Persistent DNA damage-induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated ß-galactosidase activity and enlarged γH2AX foci co-localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence-associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour-promoting behaviour.

Factors Ruling the Uptake of Silica Nanoparticles by Mesenchymal Stem Cells: Agglomeration Versus Dispersions, Absence Versus Presence of Serum Proteins.

The results of a systematic investigation of the role of serum proteins on the interaction of silica nanoparticles (NP) doped in their bulk with fluorescent molecules (IRIS Dots, 50 nm in size), with human mesenchymal stem cells (hMSCs) are reported. The suspension of IRIS Dots in bare Dulbecco-modified Eagle's medium results in the formation of large agglomerates (≈1.5 µm, by dynamic light scattering), which become progressively smaller, down to ≈300 nm in size, by progressively increasing the fetal bovine serum (FBS) content of the solutions along the series 1.0%, 2.5%, 6.0%, and 10.0% v/v. Such difference in NP dispersion is maintained in the external cellular microenvironment, as observed by confocal microscopy and transmission electron microscopy. As a consequence of the limited diffusion of proteins in the inter-NP spaces, the surface of NP agglomerates is coated by a protein corona independently of the agglomerate size/FBS concentration conditions (ζ-potential and UV circular dichroism measurements). The protein corona appears not to be particularly relevant for the uptake of IRIS Dots by hMSCs, whereas the main role in determining the internalization rate is played by the absence/presence of serum proteins in the extracellular media.

Human mesenchymal stem cells labelled with dye-loaded amorphous silica nanoparticles: long-term biosafety, stemness preservation and traceability in the beating heart.

BACKGROUND: Treatment of myocardial infarction with mesenchymal stem cells (MSCs) has proven beneficial effects in both animal and clinical studies. Engineered silica nanoparticles (SiO2-NPs) have been extensively used as contrast agents in regenerative medicine, due to their resistance to degradation and ease of functionalization. However, there are still controversies on their effective biosafety on cellular systems. In this perspective, the aims of the present study are: 1) to deeply investigate the impact of amorphous 50 nm SiO2-NPs on viability and function of human bone marrow-derived MSCs (hMSCs); 2) to optimize a protocol of harmless hMSCs labelling and test its feasibility in a beating heart model. RESULTS: Optimal cell labelling is obtained after 16 h exposure of hMSCs to fluorescent 50 nm SiO2-NPs (50 µg mL(-1)); interestingly, lysosomal activation consequent to NPs storage is not associated to oxidative stress. During prolonged culture hMSCs do not undergo cyto- or genotoxicity, preserve their proliferative potential and their stemness/differentiation properties. Finally, the bright fluorescence emitted by internalized SiO2-NPs allows both clear visualization of hMSCs in normal and infarcted rat hearts and ultrastructural analysis of cell engraftment inside myocardial tissue. CONCLUSIONS: Overall, 50 nm SiO2-NPs display elevated compatibility with hMSCs in terms of lack of cyto- and genotoxicity and maintenance of important features of these cells. The demonstrated biosafety, combined with proper cell labelling and visualization in histological sections, make these SiO2-NPs optimal candidates for the purpose of stem cell tracking inside heart tissue.

High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells.

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

Fluorescent silica nanoparticles improve optical imaging of stem cells allowing direct discrimination between live and early-stage apoptotic cells.

Highly bright and photostable cyanine dye-doped silica nanoparticles, IRIS Dots, are developed, which can efficiently label human mesenchymal stem cells (hMSCs). The application procedure used to label hMSCs is fast (2 h), the concentration of IRIS Dots for efficient labeling is low (20 µg mL(-1) ), and the labeled cells can be visualized by flow cytometry, confocal microscopy, and transmission electron microscopy. Labeled hMSCs are unaffected in their viability and proliferation, as well as stemness surface marker expression and differentiation capability into osteocytes. Moreover, this is the first report that shows nonfunctionalized IRIS Dots can discriminate between live and early-stage apoptotic stem cells (both mesenchymal and embryonic) through a distinct external cell surface distribution. On the basis of biocompatibility, efficient labeling, and apoptotic discrimination potential, it is suggested that IRIS Dots can serve as a promising stem cell tracking agent.

Risk Assessment and Risk Minimization in Nanomedicine: A Need for Predictive, Alternative, and 3Rs Strategies.

The use of nanomaterials in medicine has grown very rapidly, leading to a concern about possible health risks. Surely, the application of nanotechnology in medicine has many significant potentialities as it can improve human health in at least three different ways: by contributing to early disease diagnosis, improved treatment outcomes and containment of health care costs. However, toxicology or safety assessment is an integral part of any new medical technology and the nanotechnologies are no exception. The principle aim of nanosafety studies in this frame is to enable safer design of nanomedicines. The most urgent need is finding and validating novel approaches able to extrapolate acute in vitro results for the prediction of chronic in vivo effects and to this purpose a few European initiatives have been launched. While a "safe-by-design" process may be considered as utopic, "safer-by-design" is probably a reachable goal in the field of nanomedicine.

Silica nanoparticles actively engage with mesenchymal stem cells in improving acute functional cardiac integration.

AIM: To assess functional effects of silica nanoparticles (SiO2-NPs) on human mesenchymal stem cell (hMSC) cardiac integration potential. METHODS: SiO2-NPs were synthesized and their internalization effects on hMSCs analyzed with particular emphasis on interaction of hMSCs with the cardiac environment Results: SiO2-NP internalization affected the area and maturation level of hMSC focal adhesions, accounting for increased in vitro adhesion capacity and augmented engraftment in the myocardial tissue upon cell injection in infarcted isolated rat hearts. SiO2-NP treatment also enhanced hMSC expression of Connexin-43, favoring hMSC interaction with cocultured cardiac myoblasts in an ischemia-like environment. CONCLUSION: These findings provide strong evidence that SiO2-NPs actively engage in mediating biological effects, ultimately resulting in augmented hMSC acute cardiac integration potential.

Stem Cell Tracking with Nanoparticles for Regenerative Medicine Purposes: An Overview.

Accurate and noninvasive stem cell tracking is one of the most important needs in regenerative medicine to determine both stem cell destinations and final differentiation fates, thus allowing a more detailed picture of the mechanisms involved in these therapies. Given the great importance and advances in the field of nanotechnology for stem cell imaging, currently, several nanoparticles have become standardized products and have been undergoing fast commercialization. This review has been intended to summarize the current use of different engineered nanoparticles in stem cell tracking for regenerative medicine purposes, in particular by detailing their main features and exploring their biosafety aspects, the first step for clinical application. Moreover, this review has summarized the advantages and applications of stem cell tracking with nanoparticles in experimental and preclinical studies and investigated present limitations for their employment in the clinical setting.

The effect of bioartificial constructs that mimic myocardial structure and biomechanical properties on stem cell commitment towards cardiac lineage.

Despite the enormous progress in the treatment of coronary artery diseases, they remain the most common cause of heart failure in the Western countries. New translational therapeutic approaches explore cardiomyogenic differentiation of various types of stem cells in combination with tissue-engineered scaffolds. In this study we fabricated PHBHV/gelatin constructs mimicking myocardial structural properties. Chemical structure and molecular interaction between material components induced specific properties to the substrate in terms of hydrophilicity degree, porosity and mechanical characteristics. Viability and proliferation assays demonstrated that these constructs allow adhesion and growth of mesenchymal stem cells (MSCs) and cardiac resident non myocytic cells (NMCs). Immunofluorescence analysis demonstrated that stem cells cultured on these constructs adopt a distribution mimicking the three-dimensional cell alignment of myocardium. qPCR and immunofluorescence analyses showed the ability of this construct to direct initial MSC and NMC lineage specification towards cardiomyogenesis: both MSCs and NMCs showed the expression of the cardiac transcription factor GATA-4, fundamental for early cardiac commitment. Moreover NMCs also acquired the expression of the cardiac transcription factors Nkx2.5 and TBX5 and produced sarcomeric proteins. This work may represent a new approach to induce both resident and non-resident stem cells to cardiac commitment in a 3-D structure, without using additional stimuli.

TCR transfer induces TCR-mediated tonic inhibition of RAG genes in human T cells.

Induction of the TCR signaling pathway terminates the expression of RAG genes, and a link between this pathway and their transcriptional control is evident from the recent demonstration of their re-expression if the TCR is subsequently lost or down-regulated. Since unstimulated T cells display a steady-state level of "tonic" TCR signaling, i.e. in the absence of any antigenic stimulus, it was uncertain whether this control was exerted through ligand-dependent or ligand-independent TCR signaling. Here we demonstrate for the first time that exogenous TCR α and ß chains transferred into the human immature RAG(+) T cell line Sup-T1 by lentiviral transduction inhibit RAG expression through tonic signaling, and that this inhibition could itself be reverted by pharmacological tonic pathway inhibitors. We also suggest that mature T cells already expressing an endogenous TCR on their surface maintain some levels of plasticity at the RAG locus when their basal TCR signaling is interfered with. Lastly, we show that the TCR constructs employed in TCR gene therapy do not possess the same basal signaling transduction capability, a feature that may have therapeutic implications.

Involvement of MRE11A and XPA gene polymorphisms in the modulation of DNA double-strand break repair activity: a genotype-phenotype correlation study.

DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation-induced DNA damage, and failure to repair them results in cell death. In order to see if any associations exist between DNA repair gene polymorphisms and phenotypic profiles of DSB repair (DSBR) we performed a genotype-phenotype correlation study in 118 young healthy subjects (mean age 25.8±6.7years). Subjects were genotyped for 768 single nucleotide polymorphisms (SNPs) with a custom Illumina Golden Gate Assay, and an H2AX histone phosphorylation assay was done to test DSBR capacity. We found that H2AX phosphorylation at 1h was significantly lower in subjects heterozygous (no variant homozygotes were observed) for the XPA gene SNP rs3176683 (p-value=0.005), while dephosphorylation was significantly higher in subjects carrying the variant allele in three MRE11A gene SNPs: rs1014666, rs476137 and rs2508784 (p-value=0.003, 0.003 and 0.008, respectively). An additive effect of low-activity DNA repair alleles was associated with altered DSBR activity, as demonstrated by both H2AX phosphorylation at 1 h (p-trend <0.0001) and γH2AX dephosphorylation at 3h (p-trend <0.0001). Our study revealed that in addition to SNPs of genes that are well-established players in DSBR, non-DSBR genes, such as the XPA gene that is mainly involved in the nucleotide excision repair pathway, can also influence DSBR in healthy subjects. This suggests that successful DSBR may require both DSBR and non-DSBR mechanisms.

A novel defect in mitochondrial p53 accumulation following DNA damage confers apoptosis resistance in Ataxia Telangiectasia and Nijmegen Breakage Syndrome T-cells.

We have previously shown that whereas T-cells from normal individuals undergo accumulation of p53 and apoptosis when treated with the genotoxic agent Actinomycin D (ActD), those from Ataxia Telangiectasia (AT) and Nijmegen Breakage Syndrome (NBS) patients resist ActD-induced apoptosis [1]. We have now found similar resistance by the p53-null Jurkat T-cell line and by siRNA p53-knockdown normal T-cells. This evidence that ActD initiates a p53-dependent apoptotic responce prompted us to look for defective p53 accumulation by AT and NBS T-cells. Surprisingly the total p53 level was only slightly reduced compared to normal T cells but its intracellular localization was highly defective: p53 was poorly accumulated in the cytosol and nearly undetectable in mitochondria. In accordance with the dependence of ActD-induced apoptosis on a mitochondrial p53 function, in control T-cells specific inhibition of mitochondrial p53 translocation with µ pifithrin reduced apoptosis by 86%, whereas treatment with α pifithrin, which blocks p53-mediated transcription, had no effect. We also showed that nuclear export is not required for mitochondrial p53 translocation. Observation of an altered p53 ubiquitination pattern and Mdm2 accumulation in ActD-treated AT and NBS T-cells provided a mechanistic link to their defective extranuclear p53 localization. Our results disclose an undescribed defect in mitochondrial p53 accumulation in AT and NBS T-cells that makes them resistant to apoptosis following unrepairable DNA damage.