Schlötzer-Schrehardt Ursula
Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany.
Methods Mol Biol. 2013;1014:55-64. doi: 10.1007/978-1-62703-432-6_3.
While convincing data clearly suggest the presence of stem cells in the basal limbal epithelium in vivo, testing the proliferation, self-renewal, and differentiation capacity of stem cells relies on the development of methodologies that allow for their isolation and extensive propagation in vitro. Clonal analysis involving differentiation between short-lived transient cell clones and long-lived stem cell clones is an invaluable technique to identify stem cells in vitro, and allows cells to be expanded over multiple passages. This chapter describes a protocol for the isolation, expansion, and clonal analysis of limbal epithelial stem cells. The cultivation method described may be essential for long-term restoration of the damaged ocular surface in patients with limbal stem cell deficiency.
虽然确凿的数据清楚地表明体内角膜缘基底上皮中存在干细胞，但要测试干细胞的增殖、自我更新和分化能力，依赖于开发能够在体外对其进行分离和大量扩增的方法。涉及区分短期瞬态细胞克隆和长期干细胞克隆的克隆分析是在体外鉴定干细胞的一项宝贵技术，并且能够使细胞在多次传代中得以扩增。本章描述了一种角膜缘上皮干细胞的分离、扩增和克隆分析方法。所描述的培养方法对于角膜缘干细胞缺乏症患者受损眼表的长期修复可能至关重要。
