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The COVID-19 epidemic represents an unprecedented global health emergency, further aggravated by the lack of effective therapies. For this reason, several clinical trials are testing different off-label drugs, already approved for other pathologies. Mesenchymal stem/stromal cells (MSCs) have been tested during the last two decades for the treatment of various pathologic conditions, including acute and chronic lung diseases, both in animal models and in patients. In particular, promising results have been obtained in the experimental therapy of acute respiratory distress syndrome, which represents the most threatening complication of COVID-19 infection. Furthermore, more recently, great interest has been devoted to the possible clinical applications of extracellular vesicles secreted by MSCs, nanoparticles that convey much of the biological effects and of the therapeutic efficacy of their cells of origin. This review summarizes the experimental evidence underlying the possible use of MSCs and of MSC-EVs in severe COVID-19 infection and underlines the need to evaluate the possible efficacy of these therapeutic approaches through controlled studies under the supervision of the Regulatory Authorities. Unlabelled Image [ABSTRACT FROM AUTHOR]

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system. [ABSTRACT FROM AUTHOR]

Wharton's jelly stem cells (WJSC) from the human umbilical cord (UC) are one of the most promising mesenchymal stem cells (MSC) in tissue engineering (TE) and advanced therapies. The cell niche is a key element for both, MSC and fully differentiated tissues, to preserve their unique features. The basement membrane (BM) is an essential structure during embryonic development and in adult tissues. Epithelial BMs are well-known, but similar structures are present in other histological structures, such as in peripheral nerve fibers, myocytes or chondrocytes. Previous studies suggest the expression of some BM molecules within the Wharton's Jelly (WJ) of UC, but the distribution pattern and full expression profile of these molecules have not been yet elucidated. In this sense, the aim of this histological study was to evaluate the expression of main BM molecules within the WJ, cultured WJSC and during WJSC microtissue (WJSC-MT) formation process. Results confirmed the presence of a pericellular matrix composed by the main BM molecules—collagens (IV, VII), HSPG2, agrin, laminin and nidogen—around the WJSC within UC. Additionally, ex vivo studies demonstrated the synthesis of these BM molecules, except agrin, especially during WJSC-MT formation process. The WJSC capability to synthesize main BM molecules could offer new alternatives for the generation of biomimetic-engineered substitutes where these molecules are particularly needed. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extensively studied as therapeutic tools. Evaluation of their biodistribution is fundamental to understanding MSC-EVs' impact on target organs. In our work, MSC-EVs were initially labeled with DiR, a fluorescent lipophilic dye, and administered to BALB/c mice (2.00 × 1010 EV/mice) through the following routes: intravenous (IV), intratracheal (IT) and intranasal (IN). DiR-labeled MSC-EVs were monitored immediately after injection, and after 3 and 24 hours (h). Whole-body analysis, 3 h after IV injection, showed an accumulation of MSC-EVs in the mice abdominal region, compared to IT and IN, where EVs mainly localized at the levels of the chest and brain region, respectively. After 24 h, EV-injected mice retained a stronger positivity in the same regions identified after 3 h from injection. The analyses of isolated organs confirmed the accumulation of EVs in the spleen and liver after IV administration. Twenty-four hours after the IT injection of MSC-EVs, a stronger positivity was detected selectively in the isolated lungs, while for IN, the signal was confined to the brain. In conclusion, these results show that local administration of EVs can increase their concentration in selective organs, limiting their systemic biodistribution and possibly the extra-organ effects. Biodistribution studies can help in the selection of the most appropriate way of administration of MSC-EVs for the treatment of different diseases. [ABSTRACT FROM AUTHOR]

Early therapeutic effect of intratracheally (IT)-administered extracellular vesicles secreted by mesenchymal stem cells (MSC-EVs) has been demonstrated in a rat model of bronchopulmonary dysplasia (BPD) involving hyperoxia exposure in the first 2 postnatal weeks. The aim of this study was to evaluate the protective effects of IT-administered MSC-EVs in the long term. EVs were produced from MSCs following GMP standards. At birth, rats were distributed in three groups: (a) animals raised in ambient air for 6 weeks (n = 10); and animals exposed to 60% hyperoxia for 2 weeks and to room air for additional 4 weeks and treated with (b) IT-administered saline solution (n = 10), or (c) MSC-EVs (n = 10) on postnatal days 3, 7, 10, and 21. Hyperoxia exposure produced significant decreases in total number of alveoli, total surface area of alveolar air spaces, and proliferation index, together with increases in mean alveolar volume, mean linear intercept and fibrosis percentage; all these morphometric changes were prevented by MSC-EVs treatment. The medial thickness index for <100 mm vessels was higher for hyperoxia-exposed/sham-treated than for normoxia-exposed rats; MSC-EV treatment significantly reduced this index. There were no significant differences in interstitial/alveolar and perivascular F4/8-positive and CD86-positive macrophages. Conversely, hyperoxia exposure reduced CD163-positive macrophages both in interstitial/alveolar and perivascular populations and MSC-EV prevented these hyperoxia-induced reductions. These findings further support that IT-administered EVs could be an effective approach to prevent/treat BPD, ameliorating the impaired alveolarization and pulmonary artery remodeling also in a long-term model. M2 macrophage polarization could play a role through anti-inflammatory and proliferative mechanisms. [ABSTRACT FROM AUTHOR]

Large-scale muscle injury in humans initiates a complex regeneration process, as not only the muscular, but also the vascular and neuro-muscular compartments have to be repaired. Conventional therapeutic strategies often fall short of reaching the desired functional outcome, due to the inherent complexity of natural skeletal muscle. Tissue engineering offers a promising alternative treatment strategy, aiming to achieve an engineered tissue close to natural tissue composition and function, able to induce long-term, functional regeneration after in vivo implantation. This review aims to summarize the latest approaches of tissue engineering skeletal muscle, with specific attention toward fabrication, neuro-angiogenesis, multicellularity and the biochemical cues that adjuvate the regeneration process. [ABSTRACT FROM AUTHOR]

Abstract Introduction We investigated the effects of human amniotic fluid stem cells (hAFS) and rat adipose tissue stromal vascular fraction GFP-positive cells (rSVC-GFP) in a model of cardio-renal syndrome type II (CRSII). Methods and results RHF was induced by monocrotaline (MCT) in 28 Sprague-Dawley rats. Three weeks later, four million hAFS or rSVC-GFP cells were injected via tail vein. BNP, sCreatinine, kidney and heart NGAL and MMP9, sCytokines, kidney and heart apoptosis and cells (Cs) engraftment were evaluated. Cell-treated rats showed a significant reduction of serum NGAL and Creatinine compared to CRSII. In both hAFS and rSVC-GFP group, kidney protein expression of NGAL was significantly lower than in CRSII (hAFS p = 0.036 and rSVC-GFP p < 0.0001) and similar to that of controls. In both hAFS and rSVC-GFP treated rats, we observed cell engraftment in the medulla and differentiation into tubular, endothelial and SMCs cells. Apoptosis was significantly decreased in cell-treated rats (hAFS 14.07 ± 1.38 and rSVC-GFP 12.67 ± 2.96 cells/mm2) and similar to controls (9.85 ± 2.1 cell/mm2). TUNEL-positive cells were mainly located in the kidney medulla. Pro-inflammatory cytokines were down regulated in cell-treated groups and similar to controls. In cell-treated rats, kidney and heart tissue NGAL was not complexed with MMP9 as in CRSII group, suggesting inhibition of MMPs activity. Conclusion Cell therapy produced improvement in kidney function in rats with CRSII. This was the result of interstitial, vessel and tubular cell engraftment leading to tubular and vessel regeneration, decreased tubular cells apoptosis and mitigated pro-inflammatory milieu. Reduction of NGLA-MMP9 complexes mainly due to decrease MMPs activity prevented further negative heart remodeling. Highlights • Stem cells produce improvement in kidney function in rats with cardio-renal syndrome type II • Injected cells engraftment led to tubular and vessel regeneration • Injected cells engraftment decreased tubular cells apoptosis an mitigate pro-inflammatory milieu • Reduction of NGAL-MMP9 complexes prevented further negative heart remodeling [ABSTRACT FROM AUTHOR]

Purpose: Despite a substantial amount of literature on tissue-guided regeneration, decellularization process, repopulation time points and stem cell turnover, more in-depth study on the argument is required. Currently, there are plenty of reports involving large animals, as well as clinical studies facing cardiac repair with decellularized homografts, but no exhaustive rodent models are described. The purpose of this study was to develop such a model in rats; preliminary results are also herein reported. Material and Methods: Fresh or decellularized pulmonary homografts from wild type rats were implanted in the abdominal aorta of green fluorescent protein positive rats. Three experimental groups were build up: sham, fresh homograft recipients and decellularized homograft recipients. The homograft decellularization process was performed with three cycles of detergent-enzymatic treatment protocol. Surgical technique of pulmonary homograft implantation and postoperative ultrasonographic evaluation were also reported; gross, histology and immunohistochemistry analysis on unimplanted and postoperative homografts were also carried out. Results: The median total recipient operating time was 148 minutes, with a surgical success rate of 82%. The decellularization protocol resulted effective and showed a complete decellularization with intact extracellular matrix. At 15 days from surgery, the implanted decellularized pulmonary homografts exhibited cell repopulation in the outer media wall and partial endothelial lining in absence of rejection. Conclusions: Our technique is a feasible and reproducible model that can be fundamental for building a valid study for further exploitation on the field. Even in a short-term follow up, the decellularized pulmonary homografts showed autologous repopulation in absence of rejection. [ABSTRACT FROM AUTHOR]

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations. [ABSTRACT FROM AUTHOR]

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 medicine. [ABSTRACT FROM AUTHOR]

Recently, the use of acellular matrices in tissue engineering has become extremely significant as tissue substitute for organ/tissue reconstruction. In this clinical scenario, banking decellularised organs ready for transplantation would be mandatory for patients with emergency needs. In this work a new process based on supercritical carbon dioxide (SC-CO 2 ) drying technique was investigated for obtaining a dry/preserved decellularized oesophagus. Experiments were performed coupling a detergent enzymatic treatment with two different protocols: (i) SC-CO 2 drying; (ii) dehydration in ethanol and a subsequent SC-CO 2 drying. The efficiency of the treatments was investigated by monitoring the loss of weight of the treated samples and the maintenance of the extracellular matrix (ECM) architecture, composition and mechanical properties after rehydration. A successful dry acellular matrix was reached in a shorter time using the combined ethanol and SC-CO 2 treatment. Histological analysis reported the maintenance of the tissue matrix architecture and the collagen content for all the treated samples, while the preservation of ultrastructural features were confirmed by scanning electron microscopy (SEM). Tensile tests did not show significant differences in terms of fracture strength before and after the supercritical process. Furthermore, the scaffolds demonstrated good biocompatible properties in terms of cell culture viability in vitro . Overall, the results highlighted the potential of this novel technology to obtain a dried acellular matrix for oesophageal regeneration, preserving the extracellular matrix structure of the native tissue. [ABSTRACT FROM AUTHOR]

Introduction: Endothelial dysfunction is found in different pathologies such as diabetes and renal and heart diseases, representing one of the major health problems. The reduced vasodilation of impaired endothelium starts a prothrombotic state associated with irregular blood flow. We aimed to explore the potential of amniotic fluid stem (AFS) cells as a source for regenerative medicine in this field; for the first time, we focused on third-trimester amniotic fluid AFS cells and compared them with the already-described AFS cells from the second trimester. Methods: Cells from the two trimesters were cultured, selected and expanded in normoxia (20% oxygen) and hypoxia (5% oxygen). Cells were analysed to compare markers, proliferation rate and differentiation abilities. Endothelial potential was assessed not only in vitro--Matrigel tube formation assay, acetylated human low-density lipoprotein (AcLDL) uptake--but also in vivo (Matrigel plug with cell injection and two animal models). Specifically, for the latter, we used established protocols to assess the involvement of AFS cells in two different mouse models of endothelial dysfunction: (1) a chronic ischemia model with local injection of cells and (2) an electric carotid damage where cells were systemically injected. Results: We isolated and expanded AFS cells from third-trimester amniotic fluid samples by using CD117 as a selection marker. Hypoxia enhanced the proliferation rate, the surface protein pattern was conserved between the trimesters and comparable differentiation was achieved after culture in both normoxia and hypoxia. Notably, the expression of early endothelial transcription factors and AngiomiRs was detected before and after induction. When in vivo, AFS cells from both trimesters expanded in hypoxia were able to rescue the surface blood flow when locally injected in mice after chronic ischemia damage, and importantly AFS cells at term of gestation possessed enhanced ability to fix carotid artery electric damage compared with AFS cells from the second trimester. Conclusions: To the best of our knowledge, this is the first research work that fully characterizes AFS cells from the third trimester for regenerative medicine purposes. The results highlight how AFS cells, in particular at term of gestation and cultured in hypoxia, can be considered a promising source of stem cells possessing significant endothelial regenerative potential. [ABSTRACT FROM AUTHOR]

The main cause of low back pain is the intervertebral disc (IVD) degeneration. Designing an effective disc regeneration strategy still remains a major challenge, especially for the lack of effective self-healing capacity. Understanding the properties of IVD cells in the degenerate microenvironment could help to develop in situ regeneration strategies. The objective of the present study was to investigate the ability of degenerate cells to respond to conditions they experience physiologically in their niche in vivo, namely the presence of the hypoxic environment and trophic factors. Degenerate cells from IVD of patients operated for herniated disc were exposed to hypoxic priming and decellularized Wharton's jelly matrix (DWJM) as scaffold and trophic factors source for 48 h in culture. Cell response was evaluated in terms of cell viability, proliferation, cytoskeletal organization, migratory ability and expression of discogenic transcription factors (SOX9, TRPS1), hypoxia-inducible factor 1α (HIF-1α) and longevity transcription factor FOXO3a. The recruitment of HIF-1α at FOXO3a and SOX9 gene promoters was analyzed by Chromatin immunoprecipitation. Degenerate IVD cells were able to re-acquire the discogenic phenotype, and to re-adapt to hypoxia after exposure to hypoxic priming and DWJM. We demonstrated for the first time that HIF-1α is specifically recruited at the promoter of SOX9 and FOXO3a which are crucial for IVD homeostasis and repair. These results open new avenues to engineer IVD by demonstrating that appropriate stimuli are able to dampen the degenerated IVD cell phenotype and to promote anabolic activity in cells which are constitutively characterized by poor reparative capacity. [ABSTRACT FROM AUTHOR]

Amniotic Fluid Stem (AFS) cells are broadly multipotent fetal stem cells derived from the positive selection and ex vivo expansion of amniotic fluid CD117/c-kitpos cells. Considering the differentiation potential in vitro toward cell lineages belonging to the three germ layers, AFS cells have raised great interest as a new therapeutic tool, but their immune properties still need to be assessed. We analyzed the in vitro immunological properties of AFS cells from different gestational age in coculture with T, B, and natural killer (NK) cells. Nonactivated (resting) first trimester-AFS cells showed lower expression of HLA class-I molecules and NK-activating ligands than second and third trimester-AFS cells, whose features were associated with lower sensitivity to NK cell-mediated lysis. Nevertheless, inflammatory priming with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) enhanced resistance of all AFS cell types to NK cytotoxicity. AFS cells modulated lymphocyte proliferation in a different manner according to gestational age: first trimester-AFS cells significantly inhibited T and NK cell proliferation, while second and third trimester-AFS cells were less efficient. In addition, only inflammatory-primed second trimester-AFS cells could suppress B cell proliferation, which was not affected by the first and third trimester-AFS cells. Indolamine 2,3 dioxygenase pathway was significantly involved only in T cell suppression mediated by second and third trimester-AFS cells. Overall, this study shows a number of significant quantitative differences among AFS cells of different gestational age that have to be considered in view of their clinical application. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function. [ABSTRACT FROM AUTHOR]

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, αSMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration. [ABSTRACT FROM AUTHOR]