Klymiuk MC, Balz N, Elashry MI, Wenisch S, Arnhold S.Nanoparticles including extracellular vesicles derived from mesenchymal stem cells are of increasing interest for research and clinical use in regenerative medicine. Extracellular vesicles (EVs), including also previously named exosomes, provide a promising cell-free tool for therapeutic applications, which is probably a safer approach to achieve sufficient healing. Storage of EVs may be necessary for clinical applications as well as for further experiments, as the preparation is sometimes laborious and larger quantities tend to be gained. For this purpose, nanoparticles were obtained from mes...
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...
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, Su00f6llig 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, Bu00fcttner 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...
Gysens L, Depuydt E, Patruno M, Haspeslagh M, Spaas JH, Martens A.Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovin...
Boorman S, Hanson RR, Velloso Alvarez A, Zhong K, Hofmeister E, Boone LH.To determine the effect of concurrent versus delayed treatment with corticosteroid on equine articular tissues also treated with local anesthetic in vitro in the presence of inflammatory mediators. Methods: Controlled laboratory study. Methods: Five geldings, one mare (aged 3-18 years). Methods: From each horse, 24 synovial and 12 osteochondral explants were cultured in a 12-well plate (2 wells/group, 2 synovial and 1 osteochondral explant/well, total 216 explants in the study). Explants were stimulated in culture medium with 10 μg/ml recombinant equine interleukin-1β and 10 μg/ml tumor...
Stewart AS, Schaaf CR, Veerasammy B, Freund JM, Gonzalez LM.Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sampl...
Prasinou P, De Amicis I, Fusaro I, Bucci R, Cavallini D, Parrillo S, Caputo M, Gramenzi A, Carluccio A.Fatty acid-based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species, there has been a developing interest in fertility and sperm quality. Fatty acids, being the principal components of the membranes, play an active role in the regulation of the metabolic activities, and their role on spermiogenesis seems to be of great importance for the resulting quality of the sperm and, thus, fertility. With the application of widely used lipidomic techniques, the aim of this study was to evaluate: (a) the fatty acid cont...
Sedykh SE, Purvinsh LV, Burkova EE, Dmitrenok PS, Ryabchikova EI, Nevinsky GA.Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-C...
Rooney MF, Neto NGB, Monaghan MG, Hill EW, Porter RK.Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the () gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse , we...
Patroca da Silva S, Barbosa de Almeida Medeiros D, Ribeiro Cruz AC, Marques Franu00e7a AF, Diniz Nunes BT, Guerreiro Rodrigues DS, Pinto da Silva EV....In 2018, during the surveillance for West Nile virus (WNV) in horses with neurological clinical signs in the state of Espírito Santo (Brazil), 19 animals were investigated, and 52 biological samples were collected for WNV diagnostic. One brain sample was positive for WNV by RT-qPCR and the virus was isolated in C6/36 cell culture and sequenced. We obtained a nearly complete genome of WNV co-infected with Peruvian horse sickness virus (PHSV) in the cell culture. After confirmation of PHSV by next-generation sequencing, a new PHSV RT-qPCR protocol was developed, which was used to detect another...
Pusterla N, Vaala W, Bain FT, Chappell DE, Craig B, Schneider C, Barnett DC, Gaughan E, Papich MG.Equine protozoal myeloencephalitis (EPM) has remained a devastating neurological disease of the Americas, especially in young performance horses. Prophylactic treatment strategies with diclazuril have shown to reduce seroprevalence and titer levels to Sarcocystis neurona in healthy horses continuously exposed to the apicomplexan parasite. The goal of this study was to determine if the FDA-labeled dose of 1 mg/kg of 1.56% diclazuril (Protazil) given once weekly to healthy adult horses would achieve steady-state concentrations in plasma known to be inhibitory to S. neurona in cell culture. Five ...
Freitas NPP, Silva BDP, Bezerra MRL, Pescini LYG, Olinda RG, Salgueiro CCM, Nunes JF, Martins JAM, Neto SG, Martins LT.This study investigated platelet-rich plasma (PRP) and adipose stem cell-conditioned medium (ASC-CM) use as a strategy to accelerate tissue healing. Platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were quantified in fresh and freeze-dried PRP and ASC-CM, and a stability test was performed in the freeze-dried samples (90 and 180 days of storage). A cell proliferation test was performed using equine mesenchymal stem cell culture in reconstituted PRP gel mesh after freeze-drying. In vivo PRP, ASC-CM applications, or their association were performed in induced w...
Du00fcrrwald 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...
da Silveira JC, Veeramachaneni DN, Winger QA, Carnevale EM, Bouma GJ.Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differ...
MacDonald GH, Johnston RE.The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which mi...
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...
Garmashova N, Atasheva S, Kang W, Weaver SC, Frolova E, Frolov I.The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, approxima...
McCarthy HE, Bara JJ, Brakspear K, Singhrao SK, Archer CW.A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detec...
Worster AA, Brower-Toland BD, Fortier LA, Bent SJ, Williams J, Nixon AJ.This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish fou...
Violini S, Ramelli P, Pisani LF, Gorni C, Mariani P.Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated. Results: In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cell...
Koch TG, Heerkens T, Thomsen PD, Betts DH.There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The...
Hirst SJ, Martin JG, Bonacci JV, Chan V, Fixman ED, Hamid QA, Herszberg B, Lavoie JP, McVicker CG, Moir LM, Nguyen TT, Peng Q, Ramos-Barbu00f3n D....Increased airway smooth muscle (ASM) mass is perhaps the most important component of the airway wall remodeling process in asthma. Known mediators of ASM proliferation in cell culture models fall into 2 categories: those that activate receptors with intrinsic receptor tyrosine kinase activity and those that have their effects through receptors linked to heterotrimeric guanosine triphosphate-binding proteins. The major candidate signaling pathways activated by ASM mitogens are those dependent on extracellular signal-regulated kinase and phosphoinositide 3'-kinase. Increases in ASM mass may also...
Atasheva S, Garmashova N, Frolov I, Frolova E.Venezuelan equine encephalitis virus (VEEV) represents a continuous public health threat in the United States. It has the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that replicating VEEV interferes with cellular transcription and uses this phenomenon as a means of downregulating a cellular antiviral response. VEEV capsid protein was found to play a critical role in this process, and its approximately 35-amino-acid-long peptide, fused with green fluorescent protein, functioned as efficiently as did the entire capsid. We detected a...
Vidal MA, Robinson SO, Lopez MJ, Paulsen DB, Borkhsenious O, Johnson JR, Moore RM, Gimble JM.To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Methods: In vitro experimental study. Methods: Adult Thoroughbred horses (n=11). Methods: BM (5 horses; mean [+/-SD] age, 4+/-1.4 years) or adipose tissue (6 horses; mean age, 3.5+/-1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis+/-transforming growth factor-3 (TGFbeta(3)) and bone morphogenic factor-6 (BMP-6). Pellets harvested after 3, 7, 14, and 21 days were exam...
Gaspar D, Spanoudes K, Holladay C, Pandit A, Zeugolis D.The last decade has seen significant developments in cell therapies, based on permanently differentiated, reprogrammed or engineered stem cells, for tendon injuries and degenerative conditions. In vitro studies assess the influence of biophysical, biochemical and biological signals on tenogenic phenotype maintenance and/or differentiation towards tenogenic lineage. However, the ideal culture environment has yet to be identified due to the lack of standardised experimental setup and readout system. Bone marrow mesenchymal stem cells and tenocytes/dermal fibroblasts appear to be the cell populat...
Toupadakis CA, Wong A, Genetos DC, Cheung WK, Borjesson DL, Ferraro GL, Galuppo LD, Leach JK, Owens SD, Yellowley CE.To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. Methods: 10 adult Quarter Horses and 8 newborn Thoroughbred foals. Methods: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. Results: BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP ...
Tobaly-Tapiero J, Bittoun P, Neves M, Guillemin MC, Lecellier CH, Puvion-Dutilleul F, Gicquel B, Zientara S, Giron ML, de Thu00e9 H, Sau00efb A.Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%...
Vidal MA, Kilroy GE, Johnson JR, Lopez MJ, Moore RM, Gimble JM.To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. Methods: In vitro experimental study. Methods: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). Methods: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibr...
Yu L, Wei Y, Sun HX, Mahdi AK, Pinzon Arteaga CA, Sakurai M, Schmitz DA, Zheng C, Ballard ED, Li J, Tanaka N, Kohara A, Okamura D, Mutto AA, Gu Y....Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. To date, however, no known PSCs have demonstrated dual competency for chimera formation and direct responsiveness to primordial germ cell (PGC) specification, a unique functional feature of formative pluripotency. Here, by modulating fibroblast growth factor (FGF), transforming growth factor β (TGF-β), and WNT pathways, we derived PSCs from mice, horses, and humans (designated as XPSCs) that are permissive for dire...
De Schauwer C, Meyer E, Van de Walle GR, Van Soom A.Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lac...
Radcliffe CH, Flaminio MJ, Fortier LA.Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB c...
Shehu-Xhilaga M, Ablan S, Demirov DG, Chen C, Montelaro RC, Freed EO.The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion...
Carrade Holt DD, Wood JA, Granick JL, Walker NJ, Clark KC, Borjesson DL.Mesenchymal stem cells (MSCs) are used in both human clinical trials and veterinary medicine for the treatment of inflammatory and immune-mediated diseases. MSCs modulate inflammation by decreasing the cells and products of the inflammatory response. Stimulated equine MSCs from bone marrow (BM), adipose tissue (AT), cord blood (CB), and umbilical cord tissue (CT) inhibit lymphocyte proliferation and decrease inflammatory cytokine production. We hypothesized that equine MSCs inhibit T cell proliferation through secreted mediators and that MSCs from different tissue sources decrease T cell proli...
Barberini DJ, Freitas NP, Magnoni MS, Maia L, Listoni AJ, Heckler MC, Sudano MJ, Golim MA, da Cruz Landim-Alvarenga F, Amorim RM.Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods: The BM-MS...
Munderloh UG, Madigan JE, Dumler JS, Goodman JL, Hayes SF, Barlough JE, Nelson CM, Kurtti TJ.The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, ...
Patterson-Kane JC, Becker DL, Rich T.The equine superficial digital flexor tendon (SDFT) is a frequently injured structure that is functionally and clinically equivalent to the human Achilles tendon (AT). Both act as critical energy-storage systems during high-speed locomotion and can accumulate exercise- and age-related microdamage that predisposes to rupture during normal activity. Significant advances in understanding of the biology and pathology of exercise-induced tendon injury have occurred through comparative studies of equine digital tendons with varying functions and injury susceptibilities. Due to the limitations of in-...
Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD.There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis. Methods: Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative P...
Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF.To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Methods: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. Methods: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transdu...
Maru0119dziak M, Marycz K, Lewandowski D, Siudziu0144ska A, u015amieszek A.The aim of this work study was to evaluate the cytophysiological activity of equine adipose-derived stem cells (ASCs) cultured under conditions of static magnetic field. Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. In order to investigate the effects of magnetic field on stem cell signaling, the localization and density and content of microvesicles (MVs) as well as morphology, ultrastructure, and proliferation rate of equine ASCs were evaluated. Results showed that potential of equine adipose-derived mesenchymal stem cells was accelerated when ma...
Hoynowski SM, Fry MM, Gardner BM, Leming MT, Tucker JR, Black L, Sand T, Mitchell KE.Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton's jelly). Equine umbilical cord matrix (EUCM) cells can be ...
Ott DE, Coren LV, Sowder RC, Adams J, Nagashima K, Schubert U.Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecip...
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