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). Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0 µm or larger micropores.  Live imaging of immortalized HaCaT KCs which stably express green fluorescent protein (GFP)-labelled LifeAct (LifeAct-HaCaTs) revealed that KCs moved in a reciprocating manner with actin-rich filopodia-like structures of KCs extending into and out of the 3.0 µm micropores. HaCaT KCs cultured on 3.0 µm pored-membranes upregurated the genes associated with tissue development and cell differenciation compared with on 0.4 µm-pored membranes. These results demonstrate that KCs migrate through confined spaces in a reciprocating manner, which might help form triphasic epithelia, recapitulating wound healing processes. Overall design: LifeAct-HaCaTs were seeded on 0.4 µm- or 3.0 µm-pored PET membranes at the concentration of 5.0 x10^5 /ml and cultured for 48 hours. Cells above the membrane were then collected by a scraper. RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany).