Department of Ophthalmology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Cornea. 2011 Oct;30(10):1155-62. doi: 10.1097/ICO.0b013e318213796b.
The limbus of the cornea is said to be the niche for limbal stem cells (LSCs) and the primary source of corneal epithelial maintenance. Previously, we aimed to have shown that central human epithelial cells are capable of corneal regeneration after wounding. In this study, we aimed to investigate whether central epithelial cells in human corneas have LSC properties.
Human corneal epithelial cells were separated from the central cornea and the limbus. Isolated cells were collected for sphere-forming assay, and spheres formed subsequently were analyzed using immunohistochemistry. Fluorescence-activated cell sorting (FACS) was also used to analyze epithelial cells from central cornea, limbal rim, older donors, younger donors, and dissociated spheres. These analyses were based on cell size and Hoechst 33342 dye efflux ability, and side populations and non-side populations were isolated for colony growth measurement and sphere-forming assay.
Human central and limbal epithelial cells were capable of forming spheres, in a 1:2 ratio, that were positive for p63 immunolabeling. In FACS, central and limbal epithelial cells showed no significant difference in cell size and dye efflux ability. There were almost 10 times more large cells with good dye efflux ability from younger donors than from older donors, and the gated side population showed more than 4 times faster rate of colony growth than the non-side population. Dissociated sphere cells, however, did not follow a similar pattern to tissue-derived cells using FACS analysis. In these, there were more than twice as many large cells than small cells with good dye efflux ability.
Both limbal and central epithelial cells are capable of forming spheres in cultures that have stem cell properties. Central and limbal epithelial cells cannot be differentiated using FACS, but younger donor tissues give rise to greater numbers of large cells with high dye efflux. Therefore, results indicate that human central corneal epithelium contains cells with stem/progenitor properties, and these stem properties decline with age.
角膜缘被认为是角膜缘干细胞（LSCs）的龛位，也是角膜上皮维持的主要来源。此前，我们旨在表明，中央人上皮细胞在受伤后能够进行角膜再生。在这项研究中，我们旨在研究人眼角膜中央上皮细胞是否具有 LSC 特性。
从中央角膜和角膜缘分离出人角膜上皮细胞。收集分离的细胞进行球体形成测定，随后对形成的球体进行免疫组织化学分析。还使用荧光激活细胞分选（FACS）分析中央角膜、角膜缘缘、老年供体、年轻供体和分离球体的上皮细胞。这些分析基于细胞大小和 Hoechst 33342 染料外排能力，分离侧群和非侧群用于集落生长测量和球体形成测定。
人中央和角膜缘上皮细胞能够以 1:2 的比例形成球体，这些球体对 p63 免疫标记呈阳性。在 FACS 中，中央和角膜缘上皮细胞在细胞大小和染料外排能力方面没有显著差异。来自年轻供体的具有良好染料外排能力的大细胞数量比来自老年供体的大细胞数量多近 10 倍，门控侧群的集落生长速度比非侧群快 4 倍以上。然而，用 FACS 分析分离的球体细胞并未遵循类似的模式。在这些细胞中，具有良好染料外排能力的大细胞数量是小细胞的两倍多。
角膜缘和中央上皮细胞都能够在具有干细胞特性的培养物中形成球体。中央和角膜缘上皮细胞不能通过 FACS 区分，但年轻供体组织产生的具有高染料外排能力的大细胞数量更多。因此，结果表明人中央角膜上皮含有具有干细胞/祖细胞特性的细胞，并且这些干细胞特性随年龄增长而下降。
