TGF-β1 gene expression in cultured human keratinocytes does not decrease with biologic age

The biologic activity of cultured epithelial grafts is believed to diminish with increasing cellular age. Therefore, keratinocytes from young donors are used preferentially in the production of cultured allografts for wound treatment. However, the impact of biologic age on cytokine gene expression by human keratinocytes has not been previously investigated. In this study, transforming growth factor-beta 1 (TGF-β1) gene expression in human keratinocytes derived from normal foreskins of males ranging in age from 7 months to 82 years was analyzed. Keratinocytes were harvested from fresh specimens and cultivated in vitro on 3T3 fibroblast feeder layers through second passage. The cells were analyzed both qualitatively and semiquantitatively for TGF-β1 gene expression using three separate techniques: in situ hybridization, Northern hybridization, and competitive polymerase chain reaction. By in situ hybridization, the signal representing TGF-β1 transcript was detected in cells in all layers of the stratified cultures, and immunohistochemical staining for TGF-β1 protein was equally intense in all layers. Northern blots of total RNA extracted from the cultivated cells showed no decrease in band density with increasing biologic age. Likewise, no decrease in TGF-β1 mRNA levels with biologic age was observed using a semiquantitative polymerase chain reaction assay. These results indicate that the potential for TGF-β1 gene expression in cultured foreskin keratinocytes does not decline with increasing cellular age. The findings imply that the clinical performance of cultured grafts, at least as it relates to the elaboration of this growth factor, may not be significantly altered by the biologic age of the keratinocyte donor.