Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures

Abstract

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Improving Efficiency of Direct Pro-Neural Reprogramming: Much-Needed Aid for Neuroregeneration in Spinal Cord Injury

“…For example, the temporarily inhibition of histone deacetylase and bromodomain enhanced the kinetics of neuronal reprogramming of adult fibroblasts in a recent study [83]. Another work reported that the "epigenetic resetting" of fibroblasts by DNA demethylation (treatment with 5-azacytidine), followed by a culture in neuronal differentiating media, resulted in the upregulation of "stemness" genes (Sox2, Klf4, Nanog, and Oct4) after the demethylation and expression of neuronal lineage markers after differentiation [84]; however, a significant weakness of this study was the absence of functional tests of the supposedly reprogrammed cells.…”

Somatic Cell Reprogramming for Nervous System Diseases: Techniques, Mechanisms, Potential Applications, and Challenges

“…Mature somatic cell chromatin can be activated and altered to express specific genes. When somatic cells are reprogrammed, chromatin undergoes DNA methylation [ 171 ] and demethylation [ 172 ], which upregulates neurogenesis-related gene expression. One study found that demethylation of H3K9me3 in chromatin improved somatic cell nuclear transfer and promoted cellular reprogramming in mice and humans [ 173 ].…”

Application of Small Molecules in the Central Nervous System Direct Neuronal Reprogramming

“…The former is a two-step process, in which differentiated somatic cells are first reprogrammed into intermediated states such as induced pluripotent stem cells (iPSCs) or multipotent neural stem cells (iNSCs), followed by differentiation into neurons ( Kim et al, 2011 ; Ladewig et al, 2013 ; Liu et al, 2016 ; Yuan et al, 2020 ). The latter is the direct transformation of terminally differentiated cells into target mature cells without passing through the stages of pluripotent or multipotent cells, which is also known as transdifferentiation ( Kim et al, 2011 ; Ladewig et al, 2013 ; Liu et al, 2016 ; Mollinari et al, 2018 ; Yang et al, 2020a ; Yuan et al, 2020 ; Wang et al, 2021a ). Since direct lineage reprogramming does not go through an intermediate stem cell state, it possesses the appealing features of a low likelihood of tumor formation, no age-resetting, a high conversion speed, and an efficient cell differentiation ( Yang et al, 2011 ; Li and Chen, 2016 ; Ma et al, 2019 ; Mollinari and Merlo, 2021 ).…”