Spheroids cell cultures as a promising study model for hair follice regeneration

OPPENHEIMER, FLORENCIA MAIA; CERUTI, JM; LEIRÓS, GJ; BALAÑÁ, ME

Orlando

Congreso; International Investigative Dermatology (IID) 2018 Meeting; 2018

ESDR, JSID, SID

Dermal Papilla Cells (DPC) induction on Hair Follicle Stem Cells (HFSC) is necessary for embryonic hair follicle neogenesis and adult hair cycling. As DPC inductive ability is lost when culturing in monolayer, 3D-culture strategies are necessary to regenerate hair follicles in skin substitutes. Our aim was to test different culture conditions to: a) generate DPC spheroids with improved inductive ability. b) generate heterotypic spheroids containing adult human DPC and HFSC to be used in complete skin regeneration. Wnt signaling is essential in maintaining the hair-inducing activity of DPC. We have previously shown that, Skin sub- stitutes, containing DPC promoted early neovascularization resulting in a better healing process and graft take. Alkaline phosphatase activity (APa), the expression of Wnt ligands and angiogenic factors were evaluated comparing culture times and conditions. APa, which correlates with hair inductive potential of DPC, was 25-fold higher in spheroids than in monolayer cultured cells. VEGF and Angiogenin, which have been proved to promote skin angiogenesis and hair cycle, also showed an 8-fold higher expression in DPC spheroids. The effect of culture time of the spheroids on the expression of Wnt ligands agonists (Wnt5a, Wnt10b) and antagonist (Dkk1) was evaluated. After 48 hours, an 8-fold increase of Wnt agonists and a 4-fold decrease of Dkk1 expression was observed. Histological analyses of heterotypic spheroids of HFSC and DPC showed that in appropriate culture conditions, a core of DPC expressing a-Smooth Muscle Actin and Vimentin surrounded by an epithelium with a p63 positive basal layer. These results show the importance of the culture conditions in order to obtain DPC-spheroids with improved inductivity and heterotypic spheroids with histo- logical features reminiscent of embryonic hair-buds.