344 LOSS OF CONSTRAINTS IMPOSED BY TISSUE ENVIRONMENT ACTIVATES EARLY SIGNALS OF CELLULAR REPROGRAMMING

Pluripotency is the ability of one cell to generate every cell type of the 3 germ layers, a property typically owned by embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), with some exceptions; multilineage-differentiating stress-enduring (Muse) cells are an example. Muse cells, described as pre-existing pluripotent stem cells in mesenchymal tissues (Kuroda et al. 2010) are able to form clusters from single cells in suspension culture, express pluripotency factors and differentiate into cell types of the 3 germ layers, like ESC and iPSC. In addition, Muse cells are proposed to be the only source of cells capable to generate iPSC by current methodologies (Wakao et al. 2011). However, it is unclear whether they are normally present in adult tissue, derive from precursors stem or differentiated cells, or are induced by the in vitro conditions. In our work, we tested the hypothesis that the transition from a committed (tissue) to an uncommitted (in vitro culture) environment triggers in the cells the activation of a default gene circuitry leading to pluripotency. Adult skin fibroblasts were obtained from sheep ear biopsy (n = 3) and expanded in vitro (A) or cultured in suspension in hanging drops (B) or in nonadherent dishes (C) in MEM with 10% FBS. In a subsequent experiment, clonal expansion was attempted by culturing single suspension cells in drops of medium (D). Pluripotency was assessed analysing Oct4 and Nanog expression, using real-time PCR (mRNA) and Western blotting (protein), in cultured fibroblasts compared to whole ear biopsy (30-day-old fetus was used as positive control, CTR). Furthermore, in adherent cells (A) and in clusters obtained from suspension culture (B, C, D), Oct4 and Nanog expression was compared by immunofluorescence. We found that while in the ear biopsy not one of these pluripotency markers was expressed, in in vitro-expanded fibroblasts both mRNA and protein expression was detected; mRNA expression value (mean ± s.e.m. relative to CTR) was 0.59 ± 0.18 for Nanog and 0.2 ± 0.07 for Oct4. Moreover, fibroblasts in suspension (B, C, D) were able to form clusters [obtained from 32% (16/50) of single cells, D] similar to those normally obtained with ESC, iPSC. and Muse cells. All the clusters (B, C, D) showed a more intensive signal of Oct4 and Nanog protein compared to adherent cells by immunofluorescence. In the present work we demonstrate that adult somatic cells (skin fibroblasts) express key pluripotency factors, such as OCT4 and Nanog, in both adherent and suspension culture, after removal from the tissue (ear). We can conclude that the simple in vitro culture switches on the expression of pluripotency markers in adult somatic cells. Removal from the context of the tissue probably leads the cells to lose their tissue-specific identity and acquire a new undifferentiated one, which in an optimal condition culture could result in pluripotency. Our interpretation is that reprogramming must be an automatic, default response when differentiated cells are removed from the constraints imposed by a multicellular environment.