Influence of sp1/sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues.
OBJECTIVE
Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3).


METHODS
HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes.


RESULTS
Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro.


CONCLUSIONS
Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes.
Gaudreault
Manon
M
Oncology and Molecular Endocrinology Research Center, University Hospital Center of Laval (CHUL) Research Center, Québec, Canada.
Carrier
Patrick
P
Larouche
Kathy
K
Leclerc
Steeve
S
Giasson
Marcelle
M
Germain
Lucie
L
Guérin
Sylvain L
SL
D016475:3T3 Cells; D000328:Adult; D000368:Aged; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007231:Infant, Newborn; D051379:Mice; D008875:Middle Aged; D010957:Plasmids; D016329:Sp1 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D004268:DNA-Binding Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor