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Why Do Muse Stem Cells Present an Enduring Stress Capacity? Hints from a Comparative Proteome Analysis - PubMed

  • ️Fri Jan 01 2021

Comparative Study

Why Do Muse Stem Cells Present an Enduring Stress Capacity? Hints from a Comparative Proteome Analysis

Mustafa B Acar et al. Int J Mol Sci. 2021.

Abstract

Muse cells are adult stem cells that are present in the stroma of several organs and possess an enduring capacity to cope with endogenous and exogenous genotoxic stress. In cell therapy, the peculiar biological properties of Muse cells render them a possible natural alternative to mesenchymal stromal cells (MSCs) or to in vitro-generated pluripotent stem cells (iPSCs). Indeed, some studies have proved that Muse cells can survive in adverse microenvironments, such as those present in damaged/injured tissues. We performed an evaluation of Muse cells' proteome under basic conditions and followed oxidative stress treatment in order to identify ontologies, pathways, and networks that can be related to their enduring stress capacity. We executed the same analysis on iPSCs and MSCs, as a comparison. The Muse cells are enriched in several ontologies and pathways, such as endosomal vacuolar trafficking related to stress response, ubiquitin and proteasome degradation, and reactive oxygen scavenging. In Muse cells, the protein-protein interacting network has two key nodes with a high connectivity degree and betweenness: NFKB and CRKL. The protein NFKB is an almost-ubiquitous transcription factor related to many biological processes and can also have a role in protecting cells from apoptosis during exposure to a variety of stressors. CRKL is an adaptor protein and constitutes an integral part of the stress-activated protein kinase (SAPK) pathway. The identified pathways and networks are all involved in the quality control of cell components and may explain the stress resistance of Muse cells.

Keywords: DNA damage; mesenchymal stromal cells; oxidative stress; stem cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

In vitro characterization of Muse cells. Panel (A) The picture shows a representative image of Muse cells grown in suspension and visualized through an inverted microscope (Leica DMIL 090-135.001). The black bar corresponds to 100 µM. Panel (B) After isolation, Muse cells were grown for 7–10 days and then the SSEA-3 expression was evaluated with flow cytometry analysis. The histograms show SSEA-3-positive cells (green) and the flow cytometry control reaction (red). Panel (C) RT-PCR analysis of potency (left graph) and differentiation markers (right graph) expressed by Muse cells grown either in basal growing conditions, as floating spheres, or in spontaneous differentiating conditions on culture dishes treated with 0.1% gelatin. The graph shows mRNA expression levels of genes of interest that were normalized to GAPDH mRNA level, which was selected as an internal control. The data are expressed as arbitrary units with standard deviation. BGC: Basal growth condition; SDC: Spontaneous differentiation condition. NES: NESTIN; AFP: Alpha-fetoprotein.

Figure 2
Figure 2

Venn diagram analysis. Venn diagram showing common and specific proteins among cell lysates obtained from in vitro-generated pluripotent stem cells (iPSCs), mesenchymal stromal cells (MSCs), Muse cells, and non-Muse cells. For every experimental condition, we performed biological and technical replicates. The picture refers to proteins that were consistently present in biological and technical replicates.

Figure 3
Figure 3

Minimum IMEx Interactome Networks. The picture shows the IMEx Networks generated with nodes having a connectivity degree higher than 100 and a betweenness higher than 10,000. For each node, the size is based on its degree values, with a big size for large degree values. The color switching from violet to red is proportional to betweenness centrality values, with red indicating the highest values.

Figure 4
Figure 4

Apoptosis and senescence assays. Panel (A) The graph shows the mean percentage value of senescent cells determined by ß-galactosidase assay. For each cell type, the difference in senescence level was evaluated before and 48 h after H2O2 treatment. Data are expressed with SD (n = 3 for each experimental condition), * p < 0.05, ** p < 0.01, *** p < 0.001. Panel (B) The histogram shows the mean percentage of Annexin V-positive cells. For each cell type, the difference in senescence level was determined before and 24 h after H2O2 treatment. Data are expressed with SD (n = 3 for each experimental condition), * p < 0.05, *** p < 0.001.

Figure 5
Figure 5

Reactome analysis of overrepresented pathways. Reactome analysis of protein datasets obtained from iPSCs, MSCs, Muse cells, and non-Muse cells before and after H2O2 treatment. Data are shown by Voronoi tessellation, which gives a general pathways overview. The overrepresented pathways are depicted in yellow (p < 0.05).

Figure 6
Figure 6

Details of Reactome analysis by Voronoi tessellation (A–D). Reactome analysis of protein datasets obtained from iPSCs and Muse cells before and 24 h after H2O2 treatment. Data shown are of Voronoi tessellation in detail. The overrepresented pathways (p < 0.05) are depicted in yellow.

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