L3.      In vitro generation of human somatic cellular mass composed of dermal papilla cells and keratinocytes with GSK-3beta inhibitor using hanging drop culture system.
Shigeyoshi Fuziwara, Jiro Kishimoto; Skin Research Lab, Shisheido, Yokohama, Japan.

Epithelial - mesenchymal interaction (EMI) is a powerful driving force for the induction and regeneration of hair follicle in vivo. However, despite its significance, there has been so far no successful reconstitution of follicular bud by co-culturing epidermal cells and mesenchymal dermal papilla cells (DPs) in vitro. In an attempt on in vitro follicular formation, we have applied the hanging-drop (HD) culture system, which is used extensively for mouse embryonic stem (ES) cell aggregation and differentiation experiments, in combination with the known Wnt signaling pathway inducer, GSK-3beta inhibitor 9 (BIO). Initially, the proliferation and schematic aggregation between DPs and keratinocytes (KCs) was observed in three-dimensional interaction between DPs and KCs in HD only under BIO+ condition. After the withdrawal of BIO, these cellular masses showed autonomous growth and development in HD. Interestingly, this three-dimensional interaction was characteristic of DPs, which does not occur with normal human fibroblasts. Moreover, subsequent implantation of these cell mass into immunodeficient mice produced evidence of follicular unit reconstitution. These results showed that the HD culture system with BIO is not only applicable for in vivo hair generation, but also a candidate model system for EMI, and may represent a primitive but potential novel hair niche in vitro.