Name: the organoid reconstitution assay (ora) for the functional analysis of intestinal stem and niche cells
Uploaded: 2017-11-20T13:00:00
Description: Watch this Scientific Journal Video about The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells at JoVE.com

这项研究是由荷兰癌症协会 (凯) 资助的。EMCR 2012-5473) 和世界癌症研究基金国际 (WCRF; 2014-1181 号项目)

所有的程序都是根据当地的动物福利法和指导方针进行的。
1. 文书、文化媒体和菜肴的准备
<ol><li>高压釜1套肠剪刀、普通剪刀和无菌容器中的镊子。</li>
	<li>在37° c 的恒温箱中放置一个96井 (平底) 盘。</li>
	<li>用材料表中所列试剂制备10毫升的完整培养基。</li>
	<li>在37° c 的水浴中孵育整个培养基。</li>
	<li>把地下室的膜放在冰桶里融化。重组后的基底膜将在4° c 时变为液?...

<ol>
	<li>Barker, 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=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li><li>Sato, 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=Paneth+cells+constitute+the+niche+for+Lgr5+stem+cells+in+intestinal+crypts.">Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.</a> Nature. 469, 415-418 (2011).</li><li>Sato, 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Development+and+Disease+with+Organoids.">Modeling Development and Disease with Organoids.</a> Cell. 165, 1586-1597 (2016).</li><li>Cantrell, M. A., Kuo, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+modeling+for+cancer+precision+medicine.">Organoid modeling for cancer precision medicine.</a> Genome Med. 7, 32 (2015).</li><li>Schewe, 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=Secreted+Phospholipases+A2+Are+Intestinal+Stem+Cell+Niche+Factors+with+Distinct+Roles+in+Homeostasis,+Inflammation,+and+Cancer.">Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.</a> Cell Stem Cell. 19, 38-51 (2016).</li><li>Roth, S., 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=Paneth+cells+in+intestinal+homeostasis+and+tissue+injury.">Paneth cells in intestinal homeostasis and tissue injury.</a> PLoS One. 7, e38965 (2012).</li><li>Rodriguez-Colman, M. J., 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=Interplay+between+metabolic+identities+in+the+intestinal+crypt+supports+stem+cell+function.">Interplay between metabolic identities in the intestinal crypt supports stem cell function.</a> Nature. 543, 424-427 (2017).</li><li>Dow, L. E., 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=Apc+Restoration+Promotes+Cellular+Differentiation+and+Reestablishes+Crypt+Homeostasis+in+Colorectal+Cancer.">Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.</a> Cell. 161, 1539-1552 (2015).</li><li>Yilmaz, O. H., 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=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li><li>Igarashi, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+and+SIRT1+Cooperate+to+Foster+Expansion+of+Gut+Adult+Stem+Cells+during+Calorie+Restriction.">mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction.</a> Cell. , 436-450 (2016).</li></ol>

该方法允许对肠道干细胞生态位的两个基本成分, 即Lgr5+和氏细胞进行精细的功能分析。此方法以前已被我们和其他一些轻微修改6,10,11。在这里, 我们提出了作为一个可重复和标准化的实验室协议的过程。此外, 我们报告的影响, Apc下调在Lgr5+干细胞, 作为一个例子的可能性实施遗传 (或生物化...

肠上皮是哺乳动物体内最快速的更新组织, 因此, 它一直是大量研究的对象, 目的是在隐窝底部的成体干细胞的鉴定和功能表征。Lieberkühn, 由Lgr5基因的表达式指定, 并依赖于标准 Wnt 信号1。值得注意的是, Lgr5+干细胞是由专门的小生境细胞 (氏细胞) 所支持的, 它也依赖于其成熟度 2的 Wnt 信号。同时, 这两种细胞类型的基础上的肠道上皮内膜的自我更新和保持每日的稳态平衡: Lgr5+ 干细胞迅速分化并引起祖细胞和更专门的肠上皮细胞;氏细胞提供必要的利基因素 (如, Dll1, Wnt3, EGF) Lgr5+ 干细胞2。肠道棺的能力, 当镀前体形成组织和更新结构称为 organoids, 或 "mini-guts", 已被作为一个实验工具, 以提供洞察的过程, 如自我更新和正常和病理条件下的分化, 包括癌症4。从老鼠和人类样本中的几个组织, 包括肠道、胰腺、肝脏和肾脏, 都建立了化的培养4。这些化培养物的提取方法和生长因子是组织特异性的, 其目的是促使多分化, 并尽可能地模仿原干细胞生态位在体内.Organoids 可能有潜在的应用, 包括基因疾病的治疗, 对癌症的疗效评估, 药物毒性分析, 或器官的研究体外5。
总的来说, 从组织标本中建立的化文化的主要局限是缺乏细胞特异性。例如, 肠道 organoids 建立从整个肠道棺不允许分析的个别细胞成分包含在组织来源 (如, 整个棺包含Lgr5+, 氏, 和祖细胞)。
在这里, 我们描述了一种新的方法, 称为化, 结合了肠道的优势, 其最基本的组成部分, 即词干 (Lgr5+) 和利基 (氏) 细胞的功能分析。这是通过氏和Lgr5+单元格的独特功能实现的, 当 co-incubated 并引发 organoids2、610时, 它们之间的物理关联。我们利用这一功能, 并预处理两个细胞类型, 然后再允许他们重建 organoids。当这样做时, 每个细胞成分可以暴露在任何给定的药物, 生长因子, 生化抑制剂, 基因改造, 或化学处理之前, 重组和化形成。因此, 使用 "化验" 将允许确定特定药物治疗或基因修饰对干细胞或其生态位对应物是否有特定的影响。

<table>
	<tbody>
		<tr>
			<td>Advanced DMEM F12 *</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax *</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Final concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin *</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Final concentration: 1%</td>
		</tr>
		<tr>
			<td>Hepes *</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>Final concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant murine EGF *</td>
			<td>Thermo Fisher Scientific</td>
			<td>PMG8041</td>
			<td>Final concentration: 50 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant murine Noggin *</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Final concentration: 100 ng/ml</td>
		</tr>
		<tr>
			<td>y27632 *</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Final concentration: 10 &#181;M</td>
		</tr>
		<tr>
			<td>Jagged-1 *</td>
			<td>Anaspec Bio</td>
			<td>AS-61298</td>
			<td>Final concentration: 1 &#181;M</td>
		</tr>
		<tr>
			<td>Recombinant murine R-spondin *</td>
			<td>R&#38;D systems</td>
			<td>3474-RS-050</td>
			<td>Final concentration: 1 mg/ml</td>
		</tr>
		<tr>
			<td>Flexitube Gene solution siRNA APC</td>
			<td>Qiagen</td>
			<td>GS11789</td>
			<td>Final concentration: 100 nM</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24 APC</td>
			<td>Biolegend</td>
			<td>101814</td>
			<td></td>
		</tr>
		<tr>
			<td>c-kit PE</td>
			<td>Biolegend</td>
			<td>105808</td>
			<td></td>
		</tr>
		<tr>
			<td>BV 421 CD31</td>
			<td>Biolegend</td>
			<td>102424</td>
			<td></td>
		</tr>
		<tr>
			<td>BV 421 CD45</td>
			<td>Biolegend</td>
			<td>103134</td>
			<td></td>
		</tr>
		<tr>
			<td>BV421 TER119</td>
			<td>Biolegend</td>
			<td>116234</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14180046</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129P2-Lgr5tm1(cre/ERT2)Cle/J</td>
			<td>Jackson Laboratories</td>
			<td>008875</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding tubes</td>
			<td>Eppendorf</td>
			<td>Z666548-250EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>* The combination of these reagents constitutes the complete culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the organoid reconstitution assay (ora) for the functional analysis of intestinal stem and niche cells

小肠上皮的特点是以极快的速度流动。在哺乳动物中, 整个上皮衬里在 4-5 天内更新。成体肠道干细胞位于 Lieberkühn 棺的底部, 由Lgr5基因的表达指定, 并通过其特有的高增殖率1保持稳态。在整个小肠中, Lgr5+ 干细胞与称为氏细胞的特殊分泌细胞混合在一起。氏细胞分泌抗菌化合物 (即、溶菌酶和凹/防御), 并对肠道菌群发挥控制作用。最近, 发现了一个新的功能, 氏细胞, 即他们的能力提供利基支持的Lgr5+干细胞通过几个关键配体作为 Wnt3, EGF, 和 Dll12。
当孤立的ex 体内和培养在特定生长因子和细胞外基质成分的存在下, 整个肠道棺产生长寿命和更新3D 结构称为 organoids, 高度类似于地穴绒毛成人小肠的上皮结构3。化的文化, 当建立从整个棺, 允许研究的自我更新和分化的肠道干细胞利基, 但没有解决的贡献, 其各个组成部分, 即Lgr5+和氏细胞。
在这里, 我们描述了一种新的方法, 化分析利用的能力, 氏和Lgr5+细胞关联和形成 organoids 时共。这种方法, 这里称为 "化重构化验" (), 允许遗传和生物化学的修改氏或Lgr5+ 干细胞, 然后重组成 organoids。因此, 它允许功能分析的两个主要组成部分的肠道干细胞利基。

化重构试验允许对小肠上皮细胞的基本生态位和干细胞成分进行单独的功能分析, 这是由Apc基因的小干扰 RNA (siRNA) 证明的。
为了达到这个目的, 我们首先利用荧光活化细胞分类 () 分离 8000 Lgr5+细胞和6000氏细胞从近亲 C57BL6/J 小鼠。在图 1a中, 描述了该方法的流程图。化重组能力的评估...

Watch this Scientific Journal Video about The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells at JoVE.com

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

化重构法 (的) 对肠道干细胞和小生境细胞功能分析的研究

肠道化文化是建立在整个棺, 不允许分析自我更新和分化的细胞特有的方式。本协议描述了对排序后的词干 (Lgr5+) 和小生境 (氏) 单元的重构, 这些细胞在进行生物化学和遗传修饰和功能分析之前, 会产生 organoids。

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

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The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

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Stem Cell Biology And Renewal in Epithelial Tissue

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