体 (图 1A和1B)。健康的球体应该是大约1-2 毫米直径的9-10 天后的通道 (图 1C)。直径大于2毫米的球体会形成一个暗中心, 培养基中的生长因子将无法到达球体中心的细胞, 导致不必要的分化。健康的球体会出现半透明, 并在球体的边缘 (图 1C) 上显示伪纤毛。
在附着面上电镀球染色的目的将导致附着球体的生长 (图 2A)。这些球体将触及培养容器的表面, 并在保持一个中心球体 (图 2A) 的同时, 在容器表面延伸出一些突起。细胞的启动和分化要求细胞黏附在细胞培养板底部的盖子滑动, 例如24井板。在适当的启动步骤和加入分化培养基之后, 细胞将在培养容器的表面上扩散, 并作为相互连接的单层细胞生长 (图 2B)。
为了证实 hNSC 培养或分化细胞表型的有效性, 可以采用荧光免疫组织化学方法。巢是一种通常在神经干细胞中表达的中间灯丝, 可用于验证 hNSC 区域性 (图 3A)。为了验证分化后神经元的存在, III 类β-蛋白通常可以用来标记神经元 (图 3B)。虽然启动步骤是用来达到较高的百分比神经元后分化, 仍将有星形胶质细胞在文化。胶质纤维酸性蛋白(胶质蛋白) 可用于对星形胶质细胞进行标记, 以量化哪些百分比的分化单元成星形胶质或神经元。
我们发现, K048 线神经干细胞被感染的非洲和亚洲菌株的 ZIKV 检测荧光染色与特定 ZIKV 抗体。代表图像显示在图 4中。模拟感染没有引起 hNSC 形态学或生存率的变化 (图 4A, 4C)。ZIKV 倾向于留在感染细胞的外围区域一天后, 1 小时的感染, 但填补整个细胞在3至7天 (图 4B, 4D, 4E)。明显地, 与相同的莫伊 (0.1), 估计5% 感染率的 K048 细胞是大大低于最近报告的 ZIKV 感染率 (高达 80%) 在人类皮肤诱发神经干细胞7。这项研究, 报告80% 传染性, 使用的原型非洲菌株的 ZIKV (MR766), 可能已经适应了神经表型在140多个通道的小鼠脑细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56917/56917fig1.jpg"> 图 1: hNSC 文化的明亮字段图像.(A-C)在传代1、4和9天的 hNSC 培养基上分别有代表性的明亮场图像。缩放栏:60 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56917/56917fig2.jpg"> 图 2: 粘附区域性的明亮字段图像.(A) 在镀后7天内, 以10X 放大的贴附 hNSC 文化为代表的明亮的现场图像。(B) 具有代表性的明亮的现场图像, 在10X 放大后的粘附分化细胞, 在启动和9天的分化。缩放栏:60 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56917/56917fig3.jpg"> 图 3: hNSCs 和差异化文化的荧光图像.(A) 具有代表性的荧光图像, 在20X 放大的贴附 hNSCs 染色的核标记, DAPI (蓝色), 和干细胞标记巢(红色)。比例栏:60 µm (B) 有代表性的荧光图像, 在60X 放大的分化细胞染色的神经标记βIII-蛋白 (绿色), 星形胶质蛋白 (红色), 和核标记 DAPI (蓝色)。缩放栏:10 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56917/56917fig4.jpg"> 图 4: 人类胎儿脑源性神经干细胞 ZIKV 感染.人神经干细胞不是模拟治疗 (A 和 C), 接种1小时与非洲血统菌株的 ZIKV 在莫伊的 0.1 (B 和 D), 或与亚洲菌株 ZIKV 最近从蚊子在墨西哥 2016年 (E)。荧光染色检测神经干细胞中 ZIKV 抗体的标记, 在一个到七天后接种 (1 dpi 在 B, 7dpi 在 D, 3 dpi E)。缩放条形图10µm, 在 E. 中为5µm,<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig4large.jpg" target="_blank">请单击此处查看此图形的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>DFHGPS 媒体为一个 T75</td>
			<td>10毫升</td>
		</tr>
<tr>
<td>卟*</td>
			<td>173µL</td>
		</tr>
<tr>
<td>200毫米 l-Glutamin</td>
			<td>50µL</td>
		</tr>
<tr>
<td>10毫克/毫升胰岛素 **</td>
			<td>25µL</td>
		</tr>
<tr>
<td>20µg/毫升表皮生长因子</td>
			<td>10µL</td>
		</tr>
<tr>
<td>20µg/毫升碱性成纤维细胞生长因子</td>
			<td>10µL</td>
		</tr>
<tr>
<td>10µg/毫升白血病抑制剂因子</td>
			<td>10µL</td>
		</tr>
<tr>
<td>5毫克/毫升肝素</td>
			<td>10µL</td>
		</tr>
<tr>
<td colspan="2">* 含有100µg/毫升转铁蛋白、100µM putresine、20 nm 黄体酮和 30 nm 亚硒酸钠的混合物。 混合物由集中的存货, 并且等分被存放在-80 ° c 和 preferablly 使用在6星期之内准备。  集中储存的是10毫克/毫升转铁蛋白, 30 毫米腐, 10 µM 黄体酮和15µM 亚硒酸钠储存在-80 ˚C。</td>
		</tr>
<tr>
<td colspan="2">** 胰岛素被溶解在 0.01 N hydroen 氯化物, 过滤通过0.2 µM 低蛋白结合过滤器, 并存储在4˚C 6 周。</td>
		</tr>
</tbody></table>表 1: 新增殖培养基的生长因子。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>dPBS *</td>
			<td>1 mLa
</td>
			<td>3毫升b
</td>
		</tr>
<tr>
<td>10% 葡萄糖</td>
			<td>60µL</td>
			<td>180µL</td>
		</tr>
<tr>
<td>2.5% 胰蛋白酶</td>
			<td>10µL</td>
			<td>30µL</td>
		</tr>
<tr>
<td>dnasei **</td>
			<td>5µL</td>
			<td>15µL</td>
		</tr>
<tr>
<td colspan="3">* Dulbecco 的磷酸盐缓冲盐水</td>
		</tr>
<tr>
<td colspan="2">** Deoxyribonuclease</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">
a用于1000万单元格</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">
b用于3000万单元格</td>
			<td></td>
		</tr>
</tbody></table>表 2: 胰蛋白酶的制备。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>条件媒体</td>
			<td>1 mLa
</td>
			<td>3毫升b
</td>
		</tr>
<tr>
<td>胰蛋白酶抑制剂</td>
			<td>10µL</td>
			<td>30µL</td>
		</tr>
<tr>
<td>
a用于1000万单元格</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>
b用于3000万单元格</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 3: 胰蛋白酶抑制剂的制备。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>DFHGPS *</td>
			<td>0.7 mLa
</td>
			<td>2.1 毫升b
</td>
		</tr>
<tr>
<td>FBS ** 20%</td>
			<td>0.2 毫升</td>
			<td>0.6 毫升</td>
		</tr>
<tr>
<td>亚砜 ** 10%</td>
			<td>0.1 毫升</td>
			<td>0.3 毫升</td>
		</tr>
<tr>
<td colspan="3">* DMEM, F12, HEPES, 葡萄糖, 青霉素-链霉素</td>
		</tr>
<tr>
<td colspan="2">** 胎牛血清</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">二甲基亚砜</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">
a用于一个小瓶中的500万单元格</td>
			<td></td>
		</tr>
<tr>
<td colspan="3">
b用于三小瓶、500万细胞/小瓶</td>
		</tr>
</tbody></table>表 4: 冻结介质的制备。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>DFGHPS * 为24井板中的一井</td>
			<td>1毫升</td>
		</tr>
<tr>
<td>卟 **</td>
			<td>17.3 µL</td>
		</tr>
<tr>
<td>200毫米 l-谷氨酰胺</td>
			<td>5µL</td>
		</tr>
<tr>
<td>10毫克/毫升胰岛素</td>
			<td>2.5 µL</td>
		</tr>
<tr>
<td>20µg/毫升表皮生长因子</td>
			<td>1µL</td>
		</tr>
<tr>
<td>10µg/毫升白血病抑制因子</td>
			<td>1µL</td>
		</tr>
<tr>
<td>1毫克/毫升层粘连蛋白</td>
			<td>1µL</td>
		</tr>
<tr>
<td colspan="2">* 含 DMEM、F12、HEPES、葡萄糖、青霉素-链霉素</td>
		</tr>
<tr>
<td colspan="2">** 含100µg/毫升转铁蛋白, 100 µM putresine,</td>
		</tr>
<tr>
<td colspan="2">20纳米黄体酮和 30 nm 亚硒酸钠</td>
		</tr>
</tbody></table>表 5: 制备培养基。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>DFGHPS * 为24井板中的一井</td>
			<td>1毫升</td>
		</tr>
<tr>
<td>卟 **</td>
			<td>17.3 µL</td>
		</tr>
<tr>
<td>200毫米 l-谷氨酰胺</td>
			<td>5µL</td>
		</tr>
<tr>
<td>10毫克/毫升胰岛素</td>
			<td>2.5 µL</td>
		</tr>
<tr>
<td>20µg/毫升碱性成纤维细胞生长因子</td>
			<td>0.5 µL</td>
		</tr>
<tr>
<td>5毫克/毫升肝素</td>
			<td>0.5 µL</td>
		</tr>
<tr>
<td>1毫克/毫升层粘连蛋白</td>
			<td>1µL</td>
		</tr>
<tr>
<td colspan="2">* 含 DMEM、F12、HEPES、葡萄糖、青霉素-链霉素</td>
		</tr>
<tr>
<td colspan="2">** 含100µg/毫升转铁蛋白, 100 µM putresine,</td>
		</tr>
<tr>
<td colspan="2">20纳米黄体酮和 30 nm 亚硒酸钠</td>
		</tr>
</tbody></table>表 6: FHL 启动介质的制备。

Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

本文详细介绍了在培养人类胎儿脑神经干细胞的方法, 以及如何将它们区分成各种神经元亚型和星形胶质细胞, 重点是利用神经干细胞研究 Zika 病毒感染。

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.4K 次观看.  University of Texas Medical Branch. 本文详细介绍了在培养人类胎儿脑神经干细胞的方法, 以及如何将它们区分成各种神经元亚型和星形胶质细胞, 重点是利用神经干细胞研究 Zika 病毒感染。

视频: Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

免疫与感染

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

神经科学

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

应用脑注射策略建立小鼠 Zika 病毒诱发神经系统疾病模型: 胚胎、新生儿和成人

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Source: Salick, M. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56404">Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

在脑类器官中开发寨卡病毒感染的体外模型

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

Zika Virus Infection of Fetal Neural Stem Cells

Source: McGrath, E. L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018)
This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. J. Vis. Exp. (2018)
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Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

Source: Herrlinger, S. A. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56486">Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.</a> J. Vis. Exp. (2018)
The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

建立寨卡病毒诱导的成年小鼠神经退行性变模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult ...

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

Source: McGrath, E. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018).
This video demonstrates the method to differentiate human neural stem cells from neurospheres into neural and astrocyte progenitors using enzymatic treatment and culturing in a growth factor-rich medium, which eventually leads to maturation into specialized progenitor cells.

将人类神经干细胞分化为神经和星形胶质细胞祖细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Stock medium preparation and stem ...