Cell volume regulates terminal differentiation of cultured human epidermal keratinocytes

To gain insights into the human epidermal stem cell niche, we have previously identified micron-scale topographical substrates that regulate differentiation of spread keratinocytes. On one substrate (S1), cells interact with circular topographies and differentiation is stimulated; on the other (S2), cells interact with triangular topographies and differentiation is inhibited. Cell stiffness on S1 and S2 was similar, and nuclear localisation of the mechano-sensitive transcriptional regulator YAP1 was decreased on S1 and S2 compared to on flat substrates. However, cells on S2 exhibited reduced cell volume, leading us to explore the potential for volume-regulated differentiation. Treatment with polyethylene glycol decreased cell volume and inhibited differentiation under a range of conditions. Conversely, deionized water increased cell volume and stimulated differentiation. Bulk RNA sequencing identified several substrate-responsive genes, including aquaporins and ion channels. A membrane permeable Ca2+ chelator and an inhibitor of the water channel aquaporin 3 blocked volume-induced differentiation. These studies identify cell volume as a mechanism by which keratinocyte-niche interactions regulate terminal differentiation.