Effect of pregnancy on isolation efficiency and in vitro proliferation of equine peripheral-blood derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory potential and may be used to treat injured tissues. Pregnancy has been associated with increased MSCs in the peripheral circulation in multiple species, but to date, there are no reports on this matter in horses. This study aimed to evaluate the effect of pregnancy on isolation efficiency and proliferation capacity of equine MSCs derived from the peripheral blood (PB) of mares. Venous blood samples were collected at the 11th month of gestation and 1 month after delivery from clinically healthy Arabian mares that presented normal pregnancies. Blood samples were processed for in vitro cellular culture and hormonal and metabolic profiles. MSCs were isolated and characterized by trilineage differentiation potential, immunophenotyping, analyzed by gene sequencing and proliferation assays. The isolation of peripheral blood mononuclear cells (PBMCs) of pregnant mares were associated with higher isolation efficiency and proliferative capacity of MSCs derived from peripheral blood (PB-MSCs) recovered pre-partum than those isolated post-partum. Although fetal gender, parity, 5α-reduced pregnanes, insulin, and cortisol were shown to affect cellular proliferation, individual factors and the small population studied must be considered. This study suggests that PB-MSCs from pregnant mares could be a valuable alternative source of MSCs for therapeutic purposes.

Equine gamma herpesvirus presence and viral load are not associated with equine glandular gastric disease.

OBJECTIVE: To investigate the role of equine herpesvirus-2 (EHV-2) and equine herpesvirus-5 (EHV-5) in equine glandular gastric disease (EGGD) by visualizing and quantifying these gamma herpesviruses in EGGD-affected and normal glandular gastric mucosa of horses. A secondary objective was to describe the histopathological abnormalities in the equine gastric glandular mucosa in horses with EGGD. ANIMALS: 29 horses (n = 21 postmortem and 8 gastroscopy) categorized as normal (11), EGGD (12), or both EGGD and equine squamous gastric disease (6). METHODS: Glandular gastric mucosal samples were collected from horses by gastroscopy or postmortem. Histopathology and in situ hybridization targeting EHV-2 and EHV-5 were performed on grossly normal and abnormal glandular gastric mucosa. The number of in situ hybridization-positive cells per millimeter squared of tissue was calculated. Evaluators were blinded to groups. RESULTS: Glandular gastric tissues from horses without EGGD had higher viral loads in the mucosa than normal or abnormal tissues from EGGD horses. There was no difference in viral loads for EHV-2 or EHV-5 between grossly or endoscopically normal to abnormal gastric tissues within horses with EGGD. Lymphocytic plasmacytic gastritis was the most common histopathological abnormality, with only 3 horses having mucosal disruption (glandular ulcer or erosion). CLINICAL RELEVANCE: Equine gamma herpesviruses are unlikely to play a role in the pathophysiology of EGGD. EGGD is frequently inflammatory with occasional mucosal disruption (ulcer or erosion).

A xenotransplantation mouse model to study physiology of the mammary gland from large mammals.

Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to in vitro analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for in vivo comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species in vivo. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction in vivo. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for in vivo analysis of virtually any mammals, including large and rare mammals.

Comparative transcriptomic analysis of bovine mesenchymal stromal cells reveals tissue-source and species-specific differences.

Mesenchymal stromal cells (MSCs) have the potential to be used as therapeutics, but their efficacy varies due to cellular heterogeneity, which is not fully understood. After characterizing donor-matched bovine MSC from adipose tissue (AT), bone marrow (BM), and peripheral blood (PB), we performed single-cell RNA sequencing (scRNA-seq) to evaluate overarching similarities and differences across these three tissue-derived MSCs. Next, the transcriptomic profiles of the bovine MSCs were compared to those of equine MSCs, derived from the same tissue sources and previously published by our group, and revealed species-specific differences. Finally, the transcriptomic profile from bovine BM-MSCs was compared to mouse and human BM-MSCs and demonstrated that bovine BM-MSCs share more common functionally relevant gene expression profiles with human BM-MSCs than compared to murine BM-MSCs. Collectively, this study presents the cow as a potential non-traditional animal model for translational MSC studies based on transcriptomic profiles similar to human MSCs.

Naturally acquired equine parvovirus-hepatitis is associated with a wide range of hepatic lesions in horses.

Equine parvovirus-hepatitis (EqPV-H) is the causative agent of Theiler's disease, or severe acute hepatic necrosis, in horses. However, it is poorly understood whether EqPV-H is associated with other histologic findings in horses with clinical liver disease. The objective of this study was to examine the prevalence and severity of EqPV-H infections in diagnostic liver samples. Archived formalin-fixed, paraffin-embedded (FFPE) liver samples (n = 98) from Cornell University and University of California, Davis, collected between 2007 and 2022 were evaluated for 15 individual histologic features and by EqPV-H in situ hybridization. EqPV-H was detected in 48% (n = 47) of samples. The most common histologic features of EqPV-H-positive samples included individual hepatocyte death (n = 40, 85%), lobular infiltrates (n = 38, 80%), portal infiltrates (n = 35, 74%), and ductular reaction (n = 33, 70%). Centrilobular necrosis, portal infiltrate, and individual hepatocyte death were positively associated with high viral load. Neutrophil infiltrates, bridging fibrosis, and portal edema were negatively associated with a high viral load. Only 4 of 49 tested samples were positive for equine hepacivirus by polymerase chain reaction (PCR), but the PCR assay was unreliable for FFPE tissues. In summary, this study demonstrates that EqPV-H is common in a variety of liver pathologies and should be considered as a differential diagnosis in cases of hepatitis other than Theiler's disease.

miRNA-214-3p stimulates carcinogen-induced mammary epithelial cell apoptosis in mammary cancer-resistant species.

Mammary cancer incidence varies greatly across species and underlying mechanisms remain elusive. We previously showed that mammosphere-derived epithelial cells from species with low mammary cancer incidence, such as horses, respond to carcinogen 7, 12-Dimethylbenz(a)anthracene-induced DNA damage by undergoing apoptosis, a postulated anti-cancer mechanism. Additionally, we found that miR-214-3p expression in mammosphere-derived epithelial cells is lower in mammary cancer-resistant as compared to mammary cancer-susceptible species. Here we show that increasing miR-214 expression and decreasing expression of its target gene nuclear factor kappa B subunit 1 in mammosphere-derived epithelial cells from horses abolishes 7,12-Dimethylbenz(a)anthracene-induced apoptosis. A direct interaction of miR-214-3p with another target gene, unc-5 netrin receptor A, is also demonstrated. We propose that relatively low levels of miR-214 in mammosphere-derived epithelial cells from mammals with low mammary cancer incidence, allow for constitutive gene nuclear factor kappa B subunit 1 expression and apoptosis in response to 7, 12-Dimethylbenz(a)anthracene. Better understanding of the mechanisms regulating cellular responses to carcinogens improves our overall understanding of mammary cancer resistance mechanisms.

Bovine milk-derived cells express transcriptome markers of pluripotency and secrete bioactive factors with regenerative and antimicrobial activity.

The bovine mammary stem/progenitor cell secretome stimulates regeneration in vitro and contains proteins associated with antimicrobial defense. This has led to the exploration of the secretome as a biologic treatment for mastitis, a costly inflammation of the udder commonly caused by bacteria. This study reports on a population of bovine mammary stem/progenitor cells isolated non-invasively from milk (MiDCs). MiDCs were characterized by immunophenotyping, mammosphere formation assays, and single cell RNA sequencing. They displayed epithelial morphology, exhibited markers of mammary stem/progenitor cells, and formed mammospheres, like mammary gland tissue-isolated stem/progenitor cells. Single cell RNA sequencing revealed two sub-populations of MiDCs: epithelial cells and macrophages. Functionally, the MiDC secretome increased fibroblast migration, promoted angiogenesis of endothelial cells, and inhibited the growth of mastitis-associated bacteria, including antibiotic-resistant strains, in vitro. These qualities of MiDCs render them a source of stem cells and stem cell products that may be used to treat diseases affecting the dairy industry, including mastitis.

Use of Biologics and Stem Cells for Wound Healing in the Horse.

Treatment of skin wounds is a high priority in veterinary medicine because healthy uncompromised skin is essential for the well-being of horses. Stem cells and other biologic therapies offer benefits by reducing the need for surgical procedures and conventional antibiotics. Evidence from in vitro studies and small in vivo trials supports the use of equine stem cells and biologics for the treatment of acute and chronic cutaneous wounds. Larger clinical trials are warranted to better evaluate the regenerative and immunological responses to these treatments. Additionally, delivery methods and treatment schedules should be optimized to improve efficacy of these novel therapies.

Induced pluripotent stem cells from domesticated ruminants and their potential for enhancing livestock production.

Ruminant livestock, including cattle, sheep, goat, and buffalo, are essential for global food security and serve valuable roles in sustainable agricultural systems. With the limited availability of embryonic stem cells (ESCs) from these species, ruminant induced pluripotent stem cells (iPSCs) and iPSC-like cells provide a valuable research tool for agricultural, veterinary, biomedical, and pharmaceutical applications, as well as for the prospect of translation to human medicine. iPSCs are generated by reprogramming of adult or fetal cells to an ESC-like state by ectopic expression of defined transcription factors. Despite the slow pace the field has evolved in livestock species compared to mice and humans, significant progress has been made over the past 15 years in using different cell sources and reprogramming protocols to generate iPSCs/iPSC-like cells from ruminants. This mini review summarizes the current literature related to the derivation of iPSCs/iPSC-like cells from domesticated ruminants with a focus on reprogramming protocols, characterization, associated limitations, and potential applications in ruminant basic science research and production.

Prevalence and pathology of equine parvovirus-hepatitis in racehorses from New York racetracks.

BACKGROUND: Theiler's disease, a.k.a. equine serum hepatitis, is a devastating, highly fatal disease of horses. Equine parvovirus-hepatitis (EqPV-H) has been identified as the likely cause of this disease. While the incidence of Theiler's disease is low, the prevalence of EqPV-H DNA in horses is high, with up to 37% in some regions, suggesting that subclinical or persistent infection is common. METHODS: To determine the prevalence and pathogenicity of EqPV-H infection at New York racetracks, DNA was extracted from archived formalin-fixed, paraffin-embedded liver tissues from racehorses submitted for necropsy to the Animal Health Diagnostic Center as part of the New York State Gaming Commission-Cornell University postmortem examination program. A total of 191 liver samples from horses between 2 and 13 years old were evaluated. Extracted DNA was tested for EqPV-H using PCR and gel electrophoresis. PCR-positive samples were further assessed for tissue morphology using histology and detection of viral nucleic acid using in situ hybridization. RESULTS: Forty-two samples were PCR positive (22%). Of those, 31 samples had positive viral nucleic acid hybridization in hepatocytes with 11 samples showing positive hybridization in necrotic hepatocytes associated with inflammatory cells, indicating active hepatitis. Both individual hepatocyte necrosis and hepatitis were positively associated with EqPV-H detection (p < 0.0001 and p = 0.0005, respectively). CONCLUSION: These findings indicate that presence of EqPV-H in the liver and parvoviral-associated hepatitis are prevalent in racehorses from New York racetracks, thus warranting additional studies examining potential associations between EqPV-H infection and racehorse performance.

Nasal transmission of equine parvovirus hepatitis.

BACKGROUND: Equine parvovirus hepatitis (EqPV-H) is highly prevalent and causes subclinical to fatal hepatitis, which can occur in outbreaks. Whereas iatrogenic transmission is well documented, the mode of horizontal transmission is not known. The virus is shed in nasal, oral and fecal secretions, and PO transmission has been reported in a single horse. HYPOTHESIS/OBJECTIVE: Investigate the efficiency of PO and nasal transmission of EqPV-H in a larger cohort. METHODS: Prospective experimental transmission study. Eleven EqPV-H-negative horses were inoculated with 5 × 106 genome equivalents EqPV-H. Serum PCR and serology for EqPV-H were performed weekly and monthly, respectively. Horses first were inoculated PO, and then intranasally 8 weeks later. RESULTS: No horse became viremic or seroconverted within 8 weeks after PO inoculation. After intranasal inoculation, 5 horses became viremic within 6 to 12 weeks and seroconverted within 10 to 19 weeks. After a period without monitoring from 12 to 19 weeks postinoculation, another 5 horses were found to be viremic at 19 to 22 weeks. The second set of 5 horses could have been infected by horizontal transmission from the first 5 because of cohousing. CONCLUSIONS AND CLINICAL IMPORTANCE: We demonstrated that EqPV-H can be transmitted nasally. The prolonged eclipse phase before detectable viremia indicates biosecurity measures to control spread could be impractical.

Comparative Analysis of microRNAs that Stratify in vitro Mammary stem and Progenitor Activity Reveals Functionality of Human miR-92b-3p.

Mammary stem/progenitor cells are fundamental for mammary gland development and function. However, much remains to be elucidated regarding their function in mammals beyond the traditionally studied rodents, human, and to a lesser extent, ruminants. Due to the growing appreciation for microRNAs (miRNAs) as regulators of stem cells and their progenitors, we compared miRNA expression in mammary stem/progenitor cells from mammals with varying mammary stem/progenitor activity in vitro, in order to identify miRNA candidates that regulate stem/progenitor self-renewal and function. Mammosphere-derived epithelial cells (MDECs), which are primary cell lines enriched in mammary stem and progenitor cells, were generated from six mammalian species (i.e., cow, human, pig, horse, dog, and rat) and small RNA sequencing was performed. We identified 9 miRNAs that were significantly differentially expressed in MDEC cultures with a low versus high mammary stem/progenitor activity. miR-92b-3p was selected for functional follow-up studies, as this miRNA is understudied in primary mammary cells but has well-described gene targets that are known to regulate mammary stem/progenitor activity. Altering the expression of miR-92b-3p in MDECs from species with low stem/progenitor activity (human and cow) and those with high stem/progenitor activity (dog and rat) via inhibition and overexpression, respectively, resulted in significantly decreased mammosphere formation of human MDECs, but showed no significant effects in cow, dog, or rat MDECs. This study is the first to perform small RNA sequencing in MDECs from various mammals and highlights that conserved miRNAs can have different functions in mammary stem/progenitor cells across species.

Mesenchymal stromal cells isolated from chicken peripheral blood secrete bioactive factors with antimicrobial and regenerative properties.

Mesenchymal stromal cells (MSCs) are adult multipotent progenitor cells that have been isolated from various tissue sources of many species, primarily mammals. Generally, these cells proliferate extensively in culture and have been shown to secrete bioactive factors that contribute to healing processes by regulating inflammation, modulating immune responses, inhibiting bacterial growth, and promoting tissue regeneration. The present study reports on the isolation and characterization of MSCs from the peripheral blood (PB) of chickens. Chicken PBMSCs were characterized based on their trilineage differentiation potential and gene and protein expression of MSC-specific cell surface markers. To determine functionality, conditioned medium (CM), which contains all bioactive factors secreted by MSCs, was collected from chicken PBMSCs, and used in in vitro antimicrobial, migration, and angiogenesis assays. Chicken PBMSC CM was found to (i) inhibit the growth of planktonic Staphylococcus aureus (S. aureus), and even more significantly the methicillin-resistant S. aureus (MRSA), (ii) decrease adhesion and promote migration of fibroblasts, and (iii) support endothelial cell tube formation. Collectively, these data indicate that chicken PBMSCs secrete bioactive factors with antimicrobial and regenerative properties, and as such, provide a novel source of cell-based therapies for the poultry industry.

Induced mammary cancer in rat models: pathogenesis, genetics, and relevance to female breast cancer.

Mammary cancer, or breast cancer in women, is a polygenic disease with a complex etiopathogenesis. While much remains elusive regarding its origin, it is well established that chemical carcinogens and endogenous estrogens contribute significantly to the initiation and progression of this disease. Rats have been useful models to study induced mammary cancer. They develop mammary tumors with comparable histopathology to humans and exhibit differences in resistance or susceptibility to mammary cancer depending on strain. While some rat strains (e.g., Sprague-Dawley) readily form mammary tumors following treatment with the chemical carcinogen, 7,12-dimethylbenz[a]-anthracene (DMBA), other strains (e.g., Copenhagen) are resistant to DMBA-induced mammary carcinogenesis. Genetic linkage in inbred strains has identified strain-specific quantitative trait loci (QTLs) affecting mammary tumors, via mechanisms that act together to promote or attenuate, and include 24 QTLs controlling the outcome of chemical induction, 10 QTLs controlling the outcome of estrogen induction, and 4 QTLs controlling the outcome of irradiation induction. Moreover, and based on shared factors affecting mammary cancer etiopathogenesis between rats and humans, including orthologous risk regions between both species, rats have served as useful models for identifying methods for breast cancer prediction and treatment. These studies in rats, combined with alternative animal models that more closely mimic advanced stages of breast cancer and/or human lifestyles, will further improve our understanding of this complex disease.

Comparative Efficacy of Topical Ophthalmic Ganciclovir and Oral Famciclovir in Cats with Experimental Ocular Feline Herpesvirus-1 Epithelial Infection.

Purpose: To determine the comparative efficacy of ganciclovir ophthalmic gel and famciclovir oral tablets in cats with experimentally induced ocular feline herpesvirus-1 (FHV-1) epithelial infection. Methods: A randomized, placebo-controlled trial was performed using 16 nonvaccinated, specific pathogen-free cats with experimental FHV-1 infection induced by topical ocular inoculation. Cats received topical ganciclovir 0.15% ophthalmic gel (1 drop 3 times daily, n = 6 cats), oral famciclovir (90 mg/kg twice daily, n = 6), or topical artificial tear gel (1 drop 3 times daily, n = 4) for 14 days. Cats were monitored after inoculation for 30 days. Ophthalmic examinations were performed every 2 days and ocular disease scores calculated. In vivo confocal microscopy was performed, and corneal leukocyte infiltrates quantified. Ocular samples for FHV-1 quantitative polymerase chain reaction (qPCR) and virus isolation assays were collected every 3 days. Hemograms and serum biochemistry panels were performed at intervals. Results: Clinical ocular disease scores and corneal leukocyte infiltrates were significantly lower in the ganciclovir and famciclovir groups compared with placebo, but no significant differences were detected between the antiviral treatment groups. Ocular viral loads determined by qPCR were significantly lower in the ganciclovir group compared with the placebo group, but there were no significant differences between the other study groups. Hemograms and biochemistry panels were unremarkable. Conclusion: Topical application of ganciclovir gel 3 times daily was well-tolerated and displayed similar efficacy at reducing clinical ocular disease scores and corneal inflammation as twice daily oral famciclovir treatment in cats with experimental ocular FHV-1 infection.

A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Establishment and characterization of equine mammary organoids using a method translatable to other non-traditional model species.

Mammary organoid (MaO) models are only available for a few traditional model organisms, limiting our ability to investigate mammary gland development and cancer across mammals. This study established equine mammary organoids (EqMaOs) from cryopreserved mammary tissue, in which mammary tissue fragments were isolated and embedded into a 3D matrix to produce EqMaOs. We evaluated viability, proliferation and budding capacity of EqMaOs at different time points during culture, showing that although the number of proliferative cells decreased over time, viability was maintained and budding increased. We further characterized EqMaOs based on expression of stem cell, myoepithelial and luminal markers, and found that EqMaOs expressed these markers throughout culture and that a bilayered structure as seen in vivo was recapitulated. We used the milk-stimulating hormone prolactin to induce milk production, which was verified by the upregulation of milk proteins, most notably ß-casein. Additionally, we showed that our method is also applicable to additional non-traditional mammalian species, particularly domesticated animals such as cats, pigs and rabbits. Collectively, MaO models across species will be a useful tool for comparative developmental and cancer studies.

Small but mighty: old and new parvoviruses of veterinary significance.

In line with the Latin expression "sed parva forti" meaning "small but mighty," the family Parvoviridae contains many of the smallest known viruses, some of which result in fatal or debilitating infections. In recent years, advances in metagenomic viral discovery techniques have dramatically increased the identification of novel parvoviruses in both diseased and healthy individuals. While some of these discoveries have solved etiologic mysteries of well-described diseases in animals, many of the newly discovered parvoviruses appear to cause mild or no disease, or disease associations remain to be established. With the increased use of animal parvoviruses as vectors for gene therapy and oncolytic treatments in humans, it becomes all the more important to understand the diversity, pathogenic potential, and evolution of this diverse family of viruses. In this review, we discuss parvoviruses infecting vertebrate animals, with a special focus on pathogens of veterinary significance and viruses discovered within the last four years.

The equine mesenchymal stromal cell secretome inhibits equid herpesvirus type 1 strain Ab4 in epithelial cells.

Equid herpesvirus 1 (EHV-1) outbreaks occur when virus spreads from infected horses to in-contact horses, primarily via nasal shedding. This study evaluated the efficacy of factors secreted by equine peripheral blood derived mesenchymal stromal cells (PB-MSCs), collectively named the secretome, to inhibit the growth of EHV-1 in (i) 2D epithelial cell cultures (RK-13) in vitro, (ii) 3D equine nasal explants in vitro and (iii) an EHV-1 infection mouse model in vivo. The PB-MSC secretome was found to inhibit EHV-1 in RK-13 cells as well as in the epithelium of equine nasal explants. Although the PB-MSC secretome did not decrease overall severity of EHV-1 infection in mice, as determined by weight loss and viral titers in lungs, histological analyses indicated local reduction of EHV-1 infection in nasal epithelium. These results indicate that the PB-MSC secretome inhibits EHV-1 in epithelial cells in a context-dependent manner.

Mesenchymal stromal cell-secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes.

Mesenchymal stromal cells (MSCs) from both humans and horses, which represent a clinically relevant translation animal model for human cutaneous wound healing, were recently found to possess antimicrobial properties against planktonic bacteria, and in the case of equine MSCs, also against biofilms. This, together with previous findings that human and equine MSCs promote angiogenesis and wound healing, makes these cells an attractive approach to treat infected cutaneous wounds in both species. The anti-biofilm activities of equine MSC, via secretion of cysteine proteases, have only been demonstrated in vitro, thus lacking information about in vivo relevance. Moreover, the effects of the equine MSC secretome on resident skin cells have not yet been explored. The goals of this study were to (a) test the efficacy of the MSC secretome in a physiologically relevant ex vivo equine skin biofilm explant model and (b) explore the impact of the MSC secretome on the antimicrobial defense mechanisms of resident skin cells. Our salient findings were that secreted factors from equine MSCs significantly decreased viability of methicillin-resistant Staphylococcus aureus bacteria in mature biofilms in this novel skin biofilm explant model. Moreover, we demonstrated that equine MSCs secrete CCL2 that increases the antimicrobial activity of equine keratinocytes by stimulating expression of antimicrobial peptides. Collectively, these data contribute to our understanding of the MSC secretome's antimicrobial properties, both directly by killing bacteria and indirectly by stimulating immune responses of surrounding resident skin cells, thus further supporting the value of MSC secretome-based treatments for infected wounds.