A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

Effects of oxidative stress and neuroprotection in apoptosis in neuronal cell models

The PC12 and SH-SY5Y cell models have been proposed as potentially realistic models to investigate neuronal cell toxicity. The effects of oxidative stress (OS) caused by both H2O2 and Aβ on both cell models were assessed by several methods. Cell toxicity was quantitated by measuring cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) viability assay, an indicator of the integrity of the electron transfer chain (ETC), and cell morphology by fluorescence and video microscopy, both of which showed OS to cause decreased viability and changes in morphology. Levels of intracellular peroxide production, and changes in glutathione and carbonyl levels were also assessed, which showed OS to cause increases in intracellular peroxide production, glutathione and carbonyl levels. Differentiated SH-SY5y cells were also employed and observed to exhibit the greatest sensitivity to toxicity. The neurotrophic factor, nerve growth factor (NGF) was shown to cause protection against OS. Cells pre-treated with NGF showed higher viability after OS, generally less apoptotic morphology, recorded less apoptotic nucleiods, generally lower levels of intracellular peroxides and changes in gene expression. The neutrophic factor, brain derived growth factor (BDNF) and ascorbic acid (AA) were also investigated. BDNF showed no specific neuroprotection, however the preliminary data does warrant further investigation. AA showed a 'janus face' showing either anti-oxidant action and neuroprotection or pro-oxidant action depending on the situation. Results showed that the toxic effects of compounds such as Aβ and H2O2 are cell type dependent, and that OS alters glutathione metabolism in neuronal cells. Following toxic insult, glutathione levels are depleted to low levels. It is herein suggested that this lowering triggers an adaptive response causing alterations in glutathione metabolism as assessed by evaluation of glutathione mRNA biosynthetic enzyme expression and the subsequent increase in glutathione peroxidase (GPX) levels.

Evaluating the potential neurotoxicity of hexanedione isomers:an in vitro approach

2,5-hexanedione (2,5HD) is the neurotoxic metabolite of the aliphatic hydrocarbon n-Hexane. The isomers, 2,3-hexanedione (2,3HD) and 3,4-hexanedione (3,4HD) are used as food additives. Although the neurotoxicity of 2,5HD is well established, there are no human data of the possible toxicity of the 2,3- and 3,4- isomers. MTT and flow cytometry were utilised to determine the cytotoxicity of hexanedione isomers in neuroblastoma cells. The neuroblastoma cell lines SK-N-SH and SH-SY5Y are sufficiently neuron-like to provide preliminary assessment of the neurotoxic potential of these isomers, in comparison with toxicity towards human non-neuronal cells. Initial studies showed that 2,5HD was the least toxic in all cell lines at all times (4, 24 and 48h). Although considerably lower than for 2,5HD, in general the IC50s for the α isomers were not significantly different from each other and, besides 4h exposure, the SH-SY5Y cells were significantly more sensitive to 2,3HD and 3,4HD than the SK-N-SH cells. All three isomers caused varying degrees of apoptosis in the neuroblastoma lines, with 3,4HD more potent than 2,3HD. Flow cytometry highlighted cell cycle arrest indicative of DNA damage with 2,3- and 3,4HD. The toxicity of the isomers towards 3 non-neuronal cell lines (MCF7, HepG2 and CaCo-2) was assessed by MTT assay. All 3 hexanedione isomers proved to be cytotoxic in all non-neuronal cell lines at all time points. These data suggest cytotoxicity of 2,3- and 3,4HD (mM range), but it is difficult to define this as specific neurotoxicity in the absence of specific neurotoxic endpoints. However, the neuroblastomas were significantly more susceptible to the cytotoxic effects of the α hexanedione isomers at exposures of 4 and 24 hours, compared to non-neuronal lines. Finally, a mechanism of toxicity is suggested for the α HD isomers whereby inhibition of the oxoglutarate carrier (OGC) releases apoptosis inducing factor (AIF), causing apoptosis-like cell death.

A preliminary investigation into the impact of a pesticide combination on human neuronal and glial cell lines in vitro

Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health.

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role. © 2011 Springer-Verlag.

Human intervertebral disc aggrecan inhibits nerve growth in vitro

OBJECTIVE: To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques. METHODS: Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared. RESULTS: Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentration-dependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects. CONCLUSION: Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.

Chemical characterization and in vitro cyto- and genotoxicity of ‘legal high’ products containing Kratom (Mitragyna speciosa)

Kratom is a popular ‘legal high’ mainly constituted by alkaloids extracted from the Mitragyna speciosa plant with mitragynine (MG) as the dominant active substance. The increasing use of Kratom for recreational purposes has alerted risk assessment bodies of the lack of information on the real composition and its potential health risks. The present study aimed to determine and compare the MG composition of 13 commercial products of Kratom sold online and in “smartshops”, by gas chromatography–mass spectrometry. For the first time, the cytotoxicity induced by pure MG and Kratom, extracts was evaluated in in vitro models of human intestinal (Caco-2) and neuronal (SH-SY5Y) cells after 6 and 24 h. Genotoxicity was also evaluated in intestinal Caco-2 cells following 24 h of exposure to subtoxic concentrations using the comet assay. The obtained results revealed an inconsistency between the information (‘power’) provided in labels and the MG content. Cytotoxicity tests revealed a concentration-dependent decrease in cell viability in both cellular models, with the SH-SY5Y cells being more sensitive to the Kratom extracts. The resin and the ‘powered extracts’ were the most cytotoxic samples, with IC50 values significantly lower than the leaf extracts and pure MG (P < 0.0001 vs. leaf extracts and MG). In addition, significant DNA damage was observed in Caco-2 cells exposed to these extracts but not to pure MG, which suggests that other substances present in the extracts or interactions involving Kratom components might be responsible for the observed effects.

CD81 interacting proteins

Fertilization is a multistep and complex process culminating in the merge of gamete membranes, cytoplasmic unity and fusion of genome. CD81 is a tetraspanin protein that participates in sperm-oocyte interaction, being present at the oocyte surface. CD81 has also been implicated in other biological processes, however its specific function and molecular mechanisms of action remain to be elucidated. The interaction between CD81 and its binding partner proteins may underlie the CD81 involvement in a variety of cellular processes and modulate CD81/interactors specific functions. Interestingly, in a Yeast two Hybrid system previously performed in our lab, CD81 has emerged as a putative interactor of the Amyloid Precursor Protein (APP). In the work here described, bioinformatics analyses of CD81 interacting proteins were performed and the retrieved information used to construct a protein-protein interaction network, as well as to perform Gene Ontology enrichment analyses. CD81 expression was further evaluated in CHO, GC-1 and SH-SY5Y cell lines, and in human sperm cells. Additionally, its subcellular localization was analyzed in sperm cells and in the neuronal-like SH-SY5Y cell line. Subsequently, coimmunoprecipitation assays were performed in CHO and SH-SY5Y cells to attempt to prove the physical interaction between CD81 and APP. A functional interaction between these two proteins was accessed thought the analyses of the effects of CD81 overexpression on APP levels. A co-localization analysis of CD81 and some interactors proteins retrieved from the bioinformatics analyses, such as APP, AKT1 and cytoskeleton-related proteins, was also performed in sperm cells and in SH-SY5Y cells. The effects of CD81 in cytoskeleton remodeling was evaluated in SH-SY5Y cells through monitoring the effects of CD81 overexpression in actin and tubulin levels, and analyzing the colocalization between overexpressed CD81 and F-actin. Our results showed that CD81 is expressed in all cell lines tested, and also provided the first evidence of the presence of CD81 in human sperm cells. CD81 immunoreactivity was predominantly detected in the sperm head, including the acrosome membrane, and in the midpiece, where it co-localized with APP, as well as in the post-acrosomal region. Furthermore, CD81 co-localizes with APP in the plasma membrane and in cellular projections in SH-SY5Y cells, where CD81 overexpression has an influence on APP levels, also visible in CHO cells. The analysis of CD81 interacting proteins such as AKT1 and cytoskeletonrelated proteins showed that CD81 is involved in a variety of pathways that may underlie cytoskeleton remodeling events, related to processes such as sperm motility, cell migration and neuritogenesis. These results deepen our understanding on the functions of CD81 and some of its interactors in sperm and neuronal cells.

Study of medicinal and food plants as a source of biologically active compounds.

In the last few decades, scientific evidence has pointed out the health-beneficial effects of phenolic compounds in foods, including a decrease in risk of developing degenerative and chronic diseases, known to be caused by oxidative stress. In this frame can be inserted research carried out during my PhD thesis, which concerns the phytochemical investigation of phenolic composition in sweet cherries (Prunus avium L.), apple fruits (Malus domestica L.) and quinoa seeds (Chenopodium quinoa Willd.). The first project was focused on the investigation of phytochemical profile and nutraceutical value of fruits of new sweet cherry cultivars. Their phenolic profile and antioxidant activity were investigated and compared with those of commonly commercialized cultivars. Their nutraceutical value was evaluated in terms of antioxidant/neuroprotective capacity in neuron-like SH-SY5Y cells, in order to investigate their ability to counteract the oxidative stress and/or neurodegeneration process The second project was focused on phytochemical analysis of phenolic compounds in apples of ancient cultivars with the aim of selecting the most diverse cultivars, that will then be assayed for their anti-carcinogenic and anti-proliferative activities against the hepato-biliary and pancreatic tumours. The third project was focused on the analysis of polyphenolic pattern of seeds of two quinoa varieties grown at different latitudes. Analysis of phenolic profile and in vitro antioxidant activity of seed extracts both in their free and soluble-conjugated forms, showed that the accumulation of some classes of flavonoids is strictly regulated by environmental factors, even though the overall antioxidant capacity does not differ in quinoa Regalona grown in Chile and Italy. During the internship period carried out at the Department of Organic Chemistry at Universidad Autónoma de Madrid (UAM), it was achieved the isolation of two pentacyclic triterpenoids, from an endemic Peruvian plant, Jatropha macrantha Müll. Arg., with bio-guided fractionation technique.

Neuroprotective role of nutraceutical and phytochemical components

Neurodegenerative diseases (NDs) are characterized by a multifactorial etiology, in which oxidative stress and inflammation are the main causative factors. For this reason, increasing attention is being paid to the characterization and the identification of nutraceuticals and phytochemicals with intrinsic pleiotropic activity. Moreover, in a Circular Economy perspective, these natural compounds can be obtained also from renewable resources derived from the food industry by-products and can be used for both preventive and therapeutic purposes. The aim of this PhD program was to identify nutraceuticals and phytochemicals, both as extracts and pure compounds, and obtained from both plant and renewable sources, which due to their antioxidant and anti-inflammatory properties, were able to counteract cellular and molecular alterations that characterize NDs. Their neuroprotective potential has been evaluated in an in vitro model of neuroinflammation (the LPS-activated BV-2 microglial cell line), and/or in an in vitro model of neuronal oxidative stress (the neuron-like SH-SY5Y cell line differentiated with retinoic acid and exposed to H2O2). Four different projects, although deeply linked by the aforementioned common goal, have been discussed in this thesis: 1_ Impact of phenolic profile of different cherry cultivars on the potential neuroprotective effect in SH-SY5Y cells. 2_Anti-inflammatory activities of Spilanthol-rich essential oil from Acmella oleracea (L.). 3_Study of the anti-inflammatory activity of novel tacrine derivatives with lipids extracted from cashew nutshell liquid. 4_Coffee Silverskin (CSS) and Spent Coffee Grounds (SCG): coffee industry by-products as a promising source of neuroprotective agents. In general, it is, therefore, possible to conclude that the natural compounds studied in this thesis have been proven, due to their antioxidant and/or anti-inflammatory properties, to be valid preventive and therapeutic strategies for the treatment of NDs, to improve the life quality of these patients and of the general population by preventing and combating the onset of these deleterious diseases.

Development of advanced analytical methodologies for Alzheimer’s disease drug discovery

Advanced analytical methodologies were developed to characterize new potential active MTDLs on isolated targets involved in the first stages of Alzheimer’s disease (AD). In addition, the methods investigated drug-protein bindings and evaluated protein-protein interactions involved in the neurodegeneration. A high-throughput luminescent assay allowed the study of the first in class GSK-3β/ HDAC dual inhibitors towards the enzyme GSK-3β. The method was able to identify an innovative disease-modifying agent with an activity in the micromolar range both on GSK-3β, HDAC1 and HDAC6. Then, the same assay reliably and quickly selected true positive hit compounds among natural Amaryllidaceae alkaloids tested against GSK-3β. Hence, given the central role of the amyloid pathway in the multifactorial nature of AD, a multi-methodological approach based on mass spectrometry (MS), circular dichroism spectroscopy (CD) and ThT assay was applied to characterize the potential interaction of CO releasing molecules (CORMs) with Aβ1-42 peptide. The comprehensive method provided reliable information on the different steps of the fibrillation process and regarding CORMs mechanism of action. Therefore, the optimal CORM-3/Aβ1−42 ratio in terms of inhibitory effect was identified by mass spectrometry. CD analysis confirmed the stabilizing effect of CORM-3 on the Aβ1−42 peptide soluble form and the ThT Fluorescent Analysis ensured that the entire fibrillation process was delayed. Then the amyloid aggregation process was studied in view of a possible correlation with AD lipid brain alterations. Therefore, SH-SY5Y cells were treated with increasing concentration of Aß1-42 at different times and the samples were analysed by a RP-UHPLC system coupled with a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode. Each lipid class profiling in SH-SY5Y cells treated with Aß1-42 was compared to the one obtained from the untreated. The approach underlined some peculiar lipid alterations, suitable as biomarkers, that might be correlated to Aß1-42 different aggregation species.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Protection by polyphenol extract from olive stones against apoptosis produced by oxidative stress in human neuroblastoma cells

Objective: We evaluated the protective activity of an extract from a by-product such as olive stones, through its ability to inhibit H2O2 induced apoptosis in the SH-SY5Y human neuroblastoma cell line. Material and methods: To such end, 20,000 cells/well were cultivated and differentiation with retinoic acid was initiated. Once the cells were differentiated, apoptosis was induced with and without H2O2 extract. Finally, cDNA extraction was performed, and pro-apoptotic genes Bax and anti-apoptotic genes Bcl-2 were analyzed. Quantification of the gene expression was performed using the GAPDH gene marker. Results: Cell viability with the extract is 97.6% (SD 5.7) with 10 mg/l and 62.8% (SD 1.2) to 50 mg/l, using 10 mg/l for the biomarker assay. The retinoic acid differentiated SH-S cell line (10 µM) shows a clear apoptosis when treated with H2O2 150 µM, with a Bax/Bcl-2 ratio of 3.75 (SD 0.80) in contrast to the differentiated control cells subjected to H2O2 and with extract, which have the same ratio of 1.02 (SD 0.01-0.03). Conclusion: The olive stone extract shows anti-apoptotic activity in the provoked cell death of SH-SY5Y human neuroblastoma cells in their normal state, defending them from oxidative stress which produces a significant increase in the apoptotic gene ratio in contrast to anti-apoptotic genes (Bax/Bcl-2).

Canine mesenchymal stem cells are neurotrophic and angiogenic:an in vitro assessment of their paracrine activity

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P < 0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker βIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P < 0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action.