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V. Muthukkaruppan, P. Arpitha, N. V. Prajna, M. Srinivasan; The Isolated Human Limbal Basal Cells With High Levels of P63 Expression and Large N/C Ratio Possess Slow-Cycling Property. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6069.
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We have earlier identified a subset of limbal epithelial cells with a large N/C ratio, expressing high levels of a transcription factor p63 (IOVS, 2005). The objectives of the present study were to isolate viable human limbal basal cells in order to enrich this subset and to evaluate these cells for their slow cycling property.
The epithelial cells from the basal layer of limbus were isolated by using differential enzymatic treatment of limbal tissues. In order to assess the slow-cycling property of the unique limbal subset, limbal explant cultures were pulse label with BrdU followed by 3-weeks chase. Cytospin smears of epithelial cells after double- immunostaining for p63 along with BrdU or any one of the stem cell (SC)-related markers were subjected to two- parameter analysis. Limbal epithelial outgrowth was also studied for colony forming assay (CFE).
The isolated limbal basal cells were highly positive for ΔNp63α mRNA but expressing low Cx43 mRNA. They gave rise to large colonies with compact morphology and possess higher CFE, in contrast to the colonies derived from isolated limbal suprabasal/superficial (LS/S) cells. Furthermore, a subset with a large N/C ratio expressing high levels of p63 was observed as much as 25% among the limbal basal cell fraction, in contrast to only about 4% in the total limbal epithelial cells and was absent in the LS/S fraction. Such cells were positive for K5 and negative for Ki67, Cx43 and 14-3-3σ. Limbal explant cultures and their outgrowth showed a distinct population of small cells with a large N/C ratio expressing high levels of p63, retaining the BrdU label after 21-day chase. This is correlated with the ability of the outgrowth to form holoclone colonies, indicating migration of SCs from the explant under these culture conditions.
The above evidences confirm that the two-parameter analysis is a useful method to identify and quantify corneal epithelial SCs. The importance of the study in SC biology and in regenerative medicine will be discussed.
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