Epigenetic Erasing and Pancreatic Differentiation of Dermal Fibroblasts into Insulin-Producing Cells are Boosted by the Use of Low-Stiffness Substrate

Abstract

Several studies have demonstrated the possibility to revert differentiation process, reactivating hypermethylated genes and facilitating cell transition to a different lineage. Beside the epigenetic mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces in supporting cell plasticity and boosting differentiation. Here, we describe epigenetic erasing and conversion of dermal fibroblasts into insulin-producing cells (EpiCC), and demonstrate that the use of a low-stiffness substrate positively influences these processes. Our results show a higher expression of pluripotency genes and a significant bigger decrease of DNA methylation levels in 5-azacytidine (5-aza-CR) treated cells plated on soft matrix, compared to those cultured on plastic dishes. Furthermore, the use of low-stiffness also induces a significant increased up-regulation of ten-eleven translocation 2 (Tet2) and histone acetyltransferase 1 (Hat1) genes, and more decreased histone deacetylase enzyme1 (Hdac1) transcription levels. The soft substrate also encourages morphological changes, actin cytoskeleton re-organization, and the activation of the Hippo signaling pathway, leading to yes-associated protein (YAP) phosphorylation and its cytoplasmic translocation. Altogether, this results in increased epigenetic conversion efficiency and in EpiCC acquisition of a mono-hormonal phenotype. Our findings indicate that mechano-transduction related responsed influence cell plasticity induced by 5-aza-CR and improve fibroblast differentiation toward the pancreatic lineage.

Genome-Wide DNA Methylation Enhances Stemness in the Mechanical Selection of Tumor-Repopulating Cells

“…Previous studies have reported that mechanical stretch or soft matrix significantly decreased the DNA methylation level of critical CG sites or specific genes (Vlaikou et al, 2017;Wang et al, 2017;Pennarossa et al, 2018), and non-CG methylation may regulate gene expression according to environmental changes (Patil et al, 2014). Our results were consistent with these reports that numerous DMRs and DMGs of mCG and non-CG methylation were identified in the stage (1-day/0 h) that environment-switched from 2D rigid to 3D soft (Figure 4A and Supplementary Figure S1).…”

Shaping Pancreatic β-Cell Differentiation and Functioning: The Influence of Mechanotransduction

“…Similarly, the epigenetic erasing and conversion of dermal fibroblasts into insulin-producing cells was promoted by growing cells on low-stiffness substrates, which prevents stress fiber formation, and modulates YAP expression and distribution within cells [167].…”

New tools for cell reprogramming and conversion: Possible applications to livestock

“…Because of its powerful effects, 5-aza-CR induces global DNA hypomethylation (Christman, 2002) and gene reactivation (Jones, 1985) facilitating somatic cells switching from one phenotype to another (Glover et al, 1986;Harris et al, 2011;Taylor and Jones, 1979). A brief exposure to 5-aza-CR can convert adult skin fibroblasts and granulosa cells into different cell types Pennarossa et al, 2017Pennarossa et al, , 2014Pennarossa et al, , 2013. Such fate switch is not limited to cells belonging to the same embryonic layer but can also occur between cells belonging to different embryonic layers.…”

“…These include reduced dimensions with large nuclei, global chromatin decondensation, as well as expression of pluripotencyrelated genes such as OCT4, NANOG, ZFP42 zinc finger protein (REX1) and SRY (sex determining region Y)-box 2 (SOX2). This was achieved not only with human and mouse but also with pig and dog fibroblasts Pennarossa et al, 2017Pennarossa et al, , 2014Pennarossa et al, , 2013 and the high efficiency and robustness of the process makes it the best option for working in domestic species .…”