Canine Mesenchymal Stem Cells Research Articles

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.

In vitro generation of transplantable insulin-producing cells from canine adipose-derived mesenchymal stem cells

Canine mesenchymal stem cells (cMSCs) have potential applications for regenerative therapy, including the generation of insulin-producing cells (IPCs) for studying and treating diabetes. In this study, we established a useful protocol for generating IPCs from canine adipose mesenchymal stem cells (cAD-MSCs). Subsequently, in vitro preservation of pluronic F127-coated alginate (ALGPA)-encapsulated cAD-MSC-derived IPCs was performed to verify ready-to-use IPCs. IPCs were induced from cAD-MSCs with the modulated three-stepwise protocol. The first step of definitive endoderm (DE) induction showed that the cooperation of Chir99021 and Activin A created the effective production of Sox17-expressed DE cells. The second step for pancreatic endocrine (PE) progenitor induction from DE indicated that the treatment with taurine, retinoic acid, FGF2, EGF, TGFβ inhibitor, dorsomorphin, nicotinamide, and DAPT showed the significant upregulation of the pancreatic endocrine precursor markers Pdx1 and Ngn3. The last step of IPC production, the combination of taurine, nicotinamide, Glp-1, forskolin, PI3K inhibitor, and TGFβ inhibitor, yielded efficiently functional IPCs from PE precursors. Afterward, the maintenance of ALGPA-encapsulated cAD-MSC-derived IPCs with VSCBIC-1, a specialized medium, enhanced IPC properties. Conclusion, the modulated three-stepwise protocol generates the functional IPCs. Together, the encapsulation of cAD-MSC-derived IPCs and the cultivation with VSCBIC-1 enrich the maturation of generated IPCs.

Effect of Canine Adipose Mesenchymal Stem Cell Secretome on a Model of Second-Intention Wound Healing in the Red-Eared Slider Turtle (Trachemys scripta).

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.

Suicide gene therapy by canine mesenchymal stem cell transduced with thymidine kinase in a u-87 glioblastoma murine model: Secretory profile and antitumor activity.

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.

Smad4 and γ-secretase knock-down effect on osteogenic differentiation mediated via Runx2 in canine mesenchymal stem cells

Cell lineage determination during mesenchymal stem cell (MSCs) differentiation is a highly orchestrated process involving diverse signaling pathways and distinct classes of regulatory molecules. Bone morphogenetic protein (BMP) signaling positively influence the osteoblast lineage determination, whereas the Notch signaling may have a dimorphic action. Effective regenerative therapy for repairing bone defects requires ample knowledge of the signaling pathways responsible for the differentiation of MSCs. To elucidate the signaling pathways that drives canine bone-marrow derived MSCs towards osteogenic lineage, the current work was focused on BMP and Notch signaling. Target genes of Runx2, Smad4 and γ-secretase were silenced by short hairpin RNA (shRNA) in canine MSCs. Evaluation of the effect of gene silencing on in-vitro osteogenic differentiation potential was done by quantitative polymerase chain reaction (qPCR) for osteoblastic markers (Osteocalcin and Osteopontin) and Alizarin red S staining for the extracellular deposition of calcium. Silencing of Runx2 significantly reduced the osteocalcin and osteopontin gene expression while a similar trend was observed in the case of smad 4 silencing and their combination groups, but there was no difference found in Hey 1 expression. Runx2 and Smad4 silencing groups showed very less positive staining with Alizarin red S staining, whereas knockdown of γ-secretase and its combination groups showed reverse results as that of Runx2 and Smad4. Runx2 plays an indispensable part in directing the canine mesenchymal stem cells towards osteogenic lineage. Also, Smad-mediated BMP signaling induced the osteoblast-specific gene expression, whereas the notch pathway negatively regulated the osteogenic differentiation of canine MSCs.

Abstract 137: Dose-dependent Improvement In Stroke Outcomes Following Intra-arterial Mesenchymal Stem Cell Therapy In A Canine Endovascular Stroke Model

Introduction: Cell-based therapies for stroke remain under development and there is a lack of pre-clinical large animal data defining optimal dosing and delivery methods. Whereas safety and efficacy of intra-arterial (IA) mesenchymal stem cells (MSC) have been demonstrated in rodent models, large animal model are lacking, hampering advancement of the field and limiting mechanistic insights. Methods: An endovascular canine reversible Middle Cerebral Artery Occlusion (MCAO) model using a retractable platinum coil for 60-120 min was established. At 48-hour post-MCAO, allogeneic male canine MSCs (10-80 million) were delivered using a microcatheter in the ipsilateral upper cervical internal carotid artery of female canine subjects. Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at15-30 d post-MCAO and brains were harvested for histology and molecular analyses. Results: There was a dose-dependent reduction of infarct volume on MRI associated with improved neurological scoring and corticospinal tract caliber on Diffusion Tensor Imaging over the range of 10, 20 and 40 million MSCs vs. PBS control. This dose-range was safe and did not result in worsening of stroke volume or behavioral outcomes. In contrast, 80 million IA MSCs led to a worsened neurological score immediately following the injection and an increase in the infarct volume at 4 days post injection. Conclusion: Together these findings establish a successful preclinical canine model of stroke in which dose-dependent efficacy of IA MSC therapy is demonstrated to reduce stroke size and augment DTI. These findings have important clinical implications in designing early-stage trials of IA delivery in humans.

Effects of carrier solutions on the viability and efficacy of canine adipose-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) have favorable characteristics that render them a potent therapeutic tool. We tested the characteristics of MSCs after temporal storage in various carrier solutions, such as 0.9% saline (saline), 5% dextrose solution (DS), heparin in saline, and Hartmann’s solution, all of which are approved by the U.S. Food and Drug Administration (FDA). Phosphate-buffered saline, which does not have FDA approval, was also used as a carrier solution. We aimed to examine the effects of these solutions on the viability and characteristics of MSCs to evaluate their suitability and efficacy for the storage of canine adipose-derived MSCs (cADMSCs).ResultsWe stored the cADMSCs in the test carrier solutions in a time-dependent manner (1, 6, and 12 h) at 4 °C, and analyzed cell confluency, viability, proliferation, self-renewability, and chondrogenic differentiation. Cell confluency was significantly higher in 5% DS and lower in phosphate-buffered saline at 12 h compared to other solutions. cADMSCs stored in saline for 12 h showed the highest viability rate. However, at 12 h, the proliferation rate of cADMSCs was significantly higher after storage in 5% DS and significantly lower after storage in saline, compared to the other solutions. cADMSCs stored in heparin in saline showed superior chondrogenic capacities at 12 h compared to other carrier solutions. The expression levels of the stemness markers, Nanog and Sox2, as well as those of the MSC surface markers, CD90 and CD105, were also affected over time.ConclusionOur results suggest that MSCs should be stored in saline, 5% DS, heparin in saline, or Hartmann’s solution at 4 °C, all of which have FDA approval (preferable storage conditions: less than 6 h and no longer than 12 h), rather than storing them in phosphate-buffered saline to ensure high viability and efficacy.

A double-blinded placebo-controlled evaluation of adipose-derived mesenchymal stem cells in treatment of canine atopic dermatitis.

Mesenchymal stem cells (MSCs) have emerged as a new therapy for various immune-mediated inflammatory diseases. In this study we perform the first double-blinded, placebo-controlled evaluation of the efficacy of adipose-derived allogenic canine MSCs for the treatment of canine atopic dermatitis (cAD). Enrolled canine patients were randomly divided into placebo (PBS saline), low-dose (5 × 105 cells/kg), and high-dose (5 × 106 cells/kg) treatment groups. Each patient received three subcutaneous MSCs treatments or PBS saline at four-week intervals with injections at five sites. Patients were monitored by physical exams, pruritus visual analog scales (PVAS) signed by the primary caretaker, canine atopic dermatitis extent and severity index-4 (CADESI-4) scores by two veterinarians, and complete blood count and serum chemistry analysis along with laboratory analysis for potential biomarkers. Patients were kept off any immune-modulating drugs during the study period, and oral antibiotics and topicals were used for managing pruritus and secondary infections. The PVAS scores and the serum miR-483 levels were significantly lower in the high dose group compared to the placebo group at day90 post first-treatment. The CADESI-4 scores of the high dose group also showed downward trends. No severe adverse effects were observed in any patient in this study. The high dose MSC treatment is efficacious in alleviating the clinical signs of cAD until 30days after the last subcutaneous administration of MSCs, and miRNA-483 may be a reliable prognostic biomarker for cAD. The MSCs efficacy and potential biomarkers should be further explored by a larger scale clinical trial.

Transdifferentiation of canine mesenchymal stem cells into neuron-like cells by induction with β-mercaptoethanol

The objective of this study was to check whether β- mercaptoethanol in a culture medium can induce the neuronal differentiation of canine MSCs. The canine bonemarrow derived MSCs were first pre-inducted with 1 mM BME for 24 hrs followed by induction in a serum-free medium supplemented with 4 mM BME without FBS for another 6 days. Morphological changes in MSCs from spindle-shaped to neuron-like branching from the edges of the cells were noticed at the end of induction. These neuronlike cells were found positive for the immunophenotypic expression of different neural cell markers β-tubulin III, MAP-2 and Nestin. In RT-PCR analysis, it was also evident that the relative expressions of these representative genes were significantly higher in the differentiated cells. On the basis of our observations, it can be summarized that the BME induction of canine MSCs resulted in morphological changes that resembled neuron-like cells which were found to express the representative neuronal markers. Therefore, inducing canine MSCs with BME resulted in the generation of neuron-like cells that might be utilized for the prospective therapeutic applications in veterinary medicine.

The Expression Pattern of Surface Markers in Canine Adipose-Derived Mesenchymal Stem Cells.

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1–P3) to late (P4–P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73′s one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.

Tailored generation of insulin producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol’s efficiency.

Derivation, characterization, and in vitro cell regeneration of canine white adipose tissue-derived mesenchymal stem cells obtained from a mesenteric region

BackgroundThe study was conducted to assess the characterization, differentiation, and in vitro cell regeneration potential of canine mesenteric white adipose tissue-derived mesenchymal stem cells (AD-MSCs). The tissue was harvested through surgical incision and digested with collagenase to obtain a stromal vascular fraction. Mesenchymal stem cells isolated from the stromal vascular fraction were characterized through flow cytometry and reverse transcription-polymerase chain reaction. Assessment of cell viability, in vitro cell regeneration, and cell senescence were carried out through MTT assay, wound healing assay, and β-galactosidase assay, respectively. To ascertain the trilineage differentiation potential, MSCs were stained with alizarin red for osteocytes, alcian blue for chondrocytes, and oil o red for adipocytes. In addition, differentiated cells were characterized through a reverse transcription-polymerase chain reaction.ResultsWe observed the elongated, spindle-shaped, and fibroblast-like appearance of cells after 72 h of initial culture. Flow cytometry results showed positive expression for CD44, CD90, and negative expression for CD45 surface markers. Population doubling time was found 18–24 h for up to the fourth passage and 30±0.5 h for the fifth passage. A wound-healing assay was used to determine cell migration rate which was found 136.9 ± 4.7 μm/h. We observed long-term in vitro cell proliferation resulted in MSC senescence. Furthermore, we also found that the isolated cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages.ConclusionsMesenteric white adipose tissue was found to be a potential source for isolation, characterization, and differentiation of MSCs. This study might be helpful for resolving the problems regarding the paucity of information concerning the basic biology of stem cells. The large-scale use of AD-MSCs might be a remedial measure in regenerative medicine.

Safety of intravenous and topic eye application of canine mesenchymal stem cells secretome

Safety of intravenous and topic eye application of canine mesenchymal stem cells secretome

Comparative analysis of canine mesenchymal stem cells and bone marrow-derived mononuclear cells.

Background and aim:Mesenchymal stem cells (MSCs), which have multi-lineage differentiation potentials, are a promising source for regenerative medicine. However, the focus of study of MSCs is shifting from the characterization of the differentiation potential to their secretion potential for cell transplantation. Tissue regeneration and the attenuation of immune responses are thought to be affected by the secretion of multiple growth factors and cytokines by MSCs. However, the secretion potential of MSCs profiling remains incompletely characterized. In this study, we focused on the secretion ability related and protein mRNA expression of dog adipose tissue-derived MSCs (AT-MSC), bone marrow (BM)-derived MSCs, and BM-derived mononuclear cells (BM-MNC).Materials and Methods:Real-time polymerase chain reaction analyses revealed mRNA expression of nine growth factors and seven interleukins in these types of cells and three growth factors protein expression were determined using Enzyme-linked immunosorbent assay.Results:For the BM-MNC growth factors, the mRNA expression of transforming growth factor-β (TGF-β) was the highest. For the BM-derived MSC (BM-MSC) and AT-MSC growth factors, the mRNA expression of vascular endothelial growth factor (VEGF) was highest. BM-MSCs and AT-MSCs showed similar expression profiles. In contrast, BM-MNCs showed unique expression profiles for hepatocyte growth factor and epidermal growth factor. The three types of cells showed a similar expression of TGF-β.Conclusion:We conclude that expression of cytokine proteins and mRNAs suggests involvement in tissue repair and protection.

Intra-arterial transplantation of stem cells in large animals as a minimally-invasive strategy for the treatment of disseminated neurodegeneration

Stem cell transplantation proved promising in animal models of neurological diseases; however, in conditions with disseminated pathology such as ALS, delivery of cells and their broad distribution is challenging. To address this problem, we explored intra-arterial (IA) delivery route, of stem cells. The goal of this study was to investigate the feasibility and safety of MRI-guided transplantation of glial restricted precursors (GRPs) and mesenchymal stem cells (MSCs) in dogs suffering from ALS-like disease, degenerative myelopathy (DM). Canine GRP transplantation in dogs resulted in rather poor retention in the brain, so MSCs were used in subsequent experiments. To evaluate the safety of MSC intraarterial transplantation, naïve pigs (n = 3) were used as a pre-treatment control before transplantation in dogs. Cells were labeled with iron oxide nanoparticles. For IA transplantation a 1.2-French microcatheter was advanced into the middle cerebral artery under roadmap guidance. Then, the cells were transplanted under real-time MRI with the acquisition of dynamic T2*-weighted images. The procedure in pigs has proven to be safe and histopathology has demonstrated the successful and predictable placement of transplanted porcine MSCs. Transplantation of canine MSCs in DM dogs resulted in their accumulation in the brain. Interventional and follow-up MRI proved the procedure was feasible and safe. Analysis of gene expression after transplantation revealed a reduction of inflammatory factors, which may indicate a promising therapeutic strategy in the treatment of neurodegenerative diseases.

Transformed Canine and Murine Mesenchymal Stem Cells as a Model for Sarcoma with Complex Genomics.

Simple SummarySarcomas are rare cancers of mesenchymal origin, the majority of which are characterized by many copy number alterations, amplifications, or deletions. Because of these complex genomics, it is notoriously difficult to identify driver events of malignant transformation. In this study, we show that murine and canine mesenchymal stem cells (MSCs) can be used to model spontaneous malignant transformation towards sarcomas with complex genomics. We show that these MSCs have an abnormal karyotype, many structural variants, and point mutations at whole genome sequencing analysis, and form sarcomas after injection into mice. Our cross-species analysis reveals that p53 loss is an early event in sarcomagenesis, and it was shown that MSCs with a knock-out in Trp53 transform earlier compared to wild-type MSCs. Our study points to the importance of p53 loss in the transformation process towards sarcomas with complex genomics.Sarcomas are rare mesenchymal tumors with a broad histological spectrum, but they can be divided into two groups based on molecular pathology: sarcomas with simple or complex genomics. Tumors with complex genomics can have aneuploidy and copy number gains and losses, which hampers the detection of early, initiating events in tumorigenesis. Often, no benign precursors are known, which is why good models are essential. The mesenchymal stem cell (MSC) is the presumed cell of origin of sarcoma. In this study, MSCs of murine and canine origin are used as a model to identify driver events for sarcomas with complex genomic alterations as they transform spontaneously after long-term culture. All transformed murine but not canine MSCs formed sarcomas after subcutaneous injection in mice. Using whole genome sequencing, spontaneously transformed murine and canine MSCs displayed a complex karyotype with aneuploidy, point mutations, structural variants, inter-chromosomal translocations, and copy number gains and losses. Cross-species analysis revealed that point mutations in Tp53/Trp53 are common in transformed murine and canine MSCs. Murine MSCs with a cre-recombinase induced deletion of exon 2–10 of Trp53 transformed earlier compared to wild-type murine MSCs, confirming the contribution of loss of p53 to spontaneous transformation. Our comparative approach using transformed murine and canine MSCs points to a crucial role for p53 loss in the formation of sarcomas with complex genomics.

Biological characteristics and metabolic profile of canine mesenchymal stem cells isolated from adipose tissue and umbilical cord matrix.

Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production. Frozen-thawed samples were used for this study. No differences in mean cell proliferation were found (P>0.05). However, while AT-MSCs showed a progressive increase in doubling time over passages, UC-MSCs showed an initial post freezing-thawing latency. No differences in migration, spheroid formation ability, and differentiation potential were found (P>0.05). RT-PCR analysis confirmed the expression of CD90 and CD44, the lack of CD14 and weak expression of CD34, mostly by AT-MSCs. DLA-DRA1 and DLA-DQA1 were weakly expressed only at passage 0 by UC-MSCs, while they were expressed at different passages for AT-MSCs. There was no difference (P>0.05) in total ATP production between cell cultures, but the ratio between the “mitochondrial ATP Production Rate” and the “glycolytic ATP Production Rate” was higher (P<0.05) in AT- than in UC-MSCs. However, in both MSCs types the mitochondrial respiration was the main pathway of ATP production. Mitochondrial respiration and ATP turnover in UC-MSCs were higher (P<0.05) than in AT-MSCs, but both had a 100% coupling efficiency. These features and the possibility of increasing the oxygen consumption by a spare respiratory capacity of four (AT-MSCSs) and two (UC-MSCs) order of magnitude greater than basal respiration, can be taken as indicative of the cell propensity to differentiate. The findings may efficiently contribute to select the most appropriate MSCs, culture and experimental conditions for transplantation experiments in mesenchymal stem cell therapy for companion animals.

Immunosuppression-enhancing effect of the administration of allogeneic canine adipose-derived mesenchymal stem cells (cA-MSCs) compared with autologous cA-MSCs in vitro

BackgroundRecently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable.ObjectivesTo investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro.MethodsCanine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR.ResultscA-MSCs were expressed cell surface markers such as CD90+/44+/29+/45- and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes.ConclusionsThe administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.