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.
Euppayo T, Siengdee P, Limlenglert P, Nganvongpanit K, Watanabe G, Kasashima Y, Arai K.The self-renewal capacity of chondrocytes in osteoarthritis (OA) joints is limited, and mesenchymal stem cells (MSCs) are crucial in disease treatment. This study established an OA model from equine bone marrow-derived mesenchymal stem cells (eBMSCs). The eBMSCs were cultured and differentiated into chondrocytes to generate cartilage pellets, which were induced for 7 d with inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) to mimic OA conditions. Treated culture medium was collected to estimate enzyme activity (MMP-2, MMP-3, and MMP-9) using zymograph...
Elena ZK, Reimer C, Herrmann D, Klein C.Equine endometrial fibrosis is a leading cause of subfertility in aging mares. Fibrosis is a reparative response involving excess deposition of extracellular matrix (ECM) and increasing tissue stiffness. Augmented rigidity itself can drive fibrosis, by stimulating transition from fibroblasts to myofibroblasts. Myofibroblasts release latent transforming growth factor beta 1 (TGF-β1) from the ECM, thereby activating this profibrotic cytokine. Tissue culture polystyrene (TCP) is commonly used for in vitro experiments. The endometrium, however, is considerably softer than TCP. This study critical...
Miranda MR, Montano C, Golino V, de Chiara M, Del Prete C, Pepe G, De Biase D, Ciaglia T, Bertamino A, Campiglia P, Sommella E, Vestuto V, Pasolini MP.Wounds are common in equine practice, and often lead to complications such as infections, delayed healing and hypertrophic scarring, which can be costly and difficult to manage. Developing affordable and effective treatments has become an increasingly important focus in veterinary research. Equine advanced-platelet-rich fibrin plus (A-PRF+) demonstrates regenerative properties comparable to its human counterpart, but cellular-level investigations exploring its molecular mechanisms remain limited. This study aimed to investigate the in vitro effects of equine A-PRF + on primary fibroblast ...
Li X, Hu S, Yu Z, He F, Zhao X, Liu R.Nanoplastics (NPs) are emerging pollutants worldwide. Particularly worrisome is that although studies have reported that NPs can amplify the biotoxicity of environmental pollutants, the specific mechanism remains unclear. Here, we found that NPs, even without significant toxicity (cell survival: 99.11%), amplified the hepatocyte toxicity of Cd. Mechanistically, higher Cd uptake (Δ = 23.80%) combined with crucial intracellular desorption behavior of Cd loaded in NPs (desorption rate: 82.70%) were identified as prerequisites for NPs amplifying Cd cytotoxicity. As for toxigenic pathways, the inf...
Hannan MA, Watanabe H, Takeyama A, Yoshida S, Wudamu D, Lkhagvasuren N, Claes A, Stout TAE, Cheong SH, Haneda S, Nambo Y.This study evaluated the viability of in vitro embryo production using ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) as breeding techniques for pure and crossbred Hokkaido native ponies (n = 9). Oocytes were collected using transvaginal ultrasound-guided follicle aspiration. ICSI was performed on in vitro matured oocytes using frozen semen. Embryonic cultures were monitored using time-lapse cinematography. Blastocysts were cryopreserved and, after thawing, were transferred non-surgically into recipient mares. Over nine OPU sessions, the mean number of aspirated follicles was 2...
Cardinaux EM, Oltmanns H, Beineke A, Meißner J, Geburek F.Flexural limb deformities are a widespread condition in foals. Oxytetracycline is a common conservative treatment option with relaxing effects on the muscle-tendon-unit, potentially mediated through a matrix-metalloproteinase (MMP)-inhibitor mechanism. Its high therapeutic dose for this indication, potential negative side effects, and the guidelines for prudent use of antimicrobials make investigating alternatives desirable. In this study, the influence of substances with potentially similar mechanisms of action, however without antimicrobial properties, on viability and proliferation of juven...
Dahlgren AR, Careddu F, Norris JW, Adili R, Kropp EM, Li Q, Holinstat M, Biunno I, Balduini A, Tablin F, Shavit JA, Finno CJ.SEL1L is a well-known protein in the endoplasmic reticulum associated degradation (ERAD) pathway. While it is known to be expressed in platelets, SEL1L has never been shown to play an active role. Here we find evidence that SEL1L regulates platelet function. We first identified SEL1L through the study of Atypical Equine Thrombasthenia (AET), an autosomal recessive platelet disorder found in Thoroughbred horses. A missense variant in (c.1810A>G p.Ile604Val) was found in AET-affected horses, which we show is associated with decreased protein expression. SEL1L is intracellular in equine platelet...
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...
Battistin L, Moya LFA, Ferreira LVO, Braz AMM, Carvalho M, Golim MA, Amorim RM.Cell-based therapy using mesenchymal stem cells (MSCs) shows promise for treating several diseases due to their anti-inflammatory and immunomodulatory properties. To enhance the therapeutic potential of MSCs, in vitro priming strategies have been explored. Cannabidiol (CBD), a non-psychoactive compound derived from cannabis, may influence MSC proliferation, differentiation, and immunomodulatory properties. This study evaluates the immunomodulatory potential of equine adipose tissue-derived MSCs (EqAT-MSCs) primed with a CBD-rich cannabis extract. EqAT-MSCs (P3) were primed with CBD concentrati...
Ji Y, Xu D, Si W, Zhang Y, Khan MZ, Zhao X, Liu W.The present study investigated the host cell response to EHV-8 infection in rabbit kidney (RK-13) cells through transcriptomic and proteomic approaches. At 24 h post-infection, a total of 2118 differentially expressed genes (DEGs) were identified, with 1338 upregulated and 780 downregulated. At 48 h, 7388 DEGs were detected, with 4342 upregulated and 3046 downregulated genes. Proteomic analysis revealed 932 differentially expressed proteins (DEPs) at 24 h (364 upregulated and 568 downregulated) and 3866 DEPs at 48 h (2285 upregulated and 1581 downregulated). Of these, 237 upregulated and 336 d...
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...
Pawlina-Tyszko K, Semik-Gurgul E, Podstawski P, Herc W, Witkowski M, Ropka-Molik K.Alterations in the genes involved in the creation of the extracellular matrix (ECM) were observed in our earlier transcriptome studies of sarcoids and their cell culture model. For a complete characterization of the underlying molecular pathways, it is imperative to comprehend the involvement of ECM modifications in the oncogenic transformation of sarcoid fibroblasts. Thus, the aim of this investigation was to describe the expression patterns of a set of genes that are essential for the rearrangements of the extracellular matrix, namely collagen genes, and elucidate possible mechanisms underly...
Jones CJP, Wilsher S, Aplin JD.We report a lectin histochemical study of oviductal ampulla and isthmus of thirteen mares euthanised at anestrus (1), estrus (5), 1-day post-ovulation (5) or diestrus (2). Staining with a panel of 5 lectins recognizing N-Acetylgalactosamine (GalNAc) residues revealed mosaicism in glycan expression in some specimens of ampulla and isthmus, peaking at estrus and 1-day post-ovulation (ov+1). In the ampulla, this presented as positively stained areas of both ciliated and non-ciliated cells in otherwise negatively-stained ampullae, ranging from small foci (diestrus) to areas up to 200 microns in le...
Wójtowicz A, Sadowska A, Piotrowska-Tomala K, Szóstek-Mioduchowska A.The role of AREG in the development of fibrosis in the progression of endometrosis in mare remains unknown. We aimed to determine the effects of AREG on fibroblast functional characteristics as well as the expression of extracellular matrix (ECM)-associated genes in fibroblast derived from non-fibrotic and fibrotic equine endometria. Our findings suggest that the mechanisms associated with ECM remodeling regulated by AREG in non-fibrotic fibroblasts may be dysregulated in the progression of fibrosis in endometrosis.
Kushida C, Usui T, Tamura N, Kasashima Y, Sato K, Arai K.This study evaluated the effects of cell adhesion substrates that lead to the generation of equine-induced pluripotent stem cells (eiPSC) from embryonic skin fibroblasts by lipofection of plasmid vectors expressing five reprogramming factors. The reprogramming efficiency of cells induced on the E8 fragment of laminin-511 (eiPSC-511) was higher than that on Geltrex containing laminin-111 as a major laminin (eiPSC-111), and supplementation with a cocktail of small molecular compounds increased the number of iPSC colonies on both substrates. In the cell proliferation assay, eiPSC-511 showed highe...
Baatar T, Song D, Weng Y, Wang G, Jin L, Guo R, Li B, Dugarjaviin M.The epigenetic regulation of gene expression through the covalent modification of histones is crucial for developing germline cells. To study the regulatory role of alternative splicing (AS) of euchromatic histone lysine methyltransferase 2 (EHMT2/G9A) in spermatogenesis in Mongolian horses, this study first examines the localization of the EHMT2 gene in testicular support cells and then predicts the higher-order structures of sequences with and without AS. Two types of lentiviral vectors for overexpression were subsequently constructed for the EHMT2 gene, one with AS and one without, to infec...
Antoni KP, Natalia L, Maciej S, Leszek N, Małgorzata SS, Robert M, Maciej G, Ewa PO.Adipose tissue functions as an endocrine organ; however, excessive lipid accumulation can lead to obesity and metabolic disorders, such as Equine Metabolic Syndrome (EMS), characterized by insulin resistance, fat deposition, and increased inflammation. Despite the growing prevalence of obesity in horses, knowledge of equine adipocytes and their metabolic functions remains limited. The main objective of the study was to develop and optimize a method for isolating equine adipocytes and to characterize their metabolic activity. Using slaughterhouse-derived horse visceral adipose tissue, we develo...
Klymiuk MC, Speer J, Marco I, Elashry MI, Heimann M, Wenisch S, Arnhold S.Osteoarthritis (OA) is a common and incurable disease in humans and animals. To gain a better understanding of the pathogenesis and identify potential treatments, miRNAs will be extracted and analysed from extracellular vesicles (EVs) of equine adipose derived mesenchymal stem cells (AdMSCs). Methods: For this purpose we cultivated and pretreated AdMSCs under different conditions: interleukin 1β, shock wave, chondrogenic differentiation, chondrogenic differentiation under hypoxia, or after senescence. After treatment, EVs were harvested from the cell culture supernatants. Next-generation sequ...
Castanheira CIGD, Anderson JR, Clarke EJ, Hackl M, James V, Clegg PD, Peffers MJ.This study describes a novel technique to analyze the extracellular vesicle (EV)-derived microRNA (miRNA) crosstalk between equine chondrocytes and synoviocytes. Donor cells (chondrocytes, n = 8; synoviocytes, n = 9) were labelled with 5-ethynyl uridine (5-EU); EVs were isolated from culture media and incubated with recipient cells (chondrocytes [n = 5] were incubated with synoviocyte-derived EVs, and synoviocytes [n = 4] were incubated with chondrocyte-derived EVs). Total RNA was extracted from recipient cells; the 5-EU-labelled RNA was recovered and sequenced. Differential expression analysi...
Del Prete C, Gaspari G, Kosior MA, Merlo B, Iacono E, Longobardi C, Martino NA, Dell'Aquila ME, Damiano S, Cocchia N, Gasparrini B, Lange-Consiglio A.Over the years, regenerative therapies have emerged as promising alternatives for persistent breeding-induced endometritis. In vitro studies testing the effects of these therapies on equine endometrial cells are still scarce. This study aimed to evaluate in vitro the effect of Wharton's jelly (WJ) mesenchymal stromal/stem cell (MSCs)-derived conditioned medium (WJ-CM) and platelet-rich plasma (PRP) on equine endometrial cells, with or without lipopolysaccharide (LPS)-induced inflammation. The WJ-CM was obtained after 24 h of starvation in Ringer's lactate of WJ-MSCs and PRP was prepared using...
Heidenberger J, Reihs EI, Strauss J, Frauenlob M, Gültekin S, Gerner I, Tögel S, Ertl P, Windhager R, Jenner F, Rothbauer M.Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by cartilage degradation, chronic inflammation, and progressive joint dysfunction. Despite rising incidences driven by ageing and increasing obesity, potent treatments remain elusive, exacerbating the socioeconomic burden. OA pathogenesis involves an imbalance in extracellular matrix (ECM) turnover, mediated by inflammatory cytokines and matrix-degrading enzymes, leading to oxidative stress, chondrocyte apoptosis, and ECM degradation. Additionally, synovial inflammation (synovitis) plays a critical role in disease pro...
Chen X, Gulbahar K, Ding H, Nie C, Gao X.Horse milk is a highly valuable organic food that is a promising alternative to cow milk, exhibiting plenty of healthy and immune benefits to human. However, identification of proteins associated human wellness and underlying molecular mechanism in horse milk remain unclear. Unassigned: Label-free mass spectrometry-based protein quantification technology was employed to investigate protein composition of animal milk, including cow, goat, camel and horse milk. Prokaryotic expression and disk diffusion assay were applied to acquire and evaluate antimicrobial activity of candidate proteins. RAW2...
Arnade H, Ivie IE, Gordon J, Peroni JF.To evaluate the feasibility of short-term 3-D tissue culture in maintaining the structure and function of equine synovial explants and demonstrate that day 4 explant characteristics were not significantly different from day 0. We hypothesized that a 4-day culture period in 3-D explant culture would not significantly disrupt synovial tissue health and function compared to baseline measurements. Unassigned: Synovial explants (n = 24/horse) from healthy carpal joints of 5 horses were cultured in 12-well plates using tissue-stabilizing agar rings. Explants were evaluated in triplicate or quadrupli...
Gugole PM, Zannoni A, Forni M, Iacono E, Zambelli F, Merlo B.This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10 M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10 M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At sur...
Dubeibe-Marin DF, Borraez DF, Gomez-López DL, Velasco-Acosta DA.This study evaluated the effect of the presence and location of the corpus luteum (CL) on quantity and competence of equine cumulus-oocyte complexes (COCs). Pairs of ovaries were collected at the slaughterhouse and classified into three groups: ovaries with CL (CL+); ovaries with CL in the contralateral ovary (CL-) and ovaries without a CL (NCL). Were evaluated COC quantity, morphology, nuclear maturation rate, and intrafollicular progesterone concentration and gene expression (BAX, BMP-15, ODC1, GDF9, AURKA) in both cumulus cells and oocytes after in vitro maturation. Results showed a lower r...
Gaspari G, Lange-Consiglio A, Cremonesi F, Desantis S.Extracellular vesicles (EVs) are important mediators of cell-cell communication thanks to their ability to transfer their bioactive cargo, thus regulating a variety of physiological contexts. EVs derived from amniotic mesenchymal/stromal cells (eAMC-EVs) are internalized by equine endometrial cells (eECs) with positive effects on regenerative medicine treatments. As the cellular uptake of EVs is influenced by the glycan profile of both EVs and target cells, this study is focused on the role of surface glycans in the uptake of eAMC-EVs by recipient eECs. Equine ECs were obtained by enzymatic di...
Li D, Wang X, Yao J, Chen S.Adipose tissue-derived extracellular vesicles (ATEVs) have garnered attention for their roles in intercellular communication and regulation. This study presents the first investigation of equine adipose tissue, with ATEV being extracted concurrently during the isolation of adipose-derived mesenchymal stem cells (ADSCs). Through CCK-8 cell proliferation assays and scratch migration assays, a significant promotional effect of ATEV on ADSCs was observed, which not only accelerated the proliferation rate of the stem cells but also enhanced their migratory capacity. The application of Trypan Blue e...
Ajeeb B, Kiyotake EA, Keefe PA, Phillips JN, Hatzel JN, Goodrich LR, Detamore MS.Cartilage injuries pose significant challenges in horses and often lead to post-traumatic osteoarthritis (PTOA). Despite the advances in surgical and regenerative techniques, the result in most cases is the formation of a fibrocartilage repair tissue. Cell-based cartilage therapies are mainly focused on equine bone marrow-derived mesenchymal stem cells (eBMSCs) as they are easily accessible, and multipotent. Nonetheless, alternative allogeneic sources, for example equine umbilical cord matrix mesenchymal stromal cells (eUCMSCs), hold promise given their non-invasive and readily accessible natu...
Byambaragchaa M, Park SH, Park MH, Kang MH, Min KS.Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter ...
Beaumont RE, Smith EJ, David C, Paterson YZ, Faull E, Guest DJ.Tendon injuries are a common problem in humans and horses. There is a high re-injury rate in both species due to the poor regeneration of adult tendon and the resulting formation of scar tissue. In contrast, fetal tendon injuries undergo scarless regeneration, but the mechanisms which underpin this are poorly defined. It is also unclear if tendon cells derived from embryonic stem cells (ESCs) would aid tendon regeneration. In this study we determined the responses of adult, fetal and ESC-derived equine tenocytes to a range of cytokines, chemokines and growth factors that are upregulated follow...
Raabe O, Reich C, Wenisch S, Hild A, Burg-Roderfeld M, Siebert HC, Arnhold S.Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml)...
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...
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...
Bussche L, Harman RM, Syracuse BA, Plante EL, Lu YC, Curtis TM, Ma M, Van de Walle GR.The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailabl...
Dobbe JC, van der Meer Y, Spaan WJ, Snijder EJ.The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodime...
Youngstrom DW, Barrett JG, Jose RR, Kaplan DL.Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribo...
Hortensius RA, Harley BA.The design of biomaterials for regenerative medicine can require biomolecular cues such as growth factors to induce a desired cell activity. Signal molecules are often incorporated into the biomaterial in either freely-diffusible or covalently-bound forms. However, biomolecular environments in vivo are often complex and dynamic. Notably, glycosaminoglycans (GAGs), linear polysaccharides found in the extracellular matrix, are involved in transient sequestration of growth factors via charge interactions. Biomaterials mimicking this phenomenon may offer the potential to amplify local biomolecular...
Braun J, Hack A, Weis-Klemm M, Conrad S, Treml S, Kohler K, Walliser U, Skutella T, Aicher WK.To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs). Methods: 6 adult racing horses treated for articular Injury but otherwise healthy. Methods: AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli...
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...
Wang L, Raidal SL, Pizzirani A, Wilcox GE.A nested multiplex PCR was developed as a rapid (<12h), sensitive test for the simultaneous identification of equine herpesviruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and ...
Stewart AA, Byron CR, Pondenis H, Stewart MC.To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system. Methods: 6 healthy horses, 6 months to 5 years of age. Methods: Bone marrow-derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm(2) and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days....
Haber M, Cao Z, Panjwani N, Bedenice D, Li WW, Provost PJ.The physiologic mechanisms involving growth factors, including PDGF-BB, EGF, and TGF-beta 1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Methods: Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF-BB, EGF, TGF-...
Saito S, Ugai H, Sawai K, Yamamoto Y, Minamihashi A, Kurosaka K, Kobayashi Y, Murata T, Obata Y, Yokoyama K.Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells diffe...
Wang ZW, Nara M, Wang YX, Kotlikoff MI.The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, beta-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2'-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibit...
Cadby JA, Buehler E, Godbout C, van Weeren PR, Snedeker JG.The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency as...
Naskou MC, Sumner SM, Chocallo A, Kemelmakher H, Thoresen M, Copland I, Galipeau J, Peroni JF.Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) poo...
Marędziak M, Marycz K, Smieszek A, Lewandowski D, Toker NY.The aim of this study was to evaluate the proliferation rate and morphological changes of adipose-derived mesenchymal stem cells of canine and equine origin (Eq- and CaAdMSC). Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. Proliferation activity of cells was determined with the Alamar Blue assay. Obtained results, normalized in respect to the control culture, showed that EqAdMSC exposed to MF maintained a high proliferation status, whereas proliferation activity of CaAdMSC cultured in the presence of MF was decreased. Estimations of population doub...
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...
Watts AE, Ackerman-Yost JC, Nixon AJ.To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Tolu...
Worster AA, Nixon AJ, Brower-Toland BD, Williams J.To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. Methods: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. Methods: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated fro...
Jörg A, Henderson WR, Murphy RC, Klebanoff SJ.Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spe...
Yin X, Hu Z, Gu Q, Wu X, Zheng YH, Wei P, Wang X.Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and charact...
Reed SA, Johnson SE.Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by sport horses. The objective of the study was to identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like morphology and express the stem cell markers Oct4, SSEA-1, Tra1-60 and Tra1-81. Culture of the cells in tissue-specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins. Chondrogenic...
Broeckx S, Suls M, Beerts C, Vandenberghe A, Seys B, Wuertz-Kozak K, Duchateau L, Spaas JH.Cell-based therapies, such as treatments with mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) are thought to have beneficial effects on the clinical outcome of orthopedic injuries, but very few animal studies with large sample size are published so far. Therefore, the aim of this study was to assess the safety and report the clinical outcome of allogenic, immature or chondrogenic induced MSCs in combination with PRP for the treatment of degenerative joint disease (DJD) in 165 horses. MSCs and PRP were isolated from a 6-year-old donor horse and transplanted either in their native s...
Mobasheri A, Lewis R, Maxwell JE, Hill C, Womack M, Barrett-Jolley R.Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20-200 mmHg) activated ion channel currents in six of seven patches. ...
Wood PL, Scoggin K, Ball BA, Troedsson MH, Squires EL.Using a nontargeted lipidomics analysis of equine sperm and seminal plasma, we were able to characterize a diverse array of individual lipids including ethanolamine and choline ether lipids and seminolipids essential to membrane raft function. We also detected, for the first time in sperm, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths, which were localized to the head and not the tail of sperm. The only previous identification of OAHFAs has been in meibomian glands and their sebaceous secretions. The identities of these lipid amphiphiles were valida...
Frisbie DD, Smith RK.Stem cells have received much attention in recent times because of their potential to improve healing of othropaedic problems. This manuscript presents the genesis, issues and current state of stem cell treatment in equine medicine. Current literature supports the use of mesenchymal stem cells (MSCs) for treatment of orthopaedic problems.
Raabe O, Shell K, Fietz D, Freitag C, Ohrndorf A, Christ HJ, Wenisch S, Arnhold S.Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tensio...
Hdud IM, Mobasheri A, Loughna PT.The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process...
