Boosting and Rescuing Epidermal Superior Population from Fresh Keratinocyte Cultures

Abstract

Epidermal stem cells (EpSCs) hold great expectations in a regenerative medicine context, but innovative methods that permit to obtain a significant yield of EpSCs or stem-like epidermal cells are still required. We propose a twostep 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 rockassociated kinase inhibitor (ROCKi) treatment, and the subsequent immunomagnetic separation of the a6 high / CD71 dim 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 a6 high /CD71 dim 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.

Novel role for the testis‐enriched HSPA2 protein in regulating epidermal keratinocyte differentiation

“…Results of an assay known as the “rapid adherence to collagen‐IV” confirmed that HSPA2 deficiency correlated with impaired cell‐ECM adhesion and reduced clonogenicity of cells (Figures H and 3E). Since undifferentiated keratinocytes are more adhesive and have higher clone‐forming ability than differentiated ones, rapid adhesion assay is frequently used for separation of undifferentiated cells from a total pool of keratinocytes (Cerqueira, Frias, Reis, & Marques, ; Jones & Watt, ). We selected rapidly adherent cells (RAC) by allowing them only a brief contact with a collagen‐IV‐coated surface, unattached cells were considered non‐rapidly adherent (NRAC).…”

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

“…The regenerative ability of keratinocyte stem cells has been known since the 1980s and has been well described in numerous studies 26–30 . However, the expansion of keratinocytes in vitro for clinical use has remained challenging, particularly as the use of undefined xenogeneic materials in the treatment of patients runs counter to best practice for cell therapy, due to concerns about xenopathogens 31 .…”

IL-1β–Primed Mesenchymal Stromal Cells Improve Epidermal Substitute Engraftment and Wound Healing via Matrix Metalloproteinases and Transforming Growth Factor-β1

“…Mesenchymal stromal cells (MSCs) have become an attractive therapeutic option to improve tissue repair and treat severe skin disorders in clinical cases (Cerqueira et al, 2014;Gaur et al, 2017). Preclinical studies have highlighted their potential to reduce inflammation and promote reepithelialization and extracellular matrix remodeling through paracrine mechanisms (Chen et al, 2016;Jackson et al, 2012).…”