Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia

Epithelial cells undergo epithelial-mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal- epithelial transition; MET). We established an experimental system that reproduced three-dimensional triphasic epithelia, i.e., the original epithelium, its EMT, and MET. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-{micro}m or larger micropores. The 3.0-{micro}m-pored membrane induced an epithelial structure with three states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the triphasic epithelia. Live imaging revealed that KCs moved in a reciprocating manner, with actin-rich filopodia-like KC structures extending into and out of the 3.0-{micro}m micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry to and exit from the 3.0-{micro}m micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in a reciprocating manner, which might help form triphasic epithelia, recapitulating wound healing processes.