Increased DNA damage of adipose tissue-derived mesenchymal stem cells under inflammatory conditions

Abstract

Cells have evolved various DNA repair mechanisms to prevent DNA damage from building up. Malfunctions during DNA repair can influence cellular homeostasis because they can bring on genomic instability through the improper recognition of DNA damage or dysregulation of the repair process. Maintaining proper DNA repair is also essential for stem cells (SCs), as they provide a differentiated cell population to the living organism. SCs are regularly used in personalized stem cell therapy. Patients must be treated with specific activators to produce these SCs effectively. This report investigated the impact of treating mesenchymal stem cells (MSC) with lipopolysaccharide, tumor necrosis factor, interferon-gamma, polyinosinic acid, interleukin 1 beta, while monitoring their transcription-related response using next-generation sequencing. RNA sequencing revealed robust gene expression changes, including those of specific genes encoding proteins implicated in DNA damage response. Stem cells can effectively repair specific DNA damages; moreover, they fail to undergo senescence or cell death when genetic lesions accumulate. Here, we draw attention to an elevated DNA repair activation following MSC induction, which may be the main reason for the ineffective stem cell transplantation and may also contribute to the genetic drift that can initiate tumor formation.

Keywords: ADMSC; DNA repair; NHEJ; RNAseq; Stem cells.

Graphical abstract

Fig. 1

The schematic representation of the bioinformatic pipeline for next-generation sequencing data processing.

Fig. 2

Gene expressional changes in the stromal vascular fraction of human MSC cells following tumor necrosis factor (TNF-α), lipopolysaccharide (LPS), interleukin 1 beta (IL-1β), polyinosinic acid (PolyI:C), or interferon-gamma (IFN-γ) treatment. A) Principal component analysis of the dataset, B) Heatmap and cluster analysis of the differentially expressed genes. Red represents the upregulated genes with higher z-score, while blue indicates the downregulated genes. D1, D2, and D3 refer to the biological replicates. C) Volcano plot representation of the differentially expressed DNA repair-related genes. For visualization of the most significant DNA repair genes, we employed a cut-off value of 1 for absolute log₂ fold-change (upper horizontal dashed line) and 0.05 for statistical significance (lower horizontal dashed line). The dotted vertical line denotes to –log₁₀ (P_cut-off).

Fig. 3

The affected signaling pathways related to DNA repair in response to TNF-α, LPS, IL-1β, PolyI:C, or IFN-γ treatment. A) Schematic representation of the enriched DNA repair pathways in response to TNF-α, LPS, IL-1β, PolyI:C, or IFN-γ treatment. B) Dendrogram of the affected DNA repair pathways highlighted on panel A) where the network diagram indicates the proportions of treatments concerning various pathways. C) Enriched DNA repair-related pathways according to the KEGG database. D) The qPCR validation of the RNA-seq data the measured expression of each condition of the 13 genes are shown. The error bars represent the standard deviation.

Fig. 4

Monitoring the changes of γH2AX and 53PB1 protein levels in ADMSC cells following TNF-α, LPS, IL-1β, PolyI:C, and IFN-γ. Confocal microscopy images of patient-derived cells treated with TNF-α, LPS, IL-1β, PolyI:C, or IFN-γ and stained with A) anti-53BP1 and B) γH2AX antibody. The quantification of the foci is depicted on the right side of each panel. The values represent the mean ± standard deviation from three independent experiments (N = 100 cells in each experiment). Statistical significance in both bar charts was calculated using independent samples t-tests (P < 0.001***).