Isolation and enrichment of newborn and adult skin stem cells of the interfollicular epidermis

The interfollicular epidermis regenerates from a heterogeneous population of basal cells undergoing either self-renewal or terminal differentiation, thereby balancing cell loss in tissue turnover or in wound repair. In this chapter, we describe a reliable and simple method for isolating interfollicular epithelial stem cells from the skin of newborn mice or from tail and ear skin of adult mice using fluorescence-activated cell sorting (FACS). We also provide a detailed protocol for culturing interfollicular epidermal stem cells and to assess their proliferative potential and self-renewing ability. These techniques are useful for directly evaluating epidermal stem cell function in normal mice under different conditions or in genetically modified mouse models

In vivo reprogramming of wound-resident cells generates skin epithelial tissue

Large cutaneous ulcers are, in severe cases, life threatening(1,2). As the global population ages, non-healing ulcers are becoming increasingly common(1,2). Treatment currently requires the transplantation of pre-existing epithelial components, such as skin grafts, or therapy using cultured cells(2). Here we develop alternative supplies of epidermal coverage for the treatment of these kinds of wounds. We generated expandable epithelial tissues using in vivo reprogramming of wound-resident mesenchymal cells. Transduction of four transcription factors that specify the skin-cell lineage enabled efficient and rapid de novo epithelialization from the surface of cutaneous ulcers in mice. Our findings may provide a new therapeutic avenue for treating skin wounds and could be extended to other disease situations in which tissue homeostasis and repair are impaired

NFI transcription factors provide chromatin access to maintain stem cell identity while preventing unintended lineage fate choices

Tissue homeostasis and regeneration rely on resident stem cells (SCs), whose behaviour is regulated through niche-dependent crosstalk. The mechanisms underlying SC identity are still unfolding. Here, using spatiotemporal gene ablation in murine hair follicles, we uncover a critical role for the transcription factors (TFs) nuclear factor IB (NFIB) and IX (NFIX) in maintaining SC identity. Without NFI TFs, SCs lose their hair-regenerating capability, and produce skin bearing striking resemblance to irreversible human alopecia, which also displays reduced NFIs. Through single-cell transcriptomics, ATAC-Seq and ChIP-Seq profiling, we expose a key role for NFIB and NFIX in governing super-enhancer maintenance of the key hair follicle SC-specific TF genes. When NFIB and NFIX are genetically removed, the stemness epigenetic landscape is lost. Super-enhancers driving SC identity are decommissioned, while unwanted lineages are de-repressed ectopically. Together, our findings expose NFIB and NFIX as crucial rheostats of tissue homeostasis, functioning to safeguard the SC epigenome from a breach in lineage confinement that otherwise triggers irreversible tissue degeneration