Growth and differentiation of human lens epithelial cells in vitro on matrix

PURPOSE. To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS. HLE cells, established from 18-week prenatal lenses, were maintain ed on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, gr owth, and differentiation of the cultures were characterized by karyotyping , cell morphology, and growth kinetics studies, reverse transcription-polym erase chain reaction (RT-PCR), immunofluorescence, and Western blot analysi s. RESULTS. HLE cells had a male, human diploid (2N = 46) karyotype. The popul ation-doubling time of exponentially growing cells was 24 hours. After 15 d ays in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated express ion of alphaA- and beta B2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologic ally distinct classes of crystallin proteins (alphaA-, alphaB-, and beta B2 -crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was det ectable in exponential cultures, and levels increased after confluence. MIP 2G and gamma -crystallin protein expression was detected in confluent cultu res, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS. HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for , lens fiber cell differentiation. This in vitro model will be useful for i nvestigations of radiation-induced cataractogenesis and other studies of le ns toxicity.