Cell culture in horses involves the in vitro cultivation of equine cells under controlled conditions. This technique is employed to study various cellular processes, including growth, differentiation, and response to external stimuli, in an isolated environment. Equine cell cultures can be derived from various tissues, such as skin, muscle, or bone, and are used in a range of research applications, including genetic studies, drug testing, and disease modeling. These cultures provide a valuable platform for understanding cellular mechanisms and developing therapeutic strategies. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and findings related to cell culture in equine research.
Stewart AS, Schaaf CR, Veerasammy B, Freund JM, Gonzalez LM.Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sampl...
Prasinou P, De Amicis I, Fusaro I, Bucci R, Cavallini D, Parrillo S, Caputo M, Gramenzi A, Carluccio A.Fatty acid-based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species, there has been a developing interest in fertility and sperm quality. Fatty acids, being the principal components of the membranes, play an active role in the regulation of the metabolic activities, and their role on spermiogenesis seems to be of great importance for the resulting quality of the sperm and, thus, fertility. With the application of widely used lipidomic techniques, the aim of this study was to evaluate: (a) the fatty acid cont...
Sedykh SE, Purvinsh LV, Burkova EE, Dmitrenok PS, Ryabchikova EI, Nevinsky GA.Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-C...
Rooney MF, Neto NGB, Monaghan MG, Hill EW, Porter RK.Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the () gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse , we...
Patroca da Silva S, Barbosa de Almeida Medeiros D, Ribeiro Cruz AC, Marques França AF, Diniz Nunes BT, Guerreiro Rodrigues DS, Pinto da Silva EV....In 2018, during the surveillance for West Nile virus (WNV) in horses with neurological clinical signs in the state of Espírito Santo (Brazil), 19 animals were investigated, and 52 biological samples were collected for WNV diagnostic. One brain sample was positive for WNV by RT-qPCR and the virus was isolated in C6/36 cell culture and sequenced. We obtained a nearly complete genome of WNV co-infected with Peruvian horse sickness virus (PHSV) in the cell culture. After confirmation of PHSV by next-generation sequencing, a new PHSV RT-qPCR protocol was developed, which was used to detect another...
Pusterla N, Vaala W, Bain FT, Chappell DE, Craig B, Schneider C, Barnett DC, Gaughan E, Papich MG.Equine protozoal myeloencephalitis (EPM) has remained a devastating neurological disease of the Americas, especially in young performance horses. Prophylactic treatment strategies with diclazuril have shown to reduce seroprevalence and titer levels to Sarcocystis neurona in healthy horses continuously exposed to the apicomplexan parasite. The goal of this study was to determine if the FDA-labeled dose of 1 mg/kg of 1.56% diclazuril (Protazil) given once weekly to healthy adult horses would achieve steady-state concentrations in plasma known to be inhibitory to S. neurona in cell culture. Five ...
Freitas NPP, Silva BDP, Bezerra MRL, Pescini LYG, Olinda RG, Salgueiro CCM, Nunes JF, Martins JAM, Neto SG, Martins LT.This study investigated platelet-rich plasma (PRP) and adipose stem cell-conditioned medium (ASC-CM) use as a strategy to accelerate tissue healing. Platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were quantified in fresh and freeze-dried PRP and ASC-CM, and a stability test was performed in the freeze-dried samples (90 and 180 days of storage). A cell proliferation test was performed using equine mesenchymal stem cell culture in reconstituted PRP gel mesh after freeze-drying. In vivo PRP, ASC-CM applications, or their association were performed in induced w...
Dürrwald R, Kolodziejek J, Oh DY, Herzog S, Liebermann H, Osterrieder N, Nowotny N.(1) Background: Vaccination of horses and sheep against Borna disease (BD) was common in endemic areas of Germany in the 20th century but was abandoned in the early 1990s. The recent occurrence of fatal cases of human encephalitis due to Borna disease virus 1 (BoDV-1) has rekindled the interest in vaccination. (2) Methods: The full genomes of the BD live vaccine viruses "Dessau" and "Giessen" were sequenced and analyzed for the first time. All vaccination experiments followed a proof-of-concept approach. Dose-titration infection experiments were performed in rabbits, based on both cell culture...
Hainisch EK, Jindra C, Reicher P, Miglinci L, Brodesser DM, Brandt S.Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 10-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four pass...
Garland A, Wierenga C, McCrae P, Pearson W.The purpose of this study was to determine anti-inflammatory and/or chondroprotective effects of Equine Omega Complete (EOC) on cartilage explants stimulated with lipopolysaccharide (LPS). Explants were aseptically prepared from the intercarpal joints of 17 market-weight pigs and placed in culture at 37°C for a total of 120 hours. For the final 96 hours, explants were conditioned with a simulated digestion extract of EOC (0, 36 or 180 μL/mL), and for the final 48 hours explants were stimulated with LPS (0 or 15µg/mL). Media was removed and replaced every 24 hours. Samples from the final 48 ...
Dechêne L, Colin M, Demazy C, Fransolet M, Niesten A, Arnould T, Serteyn D, Dieu M, Renard P.Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. An equine ost...
Koch DW, Schnabel LV, Ellis IM, Bates RE, Berglund AK.Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-β2. RNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-β2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized t...
Xu Z, Hao X, Li M, Luo H.Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs prod...
Even KM, Gaesser AM, Ciamillo SA, Linardi RL, Ortved KF.Joint injury often leads to cartilage damage and posttraumatic inflammation, which drives continued extracellular matrix degradation culminating in osteoarthritis. Mesenchymal stem cells (MSCs) have been proposed as a biotherapeutic to modulate inflammation within the joint. However, concerns have been raised regarding the immunogenicity of MSCs cultured in traditional fetal bovine serum (FBS) containing media, and the potential of xenogenic antigens to activate the immune system causing rejection and destruction of the MSCs. Xenogen-free alternatives to FBS have been proposed to decrease MSC ...
Tasma Z, Hou W, Damani T, Seddon K, Kang M, Ge Y, Hanlon D, Hollinshead F, Hisey CL, Chamley LW.Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promise as off-the-shelf therapeutics; however, producing them in sufficient quantities can be challenging. In this study, MSCs were isolated from preimplantation equine embryos and used to produce EVs in two commercially available bioreactor designs. Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may...
Trachsel DS, Stage HJ, Rausch S, Trappe S, Söllig K, Sponder G, Merle R, Aschenbach JR, Gehlen H.Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASC...
Hagen A, Niebert S, Brandt VP, Holland H, Melzer M, Wehrend A, Burk J.Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine a...
Barshick MR, Gonzalez ML, Busse NI, Helsel PJ, Johnson SE.Satellite cell (SC) activation is defined as the time frame during which the stem cell becomes poised to reenter G1 of the cell cycle. The growth factors and events leading to full mitotic activation in equine SCs remain largely unknown. Insulin-like growth factor I (IGF-I), hepatocyte growth factor (HGF), and fibroblast growth factor 2 (FGF2) are sequentially transcribed during the muscle repair and recovery period following strenuous exercise in adult horses. Expression of IGF-I occurs within 24 h of the postexercise recovery period suggesting it may affect early SC actions. As a first step,...
Ząbek T, Witarski W, Semik-Gurgul E, Szmatoła T, Kowalska K, Samiec M.Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcri...
Supokawej A, Korchunjit W, Wongtawan T.The Wingless and Int-1 (WNT) and bone morphogenic protein/growth differentiation factor (BMP/GDF) signalling pathways contribute significantly to the development of the musculoskeletal system. The mechanism by which they contribute is as follows: BMP/GDF signalling usually promotes tendon differentiation, whereas WNT signalling inhibits it. We hypothesised that inhibiting WNT and subsequently stimulating BMP signalling may enhance the tenogenic differentiation of stem cells. The objective of this study was to determine whether a combination of WNT inhibitor (KY02111) and BMP12/GDF7 protein cou...
Abd-El-Aziz A, Riveroll A, Esparza-Gonsalez B, McD￮ L, Cohen AM, Fenech AL, Montelpare WJ.The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was ...
Bhutia WD, Gupta S, Rani R, Batra K, Sethi K, Kumar S, Kumar R.Trypanosoma evansi is a causative agent of chronic wasting and fatal disease of livestock and wild animals known as surra. In this study, repurposing approach based on drug target was used to investigate the efficacy of kinase inhibitors (Barasertib-HQPA, BAR and Palbociclib isethionate, PAL) and protease inhibitors (Z-pro-prolinal, Z-PRO and Leupeptin hemisulphate, LEU) against T. evansi in HMI-9 medium. BAR, PAL and Z-PRO exhibited IC values of 13.52 µM, 0.6375 µM and 63.20 µM against T. evansi in terms of growth inhibition, in the contrary, LEU failed to exhibit a significant growth i...
Adalbert R, Cahalan S, Hopkins EL, Almuhanna A, Loreto A, Pór E, Körmöczy L, Perkins J, Coleman MP, Piercy RJ.Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movem...
Malek G, Richard H, Beauchamp G, Laverty S.Focal bone microcracks with osteoclast recruitment and bone lysis, may reduce fracture resistance in racehorses. As current imaging does not detect all horses at risk for fracture, the discovery of novel serum biomarkers of bone resorption or osteoclast activity could potentially address this unmet clinical need. The biology of equine osteoclasts on their natural substrate, equine bone, has never been studied in vitro and may permit identification of specific biomarkers of their activity. Objective: (1) Establish osteoclast cultures on equine bone, (2) Measure biomarkers (tartrate resistant ac...
Thompson RE, Meyers MA, Veeramachaneni DNR, Pukazhenthi BS, Hollinshead FK.Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of t...
Gabryś J, Kij-Mitka B, Sawicki S, Kochan J, Nowak A, Łojko J, Karnas E, Bugno-Poniewierska M.The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to ...
Soukup R, Gerner I, Gültekin S, Baik H, Oesterreicher J, Grillari J, Jenner F.Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates...
Contentin R, Jammes M, Bourdon B, Cassé F, Bianchi A, Audigié F, Branly T, Velot É, Galéra P.Equine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to the cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternatives to overcome those limitations while preserving MSC restorative properties. The effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) was analyzed with indirect co-culture and/or MSC-conditioned media ...
Bartlett AP, Harman RM, Weiss JR, Van de Walle GR.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....
Walters B, Trumble TN, Wendt-Hornickle E, Kennedy M, Guedes A.Apoptosis is an important mechanism underlying chondrocyte loss in osteoarthritis that could be affected by modulation of lipid signaling via inhibition of cyclooxygenases (COX) and soluble epoxide hydrolase (sEH). Objective: To determine the impact of inhibiting COX and sEH alone or in combination on apoptosis of equine chondrocytes. Methods: Cultured primary equine chondrocytes were subjected to serum deprivation or incubation with 1 μg/ml tunicamycin for 24 h to induce apoptosis via caspase activation and endoplasmic reticulum (ER) stress, respectively. Cells were treated with the non-se...
Miyabara S, Yuda Y, Kasashima Y, Kuwano A, Arai K.Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participat...
Zayed MN, Schumacher J, Misk N, Dhar MS.Mesenchymal stromal cells (MSCs) have the capacity to differentiate into cells of mesenchymal lineage, such as chondrocytes, and have potential for use in regeneration of equine articular cartilage. MSCs instilled intra-articularly would be exposed to the inflamed environment associated with equine osteoarthritis (OA), which may compromise their function and ability to heal a cartilaginous defect. The aim of this study was to assess the ability of equine adult MSCs to differentiate into chondrocytes when stimulated with pro-inflammatory cytokines. MSCs derived from equine bone marrow (BM) and ...
Borjesson DL, Peroni JF.This article focuses on the emerging field of equine regenerative medicine with an emphasis on the use of mesenchymal stem cells (MSCs) for orthopedic diseases. We detail laboratory procedures and protocols for tissue handling and MSC isolation, characterization, expansion, and cryopreservation from bone marrow, fat, and placental tissues. We provide an overview of current clinical uses for equine MSCs and how MSCs function to heal tissues. Current laboratory practices in equine regenerative medicine mirror those in the human field. However, the translational use of autologous and allogeneic M...
Beisel CE, Edwards JF, Dunn LL, Rice NR.Transcription of pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. Four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, EIAV. Nucleotide sequence analysis demonstrated that three of these mRNAs encode known viral proteins: the envelope precursor, the product of the S2 open reading frame, and the regula...
Hasebe R, Sasaki M, Sawa H, Wada R, Umemura T, Kimura T.We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery a...
González-Fernández ML, Pérez-Castrillo S, Sánchez-Lázaro JA, Prieto-Fernández JG, López-González ME, Lobato-Pérez S, Colaço BJ, Olivera ER....OBJECTIVE To assess the ability to regenerate an equine meniscus by use of a collagen repair patch (scaffold) seeded with mesenchymal stem cells (MSCs) derived from bone marrow (BM) or adipose tissue (AT). SAMPLE 6 female Hispano-Breton horses between 4 and 7 years of age; MSCs from BM and AT were obtained for the in vitro experiment, and the horses were subsequently used for the in vivo experiment. PROCEDURES Similarities and differences between MSCs derived from BM or AT were investigated in vitro by use of cell culture. In vivo assessment involved use of a meniscus defect and implantation o...
Anderson JR, Phelan MM, Foddy L, Clegg PD, Peffers MJ.Osteoarthritis is an age-related degenerative musculoskeletal disease characterized by loss of articular cartilage, synovitis, and subchondral bone sclerosis. Osteoarthritis pathogenesis is yet to be fully elucidated with no osteoarthritis-specific biomarkers in clinical use. equine cartilage explants ( = 5) were incubated in tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)-supplemented culture media for 8 days, with the media removed and replaced at 2, 5, and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent one-d...
Dobrinski I, Smith TT, Suarez SS, Ball BA.Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC...
Nixon AJ, Lust G, Vernier-Singer M.Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffere...
Glynn ER, Londono AS, Zinn SA, Hoagland TA, Govoni KE.The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated fro...
Lee EM, Kim AY, Lee EJ, Park JK, Park SI, Cho SG, Kim HK, Kim SY, Jeong KS.Horse health has become a major concern with the expansion of horse-related industries and sports; the importance of healthy muscles for horse performance and daily activities is undisputed. Here we generated equine-induced pluripotent stem cells (E-iPSCs) by reprogramming equine adipose-derived stem cells (E-ADSCs) into iPSCs using a polycistronic lentiviral vector encoding four transcription factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and then examined their pluripotent characteristics. Subsequently, established E-iPSCs were transplanted into muscle-injured Rag/ mdx mice. The histopathology ...
Raabe MR, Issel CJ, Montelaro RC.Equine infectious anemia virus (EIAV) has been shown to infect cells of monocyte/macrophage lineage. These primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. As a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. We describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. The in vitro differentiation of these cells into mature equine macrophage was verified using various cytoc...
Donnelly KS, Giuliano EA, Sharm A, Mohan RR.To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Methods: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determi...
Pezzanite LM, Chow L, Johnson V, Griffenhagen GM, Goodrich L, Dow S.To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro. Methods: Controlled laboratory study. Methods: Equine bone-marrow-derived MSCs (three horses). Methods: MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 105 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicr...
Russell KA, Koch TG.Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. Objective: To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Methods: Randomised dose escalation study. Methods: Platelet concentrate was generated from 5 equine whol...
Kurtz BM, Singletary LB, Kelly SD, Frampton AR.In this study, Equus caballus major histocompatibility complex class I (MHC-I) was identified as a cellular entry receptor for the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This novel EHV-1 receptor was discovered using a cDNA library from equine macrophages. cDNAs from this EHV-1-susceptible cell type were inserted into EHV-1-resistant B78H1 murine melanoma cells, these cells were infected with an EHV-1 lacZ reporter virus, and cells that supported virus infection were identified by X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining. Positive cells were subjected t...
Kremer A, Ribitsch I, Reboredo J, Dürr J, Egerbacher M, Jenner F, Walles H.Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for ...
Hale BW, Goodrich LR, Frisbie DD, McIlwraith CW, Kisiday JD.To evaluate the effect of fibrin concentrations on mesenchymal stem cell (MSC) migration out of autologous and commercial fibrin hydrogels. Methods: Blood and bone marrow from six 2- to 4-year-old horses. Methods: Autologous fibrinogen was precipitated from plasma and solubilized into a concentrated solution. Mesenchymal stem cells were resuspended in fibrinogen solutions containing 100%, 75%, 50%, and 25% of the fibrinogen precipitate solution. Fibrin hydrogels were created by mixing the fibrinogen solutions with MSCs and thrombin on tissue culture plates. After incubation for 24 hours in cel...
Rakic R, Bourdon B, Hervieu M, Branly T, Legendre F, Saulnier N, Audigié F, Maddens S, Demoor M, Galera P.As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microe...
Khodadadi K, Sumer H, Pashaiasl M, Lim S, Williamson M, Verma PJ.Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining ...
Grooters AM, Whittington A, Lopez MK, Boroughs MN, Roy AF.The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4...
Kisiday JD, McIlwraith CW, Rodkey WG, Frisbie DD, Steadman JR.To evaluate the effects of single- and double-spin preparations of platelet-rich plasma (PRP) on anabolic and catabolic activities of cartilage and meniscal explants in vitro. Methods: Single- and double-spin PRP was prepared using laboratory processing or commercial kits. The cellular contents were quantified, and each PRP was mixed in equal quantities with cell culture medium and added to cartilage or meniscus explant cultures, with or without interleukin 1 β (IL-1β). Extracellular matrix synthesis was quantified over 24 hours via (35)S-sulfate and (3)H-proline incorporation, while gene ex...
Ahmed YA, Tatarczuch L, Pagel CN, Davies HM, Mirams M, Mackie EJ.Post-proliferative chondrocytes in growth cartilage are present in two forms, light and dark cells. These cells undergo hypertrophy and die by a mechanism that is morphologically distinct from apoptosis, but has not been characterized. The aims of the current study were to document the ultrastructural appearance of dying hypertrophic chondrocytes, and to establish a culture system in which the mechanism of their death can be examined. Methods: Growth cartilage from fetal and growing postnatal horses was examined by electron microscopy. Chondrocytes were isolated from epiphyseal cartilage from ...
Abraham G, Zizzadoro C, Kacza J, Ellenberger C, Abs V, Franke J, Schoon HA, Seeger J, Tesfaigzi Y, Ungemach FR.Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions. Results: Large numbers of EBEC were obtained by trypsin digestion and successfull...
Roth SP, Brehm W, Groß C, Scheibe P, Schubert S, Burk J.Transforming growth factor beta 3 (TGFβ3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGFβ3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGFβ3 in an in vitro model. TGFβ3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGFβ3 were not detected in the rinsing fluid of the TGFβ3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loa...
Mambelli LI, Winter GH, Kerkis A, Malschitzky E, Mattos RC, Kerkis I.Mesenchymal stem cells (MSCs), because of their immunomodulation and trophic activities, in addition to their capacity to regenerate damaged tissues, have potential for treatment of many diseases. The success of stem cell therapies depends, in part, on the method of cell delivery, which should provide wide cell distribution and homing in to injured sites. The objective of the present study was to developing a novel strategy for delivery of MSCs into the uterus of mares with endometrosis (degenerative alteration of uterine glands and surrounding stroma). Endometrosis was confirmed in all mares ...
Hinrichs K, Choi YH, Love LB, Varner DD, Love CC, Walckenaer BE.We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the ...
Seo JP, Tsuzuki N, Haneda S, Yamada K, Furuoka H, Tabata Y, Sasaki N.Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause equine bone marrow-derived MSC expansion. MSCs were isolated from bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences...
Paris DB, Stout TA.Cartilage and tendon injuries are a significant source of animal wastage and financial loss within the horse-racing industry. Moreover, both cartilage and tendon have limited intrinsic capacity for self-repair, and the functionally inferior tissue produced within a lesion may reduce performance and increase the risk of reinjury. Stem cells offer tremendous potential for accelerating and improving tissue healing, and adult mesenchymal stem cells (MSCs) are already used to treat cartilage and tendon injuries in horses. However, MSCs are scarce in the bone marrow isolates used, have limited poten...
Lettry V, Hosoya K, Takagi S, Okumura M.Bone marrow derived mesenchymal stem cells (MSCs) can be used to repair articular cartilage defects, these cells should be properly stimulated so that they could differentiate morphologically and hold cellular synthetic features closer to maturely differentiated chondrocytes. It is well known that tissue specific environment plays an important role in cell fate determination. Once improved isolation, proliferation and differentiation protocols have been developed, the likelihood of spontaneous differentiation of MSCs into divergent lineages will be reduced, thus increasing their value for cart...
