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Y. Hayashida, W. Li, Y.-T. Chen, H. He, S.-Y. Chen, S. C. G. Tseng; Heterogeneity of Limbal Basal Epithelial Progenitors and Their Interactions With Mesenchymal "Niche" Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):476.
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Stem cell functions are controlled by their special niche consisting of both cellular and extracellular components. Cumulative evidence indicates that not all limbal basal epithelial cells are stem cells, prompting us to further characterize limbal basal epithelial heterogeneity.
Human limbus and their dispase-isolated epithelial sheets were prepared for cross-section, flat-mount, and cytospin with additional brief trypsin/EDTA. Single cells were also fractionated by differential adhesion onto collagen I-coated dishes in serum-free KSFM medium. The cell phenotype was characterized by either single or double immunostaining to K12, pan-cytokeratins (PCK), Ki67, vimentin (Vim), p63, ABCG2, and PAX6, and by clonal growth in KSFM or SHEM with 3T3.
Cross-sections of the limbal tissue and dispase-isolated epithelial sheets showed the heterogeneous presence of Vim+ cells, some of which expressed PCK, but some did not. Cytospin preparation revealed four subpopulations, i.e., PCK-/Vim-, PCK+/Vim+ (small fraction), PCK+/Vim-, and PCK-/Vim+ cells. PCK-/Vim+ cells aggregated in clusters and were p63-, indicating that they were mesenchymal cells. Limbal basal progenitor cells could be enriched by rapid adhesion on collagen I-coated dishes, as cells adhering in 5 min were 57.9 ± 13.9% p63+ and 15.1 ± 8.6 % K12+, while those in 30 min were 27.9 ± 5.3 % p63+ and 60.4 ± 11.9 % K12+ (p<0.01, each). The rest three subpopulation, i.e., PCK-/Vim-, PCK+/Vim- and PCK+/Vim+ cells were smaller p63+/ABCG2+ epithelial progenitor cells, predominantly found in the rapid adhesion fraction collected in 5 min, and exhibited higher Ki67 and clonal growth in KSFM than those with slow adhesion collected between 5 min and 24 h (P<0.01, each). To the contrary, more clones were generated in slow adhesion than rapid adhesion (7.8 ± 1.4 % vs. 6.4 ± 1.2 %, p<0.05) using 3T3 feeder layers. Flat mount preparation of isolated limbal sheets before and after one day culturing in KSFM showed that Vim+/p63- cells were also ABCG2+/PAX6- and closely associated with clusters of Vim-/p63+/ABCG2+/PAX6+ epithelial progenitor cells at the limbal, but not corneal or conjunctival, region.
Limbal basal heterogeneity included PCK-/Vim-, PCK+/Vim- and PCK+/Vim+ epithelial progenitor cells that showed p63+, PAX+ and ABCG2+, and PCK-/Vim+/p63-/PAX6-/ABCG2+ mesenchymal (niche) cells. Close association between them in the limbal region pays the way for studying their interaction in controlling self-renewal and fate decision of limbal stem cells in the future.
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