Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PRC. 430030.
Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, USA. 33173.
Int J Med Sci. 2018 Jul 30;15(12):1260-1267. doi: 10.7150/ijms.27705. eCollection 2018.
To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded . 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. 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). 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.
为了研究是否可以分离和扩增泪小管上皮干细胞（LCESC）。通过泪道内窥镜检查了 6 名因碱烧伤或史蒂文斯-约翰逊综合征导致的角膜缘干细胞缺乏症（LSCD）患者的泪小管上皮。将尸体眼睑固定并准备进行横切，用 HE 和针对 PCK、Vim、p63α、SCF 和 c-Kit 的抗体进行染色。在手术显微镜下使用泪道探针作为指示剂分离泪小管组织，并使用胶原酶 A 进行消化。进一步用胰蛋白酶/EDTA 消化与基质紧密相关的上皮细胞簇，以获得用于在 Matrigel 包被的塑料板上在 MESCM 培养基中培养的单细胞。通过免疫染色确定 SCF、c-Kit 和 p63α 的表达。还通过计算克隆数除以接种细胞总数的百分比来测量 3T3 饲养层上的集落形成效率。在 6 个下泪小管中，有 5 个的下泪小管外观正常，上皮有 5-15 层，细胞小而紧密，基底细胞层表达 PCK、p63α、SCF 和 c-Kit。通过胶原酶 A 分离 LCESC，并在 MESCM 中获得克隆生长。LCESC 全克隆在 3T3 饲养层上的集落形成效率为 3.2%，而角膜缘干细胞（LSC）为 1.9%。在此，我们首次报道 LCESC 可以分离并具有干细胞特征，类似于 LSC。这一发现为 LSCD 患者的 LSC 重建提供了有前途的干细胞基质资源。
