Pre-aggregation of scalp progenitor dermal and epidermal stem cells activates the WNT pathway and promotes hair follicle formation in in vitro and in vivo systems

Billions of dollars are invested annually by pharmaceutical companies in search of new options for treating hair loss conditions; nevertheless, the challenge remains. One major limitation to hair follicle research is the lack of effective and efficient drug screening systems using human cells. Organoids, three-dimensional in vitro structures derived from stem cells, provide new opportunities for studying organ development, tissue regeneration, and disease pathogenesis. The present study focuses on the formation of human hair follicle organoids. Scalp-derived dermal progenitor cells mixed with foreskin-derived epidermal stem cells at a 2:1 ratio aggregated in suspension to form hair follicle-like organoids, which were confirmed by immunostaining of hair follicle markers and by molecular dye labeling assays to analyze dermal and epidermal cell organization in those organoids. The hair-forming potential of organoids was examined using an in vivo transplantation assay. Pre-aggregation of dermal and epidermal cells enhanced hair follicle formation in vivo. In vitro pre-aggregation initiated the interactions of epidermal and dermal progenitor cells resulting in activation of the WNT pathway and the formation of pear-shape structures, named type I aggregates. Cell-tracing analysis showed that the dermal and epidermal cells self-assembled into distinct epidermal and dermal compartments. Histologically, the type I aggregates expressed early hair follicle markers, suggesting the hair peg-like phase of hair follicle morphogenesis. The addition of recombinant WNT3a protein to the medium enhanced the formation of these aggregates, and the Wnt effect could be blocked by the WNT inhibitor, IWP2. In summary, our system supports the rapid formation of a large number of hair follicle organoids (type I aggregates). This system provides a platform for studying epithelial-mesenchymal interactions, for assessing inductive hair stem cells and for screening compounds that support hair follicle regeneration.