Canine Mesenchymal Stem Cells Research Articles

Effect of storage conditions on the quality of equine and canine mesenchymal stem cell derived nanoparticles including extracellular vesicles for research and therapy

Nanoparticles including extracellular vesicles derived from mesenchymal stem cells are of increasing interest for research and clinical use in regenerative medicine. Extracellular vesicles (EVs), including also previously named exosomes, provide a promising cell-free tool for therapeutic applications, which is probably a safer approach to achieve sufficient healing. Storage of EVs may be necessary for clinical applications as well as for further experiments, as the preparation is sometimes laborious and larger quantities tend to be gained. For this purpose, nanoparticles were obtained from mesenchymal stem cells from adipose tissue (AdMSC) of horses and dogs. The EVs were then stored for 7 days under different conditions (− 20 °C, 4 °C, 37 °C) and with the addition of various additives (5 mM EDTA, 25–250 µM trehalose). Afterwards, the size and number of EVs was determined using the nano tracking analyzing method. With our investigations, we were able to show that storage of EVs for up to 7 days at 4 °C does not require the addition of supplements. For the other storage conditions, in particular freezing and storage at room temperature, the addition of EDTA was found to be suitable for preventing aggregation of the particles. Contrary to previous publications, trehalose seems not to be a suitable cryoprotectant for AdMSC-derived EVs. The data are useful for processing and storage of isolated EVs for further experiments or clinical approaches in veterinary medicine.

Transcriptomic and intervention evidence reveals domestic dogs as a promising model for anti-inflammatory investigation.

Domestic dogs have great potential to expand our understanding of the determinants of aging. To understand the aging pattern of domestic dogs and evaluate whether they can be used as an aging model, we performed RNA sequencing on white blood cells from domestic dogs aged 1-9 years and treated aged dogs with classical antiaging approaches. We obtained 30 RNA sequencing libraries and identified 61 age-associated genes with dynamic changes, the majority of which were related to metabolism and immune function, which may be predominant biomarkers for aging in dogs. We next treated aged dogs with canine mesenchymal stem cells (cMSCs), nicotinamide mononucleotide, and rapamycin to determine whether and how they responded to the antiaging interventions. The results showed that these treatments can significantly reduce the level of inflammatory factors (IL-6 and TNF-α). MSCs effectively improved the heart functions of aged dogs. Three key potential age-related genes (PYCR1, CCRL2, and TOX) were reversed by MSC treatment, two of which (CCRL2 and TOX) are implicated in immunity. Overall, we profiled the transcriptomic pattern of domestic dogs and revealed that they may be a good model of aging, especially in anti-inflammatory investigations.

Optimal technique for canine mesenchymal stem cells labeling with novel SPIO, MIRB™: for MRI detection of transplanted stem cells canine stroke model

ABSTRACT Background Cell-based therapy has emerged as a promising avenue for post-stroke recovery. A significant challenge lies in tracking the distribution and engraftment of transplanted cells within the target cerebral tissue. To address this, we turn to the potential of Brain MRI detection of mesenchymal stem cells (MSCs), achieved by labeling these cells with superparamagnetic iron oxide (SPIO). This is the first report of a technique to label canine MSCs using a commercially available SPIO, Molday ION Rhodamine B (MIRB), to optimize both viability and labeling efficacy for transplantation purposes.” Method Canine MSCs were incubated with addition of different MIRB concentration from 0, 10, 20, 30 μg Fe/ml. The cellular uptake of MIRB was confirmed through the analysis of fluorescent images and flow cytometry. The morphological characteristics of MSCs were assessed via microscopic visualization. Cellular viability was evaluated using both a cellometer and flow cytometry. Result Fluorescent microscopic images of all MIRB incubated MSCs groups show >70% labeled cells with homogenous signal intensity. Notably, the morphology of MSCs remained unaltered in the 10 μg Fe/ml group compared to the control group. Furthermore, among the labeled groups, the 10 μg Fe/ml concentration exhibited the highest viability when assessed using two different flow cytometry methods (95.3%, p < 0.05). Conclusion This study successfully labels canine MSCs with MIRB. The optimal concentration of 10 μg Fe/ml demonstrates optimal viability, labeling efficacy, and preserved cellular morphology.

Impact of multiple isolation procedures on the differentiation potential of adipose derived canine mesenchymal stem cells

Impact of multiple isolation procedures on the differentiation potential of adipose derived canine mesenchymal stem cells

Immortalized Canine Adipose-Derived Mesenchymal Stem Cells Maintain the Immunomodulatory Capacity of the Original Primary Cells.

Mesenchymal stem cells (MSCs) are a promising cell source for stem cell therapy of intractable diseases in veterinary medicine, but donor-dependent cellular heterogeneity is an issue that influences therapeutic efficacy. Thus, we previously established immortalized cells that maintain the fundamental properties of primary cells, but functional evaluation had not been performed. Therefore, we evaluated the immunomodulatory capacity of the immortalized canine adipose-derived MSCs (cADSCs) in vitro and in vivo to investigate whether they maintain primary cell functions. C57BL/6J mice were treated with dextran sulfate sodium (DSS) to induce colitis, injected intraperitoneally with immortalized or primary cADSCs on day 2 of DSS treatment, and observed for 10 days. Administration of immortalized cADSCs improved body weight loss and the disease activity index (DAI) in DSS-induced colitic mice by shifting peritoneal macrophage polarity from the M1 to M2 phenotype, suppressing T helper (Th) 1/Th17 cell responses and inducing regulatory T (Treg) cells. They also inhibited the proliferation of mouse and canine T cells in vitro. These immunomodulatory effects were comparable with primary cells. These results highlight the feasibility of our immortalized cADSCs as a cell source for stem cell therapy with stable therapeutic efficacy because they maintain the immunomodulatory capacity of primary cells.

Development of a Large-Scale Pathogen Screening Test for the Biosafety Evaluation of Canine Mesenchymal Stem Cells

BackgroundThe action of mesenchymal stem cells (MSCs) is the subject of intense research in the field of regenerative medicine, including their potential use in companion animals, such as dogs. To ensure the safety of canine MSC batches for their application in regenerative medicine, a quality control test must be conducted in accordance with Good Manufacturing Practices (GMP). Based on guidance provided by the European Medicines Agency, this study aimed to develop and validate a highly sensitive and robust, nucleic acid-based test panel for the detection of various canine pathogens. Analytical sensitivity, specificity, amplification efficiency, and linearity were evaluated to ensure robust assessment. Additionally, viable spike-in controls were used to control for optimal nucleic acid extraction. The conventional PCR-based and real-time PCR-based pathogen assays were evaluated in a real-life setting, by direct testing MSC batches.ResultsThe established nucleic acid-based assays displayed remarkable sensitivity, detecting 100–1 copies/reaction of template DNA. They also exhibited high specificity and efficiency. Moreover, highly effective nucleic acid isolation was confirmed by the sensitive detection of spike-in controls. The detection capacity of our optimized and validated methods was determined by direct pathogen testing of nine MSC batches that displayed unusual phenotypes, such as reduced cell division or other deviating characteristics. Among these MCS batches of uncertain purity, only one tested negative for all pathogens. The direct testing of these samples yielded positive results for important canine pathogens, including tick-borne disease-associated species and viral members of the canine infectious respiratory disease complex (CIRDC). Notably, samples positive for the etiological agents responsible for enteritis (CPV), leptospirosis (Leptospira interrogans), and neosporosis (Neospora caninum) were also identified. Furthermore, we conducted biosafety evaluation of 12 MSC batches intended for therapeutic application. Eleven MSC batches were found to be free of extraneous agents, and only one tested positive for a specific pathogen, namely, canine parvovirus.ConclusionIn this study, we established and validated reliable, highly sensitive, and accurate nucleic acid-based testing methods for a broad spectrum of canine pathogens.

Autologous Platelet Lysate Is an Alternative to Fetal Bovine Serum for Canine Adipose-Derived Mesenchymal Stem Cell Culture and Differentiation.

The use of fetal bovine serum (FBS) in regenerative medicine raises serious ethical and scientific concerns. We have cultured and differentiated the canine mesenchymal stem cells (cMSCs) in five different media combinations of autologous platelet lysate (A-PL) and FBS; consisting of 0% A-PL and 10% FBS (M-1), 2.5% A-PL and 7.5% FBS (M-2), 5% A-PL and 5% FBS (M-3), 7.5% A-PL and 2.5% FBS (M-4), and 10% A-PL and 0% FBS (M-5). The cMSCs were evaluated for their doubling time, differentiation efficiency, and expression of CD73, CD90, CD105, and PDGFRα. The mRNA expression of NT5E, THY1, ENG, PPARγ, FABP4, FAS, SP7, BGLAP, and SPP1 was also assessed. The results indicated non-significant differences in cellular proliferation/viability; positive expression of surface markers, and PDGFRα with substantial adipo/osteogenic differentiation. The expression of adipogenic (PPARγ, FABP4, FAS), and osteogenic (SP7, BGLAP, SPP1) genes were higher (p < 0.05) in the M5 group. In conclusion, A-PL in cMSCs culture did not negatively affect cellular proliferation and viability but also enhanced their genetic potential for multilineage differentiation. Our results indicate that A-PL can be used as an alternative for FBS to develop potent cMSCs under good manufacturing practice protocol for regenerative medicine.

Immunomodulatory effects of canine mesenchymal stem cells in an experimental atopic dermatitis model.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multi-lineage cells, suggesting their future applicability in regenerative medicine and biotechnology. The immunomodulatory properties of MSCs make them a promising replacement therapy in various fields of animal research including in canine atopic dermatitis (AD), a skin disease with 10-15% prevalence. We investigated the immunomodulatory effects of MSCs in an experimental canine AD model induced by Dermatophagoides farinae extract ointment. Canine adipose tissue-derived MSCs (cAT-MSCs) were differentiated into mesodermal cell lineages at the third passage. Alterations in immunomodulatory factors in control, AD, and MSC-treated AD groups were evaluated using flow cytometric analysis, enzyme-linked immunosorbent assay, and quantitative reverse transcription PCR. In the MSC-treated AD group, the number of eosinophils decreased, and the number of regulatory T cells (Tregs) increased compared to those in the AD group. In addition, the immunoglobulin E (IgE) and prostaglandin E2 levels were reduced in the MSC-treated AD group compared to those in the AD group. Furthermore, the filaggrin, vascular endothelial growth factor, and interleukin-5 gene expression levels were relatively higher in the MSC-treated AD group than in the AD group, however, not significantly. cAT-MSCs exerted immunomodulatory effects in an AD canine model via a rebalancing of type-1 and -2 T helper cells that correlated with increased levels of Tregs, IgE, and various cytokines.

Canine amniotic membrane-derived mesenchymal stem cells ameliorate atopic dermatitis through regeneration and immunomodulation.

Mesenchymal stem cells (MSCs) are a promising tool for treating immune disorders. However, the immunomodulatory effects of canine MSCs compared with other commercialized biologics for treating immune disorders have not been well studied. In this study we investigated the characteristics and immunomodulatory effects of canine amnion membrane (cAM)-MSCs. We examined gene expression of immune modulation and T lymphocytes from activated canine peripheral blood mononuclear cell (PBMC) proliferation. As a result, we confirmed that cAM-MSCs upregulated immune modulation genes (TGF-β1, IDO1 and PTGES2) and suppressed the proliferation capacity of T cells. Moreover, we confirmed the therapeutic effect of cAM-MSCs compared with oclacitinib (OCL), the most commonly used Janus kinase (JAK) inhibitor, as a treatment for canine atopic dermatitis (AD) using a mouse AD model. As a result, we confirmed that cAM-MSCs with PBS treatment groups (passage 4, 6 and 8) compared with PBS only (PBS) though scores of dermatologic signs, tissue pathologic changes and inflammatory cytokines were significantly reduced. In particular, cAM-MSCs were more effective than OCL in the recovery of wound dysfunction, regulation of mast cell activity and expression level of immune modulation protein. Interestingly, subcutaneous injection of cAM-MSCs induced weight recovery, but oral administration of oclacitinib induced weight loss as a side effect. In conclusion, this study suggests that cAM-MSCs can be developed as a safe canine treatment for atopic dermatitis without side effects through effective regeneration and immunomodulation.

Osteogenic differentiation of adipose-derived canine mesenchymal stem cells seeded in porous calcium-phosphate scaffolds.

Engineered bone graft substitutes are a promising alternative and supplement to autologous bone grafts as treatments for bone healing impairment. Advances in human medicine extend an invitation to pursue these biomimetic strategies in animal patients, substantiated by the theory that specialized scaffolds, multipotent cells, and biological cues may be combined into a bioactive implant intended for the enhancement of tissue regeneration. This proof-of-concept study was designed to evaluate and validate the feasibility of beta-tricalcium phosphate foam scaffolds seeded with canine mesenchymal stem cells derived from adipose tissue. Cell-inoculated samples and sham controls were cultured statically for 72 hours in complete growth medium to evaluate seeding capacity, while a subset of loaded scaffolds was further induced with osteogenic culture medium for 21 days. Produced implants were characterized and validated with a combination of immunofluorescence and reflection confocal microscopy, scanning electron microscopy, and polymerase chain reaction to confirm osteogenic differentiation in tridimensional-induced samples. After 72 hours of culture, all inoculated scaffolds presented widespread yet heterogeneous surface seeding, distinctively congregating stem cells around pore openings. Furthermore, at 21 days of osteogenic culture conditions, robust osteoblastic differentiation of the seeded cells was confirmed by the change of cell morphology and evident deposition of extra-cellular matrix, accompanied by mineralization and scaffold remodeling; furthermore, all induced cell-loaded implants lost specific stemness immunophenotype expression and simultaneously upregulated genomic expression of osteogenic genes Osterix and Ostecalcin. β-TCP bio-ceramic foam scaffolds proved to be suitable carriers and hosts of canine adipose-derived MSCs, promoting not only surface attachment and proliferation, but also demonstrating strong in-vitro osteogenic potential. Although this research provides satisfactory in-vitro validation for the conceptualization and feasibility of a canine bio-active bone implant, further testing such as patient safety, large-scale reproducibility, and quality assessment are needed for regulatory compliance in future commercial clinical applications.

Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins.

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Canine umbilical cord tissue derived mesenchymal stem cells naturally express mRNAs of some antimicrobial peptides.

Antimicrobial peptides (AMPs) are naturally produced by all living organisms at a constitutive rate. They represent the first line of active defence systems against invading microorganisms, helping in innate immunity. Besides their therapeutic applications, great attention has also been given to the mesenchymal stem cells (MSCs) due to their antimicrobial activities. The study aimed to observe the mRNA expression profile of few antimicrobial peptides (AMPs) in canine MSCs during standard in vitro culture. MSCs were isolated from canine umbilical cord tissue, propagated and characterized by morphology, surface markers and tri-lineage differentiation capability. The mRNA expression of eleven commonly known antimicrobial peptides was checked by Reverse Transcriptase PCR. It has been found for the first time that canine MSCs naturally express the mRNAs of AMPs like C-X-C motif chemokine ligand 8 (CXCL8), Elafin (PI3), Hepcidin (HAMP), Lipocalin 2 (LCN2) and Secretory leukocyte protease inhibitor (SLPI). However, their expressions at protein level and, relation with antimicrobial effect of canine MSCs need to be explored.

Controlled release of canine MSC-derived extracellular vesicles by cationized gelatin hydrogels

Canine mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising form of regenerative therapy. Therapeutic application of EVs remains difficult due to the short half-life of EVs invivo and their rapid clearance from the body. We have developed cationized gelatin hydrogels that prolong the retention of EVs to overcome this problem. Canine MSCs were isolated from bone marrow. MSC-derived EVs were isolated from the culture supernatant by ultracentrifugation. Gelatin was mixed with ethylene diamine anhydrate to cationized. Distinct cross-linked cationized gelatin hydrogels were created by thermal dehydration. Hydrogels were implanted into the back subcutis of mice in order to evaluate the degradation profiles. Hydrogels with collagenase were incubated at 37°C invitro to quantize the release of EVs from hydrogels. Lipopolysaccharide (LPS)-stimulated BV-2cells were used to evaluate the immunomodulatory effect of EVs after release from the hydrogels. The cationized gelatin hydrogels suppressed EV release in PBS. More than 60% of immobilized EVs are not released from the hydrogels. The cationized hydrogels released EVs in a sustainable manner and prolonged the retention time of EVs depending on the intensity of cross-linking after degradation by collagenase. The expression of IL-1β in LPS-stimulated BV-2cells was lower in EVs released from the hydrogels than in controls. Our results indicate that the controlled release of EVs can be achieved by cationized gelatin hydrogels. The released EVs experimentally confirmed to be effective in reducing proinflammatory response. The cationized gelatin hydrogels appear to be useful biomaterials for releasing canine MSC-derived EVs for regenerative therapy.

Systemic cellular viroimmunotherapy for canine high-grade gliomas

BackgroundOncolytic viruses constitute a growing field of interest, both in human and veterinary oncology, given that they are particularly helpful for treating non-surgical tumors and disseminated cancer, such as high-grade...

Abstract IA005: On the use of stem cells to generate sarcomas in vitro models

Abstract Sarcomas are rare cancers arising in soft tissue and bone. They are difficult to diagnose and treat, with 38-48% mortality for this often young age group. It is now generally accepted that molecular changes in cancer open avenues for personalized medicine. However, to achieve this for sarcoma there are several challenges, as representative model systems are scarce. Moreover, sarcomas are heterogeneous and &amp;gt;70 sarcoma types are recognized. A simple dichotomy based on molecular pathways of simple versus complex genomics is often used as a conceptual framework. My group has used mesenchymal stem cells to generate a model for complex genome sarcomas such as osteosarcoma. Murine and canine mesenchymal stem cells (MSCs) can be used to model spontaneous malignant transformation towards sarcomas with complex genomics. These MSCs have an abnormal karyotype, many structural variants and point mutations at whole genome sequencing analysis, and form sarcomas (osteosarcoma, undifferentiated pleomorphic and spindle cell sarcoma) after injection into mice. Cross-species analysis reveals that p53 loss is an early event in sarcomagenesis, and MSCs with a knockout in Trp53 transform earlier compared to wild-type MSCs. In addition, loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: the majority of spontaneously transformed murine MSCs has alterations in p15Ink4b, p16Ink4a, or p19Arf and knockout of these genes in MSCs results in accelerated transformation. Of interest, osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor Palbociclib, suggesting that a subset of osteosarcoma patients with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6 (in total 20-23%) might benefit from CDK4/CDK6 inhibition. Bone and soft tissue tumors with a simple genome often carry chromosomal translocations. We have generated a model for pseudomyogenic hemangioendothelioma (PHE), a rare locally aggressive vascular tumor. PHE has a tumor-specific t(7;19)(q22;q13) translocation resulting in a SERPINE1-FOSB fusion. We used CRISPR/Cas9 to generate this fusion in induced pluripotent stem cells (hiPSCs), which were subsequently differentiated into ECs (hiPSC-ECs) to address this. Comparison of parental with PHE hiPSC-ECs shows invasive growth in mice after transplantation, and transcriptome analysis identifies PI3K-Akt and MAPK signaling pathways as possible therapeutic targets. The modified hiPSC-ECs thus recapitulate functional features of PHE and demonstrate how these stem cell based translocation models can be used to understand tumorigenic mechanisms and identify therapeutic targets. Citation Format: Judith V.M.G. Boveé. On the use of stem cells to generate sarcomas in vitro models [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA005.

In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album.

The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties. This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10-1, 1×10-2 potency (VAD1D2). MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test. At the concentration of 10 μL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 μL/mL presented absolute percentages of initial apoptosis twice as high as at 10 μL/mL, which was similar to the control (untreated group). The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.

MRI Tracking of Iron Oxide Labelled Canine Mesenchymal Stem Cells in Artificial Stifle Defects.

The aim of this study was to describe ultrasmall superparamagnetic iron oxides labelling of canine adipose-derived mesenchymal stem cells (AdMSCs) and the detection and semiquantitative evaluation of the labelled cells after implantation in artificial canine stifle defects using magnetic resonance imaging. Magnetic resonance imaging examinations of 10 paired (n = 20) cadaveric stifle joints were evaluated after creation of chondral defects and embedding of ultrasmall superparamagnetic iron oxides labelled canine mesenchymal stem cells. To prove the feasibility of the labelling for in vivo usage, Prussian blue staining, cell vitality tests and intralesional administration of labelled cells were conducted. Magnetic resonance imaging of ex vivo defects filled with different cell concentrations was obtained to depict the cell content semiquantitatively via signal intensity measurements (region of interest). Prussian blue staining showed that the labelling was effective. According to the vitality tests, it had no significant short-term influence on cell viability and proliferation rate. For the evaluation of the defect T2* sequences were feasible and stifle defects were visible allowing measurements of the signal intensity in all cases. Increasing the cell concentration within the chondral defects resulted in an inversely proportional, significant reduction of signal intensity according to the region of interest. Ultrasmall superparamagnetic iron oxides labelling was effective. The detection of the AdMSCs in a complex anatomical structure like the surface of the femoral condyle was possible and the T2* signal intensity of the implant region was significantly correlated with the concentration of the AdMSCs.

Topical applications of allogeneic adipose-derived mesenchymal stem cells ameliorate the canine keratoconjunctivitis sicca

BackgroundCanine keratoconjunctivitis sicca (KCS) is predominantly an immune-mediated disease. Current therapy of canine KCS is mainly by immunosuppressant, but the effectiveness was limited in some patients. In the past few years, some studies showed the results of the use of mesenchymal stem cells in treating canine KCS via periocular injections. However, the periocular injection procedure requires sedation or general anesthesia, and may lead to iatrogenic or incidental injury during the injection process. The aim of this study was to investigate the efficacy of topical allogenic canine adipose-derived mesenchymal stem cells (cAD-MSCs) in clinical patients of canine KCS.ResultsThe cAD-MSCs used in this study were characterized for their capability of tri-lineage differentiation and immunomodulatory properties. In addition, preparation methods for eye drops of cAD-MSCs was developed and its optimal preservation was tested. The canine KCS patients were recruited for clinical trial and divided into two groups based on their history of previous treatment. All patients received topical cAD-MSCs treatment once per week for 6 consecutive weeks and complete ophthalmic examinations were performed 1 week before treatment (week 0) and at 3rd, 6th, 9th weeks, respectively. The results showed that the quantity and quality of tears have improved significantly following topical cAD-MSCs treatment based on Schirmers tear test-1 and tear break-up time. More than half of all patients were found improved in the tear quantity. In particular, 56.5% of the patients that were unresponsive to prior immunosuppressant therapy had an effective increase in tear volume. The severity of clinical signs was also ameliorated according to the numeric rating scale score from both patient owners and the clinician.ConclusionTo sum up, topical cAD-MSCs may be beneficial especially in KCS patients with poor owner compliance for frequent daily use of eye drops or those who are unresponsive to immunosuppressant therapy.

A Comparative Study of Canine Mesenchymal Stem Cells Isolated from Different Sources.

Simple SummaryThe present study describes differences in the isolation yield, morphology, presence of surface markers and proliferation capacity but not in the multilineage potential of canine MSCs isolated from bone marrow, adipose tissue and amnion. Among all the MSCs analysed, AT-MSCs showed the highest isolation yield, phenotype homogeneity, proliferation capacity and osteogenic and chondrogenic potential. In addition, for BM-MSCs and AM-MSCs, we uncovered some differences that need to be considered during isolation, expansion and phenotyping prior to their possible application in targeted regenerative veterinary medicine.In this study, we provide comprehensive analyses of mesenchymal stem cells (MSCs) isolated from three types of canine tissues: bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and amniotic tissue (AM-MSCs). We compare their morphology, phenotype, multilineage potential and proliferation activity. The BM-MSCs and AM-MSCs showed fibroblast-like shapes against the spindle shape of the AT-MSCs. All populations showed strong osteogenic and chondrogenic potential. However, we observed phenotypic differences. The BM-MSCs and AT-MSCs revealed high expression of CD29, CD44, CD90 and CD105 positivity compared to the AM-MSCs, which showed reduced expression of all the analysed CD markers. Similarly, the isolation yield and proliferation varied depending on the source. The highest isolation yield and proliferation were detected in the population of AT-MSCs, while the AM-MSCs showed a high yield of cells, but the lowest proliferation activity, in contrast to the BM-MSCs which had the lowest isolation yield. Thus, the present data provide assumptions for obtaining a homogeneous MSC derived from all three canine tissues for possible applications in veterinary regenerative medicine, while the origin of isolated MSCs must always be taken into account.

Ruta Graveolens in Homeopathic Dilution Has In Vitro Antitumor Activity in Osteosarcoma Cells (U2OS) and Low Effect on Canine Mesenchymal Stem Cells (CTM)

Ruta graveolens belongs to the family Rutaceae, and it is used to treat inflammatory diseases and some cases of cancer due to its anti-inflammatory and analgesic properties. Osteosarcoma (OS) is a bone cancer that mainly affects children and adolescents and has a high index in dogs. The treatment of this cancer consists of high doses of chemotherapy, and resection of the area affected by cancer, thus making it a painful and invasive treatment for patients. Under this perspective, it is necessary to search for new forms of treatment that are less invasive and, at the same time, effective, considering that homeopathy is used as a complementary therapy and benefits the patient's quality of life. This study aimed to compare the in vitro action of the homeopathic medicine Ruta D35 on U2OS osteosarcoma cells and canine mesenchymal stem cells (MSC). Our results demonstrate that Ruta D35 has a toxic effect on U2OS cells (IC50 16.38µL/mL), reducing cell viability compared to MSC (IC50 67.38µL/mL). Therefore, Ruta D35 showed positive results in treating the OS U2OS line, demonstrating a potential activity in treating this cancer.