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W. McIntosh Ambrose, S. So, J. Elisseeff; Differences in Gene Expression of Cultured Limbal Corneal Epithelial Stem Cells With Time Suggest Plasticity of Cell Population . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3032.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the evolution of epithelial and stem cell gene expression markers over time in human limbal explant cultures, in order to assess their potential behavior for tissue reconstruction applications.
Human corneal rims from surgical specimens were cultured on plain glass dishes for 2, 3, 4, 5 and 6 weeks after which total RNA was extracted. Cultures were incubated in serum free keratinocyte supporting medium. Gene expression for the following markers was analyzed by (RT)–PCR: p63 (nuclear protein: transcription factor), integrin α9 (transmembrane protein), ABCG2 (drug resistance transporter), K3 and K12 (cornea–specific cytokeratins), connexin 43 (gap junction protein) and GAPDH (house keeping gene). p63, integrin α9 and ABCG2 have been considered as markers of ‘stemness’, while K3/K12 and connexin 43 may be regarded as corneal epithelial markers. Corneal rims were also cultured on poly–d–lysine coated plates in identical medium until confluence (∼2–3 weeks). These cultures were then divided into two groups. Cells from the first group were then detached from the culture surface and replated onto collagen coated dishes and cultured until confluence, while total RNA was extracted from the cells of the second group; this sequence was repeated and gene expression was conducted for the markers outlined above.
Continuous cultures. For K3 and K12, a gradual decrease in expression was noted from weeks 2 to 5, with some recovery observed at the final 6 week time point while stable expression of connexin 43 was seen at all time points. Strong ABCG–2 expression was observed at 2 and 3 weeks, followed by a significant reduction at the 4 week time point, then a stepwise increase though 4 and 6 weeks. This was in contrast to integrin α9 expression, which showed a gradual reduction from 2 to 6 weeks, with the most marked decrease occurring at the 4 week point. p63 expression was initially low (2 weeks), followed by complete absence at 3 weeks and then a gradual increase through 6 weeks. Detached and re–plated cultures. There was no change in K3, K12 and connexin 43 expression from P0 to P1. Integrin α9 expression was similar for both P0 and P1, while a reduction was observed from P0 and P1 for ABCG2 and p63.
These results suggest that cells cultured from limbal explants are a mixed cell population that show characteristics of both limbal stem cells and differentiated epithelial cells and whose gene expression demonstrates plasticity with time. These studies will better enable a rational choice of cell type for epithelial reconstruction.
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