Method and Analysis of High-Calcium Induced Differentiation of Human Primary Keratinocytes In Vitro

Epidermal development and regeneration rely on the stepwise differentiation of keratinocytes, the primary constituent cells of the epidermis. In the integrated epidermis, keratinocyte differentiation (KD) occurs in response to the increasing calcium gradient. Reflecting this, high-calcium incubation remains a popular approach for inducing KD in vitro. The outcome is primarily assessed by differential expression of differentiation markers compared to proliferative cells. However, it is significantly influenced by the cell models used, calcium concentration and incubation time, which often vary between studies. Such differences compromise reproducibility and comparability. In this article, we overview major parameters that shape the outcome of high-calcium induced KD. Comparative experimental data between high-calcium incubation and high-density induced differentiation underscore the need for combining additional parameters, such as microscopy based visualization with marker protein analysis for unbiased assessment of KD. These recommendations are intended to guide future research in keratinocyte biology.