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Hua-Tao Xie, Szu-Yu Chen, Scheffer C. Tseng; Reunion of Limbal Progenitors and Their Stromal Niche Cells is Critical to Maintain Stemness in a Novel Sphere Culture. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5148.
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© ARVO (1962-2015); The Authors (2016-present)
Recently we have established a new method of isolating human limbal progenitor cells and their closely associated niche cells (NCs). To examine whether their close association plays a critical role in maintaining stemness, we sought to establish a novel sphere growth starting from reunion of SC and NC.
Human corneaoscleral rim was cut into 12 segments, and each yielded a limbal sheet after digestion with 10 mg/ml Dispase II for 16 h at 4°C or a limbal cluster after digestion with 1 mg/ml collagenase A for 18 h at 37°C in ESCM . Single cells rendered by trypsin and EDTA (T/E) were seeded on a thick Matrigel in ESCM and cultured for 10 days. These resultant spheres were harvested by 10 mg/ml Dispase, rendered into single cells by T/E, and seeded at 30 cells/cm2 on 3T3 fibroblast feeder layers. Real time qPCR and immunostaining were used to measure the expression of putative limbal SC markers (delta Np63a and CK15), ESC markers (Nanog, Sox-2, and Oct-4), and corneal epithelial marker (CK12).
Compare to dispase, collagenase enriched a population of PCK-/Vim+ cells subjacent to the basement membrane. Single cells from both methods gave rise to sphere growth in a density-dependent manner with the optimal seeding density of 4x104 per cm2. At Day 1, reunion was noted between PCK+ epithelial cells and PCK-/Vim+ cells. The percentage of NC-containing spheres in dispase-isolated sheets was 15.5% (n=129), which was significantly less than 71.8% in collagenase-isolated clusters (n=177, P<0.001). At Day 10, collagenase-isolated cells (with more NCs) had a sphere-forming efficiency of 7.7 ± 0.7%, which was more than 5.1 ± 0.5% of dispase-isolated cells (with less NCs) (n=5, P<0.01). On 3T3 feeder layers, cells from spheres containing more NCs yielded a significantly higher CFE than those from spheres with less NCs (5.7±0.5% vs. 3±0.5% n=3, P<0.05). qPCR revealed higher expression of delta Np63a(P<0.05), CK15, Oct-4, and Sox-2 (all P<0.01), and lower expression of CK12 (P<0.01) in spheres containing more NCs. Double immunostaining showed that small PCK+ epithelial cells were nuclear p63a+ and Pax6+ but membranous ABCG2+ with few larger cells being cytoplasmic CK12+, and that PCK- NCs express Sox2, Nanog, and Rex1.
Sphere growth can be established by reunion between single human limbal epithelial progenitors and their NCs to promote their expression of SC markers and clonogenicity. This novel sphere culture can be used to study how SC quiescence, self-renewal, and fate decision are regulated by limbal NCs.
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