Fig. 3

Induced keratinocytes (iKCs) can differentiate in vitro. (a) To test their differentiation potential in vitro, iKCs and primary keratinocytes (1°KCs) as a control were cultured under high (0.35 mM) and low (0.07 mM) Ca²⁺ concentrations. Primary KCs cultured in high calcium flattened out and differentiated (b), in contrast to cells cultured in low calcium which remained round and undifferentiated (a). iKCs recapitulated characteristics of primary keratinocytes under these conditions, and extensive differentiation was evident after an increase in Ca²⁺ levels (c and d). Scale bars = 50 μm. (b) Expression of the differentiation markers K1 and K8, as well as expression of K14, a marker of undifferentiated keratinocytes, were measured by RT-PCR under low or high Ca²⁺ culture conditions, and relative levels of each transcript from three independent experiments were quantified as before. Error bars represent standard error between experiments. Similar to primary keratinocytes, iKCs cultured under high Ca²⁺ conditions upregulated the expression of K1 and K8 and downregulated expression of K14 in response to elevated Ca²⁺ levels