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.
Cochet M, Piumi F, Gorna K, Berry N, Gonzalez G, Danckaert A, Aulner N, Blanchet O, Zientara S, Donadeu FX, Munier-Lehmann H, Richardson J....Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplat...
Hassanien RT, Thieulent CJ, Carossino M, Li G, Balasuriya UBR.(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics ; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligi...
Hellman S, Martin F, Tydén E, Sellin ME, Norman A, Hjertner B, Svedberg P, Fossum C.Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by s...
Ivanova Z, Petrova V, Grigorova N, Vachkova E.Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore,...
Kim EY, Lee EJ, Kim RE, Kil TY, Kim MK.Amniotic fluid mesenchymal stem cells (AF-MSCs), which can be obtained from fetal tissue, reportedly have self-renewal capacity and multi-lineage differentiation potential. The aim of this study was to identify the biological characteristics of AF-MSCs and evaluate their stability and safety in long-term culture. To confirm the biological characteristics of AF-MSCs, morphology, proliferation capacity, karyotype, differentiation capacity, gene expression level, and immunophenotype were analyzed after isolating AF-MSCs from equine amniotic fluid. AF-MSCs were differentiated into adipocytes, chon...
Arroyo-Salvo C, Cogollo Villarreal MY, Clérico G, Flores Bragulat AP, Niño Vargas A, Castañeira C, Briski O, Alonso C, Plaza J, Zeledon JM....Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey ...
Nascimento C, Saraiva MVA, Pereira VM, de Brito DCC, de Aguiar FLN, Alves BG, Roballo KCS, de Figueiredo JR, Ambrósio CE, Rodrigues APR.The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X;...
Falk J, Donadeu FX.Groundbreaking work by Takahashi and Yamanaka in 2006 demonstrated that non-embryonic cells can be reprogrammed into pluripotent stem cells (PSCs) by forcing the expression of a defined set of transcription factors in culture, thus overcoming ethical concerns linked to embryonic stem cells. Induced PSCs have since revolutionized biomedical research, holding tremendous potential also in other areas such as livestock production and wildlife conservation. iPSCs exhibit broad accessibility, having been derived from a multitude of cell types and species. Apart from humans, iPSCs hold particular med...
Lessiak U, Pratscher B, Tichy A, Nell B.Anti-VEGF agents were found to have clinical implications for the successful treatment of vascular-driven diseases in humans. In this study, a detailed biological characterization of bevacizumab in a variety of in vitro assays was carried out to determine the effect of bevacizumab on equine umbilical vein endothelial cells (EqUVEC). EqUVECs were harvested from umbilical cords of clinically healthy horses and exposed to different concentrations (1, 2, 4, 6, 8 mg/mL) of bevacizumab (Avastin). Assays concerning the drug's safety (cell viability and proliferation assay) and efficacy (cell tube for...
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...
Cappelletti E, Piras FM, Sola L, Santagostino M, Petersen JL, Bellone RR, Finno CJ, Peng S, Kalbfleisch TS, Bailey E, Nergadze SG, Giulotto E.Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occu...
de la Fuente A, Scoggin C, Bradecamp E, Martin-Pelaez S, van Heule M, Troedsson M, Daels P, Meyers S, Dini P.Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes....
Watts AE.Mesenchymal stem cells (MSCs) are used as a regenerative therapy in horses for musculoskeletal injury since the late 1990s and in some regions are standard of care for certain injuries. Yet, there is no Food and Drug Administration-approved MSC therapeutic in the United States for horses. In humans, lack of regulatory approval in the United States has been caused by failure of late-phase clinical trials to demonstrate consistent efficacy, perhaps because of nonuniformity of MSC preparation and application techniques. This article discusses clinical evidence for musculoskeletal applications of ...
Legere RM, Allegro AR, Affram Y, Silveira BPD, Fridley JL, Wells KM, Oezguen N, Burghardt RC, Wright GA, Pollet J, Bordin AI, Figueiredo P....To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs). Methods: Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control. Methods: Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with ...
Velloso Alvarez A, Wooldridge AA, Fuller J, Shrader SM, Mansour M, Boone LH.No studies have evaluated the effect of culture in serum-free media (SF) vs. media supplemented with equine serum (ES) on co-culture of synovial membrane and cartilage tissue explants. The study objective was to evaluate the effects of equine serum supplementation on induced production of inflammatory and catabolic mediators from articular cartilage and synovial explants while in co-culture. Articular cartilage and synovial membrane explants were harvested from femoropatellar joints of five adult horses. Cartilage and synovial explants were harvested from the stifle of five horses, placed in c...
Beaumont RE, Smith EJ, Zhou L, Marr N, Thorpe CT, Guest DJ.We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-B) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-B inhibitors on gel contraction and IL6 secretion were measured in 3D ...
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 ...
Thompson RE, Meyers MA, Palmer J, Veeramachaneni DNR, Magee C, de Mestre AM, Antczak DF, Hollinshead FK.The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in...
De Angelis E, Barilli A, Saleri R, Rotoli BM, Ravanetti F, Ferrari F, Ferrari L, Martelli P, Dall'Asta V, Borghetti P.Due to the importance of joint disease and ostearthritis (OA) in equine athletes, new regenerative treatments to improve articular cartilage repair after damage are gaining relevance. Chondrocyte de-differentiation, an important pathogenetic mechanism in OA, is a limiting factor when differentiated articular chondrocytes are used for cell-based therapies. Current research focuses on the prevention of this de-differentiation and/or on the re-differentiation of chondrocytes by employing different strategies in vitro and in vivo. Articular chondrocytes normally live in a condition of higher osmol...
Black JB, Frampton AR.Equine herpesvirus type 1 (EHV-1) is a highly transmissible pathogen that leads to a variety of clinical disease outcomes in infected horses. A major sequela that can occur after an EHV-1 infection is a neurological disease termed equine herpesvirus myeloencephalopathy (EHM). Clinical manifestations of EHM include fever, ataxia, incontinence, and partial to full paralysis, which may ultimately lead to the euthanization of the infected horse. To develop an effective treatment strategy for EHM, it is critical that the specific virus-host interactions that lead to EHM be investigated so that safe...
Petrova V, Vachkova E.Adipose tissue is recognized as the major endocrine organ, potentially acting as a source of mesenchymal stem cells for various applications in regenerative medicine. Athletic horses are often exposed to traumatic injuries, resulting in severe financial losses. The development of adipose-derived stem cells' regenerative potential depends on many factors. The extraction of stem cells from subcutaneous adipose tissue is non-invasive, non-traumatic, cheaper, and safer than other sources. Since there is a lack of unique standards for identification, the isolated cells and applied differentiation p...
Stage HJ, Trappe S, Söllig K, Trachsel DS, Kirsch K, Zieger C, Merle R, Aschenbach JR, Gehlen H.The investigation of multipotent stem/stromal cells (MSCs) in vitro represents an important basis for translational studies in large animal models. The study's aim was to examine and compare clinically relevant in vitro properties of equine MSCs, which were isolated from abdominal (abd), retrobulbar (rb) and subcutaneous (sc) adipose tissue by collagenase digestion (ASCs-) and an explant technique (ASCs-). Firstly, we examined proliferation and trilineage differentiation and, secondly, the cardiomyogenic differentiation potential using activin A, bone morphogenetic protein-4 and Dickkopf-1. Fi...
Fujiwara Y, Kusakabe KT, Baba K, Sasaki N.Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 10/μl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time...
Andrietti ALP, Durgam SS, Naumann B, Stewart M.Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix. Unassigned: Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolate...
Belacic ZA, Sullivan SN, Rice HC, Durgam SS.To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Methods: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Methods: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via rea...
Moellerberndt J, Hagen A, Niebert S, Büttner K, Burk J.In equine medicine, the use of regenerative therapeutics has gained growing attention, but is still a new and complex field with room for improvement. Platelet lysate (PL) can be used as therapeutic agent but is also a promising supplement for the culture of multipotent mesenchymal stromal cells. To enable a targeted use of PL both in clinic and laboratory, it is crucial to learn more details on its effective ingredients. While so far, mainly growth factor components have been analyzed in platelet-based products such as PL, the current study focuses on the content of cytokines in serum, plasma...
Pechanec MY, Beall JM, Katzman S, Maga EA, Mienaltowski MJ.Tendinopathies remain the leading contributor to career-ending injuries in horses because of the complexity of tendon repair. As such, cell-based therapies like injections of adipose-derived mesenchymal stem cells (ADMSCs, or MSCs) into injured tendons are becoming increasingly popular though their long-term efficacy on a molecular and wholistic level remains contentious. Thus, we co-cultured equine MSCs with intrinsic (tendon proper) and extrinsic (peritenon) tendon cell populations to examine interactions between these cells. Gene expression for common tenogenic, perivascular, and differenti...
Fukushi N, Badr Y, Fukushi H.Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether th...
Teti G, Mazzotti E, Gatta V, Chiarini F, Alfieri ML, Falconi M.Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we ai...
Marycz K, Bourebaba N, Serwotka-Suszczak A, Mularczyk M, Galuppo L, Bourebaba L.Equine metabolic syndrome (EMS) is recognized as one of the leading cause of health threatening in veterinary medicine worldwide. Recently, PTP1B inhibition has been proposed as an interesting strategy for liver insulin resistance reversion in both equines and humans, however as being a multifactorial disease, proper management of EMS horses further necessities additional interventional approaches aiming at repairing and restoring liver functions. In this study, we hypothesized that in vitro induction of Eq_ASCs hepatogenic differentiation will generate a specialized liver progenitor-like cell...
Cabezas J, Rojas D, Navarrete F, Ortiz R, Rivera G, Saravia F, Rodriguez-Alvarez L, Castro FO.Adult stromal mesenchymal stem cells (MSCs) have been postulated as responsible for cell renewal in highly and continuously regenerative tissues such as the endometrium. MSCs have been identified in the endometrium of many species including humans, rodents, pets and some farm animals, but not in horses. The objective of this work was to isolate such cells from the endometrium of mares and to compare their main biological attributes with horse adipose-derived MSCs. Here we successfully isolated and characterized endometrial MSCs (eMSCs) from mares. Said cells showed fibroblast-like morphology, ...
Hotham WE, Thompson C, Szu-Ting L, Henson FMD.Osteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint. Objective: To isolate extracellular vesicles from bone marrow MSC and investigate their anti-inflammatory effects on chondrocytes. ...
Dhar M, Neilsen N, Beatty K, Eaker S, Adair H, Geiser D.Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood-derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. Objective: To optimise c...
Esteves CL, Sheldrake TA, Dawson L, Menghini T, Rink BE, Amilon K, Khan N, Péault B, Donadeu FX.Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in ...
Mott J, Rikihisa Y, Zhang Y, Reed SM, Yu CY.Potomac horse fever is an acute systemic equine disease caused by Ehrlichia risticii. Currently, serologic methods are widely used to diagnose this disease. However, serologic methods cannot determine whether the horse is presently infected or has been exposed to ehrlichial antigens in the past. The purpose of the present study was to compare the sensitivities of the nested PCR and cell culture with that of the indirect fluorescent-antibody (IFA) test for the diagnosis of Potomac horse fever. Blood and fecal specimens serially collected from a pony experimentally infected with E. risticii Mary...
Schuurman W, Gawlitta D, Klein TJ, ten Hoope W, van Rijen MH, Dhert WJ, van Weeren PR, Malda J.If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Objective: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Methods: Controlled laboratory study. Methods: Equine articular chondrocytes from the 3 zones were expan...
Branly T, Bertoni L, Contentin R, Rakic R, Gomez-Leduc T, Desancé M, Hervieu M, Legendre F, Jacquet S, Audigié F, Denoix JM, Demoor M, Galéra P.Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the associ...
Smith JJ, Ross MW, Smith RK.The purpose of this study was to investigate the response of suspensory ligament fibroblasts (SLF) to in vitro stimulation using acellular bone marrow (ABM), platelet rich plasma (in vitro PRP), and serum as potential treatment modalities for suspensory desmitis. Blood, bone marrow, and suspensory ligaments were collected from five horses. SLF were harvested, grown until confluent, and stimulated with various concentrations of ABM, PRP, equine serum, foetal bovine serum, and medium (control). The responses to the treatments were assessed using a combination of radio-labeling for total protein ...
Brindley MA, Maury W.Recently, it has become evident that entry of some retroviruses into host cells is dependent upon a vesicle-localized, low-pH step. The entry mechanism of equine infectious anemia virus (EIAV) has yet to be examined. Here, we demonstrate that wild-type strains of EIAV require a low-pH step for productive entry. Lysosomotropic agents that inhibit the acidification of internal vesicles inhibited productive entry of EIAV. The presence of ammonium chloride (30 mM), monensin (30 microM), or bafilomycin A (50 nM) in the medium dramatically decreased the number of EIAV antigen-positive cells. We foun...
Martinello T, Bronzini I, Maccatrozzo L, Iacopetti I, Sampaolesi M, Mascarello F, Patruno M.Mammalian adult stem cells show, in vitro, extensive differentiative ability and may represent a versatile tool for tissue regenerative purposes, even after long-term storage. Multipotent stem cells isolated from horse blood have been shown to possess the capacity to differentiate into diverse mesenchymal lineages although their full characterization is still at an early stage. The aim of this study was to examine the effects of cryopreservation on stemness characteristics of adult equine mesenchymal stem cells isolated from peripheral blood (ePB-MSC). Each sample of ePB-MSC was analyzed immed...
Stewart AS, Freund JM, Gonzalez LM.Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. Objective: To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without cli...
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...
Huang H, Lavoie-Lamoureux A, Moran K, Lavoie JP.Little is known concerning the possible contribution of T helper 2 (Th2)-type cytokines to the recruitment of neutrophils into the lung tissue. In the present study, endothelial cells from equine pulmonary arteries were cultured in the presence of recombinant equine (re) IL-4 and reIL-5, and the cytokine mRNA expression of molecules implicated in the chemotaxis and migration of neutrophils was studied using real-time RT-PCR. The functional response of reIL-4-induced endothelial cell stimulation on neutrophil migration was also studied using a chemotaxis chamber. ReIL-4 either increased the exp...
Lagutina I, Lazzari G, Duchi R, Colleoni S, Ponderato N, Turini P, Crotti G, Galli C.The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear...
Rwambo PM, Issel CJ, Hussain KA, Montelaro RC.A neutralization escape mutant (A/1 E) of equine infectious anemia virus was isolated after 13 passages in cell culture in the presence of serum containing antibodies to type- and group-specific determinants of EIAV envelope glycoproteins. Loss of neutralization by the selecting serum correlated with loss of two epitopes in the major envelope glycoprotein gp90 of A/1 E which were present in a parallel variant isolated from a persistently infected pony.
Mohanty N, Gulati BR, Kumar R, Gera S, Kumar P, Somasundaram RK, Kumar S.Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73...
Burk J, Berner D, Brehm W, Hillmann A, Horstmeier C, Josten C, Paebst F, Rossi G, Schubert S, Ahrberg AB.Tendon disease has been treated with multipotent mesenchymal stromal cells (MSCs) in the equine large-animal model with promising success. The aim of this study was to gain more insight into the fate and biodistribution of MSCs after local application into tendon lesions by long-term cell tracking in this large-animal model. Superficial digital flexor tendon lesions were induced in all limbs in six horses and injected with 10106 Molday ION Rhodamine B-labeled MSCs suspended in serum or serum alone. Follow-up was performed using low-field magnetic resonance imaging (MRI), flow cytometry, and hi...
Capomaccio S, Cappelli K, Bazzucchi C, Coletti M, Gialletti R, Moriconi F, Passamonti F, Pepe M, Petrini S, Mecocci S, Silvestrelli M, Pascucci L.Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role. Methods: EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was is...
Prado AA, Favaron PO, da Silva LC, Baccarin RY, Miglino MA, Maria DA.Isolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses. Especially in equines, these cells have being considered promising candidates for articular regeneration. Herein, we established and characterized MSCs obtained from equine SF and SM. Samples were obtained during arthroscopy and cultured using MEM (Minimum Essential Medium). MSCs were ...
Murata D, Miyakoshi D, Hatazoe T, Miura N, Tokunaga S, Fujiki M, Nakayama K, Misumi K.Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellul...
Bukowska J, Szóstek-Mioduchowska AZ, Kopcewicz M, Walendzik K, Machcińska S, Gawrońska-Kozak B.Adipose-derived stem cells (ASCs) isolated from domestic animals fulfill the qualitative criteria of mesenchymal stem cells, including the capacity to differentiate along multiple lineage pathways and to self-renew, as well as immunomodulatory capacities. Recent findings on human diseases derived from studying large animal models, have provided evidence that administration of autologous or allogenic ASCs can improve the process of healing. In a narrow group of large animals used in bioresearch studies, pigs and horses have been shown to be the best suited models for study of the wound healing ...
Xuan X, Larsen A, Ikadai H, Tanaka T, Igarashi I, Nagasawa H, Fujisaki K, Toyoda Y, Suzuki N, Mikami T.The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical ...
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...
Carter-Arnold JL, Neilsen NL, Amelse LL, Odoi A, Dhar MS.Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. Objective: To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Methods: Descriptive study of stem cell characteristics. Meth...
Franke J, Abs V, Zizzadoro C, Abraham G.Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. Results: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum (FBS) or horse serum (HS) or in serum depleted mediu...
Yuan ZQ, Gault EA, Gobeil P, Nixon C, Campo MS, Nasir L.It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental ...
Filannino A, Stout TA, Gadella BM, Sostaric E, Pizzi F, Colenbrander B, Dell'Aquila ME, Minervini F.The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging...
Zwart L, Potgieter CA, Clift SJ, van Staden V.African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designat...
Tajima M, Nakajima H, Ito Y.Equine infectious anemia (EIA) virus was observed in thin sections of infected cultured horse leukocytes by electron microscopy. The virus particles had a spherical shape and were between 80 and 120 nm in diameter. Most of them contained an electron-dense nucleoid 40 to 60 nm in diameter. They were observed to form by a process of budding from the plasma membrane and appeared to have thin surface projections. The particles described were not detected in uninfected cultured cells, and their appearance could be prevented by adding EIA immune serum to the inoculum. The implications of these findi...
