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Bernice Wright, Shengli Mi, Bo Chen, Che J. Connon; Investigation of the Relationship Between Differentiation of Limbal Epithelial Cells and Their Released Soluble Factors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1952.
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Limbal stem cell deficiency (LSCD) is a leading cause of corneal blindness. The success of the main treatment for LSCD, limbal stem cell transplantation (LSCT), is variable. Molecular mechanisms underpinning this therapy are poorly understood. This study aims to investigate mechanisms underpinning limbal stem cell differentiation, to develop strategies to modify LSCT, to reduce variations in clinical outcomes.
Limbal epithelial cells isolated from the bovine cornea (model) were cultured in serum-free medium (1, 2 and 3 days). The presence of stem or stem-like cells was determined by the colony forming efficiency (CFE) assay. Epithelial cells and culture medium (conditioned medium) were separated and total protein within these was measured. Proteins within cells and culture medium (12 µg total protein for each individual condition) were separated using tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) before total protein was visualised using a silver stain. Differentiation (cytokeratin 3 (CK3)) and undifferentiation (cytokeratin 14 (CK14)) were assessed by immunoblotting.
Limbal epithelial cells formed colonies on a mitomycin C-treated fibroblast feeder layer (CFE), demonstrating the presence of stem or stem-like cells in the population of initially isolated cells. CK14 increased at day 2 and decreased dramatically at day 3, whilst CK3 was maintained at greater levels than CK14 at day 3. These changes in CK3 and CK14 provided further evidence for the presence of limbal stem or stem-like cells. Distinct profiles of low molecular weight (8-20 kDa) proteins within conditioned medium and cells were observed. Proteins from conditioned medium are potentially growth factor-like products secreted from cells. Changes in CK3 and CK14 levels correlated well with variations in levels of 8-20 kDa proteins within conditioned medium. Greater levels of proteins (8-20 kDa) in conditioned medium were observed after 2 and 3 days than 1 day. A corresponding increase in levels of cytokeratin 14 was also observed after 2 days.
These data suggest that mechanisms of limbal epithelial cell differentiation may potentially involve the secretion of low molecular weight (8-20 kDa) proteins.
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