Human Basal and Suprabasal Keratinocytes Are Both Able to Generate and Maintain Dermo–Epidermal Skin Substitutes in Long-Term In Vivo Experiments

The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin β4 (iβ4). We compared basal iβ4(+) or suprabasal iβ4(−) keratinocytes with respect to their proliferation and colony-forming ability and their Raman spectral properties. In addition, we generated dermo–epidermal substitutes using freshly isolated and sorted basal iβ4(+) or suprabasal iβ4(−) keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal iβ4(−) keratinocytes acquire a similar proliferative capacity as basal iβ4(+) keratinocytes after two weeks of culture in vitro, with expression of high levels of iβ4 and downregulation of K10 expression. In addition, both basal iβ4(+) and suprabasal iβ4(−) keratinocytes acquire authentic self-renewing properties during the in vitro 3D-culture phase and are able to generate and maintain a fully stratified epidermis for 16 weeks in vivo. Therefore, against the leading dogma, we propose that human suprabasal keratinocytes can retro-differentiate into true basal stem cells in a wound situation and/or when in contact with the basement membrane.