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.
Sumner SM, Naskou MC, Thoresen M, Copland I, Peroni JF.Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and co...
Gastal GDA, Aguiar FLN, Alves BG, Alves KA, de Tarso SGS, Ishak GM, Cavinder CA, Feugang JM, Gastal EL.Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmen...
Esteves CL, Sheldrake TA, Mesquita SP, Pesántez JJ, Menghini T, Dawson L, Péault B, Donadeu FX.In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell population...
Suganuma K, Yamasaki S, Molefe NI, Musinguzi PS, Davaasuren B, Mossaad E, Narantsatsral S, Battur B, Battsetseg B, Inoue N.Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from...
Cymerys J, Słońska A, Tucholska A, Golke A, Chmielewska A, Bańbura MW.Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first tim...
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 ...
Ceusters J, Lejeune JP, Sandersen C, Niesten A, Lagneaux L, Serteyn D.Bone marrow and adipose tissue represent the two most commonly exploited sources of adult mesenchymal stem cells for musculoskeletal applications. Unfortunately the sampling of bone marrow and fat tissue is invasive and does not always lead to a sufficient number of cells. The present study describes a novel sampling method based on microbiopsy of skeletal muscle in man, pigs, dogs and horses. The process includes explant of the sample, Percoll density gradient for isolation and subsequent culture of the cells. We further characterized the cells and identified their clonogenic and immunomodula...
Lang HM, Schnabel LV, Cassano JM, Fortier LA.Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. Methods: Equine bone marrow-derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25-gauge [ga]) were aspirated into a 3 mL syringe and re-injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. C...
Budik S, Tschulenk W, Kummer S, Walter I, Aurich C.Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on i...
Sedykh SE, Purvinish LV, Monogarov AS, Burkova EE, Grigor'eva AE, Bulgakov DV, Dmitrenok PS, Vlassov VV, Ryabchikova EI, Nevinsky GA.Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel fi...
Vargas A, Peltier A, Dubé J, Lefebvre-Lavoie J, Moulin V, Goulet F, Lavoie JP.OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens ...
Burk J, Glauche SM, Brehm W, Crovace A, Francioso E, Hillmann A, Schubert S, Lacitignola L.Multipotent mesenchymal stromal cells (MSCs) derived from synovial fluid (SF) are considered to be a promising cell type for therapeutic applications in joint disease. However, despite their potential relevance for clinical and experimental studies, there is insufficient knowledge about SF-derived MSCs isolated from horses and sheep. In this study, cells were recovered from healthy SF and bone marrow (BM) of sheep, and from healthy and osteoarthritic SF of horses. Ovine SF-MSCs were used to assess the efficiency of intracellular labelling with quantum dots (QDs). Colony forming units, generati...
Zahedi M, Parham A, Dehghani H, Mehrjerdi HK.Application of competent cells such as mesenchymal stem cells (MSCs) for treatment of musculoskeletal disorders in equine athletes is increasingly needed. Moreover, similarities of horse and human in size, load and types of joint injuries, make horse as a good model for MSCs therapy studies. This study was designed to isolate and characterize stemness signature of equine bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: BM of three mares was aspirated and the mononuclear cells (MNCs) were isolated using density gradient. The primary MNCs were cultured and analyzed after tree passa...
Roth SP, Glauche SM, Plenge A, Erbe I, Heller S, Burk J.Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficia...
Maia L, Dias MC, de Moraes CN, de Paula Freitas-Dell'Aqua C, da Mota LS, Santiloni V, da Cruz Landim-Alvarenga F.Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis...
Cymerys J, Słońska A, Brzezicka J, Tucholska A, Chmielewska A, Rola J, Malik P, Bańbura MW.Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) ...
Colbath AC, Dow SW, Phillips JN, McIlwraith CW, Goodrich LR.The use of allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) may provide an effective alternative to autologous BMDMSCs for treatment of equine musculoskeletal injuries. However, concerns have been raised regarding the potential safety and effectiveness of allogeneic BMDMSCs. We conducted studies to assess the immunological properties of equine allogeneic BMDMSCs compared with those of autologous BMDMSCs. For assessment of inherent immunogenicity, the relative ability of allogeneic and autologous BMDMSCs to stimulate spontaneous proliferation of equine lymphocytes was compared. T...
Frondoza CG, Fortuno LV, Grzanna MW, Ownby SL, Au AY, Rashmir-Raven AM.Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE2) play major roles in the pathogenesis of osteoarthritis (OA). Although current pharmacologic treatments reduce inflammation, their prolonged use is associated with deleterious side effects prompting the search for safer and effective alternative strategies. The present study evaluated whether chondrocyte production of PGE2 can be suppressed by the combination of avocado/soybean unsaponifiables (ASU) and α-lipoic acid (LA). Design Chondrocytes from articular cartilage of equine joints were incubated for 24 hours with: (1) con...
Gurusinghe S, Hilbert B, Trope G, Wang L, Bandara N, Strappe P.Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower e...
Journal of proteomicsJanuary 3, 2017
Volume 154 102-108 doi: 10.1016/j.jprot.2016.12.015
Degroote RL, Uhl PB, Amann B, Krackhardt AM, Ueffing M, Hauck SM, Deeg CA.The membrane protein expression repertoire of cells changes in course of activation. In equine recurrent uveitis (ERU), a spontaneous autoimmune disease in horses with relapsing and ultimately blinding inner eye inflammation, CD4+ T lymphocytes are the crucial pathogenic cells activated in the periphery directly prior to an inflammatory episode. In order to find relevant changes in the membrane proteome associated to disease, we sorted CD4+ lymphocytes and compared protein abundance from the generated proteome datasets of both healthy horses and ERU cases. We detected formin like 1, a key play...
Daneshvar Amoli A, Mohebali N, Farzaneh P, Shahzadeh Fazeli SA, Nikfarjam L, Ashouri Movasagh S, Moradmand Z, Ganjibakhsh M, Nasimian A, Izadpanah M....Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymera...
Ziegler A, Everett H, Hamza E, Garbani M, Gerber V, Marti E, Steinbach F.Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies u...
Kuemmerle JM, Theiss F, Okoniewski MJ, Weber FA, Hemmi S, Mirsaidi A, Richards PJ, Cinelli P.Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 () and a G-protein regulated inducer of neurite outgrowth 3 () as specific tendon markers using RT-...
Ishikawa S, Horinouchi C, Murata D, Matsuzaki S, Misumi K, Iwamoto Y, Korosue K, Hobo S.Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are capable of self-renewal and differentiation into multiple cell lineages. Methods for cell therapy using MSCs have been developed in equine medicine. Recently, human dental pulp stem cells (DPSCs) have drawn much attention owing to their trophic factor producing ability and minimally invasive collection methods. However, there have been no reports on equine dental pulp-derived cells (eDPCs). Therefore, the aim of this study was to isolate and characterize the eDPCs from discarded wolf teeth. Plastic-adherent spindle-shaped ...
Wernersson S, Riihimäki M, Pejler G, Waern I.Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine- and human-cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of in vivo-derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of ...
Bussche L, Rauner G, Antonyak M, Syracuse B, McDowell M, Brown AMC, Cerione RA, Van de Walle GR.Signaling mechanisms that regulate mammary stem/progenitor cell (MaSC) self-renewal are essential for developmental changes that occur in the mammary gland during pregnancy, lactation, and involution. We observed that equine MaSCs (eMaSCs) maintain their growth potential in culture for an indefinite period, whereas canine MaSCs (cMaSCs) lose their growth potential in long term cultures. We then used this system to investigate the role of microvesicles (MVs) in promoting self-renewal properties. We found that Wnt3a and Wnt1 were expressed at higher levels in MVs isolated from eMaSCs compared wi...
Scharf A, Holmes SP, Thoresen M, Mumaw J, Stumpf A, Peroni J.Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 10 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migra...
Zucca E, Corsini E, Galbiati V, Lange-Consiglio A, Ferrucci F.Data obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lu...
Dias MC, Landim-Alvarenga FD, de Moraes CN, da Costa LD, Geraldini CM, de Vasconcelos Machado VM, Maia L.Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. Results: For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs dilute...
Botha M, Botes M, Loos B, Smith C, Dicks LM.Lactobacillus equigenerosi strain Le1, a natural inhabitant of the equine gastrointestinal tract, survived pH 3.0 and incubation in the presence of 1.5% (wt/vol) bile salts for at least 2 h. Strain Le1 showed 8% cell surface hydrophobicity, 60% auto-aggregation, and 47% coaggregation with Clostridium difficile C6. Only 1% of the cells adhered to viable buccal epithelial cells and invaded the cells within 20 min after contact. Preincubation of strain Le1 in a buffer containing pronase prevented adhesion to viable epithelial cells. Preincubation in a pepsin buffer delayed invasion from 20 min to...
Dutta SK, Myrup AC.A novel, simple method of infectious center assay was developed to detect and quantitate the intracellular existence of equine herpesvirus 1 and equine herpesvirus 2 in peripheral blood mononuclear cells infected in vivo and in vitro with the viruses by cocultivation of these cells with a permissive equine cell culture. The infectious center titers were correlated with the infectious virus titers. In vivo equine herpesvirus 1-infected mononuclear cells obtained from ponies experimentally infected with the virus and equine herpesvirus 2-infected mononuclear cells obtained from selected naturall...
Laviada MD, Babín M, Dominguez J, Sánchez-Vizcaíno JM.A sandwich enzyme-linked immunsorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the n...
Jackson KA, Stott JL, Horohov DW, Watson JL.The objectives of this study were to quantify the induction of equine CD23 transcripts in equine peripheral blood mononuclear cells (PBMCs) and pulmonary alveolar macrophages cultured with recombinant equine IL-4 (rEq IL-4). PBMCs were isolated from blood drawn from four healthy horses. Bronchoalveolar lavage (BAL) fluid was collected from three healthy horses and alveolar macrophages were purified using adherence to plastic for 120 min. PBMCs and alveolar macrophages were cultured using four different conditions: rEq IL-4 and LPS, LPS alone, rEq IL-4 alone and a media control. Total RNA was i...
Kol A, Walker NJ, Galuppo LD, Clark KC, Buerchler S, Bernanke A, Borjesson DL.Autologous cellular therapy products including adipose-derived stromal vascular fraction (SVF), bone marrow mononuclear cells (BMMNs), cord blood mononuclear cells (CBMNs) and platelet rich plasma are options for treatment of acute orthopaedic lesions while mesenchymal stem cells (MSCs) are culture expanded. These products may contribute to healing by secreting matrix proteins or growth factors, but they may also act on endogenous MSCs to facilitate healing. Objective: To determine the effects of cell therapy products on MSCs function in vitro. The hypothesis was that cell therapy products pro...
The Journal of physiologyFebruary 15, 1995
Volume 483 ( Pt 1), Issue Pt 1 201-209 doi: 10.1113/jphysiol.1995.sp020578
Hartmann J, Scepek S, Lindau M.1. We have investigated the granule size distributions in human and horse eosinophils by time-resolved patch-clamp capacitance measurements. 2. During exocytosis of single granules the electrical capacitance of the plasma membrane increases in discrete steps. The steps in horse cells are about six times larger than those in human cells in accordance with the difference in granule size. 3. In both species a multimodal capacitance step size distribution is observed with a first peak at 6-7 fF corresponding to granules with a diameter of about 450-500 nm and a surface area of about 0.7 microns2, ...
Fortuna PRJ, Bielefeldt-Ohmann H, Ovchinnikov DA, Wolvetang EJ, Whitworth DJ.Horses are susceptible to a number of neurotropic viruses, including West Nile virus (WNV), which is a pathogen of global significance in both horses and humans. However, there are no in vitro models with which to study infectious neuropathic diseases in the horse. In an effort to redress this, we have generated neurons from equine induced pluripotent stem cells (equiPSCs) that express a range of cortical neuron-specific markers, in addition to the membrane-bound ligand ephrin B3, which plays an important role in axon guidance as well as functioning as the receptor through which henipaviruses,...
Marlo TL, Giuliano EA, Sharma A, Mohan RR.To develop an ex vivo equine corneal organ culture model. Specifically, to assess the equine cornea's extracellular matrix and cellularity after 7 days using two different culture techniques: either (i) immersion system or (ii) air/liquid interface system, to determine the best ex vivo equine corneal model. Methods: Fourteen healthy equine corneas of various breeds. Methods: Equine corneas with 2 mm of perilimbal sclera were freshly harvested from 7 horses undergoing humane euthanasia. One corneal-scleral ring (CSR) from each horse was randomly placed in the (i) immersion condition organ cultu...
Tablin F, Walker NJ, Hogle SE, Pratt SM, Norris JW.To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. Methods: 6 clinically normal adult horses. Methods: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. R...
Gadella BM, Rathi R, Brouwers JF, Stout TA, Colenbrander B.During sexual reproduction, the sperm and oocyte must fuse before the production of a diploid zygote can proceed. In mammals such as equids, fusion depends critically on complex changes in the plasma membrane of the sperm and, not surprisingly, this membrane differs markedly from that of somatic cells. After leaving the testes, sperm cease to synthesize plasma membrane lipids or proteins, and vesicle-mediated transport stops. When the sperm reaches the female reproductive tract, it is activated by so-called capacitation factors that initiate a delicate reorientation and modification of molecul...
Ahern BJ, Schaer TP, Terkhorn SP, Jackson KV, Mason NJ, Hankenson KD.To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM). Methods: Samples obtained from 6 adult horses. Methods: Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential. Results: Aph...
Uhl PB, Szober CM, Amann B, Alge-Priglinger C, Ueffing M, Hauck SM, Deeg CA.Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from health...
Salcedo-Jiménez R, Koenig JB, Lee OJ, Gibson TWG, Madan P, Koch TG.Extracorporeal shock wave therapy (ESWT) has been shown to induce different biological effects on a variety of cells, including regulation and stimulation of their function and metabolism. ESWT can promote different biological responses such as proliferation, migration, and regenerations of cells. Recent studies have shown that mesenchymal stromal cells (MSCs) secrete factors that enhance the regeneration of tissues, stimulate proliferation and differentiation of cells, and decrease inflammatory and immune reactions. Clinically, the combination of these two therapies has been used as a treatme...
Garvican ER, Vaughan-Thomas A, Redmond C, Clegg PD.Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)-affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n = 10), and the gene expression evaluated and compared to aged-matched normal samples (n = 10). Cartilage was harvested from normal (n = 4) and OC (n = 3) j...
Fingerhut L, Yücel L, Strutzberg-Minder K, von Köckritz-Blickwede M, Ohnesorge B, de Buhr N.Equine recurrent uveitis (ERU) is a common ocular disease of horses and described as a model for human autoimmune uveitis. This immune-mediated, inflammatory condition progressively destroys the eye, ultimately leading to blindness. Genetic and autoimmune factors, next to infections with Leptospira, are discussed as key factors in the pathogenesis. Furthermore, a release of neutrophil extracellular traps (NETs) by activated neutrophils is involved. NETs are composed of decondensed chromatin and proteins that can immobilize invading pathogens. However, if NETs accumulate, they can contribute to...
Sp N, Kang DY, Kim DH, Lee HG, Park YM, Kim IH, Lee HK, Cho BW, Jang KJ, Yang YM.Cortisol is a hormone involved in stress during exercise. The application of natural compounds is a new potential approach for controlling cortisol-induced stress. Tumour suppressor protein p53 is activated during cellular stress. Succinate dehydrogenase complex subunit A () and hypoxanthine phosphoribosyl transferase 1 () are considered to be two of the most stable reference genes when measuring stress during exercise in horses. In the present study cells were considered to be in a 'stressed state' if the levels of these stable genes and the highly stress responsive gene p53 were altered. It ...
Clegg PD, Burke RM, Coughlan AR, Riggs CM, Carter SD.The cellular production by resident articular cells and infiltrating inflammatory cells of the gelatinase matrix metalloproteinases (MMP) was investigated by tissue culture methods and analysis of cell supernatants by gelatin zymography. Peripheral blood neutrophils in short term culture produced MMP-9, as did peripheral blood monocytes in culture. Isolated articular chondrocytes in monolayer culture produced both MMP-2 and MMP-9, although articular cartilage maintained as explant culture produced MMP-2 alone. Synovial fibroblasts grown in monolayer culture produced MMP-2 alone, although synov...
Cymerys J, Dzieciatkowski T, Słońska A, Bierla J, Tucholska A, Chmielewska A, Golke A, Bańbura MW.Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were i...
Thieulent C, Hue ES, Sutton G, Fortier C, Dallemagne P, Zientara S, Munier-Lehmann H, Hans A, Paillot R, Vidalain PO, Pronost S.Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiv...
Frellstedt L, Gosset P, Kervoaze G, Hans A, Desmet C, Pirottin D, Bureau F, Lekeux P, Art T.Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway...
Schneider N, Mouithys-Mickalad A, Lejeune JP, Duyckaerts C, Sluse F, Deby-Dupont G, Serteyn D.We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2...
Lee HY, Kopesky PW, Plaas A, Sandy J, Kisiday J, Frisbie D, Grodzinsky AJ, Ortiz C.To quantify the structural characteristics and nanomechanical properties of aggrecan produced by adult bone marrow stromal cells (BMSCs) in peptide hydrogel scaffolds and compare to aggrecan from adult articular cartilage. Methods: Adult equine BMSCs were encapsulated in 3D-peptide hydrogels and cultured for 21 days with TGF-β1 to induce chondrogenic differentiation. BMSC-aggrecan was extracted and compared with aggrecan from age-matched adult equine articular cartilage. Single molecules of aggrecan were visualized by atomic force microscopy-based imaging and aggrecan nanomechanical stiffness...
Li LF, Guan WJ, Hua Y, Bai XJ, Ma YH.A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analy...
Bianchessi M, Chen Y, Durgam S, Pondenis H, Stewart M.Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with ...
Szóstek AZ, Siemieniuch MJ, Galvão AM, Lukasik K, Zieba D, Ferreira-Dias GM, Skarzynski DJ.Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digesti...
Davidson TR, Chamberlain CS, Bridges TS, Spicer LJ.Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medi...
Bartholomew S, Owens SD, Ferraro GL, Carrade DD, Lara DJ, Librach FA, Borjesson DL, Galuppo LD.Stem cells derived from umbilical cord tissue (UCT) and umbilical cord blood (UCB) in human subjects and horses can be obtained in a minimally invasive fashion with successful propagation of mesenchymal stem cells (MSCs). Currently there are no detailed protocols documenting a procedure to harvest UCB and UCT safely for equine stem cell propagation. Objective: UCB and UCT could be collected without harm to mare or foal. Objective: To develop a standard and safe method for UCB and UCT collection, and prospectively to compare foal and mare health between groups of animals where tissue was and wa...
Wills JM, Watson G, Lusher M, Mair TS, Wood D, Richmond SJ.This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated i...
Aguiar C, Theoret C, Smith O, Segura M, Lemire P, Smith LC.Induced pluripotent stem cells (iPSC) have brought immense hope to cellular therapy and regenerative medicine. However, the antigenicity of iPSC has not been well documented and remains a hurdle for clinical applications. Expression of major histocompatibility complex (MHC) molecules by human and murine iPSC is downregulated, making these cells potentially safe for transplantation. No such data are available for any large animal model. Objective: To measure expression of MHC molecules on equine iPSC (eiPSC) and describe their antigenicity using intradermal testing. The hypothesis was that allo...
Renzi S, Lombardo T, Dotti S, Dessì SS, De Blasio P, Ferrari M.The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins...
