Stem-like cells obtained by dedifferentiation of gingival fibroblasts

Introduction: Faced with the limitations of artificial materials, regenerative dentistry (RD) aims to regenerate tissues, recurrently using stem cells. However, therapies based on stem cells have important limitations, such as obtaining the desired cells and the associated ethical/rejection problems. Innovative strategies for obtaining stem cells are needed, such as the use of gingival fibroblasts. Aim: The aim of this work is to apply a methodology of cellular dedifferentiation into gingival fibroblasts, hence to obtain stem-like cells, which in turn can be differentiated into cells of interest, for use in RD. Methodology: Cultures of mouse gingival fibroblasts were established using the explant methodology. The cells obtained were subjected to dedifferentiation with dedifferentiation agent (DA) (1 to 5μΜ) with and without medium change for 4 days. (1) morphological changes, using optical microscopy of live cells and stained with crystal violet, (2) cell cycle analysis by flow cytometry, (3) cell proliferation and (4) clonogenic efficiency were evaluated. Results: The DA promotes an increase in cell size, morphological changes, and cell death until 25%, as described for the methodology. There is an increase in the cell population in G2/M phase and the appearance of a tetraploid population. DA also induced a decrease in cell proliferation and clonogenic efficiency. Discussion: The morphological and genotypic changes observed are compatible with the appearance of a stem-like population. Conclusions: Gingival fibroblasts were successfully dedifferentiated using the DA. Clinical Implications: Stem-like cells obtained by gingival fibroblasts dedifferentiation, can be used in the future in dentistry regenerative procedures. Funding: FCT, Portugal (Strategic Project PEst-C/SAU/UI3282/2013 e UID/NEU/04539/2013],COMPETE-FEDER. Keywords: Cellular dedifferentiation, regenerative dentistry, dental pulp stem-like cells, gingival fibroblasts, morphological changes