Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer

Abstract

The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

“…The herein used proliferative potential proxies should also be complemented with molecular biomarker expression analyses [15,17,18,32,33,34]. In light of our results, further characterization of the STAT-, p53-, AMPKα1-, and Akt-related signaling pathways in skin epithelial cell cultures is also needed.…”

“…Therefore, it is generally preferred to use P1 to P3 keratinocytes when producing bio-engineered skin equivalents for clinical purposes [5,6,26]. Nevertheless, conducting these experiments into later passages could have yielded different results, as it has been shown that keratinocytes’ mean size, growth rate and key molecular processes can vary in later culture passages [15,18,27]. …”

“…This has led to the use of alternative feeder layer and cAMP inducer types. For example, in clinical settings, i3T3FL have been substituted with irradiated human dermal fibroblast feeder layers (iHFL), which have been shown to yield phenotypically-comparable keratinocyte cultures [14,15,16,17,18]. Indeed, it has been shown that human keratinocytes co-cultured with either iHFL or i3T3 display similar growth rates [15,17], clonogenicity [14], morphology [15] and viability [16].…”

Isolation and culture of human primary keratinocytes—a methods review

“…Keratinocyte survival and expansion may be facilitated by growth‐arrested murine feeder layer fibroblasts (3T3 cells) which support keratinocyte attachment by synthesis of extracellular matrix proteins, release growth factors, remove toxic metabolites from culture media and delay primary keratinocyte terminal differentiation …”