Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells
Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells
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DOI：
10.1242/jcs.02279
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发表时间：
2005-04-15
影响因子：
4
通讯作者：
Wolosin, JM
中科院分区：
文献类型：
--
作者：
Budak, MT;Alpdogan, OS;Wolosin, JM
当细胞群与dna结合染料Hoechst 33342孵育并进行Hoechst 33342排放的流式细胞术分析时，ABCG2/BCRP1转运体对染料的主动外排导致某些细胞出现分离队列，称为侧群（SP）。来自几种组织的干细胞已被证明具有SP表型。由于缺乏特异性表面标记物阻碍了上皮干细胞的分离和随后的生化表征，本研究试图确定SP细胞的存在和ABCG2在眼表上皮中的表达，并评估这些SP细胞是否具有与上皮干细胞相关的特征。用Hoechst 33342培养人和兔角膜缘和结膜上皮细胞，流式细胞术对细胞进行分析和分选。对分选的细胞进行多次测试，以确定分离的SP细胞是否表现出与干细胞表型一致的特征。在结膜和角膜缘上皮中发现了小于总细胞1%的对abcg2抑制剂fumitremorgin C敏感的侧群，但在无干细胞的角膜上皮中没有发现。采用免疫组化方法建立ABCG2的空间表达模式。抗原在结膜和角膜缘上皮基底细胞中检测到，但在角膜上皮中不存在。SP细胞的特点是极低的光侧散射，并且包含高比例的细胞：在组织收集之前显示缓慢循环；在培养后表现出最初的增殖延迟；并表现出克隆生成能力和对磷诱导分化的抗性；所有的特征都与干细胞表型一致。
When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to < 1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.