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Background Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing satellite cells (MuSCs) and an impairment of their activation and differentiation which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study we focused on an unexplored strategy to improve MuSC function and their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), multipotent cells that have been recognized to have a role in tissue repair in different disease models. Methods The myogenic capacity of the hAMSC secretome (CM-hAMSC) and of their extracellular vesicles (EVs) were tested on human myoblasts, muscle fibers and dystrophic muscle stem cells in terms of ability to favor MuSC activation and differentiation both in vitro and in vivo. Specifically, the regenerative ability of EV-hAMSC was assessed by histological and molecular analyses after intra-muscular injection in the mouse model of DMD –mdx-. Results We demonstrate that CM-hAMSC and EVs isolated thereof are able to directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Indeed, they significantly increased both nuclei numbers (pConclusions In this study, for the first time, we demonstrate that the hAMSC secretome (CM-hAMSC) and mainly EVs derived thereof are able to promote the muscle regeneration potential supporting proliferation and differentiation of resident muscle stem cells. This study paves the way to develop a novel regenerative treatment to counteract DMD progression based on the capacity of EV-hAMSC to reduce fibrosis and enhance myogenesis in dystrophic muscles.

Amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties demonstrated in vitro and in vivo in various diseases in which the dysregulated immune system plays a major role. The immunomodulatory and pro-regenerative effects of MSCs, among which hAMSCs lie in the bioactive factors they secrete and in their paracrine activity, is well known. The mix of these factors (i.e., secretome) can be either freely secreted or conveyed by extracellular vesicles (EV), thus identifying two components in the cell secretome: EV-free and EV fractions. This study aimed to discern the relative impact of the individual components on the immunomodulatory action of the hAMSC secretome in order to obtain useful information for implementing future therapeutic approaches using immunomodulatory therapies based on the MSC secretome. To this aim, we isolated EVs from the hAMSC secretome (hAMSC-CM) by ultracentrifugation and validated the vesicular product according to the International Society for Extracellular Vesicles (ISEV) criteria. EVs were re-diluted in serum-free medium to maintain the EV concentration initially present in the original CM. We compared the effects of the EV-free and EV fractions with those exerted by hAMSC-CM in toto on the activation and differentiation of immune cell subpopulations belonging to both the innate and adaptive immune systems.We observed that the EV-free fraction, similar to hAMSC-CM in toto, a) decreases the proliferation of activated peripheral blood mononuclear cells (PBMC), b) reduces the polarization of T cells toward inflammatory Th subsets, and induces the induction of regulatory T cells; c) affects monocyte polarization to antigen-presenting cells fostering the acquisition of anti-inflammatory macrophage (M2) markers; and d) reduces the activation of B lymphocytes and their maturation to plasma cells. We observed instead that all investigated EV fractions, when used in the original concentrations, failed to exert any immunomodulatory effect, even though we show that EVs are internalized by various immune cells within PBMC. These findings suggest that the active component able to induce immune regulation, tested at original concentrations, of the hAMSC secretome resides in factors not conveyed in EVs. However, EVs isolated from hAMSC could exert actions on other cell types, as reported by others.

Background It is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs. Methods Amniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM. Results We demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC. Conclusions The results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. Moreover, the possibility of having a lyophilized secretome that maintains its original properties would allow for a ready-to-use product with easier handling, shipping and storage. The use of a lyophilized product will also facilitate clinicians by permitting customized reconstitution volumes and methods according to the most suitable formula required by the clinical application.

Made available in DSpace on 2018-12-11T17:02:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-04-15 Administration of horse amniotic mesenchymal cells (AMCs) and their conditioned medium (AMC-CM) improves the in vivo recovery of spontaneous equine tendon lesions and inhibits in vitro proliferation of peripheral blood mononuclear cells (PBMC). This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication during tissue regeneration. In this study, the presence and type of MVs secreted by AMCs were investigated and the response of equine tendon cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract in vitro inflammation of tendon cells induced by lipopolysaccharide was studied through the expression of some proinflammatory genes such as metallopeptidase (MPP) 1, 9, and 13 and tumor necrosis factor-α (TNFα), and expression of transforming growth factor-β (TGF-β). Lastly, the immunomodulatory potential of MVs was investigated. Results show that AMCs secrete MVs ranging in size from 100 to 200 nm. An inverse relationship between concentration and time was found in their uptake by tendon cells: the maximal uptake occurred after 72 h at a concentration of 40 × 106 MVs/mL. MVs induced a downregulation of MMP1, MMP9, MMP13, and TNFα expression without affecting PBMC proliferation, contrary to CM and supernatant. Our data suggest that MVs contribute to in vivo healing of tendon lesions, alongside soluble factors in AMC-CM. Large Animal Hospital Reproduction Unit Universita degli Studi di Milano, Via dell'Universita 6 Department of Internal Medicine Molecular Biotechnology Center University of Torino Department of Veterinary Medicine University of Perugia Department of Biochemistry Biology and Genetics Universita Politecnica Delle Marche Department of Animal Reproduction and Radiology FMVZ UNESP Centro di Ricerca E. Menni Fondazione Poliambulanza Istituto Ospedaliero Department of Veterinary Medical Science Universita degli Studi di Milano Department of Animal Reproduction and Radiology FMVZ UNESP

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

Objectives To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. Design Prospective experimental study. Setting Laboratory research. Subjects C57Bl/6 mice. Interventions Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. Measurements and main results Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. Conclusions Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and containing neither proteic nor ribonucleic molecules was protective. This study indicates the profiling of protective factors that could be useful in cell-free therapeutic approaches for acute brain injury.