Growth and differentiation of human lens epithelial cells in vitro on matrix.
OBJECTIVE
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 maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis.


RESULTS
HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-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 immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, 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 investigations of radiation-induced cataractogenesis and other studies of lens toxicity.
Blakely
E A
EA
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley. Cellrex, San Francisco, USA. eablakely@lbl.gov
Bjornstad
K A
KA
Chang
P Y
PY
McNamara
M P
MP
Chang
E
E
Aragon
G
G
Lin
S P
SP
Lui
G
G
Polansky
J R
JR
D000818:Animals; D020346:Aquaporins; D015153:Blotting, Western; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D003459:Crystallins / Q000235:genetics / Q000378:metabolism; D050761:Cyclin-Dependent Kinase Inhibitor p57; D004728:Endothelium, Corneal / Q000166:cytology; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005109:Extracellular Matrix; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007621:Karyotyping; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D008562:Membrane Glycoproteins; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020346:Aquaporins; C493964:CDKN1C protein, human; D003459:Crystallins; D050761:Cyclin-Dependent Kinase Inhibitor p57; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; C030328:aquaporin 0; D016222:Fibroblast Growth Factor 2; C496348:FGFR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1