A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells.
A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells.
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一种通过与微环境细胞保持密切联系来分离人泪小管上皮干细胞的新方法
DOI：
10.7150/ijms.27705
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发表时间：
2018
影响因子：
3.6
通讯作者：
Li GG
中科院分区：
文献类型：
--
作者：
Hu W;Zhang Y;Tighe S;Zhu YT;Li GG
目的：探讨泪小管上皮干细胞（LCESC）能否在体外分离扩增。方法：采用泪道内窥镜对6例因碱烧伤或史蒂文斯·约翰逊综合征引起的角膜缘干细胞缺乏症（LSCD）患者的泪小管上皮进行检查。固定尸体眼睑，制备横切面，用HE和抗PCK、Vim、p63α、SCF和c-Kit抗体染色。在手术显微镜下，以泪管探针为指示剂分离小管组织，用胶原酶a消化，用胰蛋白酶/EDTA消化具有密切相关基质的上皮细胞群，获得单细胞，在MESCM培养基中涂有基质的塑料板上培养。免疫染色法检测SCF、c-Kit、p63α的表达。通过计算无性系数除以总播种细胞数的百分比，测定3T3饲喂层上的菌落形成效率。结果：6个泪小管中5个下小管上皮及6个泪小管上小管上皮外观视觉正常。人泪小管上皮有5 ~ 15层，其中有一个小而紧密的基底层细胞，表达PCK、p63α、SCF和c-Kit。利用胶原酶A分离LCESC，在MESCM中获得克隆生长。LCESC全息克隆在3T3饲养层上的集落形成效率为3.2%，而缘干细胞（LSC）的集落形成效率为1.9%。结论：本文首次报道了LCESCs可以被分离出来，并且具有与LSCs相似的干细胞特征。这一发现为LSCD患者的LSC重建提供了一个有希望的干细胞基质资源。
Purpose: To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded in vitro. Methods: The lacrimal canaliculus epithelium of 6 patients with limbal stem cell deficiency (LSCD) caused by alkali burn or Stevens Johnson Syndrome were examined by lacrimal endoscope. Cadaveric eyelids were fixed and prepared for cross section and stained with HE and antibodies against PCK, Vim, p63α, SCF and c-Kit. Canaliculus tissue was separated under an operating microscope using a lacrimal probe as an indicator and digested with collagenase A. The clusters of epithelial cells with closely associated stroma were further digested with Trypsin/EDTA to obtain single cells for culture on Matrigel-coated plastic plates in MESCM media. The expression of SCF, c-Kit and p63α was determined by immunostaining. The colony-forming efficiency on 3T3 feeder layers was also measured by calculating the percentage of the clone number divided by the total number cells seeded. Results: The epithelial layers of five out of six inferior lacrimal canaliculi and all the six superior lacrimal canaliculi were visually normal in appearance. Five to fifteen layers of the epithelium in the human lacrimal canaliculi were present with a small, tightly compacted basal layer of cells expressing PCK, p63α, SCF and c-Kit. LCESC were isolated by collagenase A and obtained clonal growth in MESCM. The colony-forming efficiency of LCESC holoclones on a 3T3 feeder layer was 3.2%, compared to 1.9% for those of limbal stem cells (LSC). Conclusions: Herein, we first report that LCESCs can be isolated and have stem cell characteristics, similar to those of LSCs. Such a discovery raises a promising substrate resource of stem cells for LSC reconstruction in LSCD patients.
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DOI：
10.1152/ajpendo.00172.2007
发表时间：
2007-08-01
影响因子：
5.1
作者：
Li, Jinming;Quirt, Jaclyn;Wang, Rennian
通讯作者：
Wang, Rennian
DOI：
10.1083/jcb.200703003
发表时间：
2007-06-18
期刊：
The Journal of cell biology
影响因子：
--
作者：
Barbaro V;Testa A;Di Iorio E;Mavilio F;Pellegrini G;De Luca M
通讯作者：
De Luca M
影响因子：
3.4
作者：
Kanavi, Mozhgan Rezaei;Tabeie, Faraj;AfsarAski, Sasha
通讯作者：
AfsarAski, Sasha
影响因子：
--
作者：
Dobrowolski D;Orzechowska-Wylegala B;Wowra B;Wroblewska-Czajka E;Grolik M;Szczubialka K;Nowakowska M;Puzzolo D;Wylegala EA;Micali A;Aragona P
通讯作者：
Aragona P
影响因子：
0.3
作者：
Kafle, P. A.;Singh, S. K.;Surin, L.
通讯作者：
Surin, L.