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Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton's Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated β-galactosidase (SA β-gal) activity and enhanced the transcription of TERT , encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1 , a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT , TP53 ( p53 ), and CDKN1A ( p21 ). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.

Human mesenchymal stem cells (hMSCs) are an effective tool in regenerative medicine notably for their intrinsic plentiful paracrine activity rather than differentiating properties. The hMSC secretome includes a wide spectrum of regulatory and trophic factors, encompassing several naked molecules as well as different kinds of extracellular vesicles (EVs). Among EVs, exosomes represent an intriguing population, able to shuttle proteins, transcription factors, and genetic materials, with a relevant role in cell-to-cell communication, modulating biological responses in recipient cells. In this context, the extracellular milieu can greatly impact the paracrine activity of stem cells, modifying their metabolism, and the dynamics of vesicle secretion. In the present study, we investigated the effects elicited on exosome patterning by tailored, ad hoc formulated lipid supplementation (Refeed ® ) in MSCs derived from human fetal membranes (hFM-MSCs). Wound healing experiments revealed that stem cell exposure to exosomes obtained from Refeed ® -supplemented hFM-MSCs increased their migratory capability, although the amount of exosomes released after Refeed ® supplementation was lower than that yielded from non-supplemented cells. We found that such a decrease was mainly due to a different rate of exosomal exocytosis rather than to an effect of the lipid supplement on the endocytic pathway. Endoplasmic reticulum homeostasis was modified by supplementation, through the upregulation of PKR-like ER kinase (PERK) and inositol-requiring enzyme 1α (IRE1α). Increased expression of these proteins did not lead to stress-induced, unfolded protein response (UPR)-mediated apoptosis, nor did it affect phosphorylation of p38 kinase, suggesting that PERK and IRE1α overexpression was due to augmented metabolic activities mediated by optimization of a cellular feeding network afforded through lipid supplementation. In summary, these results demonstrate how tailored lipid supplementation can successfully modify the paracrine features in hFM-MSCs, impacting both intracellular vesicle trafficking and secreted exosome number and function.

Background: The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®)., Methods: Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated., Results: A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules., Conclusions: Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients.

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.