角膜缘生态位细胞的分离和鉴定
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 使用无菌手术圆刀片刮擦并去除角膜周围的虹膜和内皮。 用手术刀片将角膜缘组织切成十二个大?…
Representative Results
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"…
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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）
Materials
| 4',6-Diamidino-2-Phenylindole | ThermoFisher | D1306 | 5μg/mL |
| Amphotericin B | Sigma | V900919 | 1.25 μg/mL |
| Anti-CD73 | Abcam | ab202122 | 1:50 |
| Bovine Serum Albumin | MERCK | A1933 | – |
| CD105 | Proteintech | 67075-1-Ig | 1:200 |
| CD105 | Abcam | ab114052 | 1:50 |
| CD90 | Proteintech | 66766-1-Ig | 1:100 |
| CD90 | Abcam | ab307736 | 1:50 |
| Cell Incubator | Shanghai Lishen | K1119K4644 | HF90(HT) |
| Centrifuge system | StatSpin | StatSpin CytoFuge 12 | – |
| Collagenase A | Roche | 10103578001 | 2 mg/mL |
| Confocal microscope | Zeiss | LSM700 | – |
| Culture plate | virya | 3500356 | 35 mm |
| DME/F-12 1:1 (1x) | cytiva | SH30023.01 | 90% |
| Donkey anti-Mouse IgG (H+L) Secondary Antibody | ThermoFisher | A16016 | 1:1000 |
| Donkey anti-rabbit IgG (H+L) Secondary Antibody | ThermoFisher | 31568 | 1:1000 |
| FACS Diva sofware | BD Biosciences | Tree Star | – |
| Flow Cytometer | BD Biosciences | Becton Dickinson LSRII | – |
| Fluorescence microscope | olympus | cx31 | |
| Gentamicin | Sigma | G1914 | 50 μg/mL |
| Hemocytometer | MERCK | Z359629 | Bright-Line |
| High-capacity cDNA Transcription Kit | ThermoFisher | 4374966 | |
| Inverted phase-contrast microscope | UOP | DSZ2000X | |
| ITS (insulin, transferrin, sodium selenite) | Sigma | I3146 | 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite |
| KnockOut SR Serum Replacement for ESCs/iPSCs | gibco | 10828-028 | 10% |
| Matrigel | BioCoat | 356234 | – |
| Pan-CK | Abcam | ab7753 | 1:1000 |
| Paraformaldehyde | NoninBio | NBS0135 | 4.00% |
| Paraformaldehyde | MKBio | MM-1505 | 4% |
| PDGFRβ | Abclonal | A1444 | 1:100 |
| Real-time fluorescence quantitative PCR instrument | Applied Biosystems | Step One Plus | – |
| Recombinant Human FGF-basic | Peprotech | 100-18B | 4 ng/mL |
| Recominant Human Leukemia Inhibitory Factor(Lif) | Peprotech | 300-05 | 10 ng/mL |
| RNeasy Mini RNA Isolation Kit | Qiagen | 74104 | – |
| SCF | Bioss | bs-0545R | 1:100 |
| SCF | Abcam | ab52603 | 1:50 |
| Stereomicroscope | ZEISS | SteREO Discovery. V8 | |
| Sterile surgical round blade | Careforde | 29500 | size 10 |
| TaqMan Gene Expression Assay Mix | Applied Biosystems | 4448489 | |
| Triton X-100 | MERCK | X100 | 0.20% |
| Trypan blue | ThermoFisher | 15250061 | 0.40% |
| Trypsin-EDTA | Genview | GP3108 | 0.25% |
| Tween 20 | MERCK | P9416 | – |
| Ultra Clean Bench | LaiTe | LT20200705 | SW-CJ-IFDG |
| Universal PCR Master Mix | Applied Biosystems | 4304437 | |
| Vim | Abcam | ab92547 | 1:100 |
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Citer Cet Article
Su, G., Wang, W., Xu, L., Liu, R., Tan, Y., Jin, B., You, C., Zhou, X., Xiong, Y., Xie, H., Li, G. Isolation and Identification of Limbal Niche Cells. J. Vis. Exp. (200), e65618, doi:10.3791/65618 (2023).