SPHEROIDS CELL CULTURES AS A PROMISING STUDY MODEL FOR HAIR FOLLICLE REGENERATION

OPPENHEIMER, FLORENCIA ; KUSINSKY, ANA GABRIELA; CERUTI, JULIETA MARÍA; LEIROS, GUSTAVO JOSÉ; BALAÑA MARÍA EUGENIA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

Dermal Papilla Cells (DPC) induction on Hair Follicle Stem Cells (HFSC) is necessary for embryonic hair follicle neogenesis and adult hair cycling. DPC could be used in tissue bioengineering to regenerate hair follicles. As DPC inductive ability is lost with successive culture passages in monolayer,3D-culture strategies are necessary for its use in skin substitutes. DPC-Spheroids were generated by seeding cells on Polyvinyl alcohol (PVA)-coated wells. Our objectives were 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 obtain hair follicle precursors to be used in complete skin regeneration.Wnt signaling pathway is essential in maintaining the hair-inducing activity of the dermal papilla. In the other hand, we have previously shown that, in composite skin substitutes, DPCs favors early neovascularization and contributes to the neovascular network maturation, resulting in a better healing process and graft take.Inductive parameters as alkaline phosphatase activity (APa), the expression of agonist (Wnt10b) and antagonist (Dkk1) Wnt ligands and angiogenic factors were evaluated comparing culture times and conditionsAPa, which correlates with hair inductive potential of DPC, was 25-fold higher in spheroid microtissues than in monolayer cultured cells. VEGF and angiogenin, soluble factors which have been proved to promote skin angiogenesis and hair cycle, showed an 8-fold higher expression in DPC spheroids than monolayer culture. 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.Heterotypic spheroids were done using different ratios of HFSC and DPC on PVA-coated wells. In one of the culture conditions, histological analyses of heterotypic spheroids showed a core of DPC expressing -Smooth Muscle Actin and Vimentin surrounded by an epithelium with a p63 positive basal layer. These results show the importance of the appropriate culture conditions in order to obtain DPC-spheroids with improved inductivity and heterotypic spheroids with histological features reminiscent of embryonic hair-buds.