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H. Thomasen, M. Pauklin, K.-P. Steuhl, D. Meller; Cell Morphology and Pluripotency Marker Expression in Limbal Epithelial Stem Cells Cultured in Different Media. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3737.
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© ARVO (1962-2015); The Authors (2016-present)
Human limbal epithelial stem cells (HLECs) are tissue specific stem cells which are located in the transitional region between the cornea and conjunctiva known as the limbus. They are capable of differentiation into corneal epithelial cells and form the reservoir for corneal epithelial cell renewal. The stemness of HLECs is maintained by factors within the limbal microenvironment, including basement membrane components, growth factors and limbal fibroblasts in the stroma. In culture without appropriate conditions HLEC tend to differentiate. Our intention was to evaluate the impact of different culture media on cell morprhology and the expression of pluripotency markers of HLECs cultured on plastic.
Limbal tissue (1 x 1 mm) from five different donors was placed into six well plates and cultivated either in supplemental hormonal epithelial medium (SHEM), n=3, or in defined keratinocyte serum-free medium (KSFM Gibco), n=3 for 18 days on plastic. Cell morphology was assessed by means of light microscopy. Expression of markers for stem cells (ABCG2, p63), pluripotency (OCT4, SOX2, NANOG, c-MYC, NESTIN, NOTCH, PAX6, KLF4) and corneal differentiation (CX43, K3) was examined via real time PCR. Results were analyzed statistically (ANOVA).
HLECs cultivated in SHEM displayed a dense cell monolayer composed of small uniform cells. Cells grown in KSFM also formed monolayer of small uniform cells with large cells inserted within. These cells were less adherent to the wells and some of them formed floating carpets. The expression of ABCG2, p63, NANOG, NOTCH and KLF4 was significantly higher in the KSFM group than in the SHEM group (p< 0,05). SOX2 expression was higher in the KSFM group, but the differences were not significant. Markers for corneal epithelial differentiation did not show significant differences in gene expression between the two groups.
Different media conditions can lead to variations in the morphology as well as gene expression profile of limbal stem cells. Defined KSFM increases expression of stem cell and pluripotency markers without affecting the expression of differentiation markers. KSFM might be a possible alternative to SHEM for the culture of limbal stem cells.
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