Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China.
Invest Ophthalmol Vis Sci. 2020 Dec 1;61(14):16. doi: 10.1167/iovs.61.14.16.
Limbal niche cells (LNCs) play a vital role in the maintenance of limbal epithelial stem/progenitor cells (LESCs). Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone (C-LNC), collagenase following dispase removal of the limbal epithelium (DC-LNC), dissection of dispase-isolated limbal epithelial sheets (D-LNC), and explant cultures of limbal stromal tissues (Ex-LNC). This study aimed to isolate LNCs using those four methods and to compare their capacity to maintain LESCs.
LNCs were isolated from the rat corneal limbus by the following methods: C-LNC, DC-LNC, D-LNC, and Ex-LNC. Quantitative real-time PCR and immunofluorescence staining were used to analyze the expression of embryonic stem cell (ESC) markers. The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor, proliferation, and differentiation markers in three-dimensional (3D) Matrigel and Transwell systems. Notch signaling of LESCs supported by different LNCs in Transwell inserts was analyzed by quantitative real-time PCR.
DC-LNCs exhibited lower expression of CK12 during isolation and expansion. Among P4-expanded LNCs, DC-LNCs expressed significantly higher levels of Sox2, Oct4, Nanog, and N-cadherin than C-LNCs, D-LNCs, and Ex-LNCs. Compared with other LNCs, DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency, greater expression of ΔNp63α and Ki67, and lower expression of CK12. DC-LNCs were also more capable of downregulating Notch signaling of LESCs.
DC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs. This study identifies an optimal method for the isolation of LNCs in tissue engineering and ocular surface reconstruction.
角膜缘基质细胞（LNCs）在维持角膜缘上皮干细胞/祖细胞（LESCs）中起着至关重要的作用。目前已经报道了四种分离和扩增 LNCs 的方法：单独用胶原酶消化（C-LNC）、在去除角膜缘上皮的Dispase 后用胶原酶消化（DC-LNC）、分离Dispase 分离的角膜缘上皮片（D-LNC）和培养角膜缘基质组织的外植体（Ex-LNC）。本研究旨在使用这四种方法分离 LNCs，并比较它们维持 LESCs 的能力。
通过以下方法从大鼠角膜缘分离 LNCs：C-LNC、DC-LNC、D-LNC 和 Ex-LNC。采用实时定量 PCR 和免疫荧光染色分析胚胎干细胞（ESC）标志物的表达。基于集落形成能力以及在三维（3D）Matrigel 和 Transwell 系统中祖细胞、增殖和分化标志物的表达，评估维持 LESCs 的能力。通过实时定量 PCR 分析不同 LNC 在 Transwell 插入物中支持 LESCs 的 Notch 信号。
在分离和扩增过程中，DC-LNC 表达 CK12 的水平较低。在 P4 扩增的 LNCs 中，与 C-LNC、D-LNC 和 Ex-LNC 相比，DC-LNC 表达 Sox2、Oct4、Nanog 和 N-钙黏蛋白的水平显著更高。与其他 LNCs 相比，DC-LNC 更有效地维持 LESCs，具有更高的全克隆形成效率、更高的ΔNp63α 和 Ki67 表达以及更低的 CK12 表达。DC-LNC 还能够下调 LESCs 的 Notch 信号。
与其他 LNCs 相比，DC-LNC 更有效地表达 ESC 标志物并维持 LESCs。本研究确定了一种在组织工程和眼表面重建中分离 LNCs 的最佳方法。
