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Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes  in vitro  allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show  KRT2  mRNA is stabilised whilst  KRT1  and  KRT10  mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact  in vitro  studies of keratinocyte differentiation.

Serum lipids, retinoic acid and phenol red differentially regulate expression of keratins K1, K10 and K2 in cultured keratinocytes