Cell viability refers to the ability of cells to survive and function within their physiological environment. In horses, assessing cell viability is an important aspect of veterinary research, particularly in understanding the effects of various treatments, diseases, and environmental factors on equine cellular health. Techniques such as flow cytometry, trypan blue exclusion, and MTT assays are commonly used to evaluate cell viability in equine studies. These methods help determine the proportion of living cells in a sample, providing insights into cellular responses to different stimuli or conditions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and implications of cell viability assessments in equine research.
Rosim DF, Rochetti AL, Xavier PLP, Qazi TJ, Pimentel ECC, da Matta Santos A, Strefezzi RF, Massoco CO, Dória RGS, Fukumasu H.Equine sarcoids are the most common skin tumors in horses and are characterized by local invasiveness, high recurrence rates, and inconsistent responses to current therapies. Oncolytic virotherapy represents a promising alternative approach that exploits defects in antiviral signaling pathways in tumor cells. Newcastle disease virus (NDV), an avian paramyxovirus that is nonpathogenic in mammals, has demonstrated tumor-selective activity in several species; however, its effects on equine sarcoids have not been previously investigated. In this study, primary cell cultures derived from equine sar...
Zhu Q, Jing Y, Zheng Y, Khan MZ, Liu A, Peng Y, Wang C.The gene family encodes highly conserved signaling molecules involved in vertebrate development, tissue homeostasis, and cell fate regulation. However, the evolutionary characteristics and potential biological functions of genes in the domestic donkey () remain poorly understood. In this study, we performed the first genome-wide identification and comprehensive characterization of the donkey gene family. A total of 19 genes were identified, consistent with the conserved repertoire reported in other mammalian species. Phylogenetic and synteny analyses revealed strong evolutionary conservat...
Capone A, Merlo B, Lanci A, Ballotta G, Iacono E.Mesenchymal stromal cells (MSCs) derived from perinatal tissues represent a promising resource for regenerative medicine due to their proliferative, immunomodulatory, and differentiation properties. Among these tissues, the umbilical cord is considered an accessible and ethically acceptable source of MSCs. This study evaluated the feasibility of whole umbilical cord cryopreservation in canine and equine species and assessed the biological properties of MSCs isolated from cryopreserved tissues compared with fresh samples. Equine Wharton's jelly and canine umbilical cord tissues were processed e...
Vanivska K, Kollárová I, Mezeyová I, Galovičová L, Dianová L, Halo M, Slanina T, Lenický M, Greń A, Reisenauer R, Massányi P.This study evaluated the effects of Trigonella foenum-graecum L. (fenugreek) seed extract on stallion spermatozoa quality in vitro, focusing on motility, vitality, and functional competence. In vitro spermatozoa analyses showed that fenugreek seed extract did not demonstrate overt cytotoxicity under the tested conditions: S1 = 1562 µg/mL; S2 = 781 µg/mL; S3 = 390.5 µg/mL; S4 = 195.25 µg/mL; S5 = 97.62 µg/mL; S6 = 48.81 µg/mL; S7 = 24.40 µg/mL. Computer-assisted semen analysis (CASA) revealed a concentration- and time-dependent Treatment × Time interac...
Su Y, Ren W, Zeng Y, Meng J, Yao X, Wang J.Skeletal muscle serves as the direct executor of movement, and the proliferation and migration capacities of its constituent cells represent the key physiological foundation determining equine endurance and speed. Previous whole-transcriptome analysis has shown that Insulin-like Growth Factor 1 () is significantly upregulated in following racing and is involved in the PI3K-Akt signaling pathway; however, its specific regulatory mechanism in equine skeletal muscle cells remains unclear. This study utilized primary equine skeletal muscle cells as a model. By constructing an overexpression plas...
Ross AC, Lumsden ES, Flood C, Dudhia J, Psifidi A, Guest DJ.Bone fractures in Thoroughbred racehorses are a major welfare problem. Genetic factors contribute to fracture risk. Cell models have previously identified 112 differentially expressed genes in bone-forming osteoblasts derived from horses at high and low genetic risk of fracture. However, 42 of these genes have no published role in bone. In this study, we identified novel roles for a subset of these genes in bone formation. Twenty-six of the 42 genes were expressed in Saos2 cells during basal culture and/or after 21 days of osteogenic culture. Five of these genes (ADSSL1, CABP1, ENO2, SPARCL1...
Muñoz-García CC, Soriano-Campos MP, Luis-Calero M, Gallardo-Soler A, González-Fernández L, Macías-García B.Mesenchymal stromal cells from equine ovarian follicular aspirates represent a promising source for cell-based therapies. However, the age of the donor mare may alter their biological properties and therapeutic potential. This study aimed to evaluate the effect of age on MSCs from ovarian follicular aspirates of young (< 10 years, n = 5) and aged (≥ 18 years, n = 5) mares. MSCs were isolated, expanded, cryopreserved at passage 3, and subsequently thawed to evaluate proliferation, immunophenotype, differentiation capacity, and gene expression profiles. Proliferation rates, total cel...
Bernad E, Serrano B, Vitoria A, Fuente S, Romero A, Vázquez FJ, Zaragoza P, Rodellar C, Cequier A, Barrachina L.Mesenchymal stem/stromal cells (MSCs) hold promise for treating different equine conditions but enter senescence during culture. Using induced pluripotent stem cells (iPSCs) to derive MSC-like cells (iMSCs) can increase cell availability and diminish the need for invasive and repeated tissue harvesting. While human iMSCs are intensively studied, research on equine iMSCs (eqiMSCs) is very limited and has focused on strategies for spontaneous differentiation to obtain these cells. The aim of this study was to obtain MSC-like cells from equine iPSCs (eqiPSCs) by directing their differentiation vi...
Magalhães VLP, Pinho FA, Souza FN, Santos KR, Santos AS, Hlavac N, Libera AMPD, Massoco CO, Barrouin-Melo SM.There are diverse uses for equine peripheral blood mononuclear cells (PBMC) in research. Studies have shown that PBMC isolation protocols can affect immune parameters of diagnostic and research assays. We aimed to assess the impact of equine blood handling techniques on cell yield, viability, and proliferation under mitogen stimulation. Whole blood (WB) samples were collected from eight healthy adult horses into heparinized tubes for PBMC isolation by comparing method 1 (M1) where freshly collected WB was layered directly over a density gradient medium for centrifugation, followed by PBMC harv...
Jaworska J, Tobolski D, Salem SE, Ropka-Molik K, Ząbek T, Szmatoła T, Kahler A, Wocławek-Potocka I, Piórkowska K, de Mestre AM.BACKGROUND: During equine implantation, two distinct trophoblast subpopulations are identified: the invasive chorionic girdle (CG) and the non-invasive allantochorion. The invasive CG trophoblast is believed to modulate the maternal immune response and facilitates embryo attachment to the endometrium, while the allantochorion contributes to placental development without breaching the endometrium. However, the mechanisms underlying differential adhesion and immune modulation by these two lineages remain poorly understood. This study aimed to investigate the regulation of biological processes un...
Del Prado Soriano-Campos M, Muñoz-García CC, Luis-Calero M, Gallardo-Soler A, González-Fernández L, Macías-García B.Mesenchymal stromal cells (MSCs) are widely used in human cell-based therapies and recent research is focused on the use of MSCs in equine regenerative medicine. Recently, MSCs have been isolated from equine follicular aspirates; however, a major concern during isolation is the potential contamination with fibroblasts, the predominant stromal cell type in many tissues. Furthermore, both cell types share the minimal criteria established by the International Society for Cellular Therapy (ISCT) for MSCs identification. Hence, our study aimed to compare the morphological characteristics, expansion...
Tehrani AH, Toth K, Osinchuk N, Dufour A, Krawetz R, Sen A, Sparks H.Adult bone marrow-derived mesenchymal stromal cells (BM-MSCs) hold significant therapeutic potential in human and veterinary medicine. Traditionally, BM-MSC cultures use fetal bovine serum (FBS) to promote growth, but this practice introduces batch variability and xenogeneic contamination that complicate clinical translation. While serum-free medium (SFM) alternatives have been optimized for human MSCs, limited options have been explored for alternative or veterinary species. Here, we compared a chemically defined, customizable SFM formulation with conventional serum-containing medium (SCM) fo...
Normélia P Morais A, Gastal GDA, Aguiar FLN, Hyde KA, Gastal MO, Lima LF, Ñaupas LVS, Flávia B Silva A, Becerra VAB, Gheno L, Curcio BR....Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 mm vs. 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), v...
de Agostini Losano JD, Guertin JE, McGraw MS, Katz L, Bordignon V, Callaham JW, Pontes JHF, Fleury P, Maserati M, Turner C, Daigneault BW.Equine in vitro fertilization (IVF) has historically been challenged due to lack of conditions that promote functional capacitation of stallion sperm. Capacitation success is often attributed to prolonged sperm incubation requirements. Advances to equine IVF are hindered by incomplete characterization of conditions that promote the functional capacitation of stallion sperm over time. Herein, we describe conditions that promote the functional capacitation of fresh, cooled, and frozen-thawed stallion sperm isolated by Percoll, Swim-Up, or Microfluidics (VetMotl). Fresh (diluted sperm) and previo...
Semik-Gurgul E, Ocłoń E, Zubel-Łojek J, Pędziwiatr R, Pawlina-Tyszko K.Sarcoids are locally invasive skin tumors in equids, associated with bovine papillomavirus. Objective: Address potential applications of cannabidiol (CBD) in veterinary medicine. We evaluated the response of equine sarcoid cells to CBD in vitro, focusing on viability, invasiveness, and matrix remodeling. Methods: Three primary sarcoid cell lines. Methods: Cells were treated with CBD (20, 6.75, 2.25, 0.75 μM) and incubated for 6, 24, 48, 72 hours. Cell viability, cytotoxicity, and apoptosis were assessed using the ApoTox-Glo Assay. Based on these results, further analyses were performed for ...
Wójtowicz A, Sadowska A, Myszczyński K, Molcan T, Kaczmarek MM, Szóstek-Mioduchowska A.Fibrosis remains incompletely understood, particularly in terms of how immune mediators shape stromal programs. We used a spontaneous large‑animal model-endometrosis (equine endometrial fibrosis) to define how interleukin‑4 (IL‑4) reprograms fibroblasts from healthy and fibrotic endometrium. Primary fibroblasts were exposed to IL‑4 (10 ng/mL) for 48 or 96 h. At 48 h, bulk transcriptomes revealed 1307 differentially expressed genes (DEGs; 648 up, 659 down) and 1271 DEGs (645 up, 626 down) in fibroblasts derived from endometria without or with endometrosis, respectively. Enrichment an...
Zanacchi A, Fruscione F, Grasselli S, Razzuoli E, Pastorino L, Di Lisa D.Developing advanced preclinical in vitro cancer models is essential for understanding tumor biology, improving drug discovery, and enhancing the clinical translation of new therapies. Papillomaviruses (PVs) cause diverse lesions in humans and animals; notably, bovine papillomavirus (BPV) is the main cause of equine sarcoid, the most common skin tumor in horses. Its pathogenesis involves viral infection, fibroblast transformation, and extracellular matrix (ECM) remodeling. These processes highlight the limitations of conventional two-dimensional (2D) culture systems, which fail to replicate the...
Sabzpoosh M, Hoveizi E, Gooraninejad S.This study aimed to extract and isolate endometrial stromal cells from Arabian mares and investigate their growth and differentiation potential. Endometrial biopsies were obtained from three healthy 6-year-old Arabian mares using a standardized, minimally invasive protocol. The isolated cells were characterized using flow cytometry and differentiation analysis. Flow cytometry revealed mesenchymal markers CD90 (95.2%) and CD105 (97.4%) and hematopoietic markers CD34 (1.17%) and CD45 (0.339%). The cells exhibited differentiation potential into adipocytes, osteoblasts, and chondrocytes. The findi...
Parker JL, Berghaus LJ, Gordon J, Mathews L, Akinde MA, Hall DB, Peroni JF.Mesenchymal stromal cells (MSCs) support wound healing through immunomodulation and by promoting tissue organization. Cell tracking studies demonstrate that MSCs must 'home' to injuries in order to participate in reparative processes. It has been reported that in vivo cell tracking labels, including superparamagnetic iron oxide particles (SPIOs), can alter MSC homing. Homing is thought to occur in coordinated steps including tethering, activation, arrest, diapedesis, and migration, guided by chemotactic stimuli. Equine distal limb injuries, which are commonly treated with MSCs, possess a disti...
DI Stefano DA, Coccoluto L, Panina-Bordignon P, Brambilla E, Ruffini F, Murtaj V, Orlando F, Colombo M, Frigerio C, DI Bona A, Recupero D, Morroni M....Bone grafting is a surgical procedure that involves the use of bone tissue or bone substitutes to repair damaged bone. In dentistry and maxillofacial surgery, bone graft substitutes from various sources are commonly used. Given their critical role in clinical outcomes, it is essential to thoroughly investigate the biological and mechanical properties of these materials. Methods: In this in vitro study, we evaluated the biological properties of two equine-derived bone graft substitutes in comparison to β-tricalcium phosphate. The materials included one equine-derived graft containing hydrolyze...
Bourebaba N, Domagała J, Bourebaba L.Equine metabolic syndrome (EMS) is associated with chronic low-grade inflammation, disruptions in mitochondrial dynamics and function, and an increased risk of developing laminitis. Recent research has highlighted that reduced levels of sex hormone-binding globulin (SHBG) in the bloodstream are linked to higher susceptibility to obesity, insulin resistance, and diabetes, potentially contributing to broader metabolic imbalances. This study aimed to evaluate whether exogenously administered SHBG could protect adipose-derived stem cells from horses affected by EMS (EqASC) against mitochondrial dy...
Bungărdean D, Pall E, Daradics Z, Popescu M, Tripon MA, Lupșan AF, Crecan CM, Morar IA, Nicolescu A, Bora FD, Marcus I.Musculoskeletal disorders are a major cause of lameness in horses, often necessitating innovative regenerative strategies to restore joint function and improve quality of life. This study investigated the effects of platelet-rich plasma (PRP), ozonized PRP, hyaluronic acid, paracetamol, and polyacrylamide hydrogel (NOLTREX) on the behavior of mesenchymal stem cells (MSCs) derived from equine synovial fluid. Synovial fluid samples were collected under strict cytological criteria to ensure viability, followed by in vitro expansion and phenotypic characterization of MSCs. Cultures were supplement...
Witkowska-Piłaszewicz O, Nowicka-Kazmierczak M, Pietrzak P, Marycz K.Skeletal muscle satellite cells ( SCs), essential for muscle regeneration, are a valuable model for studying exercise-induced stress relevant to human athletes. This study examined the effects of two natural compounds-chlorogenic acid (CGA) and isovanillic acid 3-O-sulfate (IVAS)-increasingly recognized as components of modern, nature-based recovery strategies. Their combination (Hybrid) was also tested on equine model of skeletal muscle satellite cells (ESCs) exposed to heat shock (40 °C, 1 h), mimicking exercise stress. Cells were treated with CGA (0.005%), IVAS (0.0005%), or both for 24...
Méndez-Pérez L, Ibáñez BO, Rodríguez S, Sen Wong Y, Caamaño D, Navarrete FI, Cabezas J, Mançanares AC, Escudero C, Rodríguez-Álvarez L....Endometrial fibrosis in mares compromises fertility through aberrant extracellular matrix deposition and sustained myofibroblast activation. Conventional interventions fail to reverse these pathological alterations, necessitating innovative, mechanism-focused therapies. In this study, we pioneered the use of prostaglandin E2 (PGE2) preconditioning of equine endometrial-derived mesenchymal stem cells (ET-eMSCs) and their extracellular vesicles (EVs) to target fibrotic processes directly. ET-eMSCs were isolated from mare endometrial biopsies pretreated with PGE2 to enhance their anti-fibrotic se...
Sawicki S, Bugno-Poniewierska M, Żurowski J, Szmatoła T, Semik-Gurgul E, Bochenek M, Karnas E, Gurgul A. Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their ability to secrete paracrine factors that modulate tissue repair. Extracellular vesicles (EVs) released by MSCs contain bioactive molecules (e.g., mRNAs, miRNAs, proteins) and play a key role in intercellular communication. This study compared the transcriptomic profiles (mRNA and miRNA) of equine MSCs derived from adipose tissue (AT-MSCs), bone marrow (BM-MSCs), and ovarian fibroblasts (as a differentiated control). Additionally, miRNAs present in EVs secreted by these cells were characterized using nex...
Orzoł D, Kępska M, Zyzak M.Equine metabolic syndrome (EMS) is a prevalent endocrine disorder associated with insulin dysregulation, oxidative stress, and impaired regenerative capacity of adipose-derived stem cells (ASCs). The aim of this study was to evaluate the effects of orientin-a plant-derived flavonoid with known antioxidant properties-on equine ASCs (EqASCs) derived from both clinically healthy and diagnosed EMS-affected mares. EqASCs were treated with orientin to evaluate its biological effects. The analysis included key cellular functions such as proliferative capacity, viability, apoptosis, oxidative stress, ...
Hernández-Avilés C.Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author's laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm ...
Koch DW.Our understanding of tendon homeostasis and repair following injury has made great strides through basic science, clinical veterinary medicine, and preclinical translational research. We have now gleaned a greater understanding of the cellular and matrix dynamics that are being orchestrated within the tendon, which provide therapeutic opportunities. This article will focus on how utilizing the horse and equine tissues have advanced our understanding of tendinopathy and the cellular and matrix dynamics at play while also identifying continued gaps in our knowledge where the horse as a veterinar...
Zamboulis DE, Thorpe CT.The high stresses and strains experienced by the superficial digital flexor tendon (SDFT) make it prone to age-related injury. The SDFT exhibits cellular and structural specializations, localized to the interfascicular matrix (IFM), enabling it to resist its extreme mechanical environment. With aging, these specializations are lost, impacting the ability of the tendon to withstand repeated loading. Within the IFM, there is loss of mechanical properties and organization accompanying cell senescence and a decreased resolution of inflammation. Developing therapeutics to reverse these changes may,...
Contentin R, Jehl C, Commenchail K, Legendre F, Galéra P, Cassé F, Demoor M.Osteoarthritis (OA) affects millions of people globally, causing irreversible cartilage damage, chronic inflammation, and progressive joint dysfunction. Similarly, horses can develop OA spontaneously or due to their athletic careers, influenced by mechanical and biochemical factors. Current treatments primarily focus on symptom relief without promoting cartilage regeneration. In line with the 3Rs principles (refine, reduce, replace), the development of OA models is essential for advancing new therapeutic approaches against OA. In response to this need, the present study aimed to develop an m...
Stone KC, Mercer RR, Gehr P, Stockstill B, Crapo JD.Allometric studies have shown that lung volume, alveolar surface area, and diffusing capacity increase proportionally with body weight across a broad range of mammalian species. Changes in the number of cells and in average cell size and surface areas with increasing body weight have not been defined. We speculated that cell size is determined more by cell function than by species and body weight. To test this hypothesis, nine species ranging in size from shrew (2 to 3 g) to horse (510 kg) were studied. Random sites from the distal alveolar region of each species were analyzed using morphometr...
Caliari SR, Harley BA.Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freez...
Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC.The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm ...
Vidal MA, Walker NJ, Napoli E, Borjesson DL.Mesenchymal stem cells (MSCs) from adult and neonatal tissues are intensively investigated for their use in regenerative medicine. The purpose of this study was to compare the onset of replicative senescence in MSCs isolated from equine bone marrow (BMSC), adipose tissue (ASC), and umbilical cord tissue (UCMSC). MSC proliferation (cell doubling), senescence-associated β-galactosidase staining, telomere length, Sox-2, and lineage-specific marker expression were assessed for MSCs harvested from tissues of 4 different donors. The results show that before senescence ensued, all cell types prolife...
Hondalus MK, Mosser DM.Rhodococcus equi is a facultative intracellular bacterium of macrophages that can cause serious pneumonia in both young horses and immunocompromised people. Essential to understanding rhodococcus pathogenesis is a quantitative documentation of the intracellular events that follow macrophage phagocytosis of the organism. By using a bacterial immunofluorescence staining assay, we verified the intracellular survival and replicative potential of R. equi in both murine peritoneal macrophages and equine alveolar macrophages in vitro. Following an initial lag period of 6 to 12 h, the intracellular nu...
Gaspar D, Spanoudes K, Holladay C, Pandit A, Zeugolis D.The last decade has seen significant developments in cell therapies, based on permanently differentiated, reprogrammed or engineered stem cells, for tendon injuries and degenerative conditions. In vitro studies assess the influence of biophysical, biochemical and biological signals on tenogenic phenotype maintenance and/or differentiation towards tenogenic lineage. However, the ideal culture environment has yet to be identified due to the lack of standardised experimental setup and readout system. Bone marrow mesenchymal stem cells and tenocytes/dermal fibroblasts appear to be the cell populat...
Ranera B, Lyahyai J, Romero A, Vázquez FJ, Remacha AR, Bernal ML, Zaragoza P, Rodellar C, Martín-Burriel I.Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in equine bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by ...
Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R, Lazzari G.Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological rec...
Marycz K, Kornicka K, Basinska K, Czyrek A.Currently, equine metabolic syndrome (EMS), an endocrine disease linked to insulin resistance, affects an increasing number of horses. However, little is known about the effect of EMS on mesenchymal stem cells that reside in adipose tissue (ASC). Thus it is crucial to evaluate the viability and growth kinetics of these cells, particularly in terms of their application in regenerative medicine. In this study, we investigated the proliferative capacity, morphological features, and accumulation of oxidative stress factors in mesenchymal stem cells isolated from healthy animals (ASCN) and horses s...
Dudhia J, Scott CM, Draper ER, Heinegård D, Pitsillides AA, Smith RK.Age-associated and degenerative loss of functional integrity in soft tissues develops from effects of cumulative and subtle changes in their extracellular matrix (ECM). The highly ordered tendon ECM provides the tissue with its tensile strength during loading. As age and exercise collide in the high incidence of tendinopathies, we hypothesized that aged tendons fail due to cumulative damage resulting from a combination of diminished matrix repair and fragmentation of ECM proteins induced by prolonged cyclical loading, and that this is an active cell-mediated process. We developed an equine ten...
Aguilar PV, Weaver SC, Basler CF.Eastern equine encephalitis virus (EEEV) causes sporadic but often severe cases of human and equine neurological disease in North America. To determine how EEEV may evade innate immune responses, we screened individual EEEV proteins for the ability to rescue the growth of a Newcastle disease virus expressing green fluorescent protein (NDV-GFP) from the antiviral effects of interferon (IFN). Only expression of the EEEV capsid facilitated NDV-GFP replication. Inhibition of the antiviral effects of IFN by the capsid appears to occur through a general inhibition of cellular gene expression. For ex...
Guest DJ, Smith MR, Allen WR.Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP...
Guest DJ, Smith MR, Allen WR.Injury to the superficial digital flexor tendon (SDFT) is common in racing and sport horses and poor tendon regeneration leads to high reinjury rates. Autologous mesenchymal stromal cells (MSCs) are being used clinically to improve tendon regeneration but they have some practical limitations. Embryonic stem cells (ESCs) may overcome these limitations but their fate following injection into the damaged SDFT is unknown. Objective: To inject MSCs and ESCs into distinct areas of damage in the SDFT and monitor their survival over a 3 month period. Methods: MSCs and ESCs expressing different reporte...
Ball BA.Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and f...
Plaza Davila M, Martin Muñoz P, Tapia JA, Ortega Ferrusola C, Balao da Silva C C, Peña FJ.Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation...
Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF.To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Methods: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. Methods: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transdu...
Berglund AK, Schnabel LV.Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. Objective: To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs. Methods: Experimental controlled study. Methods: Antisera previously collected at Days 0, 7, 14 an...
Rosser J, Bachmann B, Jordan C, Ribitsch I, Haltmayer E, Gueltekin S, Junttila S, Galik B, Gyenesei A, Haddadi B, Harasek M, Egerbacher M, Ertl P....In this work, we describe a microfluidic three-dimensional (3D) chondrocyte culture mimicking in vivo articular chondrocyte morphology, cell distribution, metabolism, and gene expression. This has been accomplished by establishing a physiologic nutrient diffusion gradient across the simulated matrix, while geometric design constraints of the microchambers drive native-like cellular behavior. Primary equine chondrocytes remained viable for the extended culture time of 3 weeks and maintained the low metabolic activity and high Sox9, aggrecan, and Col2 expression typical of articular chondrocytes...
De Schauwer C, Piepers S, Van de Walle GR, Demeyere K, Hoogewijs MK, Govaere JL, Braeckmans K, Van Soom A, Meyer E.During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate posit...
Huser CA, Davies ME.Chondrocyte apoptosis is an important factor in the progression of osteoarthritis. This study aimed to elucidate the mechanisms involved upstream of caspase 9 activation and, in particular, calcium signaling and mitochondrial depolarization. Methods: Articular cartilage explants obtained from healthy horses were subjected to a single impact load (500-gm weight dropped from a height of 50 mm) and cultured in vitro for up to 48 hours. Chondrocyte death was quantified by the TUNEL method. Release of proteoglycans was determined by the dimethylmethylene blue assay. Weight change was measured, and ...
Caliari SR, Harley BA.Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-de...
de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, Deffune E.The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique...
Sieme H, Oldenhof H, Wolkers WF.Native sperm is only marginally stable after collection. Cryopreservation of semen facilitates transport and storage for later use in artificial reproduction technologies, but cryopreservation processing may result in cellular damage compromising sperm function. Membranes are thought to be the primary site of cryopreservation injury. Therefore, insights into the effects of cooling, ice formation and protective agents on sperm membranes may help to rationally design cryopreservation protocols. In this review, we describe membrane phase behaviour of sperm at supra- and subzero temperatures. In a...
Marycz K, Kornicka K, Grzesiak J, Śmieszek A, Szłapka J.Equine metabolic syndrome (EMS) is mainly characterized by insulin resistance, obesity, and local or systemic inflammation. That unfriendly environment of adipose tissue has huge impact on stem cells population (ASC) residing within. In the present study, using molecular biology techniques and multiple imaging techniques (SEM, FIB-SEM, and confocal microscopy), we evaluated the impact of EMS on ASC viability and chondrogenic differentiation. Moreover, we visualized the mitochondrial network and dynamics in ASC and ASC during control and chondrogenic conditions. In control conditions, ASC were ...
Baer A, Lundberg L, Swales D, Waybright N, Pinkham C, Dinman JD, Jacobs JL, Kehn-Hall K.Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by car...
Stewart MC, Stewart AA.This article provides an overview of mesenchymal stem cell (MSC) biology. In the first section, the characteristics that are routinely used to define MSCs-adherence, proliferation, multi-lineage potential, and "cluster of differentiation" marker profiles-are discussed. In the second section, the major tissues and body fluids that are used as sources for equine MSCs are presented, along with the comparative biologic activities of MSCs from specific locations. Finally, the current understanding of the mechanisms by which MSCs influence repair and regeneration are discussed, with an emphasis on t...
Mocchi M, Dotti S, Bue MD, Villa R, Bari E, Perteghella S, Torre ML, Grolli S.Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and hors...
Gravance CG, Garner DL, Baumber J, Ball BA.The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Arnhold S, Elashry MI, Klymiuk MC, Geburek F.Adipose tissue-derived mesenchymal stem cells (ASCs) offer a promising cell source for therapeutic applications in musculoskeletal disorders. The appropriate selection of ASCs from various fat depots for cell-based therapy is challenging. The present study aims to compare stemness and multipotency of ASCs derived from retroperitoneal (RP), subcutaneous (SC), and lipoma (LP) fat to assess their usefulness for clinical application. Equine ASCs from the three fat tissue sources were isolated and characterized. The cell viability, proliferation, and self-renewal were evaluated using MTT, sulforhod...
