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.
Watt B, Tenza D, Lemmon MA, Kerje S, Raposo G, Andersson L, Marks MS.PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)--which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core--are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show...
Coli A, Nocchi F, Lamanna R, Iorio M, Lapi S, Urciuoli P, Scatena F, Giannessi E, Stornelli MR, Passeri S.The amnion is a particular tissue whose cells show features of multipotent stem cells proposed for use in cellular therapy and regenerative medicine. From equine amnion collected after the foal birth we have isolated MSCs (mesenchymal stem cells), namely EAMSCs (equine amnion mesenchymal stem cells), from the mesoblastic layer. The cells were grown in α-MEM (α-modified minimum essential medium) and the effect of EGF (epidermal growth factor) supplementation was evaluated. To assess the growth kinetic of EAMSCs we have taken into account some parameters [PD (population doubling), fold increas...
Parker RA, Clegg PD, Taylor SE.To investigate the effects of commonly used antibiotics on cell viability and gene expression of equine bone marrow-derived mesenchymal stromal cells (MSC) in vitro. Methods: Bone marrow-derived MSC were cultured in media containing gentamicin, amikacin, penicillin, enrofloxacin or ceftiofur at concentrations of 50, 100, 200 and 500 µg/ml. The alamarBlue fluorescence assay was used to assess cell viability over 48 h. After 5 days the cells were released and lysed prior to RNA extraction and reverse transcription. RNA levels were assessed using spectrophotometry and quantitative PCR was used...
Taylor SE, Clegg PD.Mesenchymal stromal cells (MSC) are derived from adult mesenchymal tissues and have the ability to undergo differentiation into bone, cartilage, and fat, and have therefore attracted great interest in regenerative medicine. Many isolation and culture methods have been described, making comparison between laboratories and quality-control protocols difficult. A uniform protocol to characterize equine MSC has recently been proposed, aiming to introduce consistency across the equine stem cell research field. This article reviews the published techniques for collection and propagation of equine MSC...
Nobert KM.This article provides an overview of the US Food and Drug Administration's current and potential regulation of veterinary regenerative medicine and the various products used in the practice. This article also discusses several of the potential enforcement risks associated with the commercialization of such therapies and products and offers the reader strategies for mitigating those risks. Finally, the article concludes with a review of an important and ongoing court battle that focuses on the marketing and promotion of cellular-based therapies for humans that could have a significant impact on...
Stewart MC.This article focuses on current issues facing cell-based therapies in equine practice and future studies validating the use of stem cells and related biologic therapies for the treatment of musculoskeletal conditions in the horse. Issues raised include the characterization and use of tissue- and anatomic location-specific mesenchymal stem cell (MSC) sources, the putative advantages and feasibility of allogeneic embryonic stem cell and MSC products, the technical advantages and performance of cell-based biologic agents that do not require extensive ex vivo manipulation, the regulation of MSC ho...
Peroni JF, Borjesson DL.The recent interest in equine stem cell biology and the rapid increase in experimental data highlight the growing attention that this topic has been receiving over the past few years. Within the field of stem cell biology, the relevance of immunobiology is of particular intrigue. It appears that optimal and effective stem cell therapy for equine patients will require a thorough analysis of the immune properties of stem cells as well as their response to immune mediators. The main goal of this review is to discuss the biology of adult mesenchymal stem cells in the context of immunology.
De Schauwer C, Meyer E, Cornillie P, De Vliegher S, van de Walle GR, Hoogewijs M, Declercq H, Govaere J, Demeyere K, Cornelissen M, Van Soom A.Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation meth...
Ibrahim S, Steinbach F.Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Mensing N, Gasse H, Hambruch N, Haeger JD, Pfarrer C, Staszyk C.The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC. Tissue samples were obtained from four healthy horses. Primary cell populations were harvested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtoo...
Pascucci L, Curina G, Mercati F, Marini C, Dall'Aglio C, Paternesi B, Ceccarelli P.In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 an...
Klier J, May A, Fuchs S, Schillinger U, Plank C, Winter G, Gehlen H, Coester C.Recurrent airway obstruction (RAO) in horses has become a common problem in stabled horses in industrialized countries and deserves new therapeutic strategies. CpG-oligodeoxynucleotides (CpG-ODNs) were developed as effective immunostimulating agents to induce a Th2/Th1 shift. These agents showed a beneficial therapeutic effect in allergic diseases with predominant Th2 immunoresponse. CpG-ODN delivery by gelatin nanoparticles (GNPs) resulted in enhanced cellular uptake in murine and human in vitro studies and was a starting point for the present trial. The aim of this study was to identify an o...
Schwarz C, Leicht U, Drosse I, Ulrich V, Luibl V, Schieker M, Röcken M.Adult stem cells are of particular interest for the therapeutic approach in the field of regenerative medicine. Due to their ease of harvest, adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source that has become increasingly popular. Critical aspects of applied cell therapies are the circumstances of transport from the laboratory towards the site of operation and cell delivery into the desired area. With regard to these issues, agarose-hydrogel was analyzed as a cell carrier matrix of equine and canine ASCs in vitro, which can be used for minimally invasive applicati...
Wu W, Blythe DC, Loyd H, Mealey RH, Tallmadge RL, Dorman KS, Carpenter S.Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV(PND-1)) and 5 (EIAV(PND-5)), respectively; however, the neutralization-resistant EIAV(PND-5) variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free ...
Henderson SE, Santangelo KS, Bertone AL.To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow-derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures. Methods: Articular cartilage and BMDMSCs from 5 clinically normal horses. Methods: Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10 μM or LE135 at concentrations of 0, 0.1, 1, or 10 μM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondr...
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...
Corradetti B, Lange-Consiglio A, Barucca M, Cremonesi F, Bizzaro D.Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Methods: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes...
Dougherty SS, Santangelo KS, Bertone AL.To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses. Methods: Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses. Methods: Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested...
Raabe O, Shell K, Würtz A, Reich CM, Wenisch S, Arnhold S.Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic linea...
Yoon MJ, Roser JF.Localization of IGF-I and IGF-IR were observed in Leydig cells of horses using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. Previous studies in other species have indicated that IGF-I increases basal and/or LH/hCG-induced testosterone production. The objectives of this study were to (1) test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and (2) determine if this effect is reproductive stage-dependent. Testes were collected from five pubertal (1.1±0.1 year; 1-1.5 year) and eight post...
Eberhardt C, Amann B, Stangassinger M, Hauck SM, Deeg CA.Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion techniqu...
Oldenhof H, Blässe AK, Wolkers WF, Bollwein H, Sieme H.The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1...
Watts EJ, Rose MT.Wounds on the limbs of horses are notoriously difficult to heal, with over production of TGFβ1 thought to be responsible for excessive scarring; in contrast, wounds in the oral cavity heal rapidly with minimal scarring. This experiment aimed to determine the effect of TGFβ1 on the production of mRNA and proteins for various extracellular matrix components by two equine fibroblast cell lines isolated from the oral mucosa and distal limb. Fibronectin mRNA was up-regulated by TGFβ1 in the limb but not the oral cells. TGFβ1 increased the ratio of mRNA for collagen types I-III for the oral cell...
Medina-Torres CE, Mason SL, Floyd RV, Harris PA, Mobasheri A.The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findin...
Pedreros M, Ratto M, Guerra M.In the present study, we analysed the molecular mechanism(s) by which melatonin directly affects ovarian function in the mare. In Experiment 1, follicles and corpora lutea (CL) were collected from slaughterhouse ovaries and analysed for melatonin (MT(1)) receptor mRNA and protein. In Experiment 2, CL were collected from slaughterhouse ovaries and cultured in Dulbecco's modified Eagle's medium-F12 medium (control medium) supplemented with 50 ng mL(-1) equine chorionic gonadotrophin (eCG), 1 nM-1 μM melatonin, 1 μM forskolin or 1 μM luzindole. Explants were cultured for 3 h in the presence of...
Hoffmann N, Oldenhof H, Morandini C, Rohn K, Sieme H.Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is expo...
Tanaka K, Yamada M, Tamiya-Koizumi K, Kannagi R, Aoyama T, Hara A, Kyogashima M.Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not ...
Hurley DJ, Berghaus LJ, Hurley KA, Moore JN.To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. Methods: Mononuclear cells separated from blood samples from 8 horses. Methods: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activ...
Nagy K, Sung HK, Zhang P, Laflamme S, Vincent P, Agha-Mohammadi S, Woltjen K, Monetti C, Michael IP, Smith LC, Nagy A.The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapi...
Willis P, Caudle AB, Fayrer-Hosken RA.Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oo...
Glade MJ.Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, conce...
Salter MM, Seeto WJ, DeWitt BB, Hashimi SA, Schwartz DD, Lipke EA, Wooldridge AA.To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. Methods: Jugular venous blood samples from 24 adult horses. Methods: Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (C...
Brazil TJ, Dixon PM, Haslett C, Murray J, McGorum BC.The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained respo...
Ellenberger MA, Kaeberle ML, Roth JA.A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic res...
Seo JP, Tsuzuki N, Haneda S, Yamada K, Furuoka H, Tabata Y, Sasaki N.A three dimensional scaffold is essential in mesenchymal stem cells (MSCs) delivery in cell-based therapy for facilitating cell adherence, migration, proliferation, and differentiation. The objectives of this study were to evaluate the possibility of β-tricalcium phosphate incorporated gelatin sponges (Gelatin/β-TCP sponge) as scaffolds for equine MSCs and to examine the effects of seeding density and seeding method on the proliferation of equine MSCs in the Gelatin/β-TCP sponges. Mononuclear cells and MSCs isolated from bone marrow were seeded into Gelatin/β-TCP sponges at different densi...
Crump AL, Davis W, Antczak DF.A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23...
McD￮ LA, Anderson GI, Wright GM, Ryan DA.Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted....
Madden CR, Shih DS.Sequential passage of the tissue culture-adapted prototype strain of EIAV in fetal donkey dermal (FDD) cell cultures generated a virus stock which exhibits cytopathic effects in FDD cell cultures. In this study, the effects of the long terminal repeat (LTR) region on virus replication and cytopathogenicity were examined. The FDD-adapted virus LTR was found to contain a number of base pair mutations and a large insertion within the U3 region in comparison with the previously characterized LTR, lambda12. Transient gene expression studies showed that basal promoter activity, in FDD cell cultures,...
Golke A, Cymerys J, Słońska A, Dzieciatkowski T, Chmielewska A, Tucholska A, Bańbura MW.Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of v...
Brown BN, Siebenlist NJ, Cheetham J, Ducharme NG, Rawlinson JJ, Bonassar LJ.Normal laryngeal function has a large impact on quality of life, and dysfunction can be life threatening. In general, airway obstructions arise from a reduction in neuromuscular function or a decrease in mechanical stiffness of the structures of the upper airway. These reductions decrease the ability of the airway to resist inspiratory or expiratory pressures, causing laryngeal collapse. We propose to restore airway patency through methods that replace damaged tissue and improve the stiffness of airway structures. A number of recent studies have utilized image-guided approaches to create cell-...
Ferreira LVO, Kamura BDC, Oliveira JPM, Chimenes ND, Carvalho M, Santos LAD, Dias-Melicio LA, Amorim RL, Amorim RM.Schwann cells (SCs) are essential for the regenerative processes of peripheral nerve injuries. However, their use in cell therapy is limited. In this context, several studies have demonstrated the ability of mesenchymal stem cells (MSCs) to transdifferentiate into Schwann-like cells (SLCs) using chemical protocols or co-culture with SCs. Here, we describe for the first time the in vitro transdifferentiation potential of MSCs derived from equine adipose tissue (AT) and equine bone marrow (BM) into SLCs using a practical method. In this study, the facial nerve of a horse was collected, cut into ...
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 ...
Heydrick FP, Comer JF, Wachter RF.Phospholipid analyses of Venezuelan equine encephalitis virus showed that virus propagated in L-cell monolayers had a higher sphingomyelin content and a lower phosphatidylcholine content than virus grown in chick fibroblast monolayers. Virus of L-cell origin also was found to possess greater thermal stability than virus derived from the chick fibroblast cell.
Negri S, Fila C, Farinato S, Bellomi A, Pagliaro PP.The scope of our study is to evaluate the possibility of cultivating and expanding human chondrocytes and seeding them on pure equine type I collagen support. Our results show that human articular cartilaginous cells can multiply and grow on type I collagen substrate with production of extracellular matrix. This type of chondrocyte culture on a support can be used for repairing cartilaginous lesions since they show a correct morphology (evaluated by cytological and histological methods) and a suitable differentiation and phenotype as shown by Alcian PAS staining to indicate the presence of muc...
Casaite V, Bruzyte S, Bukauskas V, Setkus A, Morozova-Roche LA, Meskys R.Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purificati...
Svala E, Thorfve AI, Ley C, Henriksson HK, Synnergren JM, Lindahl AH, Ekman S, Skiöldebrand ES.To determine the effects of interleukin (IL)-6 and IL-1β stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. Methods: Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. Methods: Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1β for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with ...
Wattrang E, Berg M, Magnusson M.This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, c...
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 ...
Staszyk C, Gasse H.Fibroblasts and cementoblasts in the periodontal ligament (PDL) of equine cheek teeth were harvested, and monocultures were obtained by means of a "selective detachment" procedure. Cells were characterized by morphological criteria and by immunostaining for vimentin, FVIII, pan-cytokeratin, smooth muscle actin, and pro-collagen. Cementogenic potential of the cells was determined by immunostaining for osteopontin and by histochemical detection of alkaline phosphatase. Equine periodontal fibroblasts (EPF) were spindle-shaped and polygonal. Equine dental cementoblasts (EDC) grew in cobblestone-li...
Lin YZ, Yang F, Zhang SQ, Sun LK, Wang XF, Du C, Zhou JH.Equine lentivirus receptor 1 (ELR1) has been identified as the sole receptor for equine infectious anemia virus (EIAV) and is a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously described membrane-associated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs). One major spliced species (ELR1-IN) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE) has a...
Chen Y, Caporali E, Stewart M.Bone morphogenetic protein 2 (BMP-2) is critical for skeletal and cartilage development, homeostasis and repair. This study was conducted to clone and characterize equine BMP-2, develop expression constructs for equine BMP-2, and to determine whether BMP-2 can stimulate chondrogenesis of equine synovial membrane-derived progenitor cells (SMPC). Methods: Equine BMP-2 cDNA was amplified from chondrocyte RNA, and then transferred into an expression plasmid and adenoviral vector. Effective expression of equine BMP-2 was confirmed using a BMP reporter cell line. SMPC were isolated from synovium, ex...
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...
Morales N, Henriquez C, Sarmiento J, Uberti B, Moran G.Neutrophils are terminally differentiated innate effector cells at the first line of host defense. Neutrophil migration within tissues is complex and involves several steps, during which these cells must be able to interpret a variety of chemical and physical signals. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including equine asthma. Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflamma...
Varner DD, Bowen JA, Johnson L.The onset of sperm capacitation/acrosome reaction was evaluated using heparin. Equine semen was incubated at 38 degrees C for 4.5 h in culture medium with and without 10 micrograms/mL heparin and with and without 0.1 microM of Ca2+ ionophore. Sperm acrosome reaction was detected using chlortetracycline fluorescence (CTC) and transmission electron microscopy (TEM). The CTC assay provided staining patterns that corresponded with the capacitation/acrosome reaction in other mammalian species (man, mouse, guinea pig). The percentages of incapacitated sperm (PUC), capacitated acrosome-intact sperm (...
Edmonds RE, Garvican ER, Smith RK, Dudhia J.To provide evidence to support recommendations regarding the co-administration of drugs with mesenchymal stem cell (MSC) therapy. Objective: To determine the influence of sedatives, local anaesthetic and corticosteroids on MSC viability and proliferation, in comparison to somatic cells derived from tendon (TDCs). Methods: In vitro cell culture. Methods: MSCs (n = 3) and TDCs (n = 2) were cultured in media containing a clinically relevant dose range of xylazine, romifidine, detomidine and butorphanol, mepivacaine, methylprednisolone, or triamcinolone acetonide. Cell viability in suspension cult...
Stefanski A, Mevissen M, Möller AM, Kuehni-Boghenbor K, Schmitz A.In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for...
Milczek-Haduch D, Żmigrodzka M, Witkowska-Piłaszewicz O.Significant systemic metabolic benefits result from even a single exercise session by activating multiple metabolic and signaling pathways within the organism. Among these mechanisms, extracellular vesicles (EVs) play a critical role by delivering their molecular cargo to neighboring or distant cells, thereby influencing cellular metabolism and function. As research progresses, EVs represent an exciting frontier in exercise science and fitness adaptation processes. There is increasing interest in understanding the physiology of EVs as signaling particles and their use as minimally invasive dia...
Samie KA, Kowalewski MP, Schuler G, Gastal GDA, Bollwein H, Scarlet D.In horses, the mechanisms behind ovarian follicle growth and oocyte maturation remain largely unknown. In other species, oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been related to the acquisition of developmental competence and to interaction with granulosa cells for the regulation of follicle development. This study assessed the expression and localization of GDF9 in the equine ovary, and its possible relationship with granulosa cell function. Results: Using custom-made antibodies, GDF9 protein was localized in oocytes from th...
Heimann M, Elashry MI, Klymiuk MC, Eldaey A, Wenisch S, Arnhold S.The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco's Modified Eagle's Medium, 10 μg/mL insulin, 0.55 μg/mL transferrin, 6.8 ng sodium selenite, 1 μM dexamethasone, and 1-5 μM of rosiglitazo...
