感谢王伟、徐玲娟、刘荣对这项工作的指导，感谢谭永瑶、金碧辉、尤春秀和李贵刚提供部分材料，感谢苏冠宇撰写手稿，感谢周小、熊一红、谢华涛对稿件的校正，感谢李贵刚的全力指导。本研究得到了国家自然科学基金（82070936、81470606、81570819）、湖北省卫生与计划生育科研项目（No.WJ2017M073）、同济医院转化医学研究十大项目（No.2016ZHYX20）、武汉市医学青年先锋培养项目（No.2015whzqnyxggrc10）、全球人才招聘计划（G2022154028L）、湖北省国家卫健委2022年度项目（WJ2021ZH0005）、湖北省财政部学科建设基金（42000022815T000000102）

年龄在50至60岁之间的供体的角膜缘组织来自同济医院（中国武汉）红十字眼库。该协议得到了同济伦理委员会的批准，并按照《赫尔辛基宣言》进行。
1. 隔离
<ol><li>从中期角膜储存培养基中获取角膜缘组织，并在超洁净工作台上在无菌条件下操作。</li>
	<li>使用无菌手术圆刀片刮擦并去除角膜周围的虹膜和内皮。</li>
	<li>用手术刀片将角膜缘组织切成十二个大小相等的碎片，角膜和巩膜两侧留有 1 毫米。</li>
	<li>将角膜缘组织转移到 3.5 cm 培养板中，加入 1 mL 胶原酶 A（2 mg/mL，在 DF-12 培养基中），浸入所有组织进行消化。</li>
	<li>将板放入37°C细胞培养箱中，消化角膜缘组织18小时。 注意:后续程序通常在第二天进行。</li>
	<li>准备5%基底膜基质（基质胶）包被的6孔培养板。<ol><li>在孔的底部涂覆5%基底膜基质（溶解在MESCM [表1]中）。</li>
		<li>在灭菌袋中准备 200 μL 和 1 mL 吸头、一个 15 mL 离心管和一个 6 孔板。</li>
		<li>将灭菌袋（包括吸头和离心管）和新的6孔板置于-20°C或-80°C环境中20分钟。在4°C的冰箱中解冻基底膜基质。</li>
		<li>从-20°C或-80°C环境中取出预冷的吸头、离心管和6孔板，然后在超净工作台上进行以下操作。</li>
		<li>使用 200 μL 吸头将 50 μL 5% 基底膜基质转移到 1 mL MESCM 中，并轻轻均匀地混合。</li>
		<li>使用预冷的 1 mL 吸头将 5% 基底膜基质（溶解在 MESCM 中）转移到 6 孔培养板的一个孔中。</li>
		<li>轻轻水平摇动6孔板，将6孔培养板置于37°C细胞培养箱中约1小时，然后进行下一步。</li>
	</ol></li>
	<li>消化18小时后，取出胶原酶A消化的角膜缘组织，只剩下一些可见的小簇和未消化的巩膜组织。 注:高倍率显微镜揭示了一个由许多密集堆积的细胞（包括LNC）组成的簇。</li>
	<li>使用200μL移液器吸头在立体显微镜下从未消化的巩膜组织中分离簇。</li>
	<li>将簇转移到3.5cm培养板中，并将它们浸入0.25%TE中，然后将板置于细胞培养箱中15分钟。</li>
	<li>加入 1 mL MESCM 和 10% 敲除血清以停止细胞消化，并使用 1 mL 移液器吸头轻轻上下移液以将簇重悬到单个细胞中。</li>
	<li>将细胞悬液转移到 15 mL 离心管中，并以 200 x g 离心 5 分钟。</li>
	<li>小心地除去上清液，不要干扰细胞沉淀，然后加入 1 mL MESCM 以重悬细胞。</li>
	<li>上下移液2-3次，直至底部细胞沉淀均匀悬浮于MESCM中，使用1mL移液器吸头，取20μL细胞悬液进行细胞计数。</li>
	<li>用 1 mL 移液器吸头将细胞悬液转移到 5% 基底膜基质包被的 6 孔板中，并将 MESCM 的体积增加到 2.5 mL。</li>
	<li>水平轻轻摇动 6 孔板以均匀分布细胞。</li>
	<li>成像和记录后将细胞转移到培养箱中。每天观察和拍摄细胞，每3-4天更换一次培养基。</li>
</ol>2. LNC的鉴定
<ol><li>免疫荧光染色<ol><li>在 37°C 细胞培养箱中使用 1 mL 0.25% TE 消化板底部的 LNC 5 分钟。</li>
		<li>通过加入 1 mL MESCM（含有敲除血清）终止消化，将细胞悬液以 200 x g 离心 5 分钟，并小心吸出上清液。</li>
		<li>加入 1 mL MESCM 以重悬悬于悬浮液中的细胞。为 4 张载玻片（20 μL/载玻片）制备 80 μL 细胞悬液（每张载玻片 4 ×10 个 3 个 细胞），并按照制造商的说明使用离心机系统将细胞沉积到显微镜载玻片上。</li>
		<li>使用4%多聚甲醛固定细胞（在步骤3中粘附在载玻片上）约10分钟，然后用0.2%Triton X-100在PBS中透化载玻片上的细胞15分钟。</li>
		<li>用2%牛血清白蛋白（BSA）封闭细胞1小时，然后将它们与一抗（Pan-CK [1:1000]，Vim [1:100]，CD90 [1:100]，CD105 [1:200]，SCF [1:100]，PDGFRβ [1:100]）在振荡器上在4°C下孵育过夜。 孵育后，通过用PBST（PBS + 0.1%吐温20）洗涤（5分钟/洗涤，3x）载玻片除去未结合的一抗。</li>
		<li>将相应的二抗（驴抗小鼠 IgG 二抗 TRITC [1:1000]、驴抗兔 IgG FITC [1:500]、驴抗小鼠 IgG 488 [1:300]、驴抗兔 IgG 594 [1:400]）与合适的 IgG 非特异性 IgG 抗体孵育 1 小时。使用PBST（5分钟/洗涤，3x）除去未结合的二抗。</li>
		<li>用 5 μg/mL DAPI（4'，6-二脒基-2-苯基吲哚）对细胞核进行复染并密封载玻片。</li>
		<li>使用荧光显微镜（放大倍率:400倍;曝光时间:50 毫秒）。</li>
	</ol></li>
	<li>实时荧光定量聚合酶链反应（qPCR）<ol><li>按照制造商的说明，使用RNA分离试剂盒提取总RNA。</li>
		<li>使用高容量 cDNA 转录试剂盒将 1-2 μg 总 RNA 逆转录为 cDNA。</li>
		<li>在含有cDNA（100ng），TaqMan基因表达检测试剂盒（1μL），通用PCR预混液（10μL）和ddH 2O（至20μL）的20μL溶液中进行逆转录（42°C，2分钟;50°C，85分钟）和qPCR（95°C，5分钟;95°C，10秒，40个循环）。</li>
		<li>使用比较 CT 技术 （ΔΔCT）4,9 检查相对基因表达。</li>
	</ol></li>
</ol>3. LNC的表征
<ol><li>细胞计数（血细胞计数器）<ol><li>在37°C细胞培养箱中使用0.25%TE从板底部消化LNC5分钟。</li>
		<li>通过加入 1 mL MESCM（含有敲除血清）终止消化，将细胞悬液以 200 x g 离心 5 分钟，并小心吸出上清液。</li>
		<li>加入 1 mL MESCM 以重悬细胞，然后在 0.5 mL 离心管中取 10 μL 细胞悬液。加入等体积的0.4%台盼蓝染色液。</li>
		<li>将 10 μL 染色的细胞悬液放在血细胞计数器的计数区域，并用盖玻片盖住进行计数。 注意:五个中间正方形区域中的单元格数定义为"A";1mL中的细胞数计算为5A×10,4×2。</li>
	</ol></li>
	<li>使用流式细胞术检查 LNC 和 BMMSC 标志物15<ol><li>使用流式细胞术15 检测 LNC 和 BMMSC 中 SCF、CD73、CD90 和 CD105 的表达（2-4 ×10 6 个事件/样品，从每个样品中获得 2,000 个细胞，总共 550,000 个同时设门的细胞）。</li>
		<li>根据试剂盒说明，使用预标记抗体（SCF [1:50]、CD70 [1:50]、CD90 [1:50]和CD105 [1:50]）在封闭缓冲液（3%BSA和0.05%吐温-20的PBS）中收集和染色细胞15分钟。</li>
		<li>使用流式细胞仪进行荧光激活细胞分选 （FACS） 分析。在这项研究中，使用了 FACS Diva 软件和 FlowJo 软件。对于每个样品，记录 10,000 个事件并设门并分析活细胞。</li>
	</ol></li>
</ol>

分离、培养和鉴定人角膜缘生态位细胞的方案

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+mucosa+tissue+equivalents+for+the+treatment+of+limbal+stem+cell+deficiency.">Oral mucosa tissue equivalents for the treatment of limbal stem cell deficiency.</a> Advanced Biosystems. 4 (7), e1900265 (2020).</li><li>Yu, D., Chen, M., Sun, X., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+induced+pluripotent+stem+cells+into+corneal+epithelial-like+cells.">Differentiation of mouse induced pluripotent stem cells into corneal epithelial-like cells.</a> Cell Biology International. 37 (1), 87-94 (2013).</li><li>Zeppieri, 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=Adipose-derived+stem+cells+for+corneal+wound+healing+after+laser-induced+corneal+lesions+in+mice.">Adipose-derived stem cells for corneal wound healing after laser-induced corneal lesions in mice.</a> Journal of Clinical Medicine. 6 (12), 115 (2017).</li><li>Kumar, A., Kumar, V., Rattan, V., Jha, V., Bhattacharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+cues+modulate+the+neurogenic+potential+of+bone+marrow+and+dental+stem+cells.">Secretome cues modulate the neurogenic potential of bone marrow and dental stem cells.</a> Molecular Neurobiology. 54 (6), 4672-4682 (2017).</li><li>Hayashi, R., 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=Coordinated+generation+of+multiple+ocular-like+cell+lineages+and+fabrication+of+functional+corneal+epithelial+cell+sheets+from+human+iPS+cells.">Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.</a> Nature Protocols. 12 (4), 683-696 (2017).</li><li>Guo, 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=Limbal+niche+cells+are+a+potent+resource+of+adult+mesenchymal+progenitors.">Limbal niche cells are a potent resource of adult mesenchymal progenitors.</a> Journal of Cellular and Molecular Medicine. 22 (7), 3315-3322 (2018).</li><li>Wang, W., 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=Differential+gene+expression+between+limbal+niche+progenitors+and+bone+marrow+derived+mesenchymal+stem+cells.">Differential gene expression between limbal niche progenitors and bone marrow derived mesenchymal stem cells.</a> International Journal of Medical Sciences. 17 (4), 549-557 (2020).</li><li>Gonz&#225;lez, S., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+native+limbal+stromal+cells+increases+the+expansion+efficiency+of+limbal+stem/progenitor+cells+in+culture.">Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture.</a> Experimental Eye Research. 116, 169-176 (2013).</li><li>Funderburgh, M. L., Du, Y., Mann, M. M., SundarRaj, N., Funderburgh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX6+expression+identifies+progenitor+cells+for+corneal+keratocytes.">PAX6 expression identifies progenitor cells for corneal keratocytes.</a> FASEB Journal. 19 (10), 1371-1373 (2005).</li><li>Funderburgh, J. L., Funderburgh, M. L., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+limbal+stroma.">Stem cells in the limbal stroma.</a> Ocular Surface. 14 (2), 113-120 (2016).</li><li>Chen, S. Y., Hayashida, Y., Chen, M. Y., Xie, H. T., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+isolation+method+of+human+limbal+progenitor+cells+by+maintaining+close+association+with+their+niche+cells.">A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells.</a> Tissue Engineering. Part C, Methods. 17 (5), 537-548 (2011).</li><li>Sato, T., 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=SnapShot:+Growing+organoids+from+stem+cells.">SnapShot: Growing organoids from stem cells.</a> Cell. 161 (7), 1700-1701 (2015).</li></ol>

所有作者均未披露任何相关的财务信息。

角膜透明度通常通过角膜基质中小纤维（直径 25-30 nm）的规则排列和分布来维持，这对于正常视力至关重要16。全世界有 2.53 亿视障者，其中 3600 万人是盲人17.世界卫生组织 （WHO） 认为角膜盲是人类视力最严重的危害之一，占全球所有失明的 5.1%16。CES正常的角膜上皮缺损可以快速愈合而不会留下疤痕18。据估计，全球有超过...

角膜上皮干细胞缺乏症（CESD），也称为角膜缘干细胞缺乏症（LSCD）1，角膜上皮再生（CES）的发病率因角膜感染和损伤而变得越来越紧迫。如果治疗不当，CESD可能导致失明，需要角膜移植。因此，CES再生变得越来越重要。有一组称为角膜缘生态位细胞 （LNC） 的支持细胞为 CES 功能提供必要的支持。角膜缘基质干细胞首先由Polisetty等2分离，Xie等3鉴定为LNC，定位于角膜缘的角膜缘上皮和角膜缘的基质。LNCs是角膜边缘的关键支持干细胞，具有骨髓来源的MSCs（BMMSCs）的功能，可诱导发育成角膜上皮细胞和角膜基质细胞等3,4,5,6,7。先前的研究表明，LNCs的干细胞质量比BMMSCs8更原始，BMMSCs8已经在临床上被广泛使用。LNC甚至可能成为继MSC之后的下一个可行选择，特别是用于治疗CESD。作为CES的重要支持细胞，LNC也是源自角膜缘"生态位"结构的干细胞。LNC 可能在成熟角膜上皮细胞 （MCEC） 向 CES9 的去分化中发挥关键作用。然而，对LNCs的研究仍然相对不足，对LNCs的术语、分离、纯化、鉴定和特性尚未达成共识。一些研究人员将 LNC 命名为角膜缘活检来源的基质干细胞10、角膜缘间充质干细胞11、角膜缘成纤维细胞干细胞 12 和角膜缘间充质基质细胞13。由于LNCs的生长特性尚未详细描述，且由于其科学和临床应用前景广阔，并且可能是未来最重要的临床工具之一，因此有必要总结LNCs的分离、纯化、鉴定和特性。
根据先前的一项研究14，LNC主要存在于角膜缘上皮下和角膜缘的基质中。该方案包括使用胶原酶A处理角膜缘组织，获得由LEPC和LNC组成的簇，并将其消化成含有0.25%胰蛋白酶-EDTA（TE）的单细胞。然后将LNCs选择性地培养在改良的胚胎干细胞培养基（MESCM）中进行纯化。本文报道的方案简单，在大量获得人LNCs方面具有很高的效率。
视频中记录了LNC分离、培养和鉴定的详细过程，供对LNC研究感兴趣的科学家使用，必要时可以方便地重复。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>5&#956;g/mL</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>V900919</td>
			<td>1.25 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-CD73</td>
			<td>Abcam</td>
			<td>ab202122</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MERCK</td>
			<td>A1933</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CD105</td>
			<td>Proteintech</td>
			<td>67075-1-Ig</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>CD105</td>
			<td>Abcam</td>
			<td>ab114052</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>Proteintech</td>
			<td>66766-1-Ig</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>Abcam</td>
			<td>ab307736</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Shanghai Lishen</td>
			<td>K1119K4644</td>
			<td>HF90(HT)</td>
		</tr>
		<tr>
			<td>Centrifuge system</td>
			<td>StatSpin&#160;</td>
			<td>StatSpin CytoFuge 12</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10103578001</td>
			<td>2 mg/mL</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss&#160;</td>
			<td>LSM700</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Culture plate</td>
			<td>virya</td>
			<td>3500356</td>
			<td>35 mm</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)&#160;</td>
			<td>cytiva</td>
			<td>SH30023.01</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>A16016</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>31568</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>FACS Diva sofware</td>
			<td>BD Biosciences</td>
			<td>Tree Star</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>Becton Dickinson LSRII</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>olympus</td>
			<td>cx31&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1914</td>
			<td>50 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>MERCK</td>
			<td>Z359629</td>
			<td>Bright-Line</td>
		</tr>
		<tr>
			<td>High-capacity cDNA Transcription Kit</td>
			<td>ThermoFisher</td>
			<td>4374966</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase-contrast microscope</td>
			<td>&#160;UOP</td>
			<td>DSZ2000X</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS (insulin, transferrin, sodium selenite)</td>
			<td>Sigma</td>
			<td>I3146</td>
			<td>5 &#956;g/mL insulin, 5 &#956;g/mL transferrin, 5 ng/mL sodium selenite</td>
		</tr>
		<tr>
			<td>KnockOut SR Serum Replacement for ESCs/iPSCs</td>
			<td>gibco</td>
			<td>10828-028</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BioCoat</td>
			<td>356234</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pan-CK</td>
			<td>Abcam</td>
			<td>ab7753</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>NoninBio</td>
			<td>NBS0135</td>
			<td>4.00%</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MKBio</td>
			<td>MM-1505</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>PDGFR&#946;</td>
			<td>Abclonal</td>
			<td>A1444</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Real-time fluorescence quantitative PCR instrument</td>
			<td>Applied Biosystems</td>
			<td>Step One Plus</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>4 ng/mL</td>
		</tr>
		<tr>
			<td>Recominant Human Leukemia Inhibitory Factor(Lif)</td>
			<td>Peprotech</td>
			<td>300-05</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini RNA Isolation Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>-</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>Bioss</td>
			<td>bs-0545R</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>Abcam</td>
			<td>ab52603</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>ZEISS</td>
			<td>SteREO Discovery. V8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical round blade</td>
			<td>Careforde</td>
			<td>29500</td>
			<td>size 10</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Mix</td>
			<td>Applied Biosystems</td>
			<td>4448489</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>MERCK</td>
			<td>X100</td>
			<td>0.20%</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFisher</td>
			<td>15250061</td>
			<td>0.40%</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Genview</td>
			<td>GP3108</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>MERCK</td>
			<td>P9416</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ultra Clean Bench</td>
			<td>LaiTe</td>
			<td>LT20200705</td>
			<td>SW-CJ-IFDG</td>
		</tr>
		<tr>
			<td>Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Vim&#160;</td>
			<td>Abcam</td>
			<td>ab92547</td>
			<td>1:100</td>
		</tr>
	</tbody>
</table>

isolation and identification of limbal niche cells

在这里，我们报告了分离和鉴定角膜缘生态位细胞（LNC）的标准程序。从眼库获得的角膜缘组织用于LNCs分离。在无菌条件下将组织分成12块，并在37°C下在细胞培养箱中消化18小时，使用胶原酶A获得具有LNC和角膜缘上皮祖细胞的细胞簇。使用0.25%胰蛋白酶-EDTA在37°C下进一步消化细胞簇15分钟以获得单细胞，然后在涂有5%基质凝胶的塑料表面上在改良的胚胎干细胞培养基（MESCM）中培养。细胞在 70% 汇合时传代，并使用免疫荧光、实时定量 PCR （qPCR） 和流式细胞术鉴定 LNC。原代 LNC 被分离并传代超过 12 次。从P4到P6的LNCs增殖活性最高。LNCs表达的干细胞标志物高于BMMSC（SCF、Nestin、Rex1、SSEA4、CD73、CD90、MSX1、P75NTR和PDGFRβ）。结果显示，P4 LNCs均均匀表达VIM、CD90、CD105和PDGFRβ，但不均匀表达Pan-CK，可作为LNCs鉴定的标志物。 流式细胞分析显示，约95%、97%、92%和11%的LNCs分别表达CD73、CD90、CD105和SCF，而在BMMSCs中分别表达68%、99%、20%和3%。LNCs分离鉴定的标准工艺可为LNCs的广泛应用提供可靠的实验室依据。

LNC的成长 如上所述，根据胶原酶A（2mg / mL）消化角膜巩膜边缘组织的酶切方法成功分离LNC（图1）。与先前报道的研究3一致，在胶原酶A消化后，在显微镜下观察到毛毛虫样簇（图2）。纺锤体细胞的比例随着细胞传代而逐渐增加。纺锤形细胞可以在包被的 5% 基底膜基质板上生长，这与在没有包被基底膜基质<sup class="xref"...

Watch this Scientific Journal Video about Standardizing Limbal Niche Cell LNC Isolation and Characterization to Support Widespread LNC Research at JoVE.com

作者聚焦:标准化角膜缘生态位细胞 （LNC） 分离和表征以支持广泛的 LNC 研究 

在这里，我们提出了一种分离和鉴定人类角膜缘生态位细胞的方案。

角膜缘生态位细胞的分离和鉴定

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

我们的研究重点是重建角膜缘生态位，以治疗角膜缘干细胞缺乏症并恢复患者的视力。我们正试图回答角膜缘生态位细胞是否是支持角膜缘干细胞功能的关键细胞，如果是，它是如何工作的。最近的研究报告称，从骨髓中移植间充质干细胞的结膜下移植可预防碱烧伤眼睛的角膜缘干细胞缺乏症。目前用于推进我们研究的技术之一是单细胞测序，它可以在单细胞水平上分析关键信号通路活性，以进行角膜缘生态位细胞表征。尽管LNC有潜力成为未来重要的临床工具，但其生长特性尚未得到过多的探索。考虑到LNC在科学和临床上的应用前景广阔，有必要对LNC的分离、纯化、鉴定和特性进行总结。

isolation-and-identification-of-limbal-niche-cells

Research

JoVE Journal

Medicine

Isolation and Identification of Limbal Niche Cells

905 次观看.  Huazhong University of Science and Technology. 我们的研究重点是重建角膜缘生态位，以治疗角膜缘干细胞缺乏症并恢复患者的视力。我们正试图回答角膜缘生态位细胞是否是支持角膜缘干细胞功能的关键细胞，如果是，它是如何工作的。最近的研究报告称，从骨髓中移植间充质干细胞的结膜下移植可预防碱烧伤眼睛的角膜缘干细胞缺乏症。目前用于推进我们研究的技术之一是单细胞测序，它可以在单细胞水平上分?...

视频: 作者聚焦:标准化角膜缘生态位细胞 （LNC） 分离和表征以支持广泛的 LNC 研究 

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

无标记的分离和细胞的富集通过非接触式介电泳

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

一个简单的机械过程来创建在小鼠角膜缘干细胞缺乏症

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...