343 DIRECT EXPRESSION OF PLURIPOTENCY MARKERS IN CULTURED SOMATIC CELLS BY SMALL REPROGRAMMING MOLECULES

Abstract

The differentiate state of the cell may be reversed by a process called reprogramming. To date, a totipotent status is conferred to a somatic cell by nuclear transfer (SCNT) and a condition of pluripotency is conferred by induced expression of defined factors (iPSC). While the restoration of full totipotency by SCNT is rarely achieved, pluripotency by Yamanaka's factors (Oct4, c-myc, Sox-2, Klf4) is inducible, although with low efficiency, in a large set of cell in different animal models. However, the isolation of iPSC requires complex technical skills and time-consuming protocols. In our laboratory we have observed that the simple expansion of fibroblasts in culture switches on pluripotency markers such as Oct4 and Nanog (Anzalone et al. 2015 Reprod. Fertil. Dev. IETS Abstract 344). CHIR99021 is a small molecule, targeting the Wnt/β-catenin signalling pathway, which is used for stem cell culture (Li et al. 2009). CHIR99021 acts as selective inhibitor of both isoform of GSK3 α/β regulating cellular proliferation and differentiation. In this work we tested the hypothesis that the exposure to a small reprogramming molecule (CHIR99021) induces pluripotency marker expression in primary cultures of somatic cells. Sheep and mouse primary fibroblasts cultured in low oxygen and induced to enter GO (low serum, 0.5% FBS for 5 days, <3% cell proliferation in our conditions) were treated with different CHIR concentrations (from 2.5 to 5 µM) for different time periods (from 1 to 5 days) in order to test the proper concentration and to exclude any cytotoxic effects. Nuclear reprogramming was assessed in treated and control cells by analysing β-catenin and oct4, nanog, sox2, klf4, and c-myc expression by immuno-detection and PCR. We found that CHIR interferes with β-catenin pathway in both sheep and mouse fibroblast in a time- and dose-dependent manner; the best results were obtained using 3 µM of CHIR for 3 days. Western blot analysis confirmed that CHIR treatment leads to an increased cellular level of β-catenin; furthermore, pluripotency marker expression (protein and mRNA) was increased (P = 0.023 nonparametric Mann-Whitney test) in CHIR-treated cells compared to controls. These observations, confirmed in both the experimental models, indicate that treatment with a small molecule inhibitor interfering with glucose metabolism induces the expression of pluripotency marker in somatic cells.