Isolation of human keratinocyte stem cells and high-throughput screening approach for their characterization

In the last three decades, regenerative medicine has opened new horizons for the in vitro reconstruction of epithelial tissues and gene therapy treatment of skin disorders involving the use of adult keratinocyte stem cells (KSCs). Although the ability to identify and isolate these cells represents an important prerequisite for the development of these approaches, molecular markers and their precise in vivo localization are still lacking. In order to define genes involved in the control of stemness and commitment of KSCs, we developed a non-invasive, stem cell-preserving magnetic micro beads based method in order to obtain a KSCs enriched population for high throughput screening experiments. After 3T3 murine fibroblast feeder layer depletion from our keratinocyte cultures, we isolated a subpopulation of basal epithelial cells on the basis of the different expression levels of the a6β4 integrin. By using different approaches, including clonal analysis and p63 bright cells quantification, we clearly showed that a6β4 integrin bright cells have greater growth potential and clonogenic capacity compared to the remaining cell fraction and they include the KSCs population. Comparing gene expression profile of a KSCs-enriched and a terminally differentiated cell population coming from the same original primary cell culture we defined a set of genes most probably involved in stemness maintenance. Ongoing gene profiling on single clone type will allow us to validate this gene signature and to start functional studies on selected genes. Extending this approach to different ectodermal derived tissues will provide a genome wide signature of the molecular pathways underlying self-renewal, commitment and differentiation of KSCs