5-Aza Exposure Improves Reprogramming Process Through Embryoid Body Formation in Human Gingival Stem Cells.

Francesca Diomede,Ilaria Merciaro,Giuseppe Pizzicannella,Jacopo Pizzicannella,Adriano Piattelli,Monica D’Orazio,Nicoletta Zini,Oriana Trubiani

doi:10.3389/fgene.2018.00419

Abstract

Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells. They are used as an in vitro model to evaluate early extraembryonic tissue formation and differentiation process. In the adult organisms, cell differentiation is controlled and realized through the epigenetic regulation of gene expression, which consists of various mechanisms including DNA methylation. One demethylating agent is represented by 5-Azacytidine (5-Aza), considered able to induce epigenetic changes through gene derepression. Human gingival mesenchymal stem cells (hGMSCs), an easily accessible stem cells population, migrated from neural crest. They are particularly apt as an in vitro study model in regenerative medicine and in systemic diseases. The ability of 5-Aza treatment to induce hGMSCs toward a dedifferentiation stage and in particular versus EBs formation was investigated. For this purpose hGMSCs were treated for 48 h with 5-Aza (5 μM). After treatment, hGMSCs are organized as round 3D structures (EBs-hGMSCs). At light and transmission electron microscopy, the cells at the periphery of EBs-hGMSCs appear elongated, while ribbon-shaped cells and smaller cells with irregular shape surrounded by extracellular matrix were present in the center. By RT-PCR, EBs-hGMSCs expressed specific transcription markers related to the three germ layers as MAP-2, PAX-6 (ectoderm), MSX-1, Flk-1 (mesoderm), GATA-4, and GATA-6 (endoderm). Moreover, in EB-hGMSCs the overexpression of DNMT1 and ACH3 other than the down regulation of p21 was detectable. Immunofluorescence staining also showed a positivity for specific etodermal and mesodermal markers. In conclusion, 5-Aza was able to induce the direct conversion of adult hGMSCs into cells of three embryonic lineages: endoderm, ectoderm, and mesoderm, suggesting their possible application in autologous cell therapy for clinical organ repair.

Highlights

MATERIALS AND METHODSMesenchymal stem cells (MSCs) are adult and multipotent stem cells described by Friedenstein et al (1970) as plasticadherent cells with fibroblast-like morphology, possessing selfrenewal and tissue regeneration abilities (Huang et al, 2009)
One of the most important mechanisms that regulates epigenetic modification of the genome is DNA methylation (Selvaraj et al, 2010). 5Azacytidine (5-Aza) is a DNA demethylating agent, in particular, it plays a role as the inhibitor of DNA methyltransferase (DNMT) and it is involved in cell growth and differentiation (Abbey and Seshagiri, 2013)
The multipotent potential of Human gingival mesenchymal stem cells (hGMSCs) for differentiation in osteogenic, adipogenic, chondrogenic, and neurogenic lineages was determined by staining with Alizarin Red S, Oil Red O, Alcian blue and βIII-tubulin, respectively, after culture in specific induction media (Figures 2A1,B1,C1,D1)

Summary

Introduction

MATERIALS AND METHODSMesenchymal stem cells (MSCs) are adult and multipotent stem cells described by Friedenstein et al (1970) as plasticadherent cells with fibroblast-like morphology, possessing selfrenewal and tissue regeneration abilities (Huang et al, 2009). DNA methylation is an important mechanism involved in maintaining pluripotency and self-renewal of stem cells. 5-Aza can be used to induce stem cell differentiation in MSCs from bone marrow and adipose tissue but its role on stem cells remains contradictory (Rangappa et al, 2004; Xu et al, 2004). Oral stem cells have been isolated and characterized from exfoliated deciduous teeth (SHEDs) (Miura et al, 2003), periodontal ligament (PDLSCs) (Trubiani et al, 2016), dental follicle progenitor cells (DFPCs) (Morsczeck et al, 2005), apical papilla (SCAPs) (Sonoyama et al, 2008), dental pulp (DPSCs) (Ashri et al, 2015), and gingival tissue [Human gingival mesenchymal stem cells (hGMSCs)] (Diomede et al, 2018)

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