Using 3D Culture to Investigate the Role of Mechanical Signaling in Keratinocyte Stem Cells

The ability to grow keratinocyte stem cells (KSCs) in 3D culture is an important step forward for investigating the physiological properties of these cells. In the epidermis, KSCs are subject to various types of mechanical stress. To study the effects of mechanical stress on KSCs, monolayer cultures are limited as the KSCs can only form cell–cell contacts in one plane and to prevent differentiation, KSCs are grown in low (0.05 mM) calcium, which impairs formation of calcium-dependent adhesion structures such as desmosomes. This is in contrast to how KSCs are found in the epidermis in vivo, where they are connected on all sides by other cells, allowing them to form a more organized cytoskeleton. The cytoskeleton is essential for transducing mechanical signals between cells, and this cannot be accurately reproduced in monolayer cultures, where the cells do not have the same level of organization or connections. We describe a technique which allows the generation of large numbers of uniformly sized cell aggregates using cultured murine KSCs. These aggregates are produced using physiological calcium concentrations (1.2 mM), allowing the cells within the aggregates to form calcium-dependent contacts with other cells on all sides, resulting in the reorganization of the cytoskeleton, integrating the cells within each aggregate. Within the aggregates, KSCs retain stem cell properties, such as p63 expression, despite the increased calcium concentration and show activation of the mitogen-activated protein kinase ERK upon stretch. KSC aggregates can be manipulated further and provide a more physiologically relevant model for studying mechanical signaling in KSCs.