7 research outputs found
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Investigation into the therapeutic potential of a clinically compliant amniotic fluid-derived stem cell conditioned media
Necrotising enterocolitis (NEC) is a devastating disease that can affect pre-term infants and if survived can lead to long term intestinal damage. Currently a variety of antibiotics and in extreme cases surgery is required to manage the disease. However, no treatment exists that attenuates the widespread cell death and suppresses the innate pro-inflammatory response whilst promoting
regeneration in the intestine thus reducing long term damage and the need for surgery. The discovery of stem cells, in particular multipotent stem cells (MSCs) has shown them to have potent regenerative therapeutic properties. In particular, C-kit+ foetal derived MSCs from amniotic fluid (amniotic fluid stem cells - AFSCs) when introduced into a rat model of NEC increased the survival of animals affected whilst also promoting the repair of damaged epithelial layers. Nevertheless, the
ability of stem cells to promote regeneration was independent of transplanted cell engraftment. This
led to a paradigm shift in which it was proposed that regeneration was via the secretion of factors acting in a paracrine manner with target cells. This was proven by the introduction of stem cell conditioned media (CM), which mimicked the beneficial effect seen when whole MSCs were introduced. However, much of the MSC CM is produced from non-stressed MSCs. Stressing MSCs has been shown to increase the secretion of paracrine factors and the extracellular vesicles that contain them. Creation of an AFSC CM (AF-CM) under conditions of stress using a protocol created by industrial partners was therefore hypothesised to produce a more potent AF-CM beneficial for regeneration and in particular for the treatment of NEC. Here we characterise the AF-CM under conditions of stress but also in a clinically compliant manner. The AF-CM was characterised for protein and RNA content, which elucidated to a multitude of proteins present but interestingly showed that the majority of RNA present was miRNA. In vitro investigation into AF-CM treatment
also showed significant improvements in cellular proliferation and migration capacity whilst protecting from senescence. Furthermore, I showed that AF-CM treatment accelerated the regeneration of skeletal muscle fibres following an established mouse model of acute cardiotoxin damage in vivo, of which could be partly attributed to increased blood vasculature
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Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease
Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage
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Protein and molecular characterization of a clinically compliant amniotic fluid stem cell derived extracellular vesicle fraction capable of accelerating muscle regeneration through the enhancement of angiogenesis
The secretome of human amniotic fluid stem cells (AFSC) has great potential as a therapeutic agent in regenerative medicine. However it must be produced in a clinically compliant manner before it can be used in humans. Here we developed a means of producing a biologically active secretome from AFSCs that is free of all exogenous molecules. We demonstrate that the full secretome is capable of promoting stem cell proliferation, migration and protection of cells against senescence. Furthermore, it has significant anti-inflammatory properties. Most importantly we show that it promotes tissue regeneration in a model of muscle damage. We then demonstrate that the secretome contains extracellular vesicles (EV) that harbour much but not all the biological activity of the whole secretome. Proteomic characterisation of the EV and free secretome fraction show the presence of numerous molecules specific to each fraction that could be key regulators of tissue regeneration. Intriguingly we show that the EVs only contain miRNA and not mRNA. This suggests tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than the imposition of new, signalling pathways. The EVs harbour significant anti-inflammatory activity as well as promoting angiogenesis; the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury
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Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins
Abstract
Background
The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair.
Methods
Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source.
Results
Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage.
We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo.
Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome.
Conclusions
Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation
Chemotherapy-induced cachexia dysregulates hypothalamic and systemic lipoamines and is attenuated by cannabigerol
Background: Muscle wasting, anorexia, and metabolic dysregulation are common sideâeffects of cytotoxic chemotherapy, having a doseâlimiting effect on treatment efficacy, and compromising quality of life and mortality. Extracts of Cannabis sativa, and analogues of the major phytocannabinoid Î9âtetrahydrocannabinol, have been used to ameliorate chemotherapyâinduced appetite loss and nausea for decades. However, psychoactive sideâeffects limit their clinical utility, and they have little efficacy against weight loss. We recently established that the nonâpsychoactive phytocannabinoid cannabigerol (CBG) stimulates appetite in healthy rats, without neuromotor sideâeffects. The present study assessed whether CBG attenuates anorexia and/or other cachectic effects induced by the broadâspectrum chemotherapy agent cisplatin. Methods: An acute cachectic phenotype was induced in adult male Listerâhooded rats by 6 mg/kg (i.p.) cisplatin. In total 66 rats were randomly allocated to groups receiving vehicle only, cisplatin only, or cisplatin and 60 or 120 mg/kg CBG (po, b.i.d.). Feeding behavior, bodyweight and locomotor activity were recorded for 72 hours, at which point rats were sacrificed for postâmortem analyses. Myofibre atrophy, protein synthesis and autophagy dysregulation were assessed in skeletal muscle, plasma metabolic profiles were obtained by untargeted 1HâNMR metabonomics, and levels of endocannabinoidâlike lipoamines quantified in plasma and hypothalami by targeted HPLCâMS/MS lipidomics. Results: CBG (120 mg/kg) modestly increased food intake, predominantly at 36â60hrs (p<0.05), and robustly attenuated cisplatinâinduced weight loss from 6.3% to 2.6% at 72hrs (p<0.01). Cisplatinâinduced skeletal muscle atrophy was associated with elevated plasma corticosterone (3.7 vs 13.1ng/ml, p<0.01), observed selectively in MHC type IIx (p<0.05) and IIb (p<0.0005) fibres, and was reversed by pharmacological rescue of dysregulated Akt/S6âmediated protein synthesis and autophagy processes. Plasma metabonomic analysis revealed cisplatin administration produced a wideâranging aberrant metabolic phenotype (Q2ƶ=0.5380, p=0.001), involving alterations to glucose, amino acid, choline and lipid metabolism, citrate cycle, gut microbiome function, and nephrotoxicity, which were partially normalized by CBG treatment (Q2ƶ=0.2345, p=0.01). Lipidomic analysis of hypothalami and plasma revealed extensive cisplatinâinduced dysregulation of central and peripheral lipoamines (29/79 and 11/26 screened, respectively), including reversible elevations in systemic Nâacyl glycine concentrations which were negatively associated with the antiâcachectic effects of CBG treatment. Conclusions: Endocannabinoidâlike lipoamines may have hitherto unrecognized roles in the metabolic sideâeffects associated with chemotherapy, with the Nâacyl glycine subfamily in particular identified as a potential therapeutic target and/or biomarker of anabolic interventions. CBGâbased treatments may represent a novel therapeutic option for chemotherapyâinduced cachexia, warranting investigation in tumourâbearing cachexia models
Additional file 2: of Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins
Figure S2. (A) The secretome generated from fibroblast cells under identical conditions to ADSC contains a significantly lower amount of total protein. (B-C) Characterising the fibroblast EVs using NTA found a significantly lower concentration per mL, with a similar size range to ADSC EVs