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S. G. Tseng, Y. Hayashida, W. Li, Y.-T. Chen, H. He, S.-Y. Chen; Limbal Epithelial Progenitors Lie Deeper Than Dispase Can Isolate and Require Contact With Limbal Niche Cells for Clonal Growth. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4439.
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Limbal epithelial stem cells (SC) are located exclusively in the limbal basal layer, and their functions are presumably under the regulation by the limbal niche. The physical closeness between limbal epithelial SCs and their adjacent "niche cells" has not been demonstrated, nor has the dependence of limbal SC functions on such close interactions.
Single epithelial cells obtained from dispase-isolated human limbal epithelial sheets were fractionated by rapid adhesion on collagen I-coated dishes for 12 min before being cultured in KSFM medium. Cells isolated from the remaining limbal stroma by collagenase were briefly treated with or without 0.25% trypsin/EDTA. Their cell phenotypes were characterized by single or double immunostaining to pan-cytokeratins (PCK), vimentin (Vim), p63, and ABCG2, and by clonal growth on plastic in a serum-free KSFM medium or on NIH/3T3 feeder layers in SHEM medium.
Histology and immunostaining to PCK of cross-sections confirmed complete separation of the limbal epithelial sheet from the underlying stroma by dispase digestion. Dispase-isolated epithelial sheets indeed contained progenitor cells that could readily exhibit clonal growth on 3T3 feeder layers, but could only do so in KSFM when rapidly adherent cells were first selected. To our surprise, two kinds of clusters made of small PCK+/Vim-/ABCG2+ epithelial aggregates or Vim+/ABCG2+ cells that were in close contact with PCK-/Vim+ cells, were found in the collagenase-digested remaining limbal stroma. These clusters generated vivid and better clonal growth, which contained primarily smaller PCK+ epithelial cells with abundant expression of p63 and ABCG2 and Vim+ spindle mesenchymal cells at day 10 in KSFM. In contrast, fewer and larger PCK+ epithelial cells without Vim+ cells were generated by the rapid adherent fraction of isolated epithelial sheets. These clusters also generated better clonal grwoth on 3T3 feeder layers than those from epithelial sheets. Once such cell clusters were dissociated by trypsin/EDTA, clonal growth was abolished in KSFM and markedly reduced in 3T3.
These results indicated that limbal epithelial progenitor cells might lie deeper than dispase can isolate, and they are in close contact with mesenchymal (niche) cells, both expressing ABCG2. Vivid clonal growth is elicited in KSFM without feeder layers when such close contact is maintained, but abolished when it is dissociated.
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