Boosting and Rescuing Epidermal superior population from fresh keratinocyte cultures

Epidermal stem cells (EpSCs) hold great expectations in a regenerative medicine context, but innovative methods that permit to obtain significant yield of EpSCs or stem-like epidermal cells are still required. We propose a two-step strategy to obtain a superior epidermal stem-like cell fraction among primary keratinocytes (KCs) isolated from adult human skin. The approach is based on the combination of rapid adherence to collagen IV with the rock associated kinase inhibitor (ROCKi) treatment, and the subsequent immunomagnetic separation of the α6high/CD71dim cell subset. The combined collagen IV and ROCKi treatment showed not only to enhance cells clonogenic capacity, but also to induce an early epidermal phenotypic signature, along with the diminished expression of late differentiation associated markers. More importantly, collagen IV and the ROCKi efficiently promoted a synergized effect over α6high/CD71dim expression, boosting the number of highly proliferative KCs stem-like cells as demonstrated by the expression of ki67. This cell fraction showed a superior ability to generate a 3D stratified epithelium formed by cells with successive differentiation phenotypes. Overall, this strategy indulged the possibility to uncover, among adult KCs, a superior epidermal cell population with stem-like proliferation capacity and early differentiation degree to be used in numerous skin regeneration approaches.