Regenerative medicine is extensively interested in developing cell therapies using mesenchymal stem cells (MSCs), with applications to several aging-associated diseases. For successful therapies, a substantial number of cells are needed, requiring extensive ex vivo cell expansion. However, MSC proliferation is limited and it is quite likely that long-term culture evokes continuous changes in MSCs. Therefore, a substantial proportion of cells may undergo senescence. In the present review, we will first present the phenotypic characterization of senescent human MSCs (hMSCs) and their possible consequent functional alterations. The accumulation of oxidative stress and dysregulation of key differentiation regulatory factors determine decreased differentiation potential of senescent hMSCs. Senescent hMSCs also show a marked impairment in their migratory and homing ability. Finally, many factors present in the secretome of senescent hMSCs are able to exacerbate the inflammatory response at a systemic level, decreasing the immune modulation activity of hMSCs and promoting either proliferation or migration of cancer cells. Considering the deleterious effects that these changes could evoke, it would appear of primary importance to monitor the occurrence of senescent phenotype in clinically expanded hMSCs and to evaluate possible ways to prevent in vitro MSC senescence. An updated critical presentation of the possible strategies for in vitro senescence monitoring and prevention constitutes the second part of this review. Understanding the mechanisms that drive toward hMSC growth arrest and evaluating how to counteract these for preserving a functional stem cell pool is of fundamental importance for the development of efficient cell-based therapeutic approaches.

In the last decade, many papers highlighted that the histone variant H2AX and its phosphorylation on Ser 139 (γH2AX) cannot be simply considered a specific DNA double-strand-break (DSB) marker with a role restricted to the DNA damage response, but rather as a ‘protagonist’ in different scenarios. This review will present and discuss an up-to-date view regarding the ‘non-canonical’ H2AX roles, focusing in particular on possible functional and structural parts in contexts different from the canonical DNA DSB response. We will present aspects concerning sex chromosome inactivation in male germ cells, X inactivation in female somatic cells and mitosis, but will also focus on the more recent studies regarding embryonic and neural stem cell development, asymmetric sister chromosome segregation in stem cells and cellular senescence maintenance. We will discuss whether in these new contexts there might be a relation with the canonical DNA DSB signalling function that could justify γH2AX formation. The authors will emphasize that, just as H2AX phosphorylation signals chromatin alteration and serves the canonical function of recruiting DSB repair factors, so the modification of H2AX in contexts other than the DNA damage response may contribute towards creating a specific chromatin structure frame allowing ‘non-canonical’ functions to be carried out in different cell types.

The potentialities to apply mesenchymal stem cells (MSCs) in regenerative medicine have been extensively studied over the last decades. In the cardiovascular disease (CVD) field, MSCs-based therapy is the subject of great expectations. Its therapeutic potential has been already shown in several preclinical models and both the safety and efficacy of MSCs-based therapy are being evaluated in humans. It is now clear that the predominant mechanism by which MSCs participate in heart tissue repair is through a paracrine activity. Via the production of a multitude of trophic factors endowed with different properties, MSCs can reduce tissue injury, protect tissue from further adverse effects, and enhance tissue repair. The present review discusses the current understanding of the MSCs secretome as a therapy for treatment of CVD. We provide insights into the possible employment of the MSCs secretome and their released extracellular vesicles as novel approaches for cardiac regeneration that would have certain advantages over injection of living cells.

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.

Phosphorylation of histone H2AX (cH2AX) 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 cH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal cH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining selfrenewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of cH2AX. We show that basal cH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with selfrenewal-enhancing small molecules. We observe that selfrenewal activity is highly compromised in H2AX2/2 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.