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M.L. Funderburgh, M.M. Mann, J.L. Funderburgh; Clonal Growth of Keratocytes: Identification of Replicative Progenitor Cells from the Corneal Stroma . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4220.
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Purpose: Growth of keratocytes cultured in serum-containing media produces a fibroblastic cell morphology and loss of secretion of the extracellular matrix components required for corneal transparency. However, mitotic activity per se does not appear to be the determining factor in this loss of keratocyte phenotype. This study was designed to (1) define culture conditions under which primary keratocytes can be propagated in vitro while continuing to express markers of their differentiated state and, (2) determine if the stroma-specific gene expression is maintained in clonal lines derived from individual keratocytes. Methods: Primary bovine keratocytes were cultured on substrata of plastic, collagen or fibronectin in media containing 2% fetal bovine serum and combinations of growth factors. Clonal growth was defined by limiting dilution. Keratocyte phenotype was determined by real-time PCR detection of keratocan and aldehyde dehydrogenase (ALDH) transcripts and by immunoblotting of keratan sulfate. Results: Mixtures of growth factors in reduced serum were screened to define a ‘Cloning Medium’ (CM) in which growth rate and cloning efficiency of primary bovine keratocytes were maximized. Primary keratocytes grown as fibroblasts in fetal bovine serum secreted little keratan sulfate and expressed ALDH and keratocan mRNA levels 200- and 50- fold below that of quiescent primary keratocytes. Primary and low-passage keratocytes grown in CM, however, exhibited dendritic morphology, secreted keratan sulfate, and had mRNA pools for keratocan and ALDH similar to those of quiescent primary keratocytes. About one in each 30 primary keratocytes grew clonally in CM, with a cloning efficiency independent of substratum. After a million-fold expansion (20 population doublings) keratocyte clones exhibited a range of stable phenotypes, but approximately 10% continued to express high levels of ALDH and keratocan. Clones have been expanded through 35 cumulative population doublings. Conclusions: The corneal stroma contains a population of precursor cells that maintain a potential for repopulation of the stroma with differentiated keratocytes. Some of these cells have a capacity for extended replication in vitro without loss of phenotype. These results have implications significant to the design of therapies for scar-free wound healing and in development of cell lines for use in ocular tissue engineering.
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