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Mesenchymal stem cell (MSC) secretome refers to a variety of bioactive compounds that represents the more important pathway by which MSCs participate in tissue regeneration. Many of these compounds have shown variable functional activity even across nonmammalian vertebrate species, although MSCs in turtles have not yet been described. Canine adipose MSC secretome has been successfully used experimentally in skin healing. Our aim was to conduct a blinded controlled study to evaluate the effect of canine adipose MSC secretome (cS-MSC) as an alternative for the healing of soft skin, second intention wounds of red-eared slider turtles (Trachemys scripta). Under general anesthesia, one circular, 6-mm full thickness wound was made in each rear leg of 14 females. After randomization, cS-MSC was injected subcutaneously around one wound at days 1, 7, and 14, whereas the other wound acted as control. Biopsies from three animals' wounds were obtained at days 21, 28, 42, and 63. Differences in mean wound retraction at days 21 (n=14) and 28 (n=11) were statistically nonsignificant. The clinical and histopathologic scores performed blind by two different investigators were similar for treated and control wounds. In conclusion, we could not detect a significant functional activity of cS-MSC on wound healing of Trachemys scripta.

The two main sources of mesenchymal stem cell (MSCs) in the canine species are bone marrow (cBM-MSCs) and adipose tissue (cAd-MSCs). The secretion of multitude bioactive molecules, included under the concept of secretome and found in the cultured medium, play a predominant role in the mechanism of action of these cells on tissue regeneration. Although certain features of its characterization are well documented, their secretory profiles remain unknown. We described and compared, for the first time, the secretory profile and exosomes characterization in standard monolayer culture of MSCs from both sources of the same donor as well as its immunomodulatory potential. We found that despite the similarity in surface immunophenotyping and trilineage differentiation, there are several differences in terms of proliferation rate and secretory profile. cAd-MSCs have advantages in proliferative capacity, whereas cBM-MSCs showed a significantly higher secretory production of some soluble factors (IL-10, IL-2, IL-6, IL-8, IL-12p40, IFN-γ, VEGF-A, NGF-β, TGF-β, NO and PGE2) and exosomes under the same standard culture conditions. Proteomics analysis confirm that cBM-MSCs exosomes have a greater number of characterized proteins involved in metabolic processes and in the regulation of biological processes compared to cAd-MSCs. On the other hand, secretome from both sources demonstrate similar immunomodulatory capacity when tested in mitogen stimulated lymphocyte reaction, but not in their exosomes at the dose used. Considering that the use of secretome open as a new therapeutic strategy for different diseases, without the need to implant cells, those biological differences should be considered, when choosing the MSCs source, for either cellular implantation or direct use of secretome for a specific clinical application.

Simple Summary Feline chronic gingivostomatitis (FCGS) is a common pathology in cats, related to an aberrant immune response. The cause of FCGS remains elusive, despite extensive investigations. A multitude of conditions and infectious agents have been related, without proof of causation, as follows: virus, bacteria, environmental stress, hypersensitivity, etc. In recent years, therapies based on feline adipose-derived mesenchymal stem cells (fAd-MSC) have become an interesting alternative for the treatment of different complex pathologies in cats. Mesenchymal stem cells secrete a wide variety of therapeutic elements, such as bioactive molecules and extracellular vesicles, such as exosomes. It is essential to characterize these elements, to better understand their mechanisms of action. In this study, we show, for the first time, that the proteomic profile of fAd-MSC-derived exosomes, from calicivirus-positive patients with severe FCGS, is altered. Using bioinformatic tools, we have demonstrated the existence of different proteins in the exosomes from diseased patients, responsible for an altered biological effect. In addition, the exosomes do not only experience changes in their cargo, but are also produced in larger quantities. This study might contribute to the better prediction of the clinical outcomes of mesenchymal stem cell treatments in veterinary patients with immune-mediated diseases, such as FCGS. Abstract Feline chronic gingivostomatitis (FCGS) is a pathology with a complicated therapeutic approach and with a prevalence between 0.7 and 12%. Although the etiology of the disease is diverse, feline calicivirus infection is known to be a predisposing factor. To date, the available treatment helps in controlling the disease, but cannot always provide a cure, which leads to a high percentage of refractory animals. Mesenchymal stem cells (MSCs) play a pivotal role in the homeostasis and reparation of different tissues and have the ability to modulate the immune system responses. This ability is, in part, due to the capacity of exosomes to play a part in intercellular cell communication. However, the precise role of MSC-derived exosomes and their alterations in immunocompromised pathologies remains unknown, especially in veterinary patients. The goal of this work was to analyze the proteomic profile of feline adipose tissue-derived MSCs (fAd-MSCs) from calicivirus-positive FCGS patients, and to detect possible modifications of the exosomal cargo, to gain better knowledge of the disease’s etiopathogenesis. Using high-resolution mass spectrometry and functional enrichment analysis with Gene Ontology, exosomes isolated from the fAd-MSCs of five healthy cats and five calicivirus-positive FCGS patients, were pooled and compared. The results showed that the fAd-MSCs from cats suffering from FCGS not only had a higher exosome production, but also their exosomes showed significant alterations in their proteomic profile. Eight proteins were exclusively found in the exosomes from the FCGS group, and five proteins could only be found in the exosomes from the healthy cats. When comparing the exosomal cargo between the two groups, significant upregulation of 17 and downregulation of 13 proteins were detected in the FCGS group compared to the control group. These findings shed light on new perspectives on the roles of MSCs and their relation to this disease, which may help in identifying new therapeutic targets and selecting specific biomarkers.

The study of cancer biology should be based around a comprehensive vision of the entire tumor ecosystem, considering the functional, bioenergetic and metabolic state of tumor cells and those of their microenvironment, and placing particular importance on immune system cells. Enhanced understanding of the molecular bases that give rise to alterations of pathways related to tumor development can open up new therapeutic intervention opportunities, such as metabolic regulation applied to immunotherapy. This review outlines the role of various oncometabolites and immunometabolites, such as TCA intermediates, in shaping pro/anti-inflammatory activity of immune cells such as MDSCs, T lymphocytes, TAMs and DCs in cancer. We also discuss the extraordinary plasticity of the immune response and its implication in immunotherapy efficacy, and highlight different therapeutic intervention possibilities based on controlling the balanced systems of specific metabolites with antagonistic functions.

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans.

Limbal stem cells (LSCs) are a quiescent cell population responsible for the renewal of the corneal epithelium. Their deficiency is responsible for the conjunctivization of the cornea that is seen in different ocular pathologies, both in humans and in the canine species. The canine species represents an interesting preclinical animal model in ocular surface pathologies. However, the role of LSCs in physiological and pathological conditions in canine species is not well understood. Our objective was to characterize for the first time the soluble factors and the proteomic profile of the secretome and exosomes of canine LSCs (cLSCs). In addition, given the important role that fibroblasts play in the repair of the ocular surface, we evaluated the influence of the secretome and exosomes of cLSCs on their proliferation in vitro. Our results demonstrated a secretory profile of cLSCs with high concentrations of MCP-1, IL-8, VEGF-A, and IL-10, as well as significant production of exosomes. Regarding the proteomic profile, 646 total proteins in the secretome and 356 in exosomes were involved in different biological processes. Functionally, the cLSC secretome showed an inhibitory effect on the proliferation of fibroblasts in vitro, which the exosomes did not. These results open the door to new studies on the possible use of the cLSC secretome or some of its components to treat certain pathologies of the ocular surface in canine species.

Background: Canine colostrum milk (CCM) is a specific secretion of the mammary gland fundamental for the survival of the newborn. It has many described components (immunoglobulins, proteins or fat), but small vesicles named exosomes are largely unknown. Results: A characterization of the CCM exosomes has been performed. Exosome concentrations were abundant in CCM and appeared with characteristic cup-shaped morphology and well-defined round vesicles. Their size distribution was between 37−140 nm and western blot analysis showed positive expression of specific exosomal markers. Proteomic analysis revealed a total of 826 proteins in exosomes cargo. We also found that exosomes modified proliferation and secretory profiles in canine mesenchymal stem cells derived from bone marrow (cBM-MSCs) and adipose tissue (cAd-MSCs). Besides, CCM exosomes demonstrated a potent antioxidant effect on canine fibroblasts in culture. Conclusions: Our findings highlight, for the first time, the abundant presence of exosomes in CCM and their ability to interact with mesenchymal stem cells (MSCs). The addition of exosomes to the two types of MSCs in culture resulted in specific secretory profiles with functions related to angiogenesis, migration and chemotaxis of immune cells. In particular, the cAd-MSCs secretory profile showed a higher potential in adipose tissue development and neurogenesis, while cBM-MSCs production was associated with immunity, cell mobilization and hematopoiesis. Finally, exosomes also presented antioxidant capacity on fibroblasts against reactive oxygen species activity within the cell, demonstrating a fundamental role in the development and maturation of the puppy in the early stages of its life.

Background Colostrum is a specific secretion of the mammary gland fundamental for the survival of the puppy during the first weeks after birth. It contains important bioactive molecules involved in the passive immunity and the maturation of various organs, highlighting small vesicles named exosomes. Exosomes have not been described yet in canine colostrum milk, where its role is crucial in dam-newborn communication for the development of the neonate. Results Exosomes were abundant in canine colostrum milk and appeared with a characteristic cup-shaped morphology and well-defined round vesicles. Their size distribution was between 37−140 nm and western blot analysis showed an expression of specific exosomal markers. Proteomic analysis revealed a total of 826 proteins in exosomes cargo. We also found that exosomes modified proliferation and secretory profile in canine mesenchymal stem cells derived from bone marrow and adipose tissue, differently according to their origin. Besides, the exosomes of canine colostrum demonstrated a potent antioxidant effect on canine fibroblast. Conclusions We described for the first time the isolation and characterization of milk exosomes from canine colostrum. Our findings highlight the abundant presence of exosomes in the colostrum of the canine species and contribute to explain their important antioxidant capacity after parturition and their role in the modulation of cell development and tissue differentiation in the newborn, where mesenchymal stem cells seem to play a key role.

Background: Colostrum is a specific secretion of the mammary gland fundamental for the survival of the puppy during the first weeks after birth. It contains important bioactive molecules involved in the passive immunity and the maturation of various organs, highlighting small vesicles named exosomes. Exosomes have not been described yet in canine colostrum milk, where its role is crucial in dam-newborn communication for the development of the neonate. Results: Exosomes were abundant in canine colostrum milk and appeared with a characteristic cup-shaped morphology and well-defined round vesicles. Their size distribution was between 37−140 nm and western blot analysis showed an expression of specific exosomal markers. Proteomic analysis revealed a total of 826 proteins in exosomes cargo. We also found that exosomes modified proliferation and secretory profile in canine mesenchymal stem cells derived from bone marrow and adipose tissue, differently according to their origin. Besides, the exosomes of canine colostrum demonstrated a potent antioxidant effect. Conclusions: We described for the first time the isolation and characterization of milk exosomes from canine colostrum. Our findings highlight the abundant presence of exosomes in the colostrum of the canine species and contribute to explain their important antioxidant capacity after parturition and their role in the modulation of cell development and tissue differentiation in the newborn, where mesenchymal stem cells seem to play a key role.

Canine atopic dermatitis (AD) is a common skin disease with a 10–15 per cent prevalence. Current treatments vary in their efficacy and safety. The immunomodulatory properties of mesenchymal stem cells (MSCs) make them a promising alternative treatment. The aim of this study was to evaluate the therapeutic efficacy and safety of allogeneic canine adipose MSCs (cAd-MSCs) in dogs with refractory AD. Twenty-six dogs, suffering from AD for at least 12 months, not responding to conventional therapy, received an intravenous dose of 1.5×10 6 cAd-MSCs/kg bodyweight. Clinical signs, haematological and biochemistry profiles, and AD severity were assessed in a six-month follow-up using a validated scoring system (Canine Atopic Dermatitis Extent and Severity Index, version 4 (CADESI-04)). The degree of pruritus was quantified using a validated visual analogue scale, and also owner’s global assessment of treatment efficacy. Twenty-two animals completed the study. Pruritus and CADESI-04 scores decreased significantly after one week or month of treatment, respectively, and remained stable for six months. Owner’s global assessment score was 2.15±1.15 for all the animals in the study. In conclusion, systemic administration of allogeneic cAd-MSCs appeared to be a simple therapy with positive outcome in the remission of clinical signs for AD refractory to conventional medications, for at least six months and with no adverse events.

Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is an immune-mediated multifactorial disease, with high level of prevalence in humans and dogs. Our aim in this study was to investigate the therapeutic effects of allogeneic adipose-derived mesenchymal stromal cells (Ad-MSCs) implanted around the lacrimal glands in 12 dogs (24 eyes) with KCS, which is refractory to current available treatments. Schirmer tear test (STT) and ocular surface integrity were assessed at 0 (before treatment), 3, 6, and 9 months after treatment. Average STT values and all clinical signs showed a statistically significant change(P<0.001)during the follow-up with reduction in all ocular parameters scored: ocular discharge, conjunctival hyperaemia, and corneal changes, and there were no signs of regression or worsening. Implanted cells were well tolerated and were effective reducing clinical signs of KCS with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that implantation of allogeneic Ad-MSCs around lacrimal glands has been found as an effective therapeutic alternative to treat dogs with KCS. These results could reinforce a good effective solution to be extrapolated to future studies in human.

Background Feline eosinophilic keratitis (FEK) is a chronic keratopathy caused by a suspected immune mediated response to an unknown antigenic stimulus. The purpose of this study was to investigate the safety and therapeutic effects of allogeneic feline adipose-derived mesenchymal stromal cells (fAd-MSCs) implanted subconjunctival around the ocular surface lesion in five cats with FEK refractory to current available treatments. Results FEK was diagnosed by clinical appearance and evidence of eosinophil and/or mast cells in corneal cytology. Each animal was treated with two applications of 2 × 106 million of fAd-MSCs 2 months apart. Ocular surface integrity was assessed before treatment and at 1, 3, 6 and 11 months after treatment. Clinical signs showed a significant change during the follow-up with resolution of the corneal and conjunctiva lesions and there were no signs of regression or worsening. Conclusions Implanted cells were well-tolerated and effective reducing clinical signs of FEK with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that local implantation of allogeneic fAd-MSCs has been found as an effective therapeutic alternative to treat cats with FEK.