Engineered EVs for Oxidative Stress Protection

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.Fil: Tolomeo, Anna Maria. Università di Padova; ItaliaFil: Quarta, Santina. Università di Padova; ItaliaFil: Biasiolo, Alessandra. Università di Padova; ItaliaFil: Ruvoletto, Mariagrazia. Università di Padova; ItaliaFil: Pozzobon, Michela. Università di Padova; ItaliaFil: De Lazzari, Giada. Università di Padova; ItaliaFil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Turato, Cristian. Università di Padova; ItaliaFil: Arrigoni, Giorgio. Università di Padova; ItaliaFil: Pontisso, Patrizia. Università di Padova; ItaliaFil: Muraca, Maurizio. Università di Padova; Itali

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1β, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.Fil: Tolomeo, Anna Maria. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Università di Padova; Italia. Consorzio per la Ricerca Sanitaria; ItaliaFil: Castagliuolo, Ignazio. Università di Padova; ItaliaFil: Piccoli, Martina. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Grassi, Michele. Università di Padova; ItaliaFil: Magarotto, Fabio. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Università di Padova; ItaliaFil: De Lazzari, Giada. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Consorzio per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Consorzio per la Ricerca Sanitaria; Italia. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Caicci, Federico. Università di Padova; ItaliaFil: Franzin, Chiara. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Scarpa, Melania. Veneto Institute of Oncology; ItaliaFil: Macchi, Veronica. Università di Padova; ItaliaFil: De Caro, Raffaele. Università di Padova; Italia. Consorzio Per la Ricerca Sanitaria; ItaliaFil: Angriman, Imerio. Università di Padova; ItaliaFil: Viola, Antonella. Università di Padova; ItaliaFil: Porzionato, Andrea. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Pozzobon, Michela. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Università di Padova; ItaliaFil: Muraca, Maurizio. Università di Padova; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Itali

Next-generation sequencing approach to hyperCKemia: A 2-year cohort study

Next-generation sequencing (NGS) was applied in molecularly undiagnosed asymptomatic or paucisymptomatic hyperCKemia to investigate whether this technique might allow detection of the genetic basis of the condition

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.</p

A systems-level analysis highlights microglial activation as a modifying factor in common forms of human epilepsy

The common human epilepsies are associated with distinct patterns of reduced cortical thickness, detectable on neuroimaging, with important clinical consequences. To explore underlying mechanisms, we layered MRI-based cortical structural maps from a large-scale epilepsy neuroimaging study onto highly spatially-resolved human brain gene expression data, identifying >2,500 genes overexpressed in regions of reduced cortical thickness, compared to relatively-protected regions. The resulting set of differentially-expressed genes shows enrichment for microglial markers, and in particular, activated microglial states. Parallel analyses of cell-specific eQTLs show enrichment in human genetic signatures of epilepsy severity, but not epilepsy causation. Post mortem brain tissue from humans with epilepsy shows excess activated microglia. In an experimental model, depletion of activated microglia prevents cortical thinning, but not the development of chronic seizures. These convergent data strongly implicate activated microglia in cortical thinning, representing a new dimension for concern and disease modification in the epilepsies, potentially distinct from seizure control

Integration of transcriptional programming and micro-technologies for in vitro modelling of neural development

The goal of this thesis is the downscaling of neuronal differentiation, starting from hiPSCs, using microfluidic and transcriptional programming technologies. The microfluidic lab-on-chip platform, used for the validation of neuronal induction protocols, offers the possibility to work in a context of tight control of culture conditions, high-throughput, efficient delivery of soluble factors. Instead, the induced pluripotent stem cells (iPSCs) technology allows to reprogram somatic cells, such as skin fibroblasts, to an embryonic-like phenotype and to obtain, in this way, a clonal expansion of undifferentiated cells that can then be differentiated in the desired phenotype (e.g. neuronal lineage). The combination between cell culture in microfluidics and iPSCs provides a very important contribution in the understanding of those unknown molecular mechanisms, responsible for specific pathologies, which could be useful for finding effective therapies. This work finds its basis and strong motivation in a contest that, to date, does not provide exhaustive in vitro or in vivo models, because a large amount of neurological diseases and human brain studies are performed on post-mortem biopsies or on tissues collected at very late disease stages. The animal models, instead, could represent a possibility to understand some neurological mechanisms, but they are limited and sometimes do not fully recapitulate the patient phenotype. In this scenario, we have focused our attention on the downscaling of in vitro neuronal differentiation protocols starting from hiPSCs, taking advantage first of all of lentiviral vectors for the overexpression of a proneural transcription factors, Ngn2, After the definition of the best culture condition, we focus our attention on the regulation of the expression pattern of Ngn2 in hiPSCs. Ngn2 is characterized by an oscillatory expression, in the early stages of the neurogenesis, and becomes constantly expressed on mature neurons. So, we evaluated weather we could reproduce in vitro this oscillatory expression pattern. Furthermore, since the generation of neurons with high efficiency is influenced by the correct delivery of exogenous factors to the cells with a precise timing, we developed a protocol of neuronal induction using an automated microfluidic platform. Lastly, to avoid any genetic aberration caused by lentiviral vectors and to have a system that could be easily modulated in term of dose and frequency of administration, we induced the generation of neurons by introducing synthetic modified mRNA encoding for Ngn2 into hiPSCs and coupled this method with microfluidic technology. We focused on increasing the differentiation efficiency of neuronal differentiation, working on cellular signallings that play important roles during the in vivo development of the central nervous system.L'obiettivo di questa tesi è il downscaling di protocolli di differenziazione neuronale, a partire da hiPSC, utilizzando tecnologie di programmazione trascrizionale, in microfluidica. La piattaforma microfluidica lab-on-chip, utilizzata per la validazione dei protocolli, offre la possibilità di lavorare in un contesto di stretto controllo delle condizioni della coltura cellulare, come ad esempio la consegna efficiente di fattori solubili. Invece, la tecnologia delle cellule staminali pluripotenti indotte (iPSCs) consente di riprogrammare le cellule somatiche, come i fibroblasti, in un fenotipo simile a quello embrionale e di ottenere, in questo modo, un'espansione clonale di cellule indifferenziate che possono quindi essere differenziate nel fenotipo desiderato (ad esempio quello neuronale). La combinazione tra coltura cellulare in microfluidica e iPSC fornisce un contributo molto importante nella comprensione di quei meccanismi molecolari, responsabili di patologie specifiche, utili all’individuazione di efficaci terapie. Questo lavoro trova le sue basi ed una forte motivazione in un contesto che, ad oggi, non fornisce modelli esaurienti in vitro o in vivo, perché una grande quantità di malattie neurologiche e studi sul cervello umano vengono eseguiti su biopsie post-mortem o su tessuti raccolti a fasi della malattia molto tardive. I modelli animali, invece, potrebbero rappresentare una possibilità di comprendere alcuni meccanismi neurologici, ma sono limitati e talvolta non ricapitolano completamente il fenotipo del paziente. In questo scenario, abbiamo focalizzato la nostra attenzione sul downscaling dei protocolli di differenziazione neuronale in vitro a partire da hiPSC, sfruttando prima di tutto i vettori lentivirali per la sovraespressione di fattori di trascrizione, nel nostro caso la Neurogenina 2, un gene proneurale. Inoltre, abbiamo usato questo approccio con una prospettiva innovativa: regolando la durata e la frequenza dell’espressione di Ngn2, attraverso la somministrazione controllata di doxiciclina, è possibile imitare, in vitro, il profilo oscillatorio di Ngn2 osservato in vivo. In vivo, infatti, i progenitori neurali sono caratterizzati da un pattern di espressione di Ngn2 oscillatorio, mentre i neuroni mostrano un'espressione sostenuta e costante di Ngn2. Inoltre, poiché la generazione di neuroni, con un’elevata efficienza, è influenzata da un corretto rilascio di fattori esogeni alle cellule, in modo time-dependent, abbiamo sviluppato un protocollo di induzione neuronale utilizzando una piattaforma microfluidica automatizzata. Infine, per evitare qualsiasi aberrazione genetica causata dai vettori lentivirali e per avere un sistema che potrebbe essere facilmente modulato in termini di dose e frequenza di somministrazione, abbiamo indotto la generazione di neuroni, in hiPSC, introducendo sintetici mRNA modificati codificanti per Ngn2 e accoppiato questo metodo con la tecnologia microfluidica. Ci siamo, quindi, concentrati sull'aumento dell'efficienza di differenziazione neuronale, lavorando sul signaling cellulare, importante per lo sviluppo in vivo del sistema nervoso centrale

Diverging concepts and novel perspectives in regenerative medicine

Regenerative medicine has rapidly evolved, due to progress in cell and molecular biology allowing the isolation, characterization, expansion, and engineering of cells as therapeutic tools. Despite past limited success in the clinical translation of several promising preclinical results, this novel field is now entering a phase of renewed confidence and productivity, marked by the commercialization of the first cell therapy products. Ongoing issues in the field include the use of pluripotent vs. somatic and of allogenic vs. autologous stem cells. Moreover, the recognition that several of the observed beneficial effects of cell therapy are not due to integration of the transplanted cells, but rather to paracrine signals released by the exogenous cells, is generating new therapeutic perspectives in the field. Somatic stem cells are outperforming embryonic and induced pluripotent stem cells in clinical applications, mainly because of their more favorable safety profile. Presently, both autologous and allogeneic somatic stem cells seem to be equally safe and effective under several different conditions. Recognition that a number of therapeutic effects of transplanted cells are mediated by paracrine signals, and that such signals can be found in extracellular vesicles isolated from culture media, opens novel therapeutic perspectives in the field of regenerative medicin