ANALYSIS OF HOMOTYPIC AND HETEROTYPIC SPHEROIDS CELL CULTURE CONDITIONS AS A PROMISING TOOL FOR HAIR FOLLICLE REGENERATION ​

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

Buenos Aires

Congreso; LXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2017

REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS

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. However, its inductive ability is lost with successive culture passages. It was shown that culturing DPC asspheroids could restore their inductivity. Our objectives were to test different culture conditions 1) to generate DPC spheroids in order to improve their inductive signatures, 2) to generate heterotypic spheroids containing adult human DPC and HFSC in order to find histological characteristics similar to hair follicle precursors.DPC-spheroids were done by seeding cells on Polyvinyl alcohol (PVA)-coated wells. 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 conditions. Heterotypic spheroids were doneusing different ratios of HFSC and DPC on PVA-coated wells. Histological studies were performed. In DPC-spheroids, APa was 20-fold higher than in monolayer cultures. Moreover, DPC-spheroid showed a 4-fold increase of APa after 72hs in culture, and a 12-fold increase after 96hs, both compared to APa observed after 48hs. After 48 hours spheroids showed a 4-fold increase of Wnt10b and a 4-fold decrease of Dkk1 expression. DPC spheroids showed an 8-fold higher VEGF and Angiogenin expression than DPC monolayer cultures. In one of the culture conditions, heterotypic spheroids showed a core of DPC expressing a-Smooth Muscle Actin and Vimentin surrounded by an epithelium with a p63 positive basal layer. Protrusions similar to those of embryonic hair follicles precursors were also seen. 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 ofembryonic hair-buds.