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The effectiveness and safety of allogeneic mesenchymal stem/stromal cells (MSCs) can be affected by patient's immune recognition. Thus, MSC immunogenicity and their immunomodulatory properties are crucial aspects for therapy. Immune responses after allogeneic MSC administration have been reported in different species, including equine. Interactions of allogenic MSCs with the recipient's immune system can be influenced by factors like matching or mismatching for the major histocompatibility complex (MHC) between donor-recipient, and by the levels of MHC expression in MSCs. The latter can vary upon MSC inflammatory exposure or differentiation, such as chondrogenic induction, making both priming and differentiation interesting therapeutic strategies. This study investigated the systemic in vivo immune cellular response against allogeneic equine MSCs in these situations. Either MSCs in basal conditions (MSC-naïve), pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were repeatedly administered subcutaneously into autologous, MHC-matched or MHC-mismatched allogeneic equine recipients. At different time-points after each administration, lymphocytes were obtained from recipient horses and exposed in vitro to the same type of MSCs to assess the proliferative response of different T cell subsets (cytotoxic, helper, regulatory), B cells, and interferon gamma (IFNγ) secretion. Higher proliferative response of helper and cytotoxic T lymphocytes and IFNγ secretion was observed in response to all types of MHC-mismatched MSCs over MHC-matched ones. MSC-primed produced the highest immune response, followed by MSC-naïve, and MSC-chondro. However, MSC-primed activated Treg and had a mild effect on B cells, and the response after their second administration was similar to the first one. On the other hand, both MSC-chondro and MSC-naïve barely induced Treg response but promoted B lymphocyte activation, and proportionally induced a higher cell response after the second administration. In conclusion, both the type of MSC conditioning and the MHC compatibility influenced systemic immune recognition of equine MSCs after single and repeated administrations, but the response was different. Selecting MHC-matched donors would be particularly recommended for MSC-primed and repeated MSC-naïve administrations. While MHC-mismatching in MSC-chondro would be less critical, B cell response should not be ignored. Comprehensively investigating the in vivo immune response against equine allogeneic MSCs is crucial for advancing veterinary cell therapies.
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OBJECTIVES: The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability. METHODS: Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P2, P4 and P6, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P2, and cells were immunophenotyped in P4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P2, P4 and P6. RESULTS: The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P2 to P6. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45-, CD90+ and CD44+. Structural and numerical cytogenetic aberrations were observed in P2-P4. CONCLUSIONS AND RELEVANCE: In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.
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Pulpa Dental , Células Madre , Gatos , Animales , Diferenciación Celular , Citometría de Flujo/veterinaria , Células Cultivadas , Proliferación CelularRESUMEN
The immunomodulatory properties of equine mesenchymal stem cells (MSCs) are important for their therapeutic potential and for their facilitating role in their escape from immune recognition, which may also be influenced by donor-recipient major histocompatibility complex (MHC) matching/mismatching and MHC expression level. Factors such as inflammation can modify the balance between regulatory and immunogenic profiles of equine MSCs, but little is known about how the exposure to the immune system can affect these properties in equine MSCs. In this study, we analyzed the gene expression and secretion of molecules related to the immunomodulation and immunogenicity of equine MSCs, either non-manipulated (MSC-naive) or stimulated by pro-inflammatory cytokines (MSC-primed), before and after their exposure to autologous or allogeneic MHC-matched/-mismatched lymphocytes, either activated or resting. Cytokine priming induced the immunomodulatory profile of MSCs at the baseline (MSCs cultured alone), and the exposure to activated lymphocytes further increased the expression of interleukin 6 (IL6), cyclooxygenase 2, and inducible nitric oxide synthase, and IL6 secretion. Activated lymphocytes were also able to upregulate the regulatory profile of MSC-naive to levels comparable to cytokine priming. On the contrary, resting lymphocytes did not upregulate the immunomodulatory profile of equine MSCs, but interestingly, MSC-primed exposed to MHC-mismatched lymphocytes showed the highest expression and secretion of these mediators, which may be potentially linked to the activation of lymphocytes upon recognition of foreign MHC molecules. Cytokine priming alone did not upregulate the immunogenic genes, but MSC-primed exposed to activated or resting lymphocytes increased their MHC-I and MHC-II expression, regardless of the MHC-compatibility. The upregulation of immunogenic markers including CD40 in the MHC-mismatched co-culture might have activated lymphocytes, which, at the same time, could have promoted the immune regulatory profile aforementioned. In conclusion, activated lymphocytes are able to induce the equine MSC regulatory profile, and their effects seem to be additive to the priming action. Importantly, our results suggest that the lymphocyte response against MHC-mismatched MSC-primed would promote further activation of their immunomodulatory ability, which eventually might help them evade this reaction. Further studies are needed to clarify how these findings might have clinical implications in vivo, which will help developing safer and more effective therapies.
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Immunomodulation and immunogenicity are pivotal aspects for the therapeutic use of mesenchymal stem cells (MSCs). Since the horse is highly valuable as both a patient and translational model, further knowledge on equine MSC immune properties is required. This study analysed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory-immunogenicity balance. Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autologous or allogeneic MHC-matched/mismatched lymphocytes in immune-suppressive assays (immunomodulation) and in modified one-way mixed leukocyte reactions (immunogenicity). After co-culture, frequency and proliferation of T cell subsets and B cells were assessed by flow cytometry and interferon-É£ (IFNÉ£) secretion by ELISA. MSC-primed showed higher regulatory potential by decreasing proliferation of cytotoxic and helper T cells and B cells. However, MHC-mismatched MSC-primed can also activate lymphocytes (proliferative response and IFNÉ£ secretion), likely due to increased MHC-expression. MSC-chondro maintained their regulatory ability and did not increase their immunogenicity, but showed less capacity than MSC-primed to induce regulatory T cells and further stimulated B cells. Subsequent in vivo studies are needed to elucidate the complex interactions between MSCs and the recipient immune system, which is critical to develop safe and effective therapies.
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The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel's proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.
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The differentiation ability of mesenchymal stem cells (MSCs) initially raised interest for treating musculoskeletal injuries in horses, but MSC paracrine activity has widened their scope for inflammatory and immune-mediated pathologies in both equine and human medicine. Furthermore, the similar etiopathogenesis of some diseases in both species has advanced the concept of "One Medicine, One Health". This article reviews the current knowledge on the use of MSCs for equine pathologies beyond the locomotor system, highlighting the value of the horse as translational model. Ophthalmologic and reproductive disorders are among the most studied for MSC application. Equine asthma, equine metabolic syndrome, and endotoxemia have been less explored but offer an interesting scenario for human translation. The use of MSCs in wounds also provides a potential model for humans because of the healing particularities in both species. High-burden equine-specific pathologies such as laminitis have been suggested to benefit from MSC-therapy, and MSC application in challenging disorders such as neurologic conditions has been proposed. The available data are preliminary, however, and require further development to translate results into the clinic. Nevertheless, current evidence indicates a significant potential of equine MSCs to enlarge their range of application, with particular interest in pathologies analogous to human conditions.
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The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, has not been described thus far. We show the presence of telocytes in the equine inter-fascicular tendon matrix near blood vessels. These telocytes have characteristic telopodes, which are composed of alternating dilated portions (podoms) and thin segments (podomers). Additionally, we demonstrate the presence of the primary cilium in telocytes and its ability to release exosomes. The location of telocytes is similar to that of tendon stem cells. The telocyte-blood vessel proximity, the presence of primary immotile cilia and the release of exosomes could have special significance for tendon homeostasis.
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Caballos/anatomía & histología , Telocitos/ultraestructura , Tendones/ultraestructura , Tenocitos/ultraestructura , AnimalesRESUMEN
BACKGROUND: Antibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor's ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular administration of stimulated MSCs (MSC-primed) and unstimulated MSCs (MSC-naïve) in pathologic joints. METHODS: From available stored samples from a previous in vivo study, cells from one donor and serially collected sera (five time-points) from three groups of recipients were used based on their ELA haplotypes to perform microcytotoxicity assays: Group 1 recipients mismatched with the donor that received MSC-naïve (naïve-mismatched recipients); Group 2 recipients mismatched with the donor that received MSC-primed (primed-mismatched recipients); Group 3 recipients halfmatched with the donor (sharing 1/2 haplotypes) that received MSC-primed (primed-halfmatched recipients). Sera from recipients (neat, 1:2 and 1:16 dilution) were tested against target cells from the donor (cryopreserved and expanded MSC-naïve and MSC-primed) or from one animal presenting the same ELA haplotypes than the donor (fresh peripheral blood lymphocytes as control). RESULTS: One to three weeks after first MSC administration, all recipient groups produced allo-antibodies regardless of MSC received (naïve or primed) and matching degree with donor. However, secondary response after MSC re-exposure was less evident in halfmatched recipients (MSC-primed) than in mismatched ones (both MSC-naïve and MSC-primed). Recipients of MSC-primed (both mismatched and halfmatched) tended towards developing lower antibody response than MSC-naïve recipients in vivo, but MSC-primed were targeted to death in higher percentage in vitro in the microcytoxicity assay. CONCLUSIONS: After first intraarticular allogeneic administration, the immunomodulatory profile of MSC-primed would have led to lower antibody production, but these antibodies would target more easily MSC-primed after second injection (re-exposure), likely because of their higher MHC expression.
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Inmunomodulación/inmunología , Leucocitos/inmunología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Caballos , Masculino , OsteoartritisRESUMEN
INTRODUCTION: Patellar tendon overuse injuries are common in athletes. Imaging may show a change in tissue structure with tendon thickening and disruption of the intratendinous substance. We wish to test the hypothesis that both autologous bone marrow expanded mesenchymal stem cells and autologous leukocyte-poor platelet-rich plasma (LP-PRP) implanted into the area of the disrupted tendinopathic patellar tendon will restore function, but tendon regeneration tissue will only be observed in the subjects treated with autologous bone marrow expanded mesenchymal stem cells. METHODS AND ANALYSIS: This is a single-centre, pilot phase I/II, double-blinded clinical trial with randomisation with active control. Twenty patients with a diagnosis of patellar tendinopathy with imaging changes (tendon thickening and disruption of the intratendinous substance at the proximal portion of the patellar tendon) will be randomised in a 1:1 ratio to receive a local injection of either bone-marrow autologous mesenchymal stem cells (MSC), isolated and cultured under GMP at The Institute of Biology and Molecular Genetics (IBGM) (Spain) or P-PRP. The study will have two aims: first, to ascertain whether a clinically relevant improvement after 3, 6 and 12 months according to the visual analogue scale (VAS), Victorian Institute of Sport Assessment for patellar tendons (VISA-P) and dynamometry scales (DYN) will be achieved; and second, to ascertain whether the proposed intervention will restore tendon structure as determined by ultrasonography (US), Doppler ultrasonography (DUS), and innovative MRI and ultrasound techniques: Magnetic Resonance T2 FAT SAT (UTE, Ultrashort Echo TE) sequence and Ultrasound Tissue Characterization (UTC). Patients who are randomised to the P-PRP treatment group but do not achieve a satisfactory primary endpoint after 6 months will be offered treatment with MSC. TRIAL REGISTRATION: NCT03454737.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Ligamento Rotuliano , Tendinopatía/terapia , Adolescente , Adulto , Ensayos Clínicos Fase I como Asunto/métodos , Ensayos Clínicos Fase II como Asunto/métodos , Método Doble Ciego , Humanos , Imagen por Resonancia Magnética , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Persona de Mediana Edad , Dimensión del Dolor/métodos , Ligamento Rotuliano/diagnóstico por imagen , Selección de Paciente , Proyectos Piloto , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Proyectos de Investigación , Tendinopatía/diagnóstico por imagen , Resultado del Tratamiento , Ultrasonografía , Adulto JovenRESUMEN
Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591â¯×â¯103 platelets/µL) or basal PL (BPL; 177â¯×â¯103 platelets/µL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (Pâ¯<â¯0.001) in the MTT assay. Post-cryopreserved viability was similar between cells cultured and preserved in FBS and CPL at all time-points. Gene expression of MSC characterization markers was similar among the three conditions. The gene expression of the immunogenic markers MHC-I, MHC-II and CD40 was slightly (non-significant) increased in CPL condition compared to FBS and BPL. The CPL condition showed higher expression of the genes coding for the immunomodulatory molecules VCAM-1 (non-significant) and IL-6 (Pâ¯<â¯0.05), and similar for COX-2; whereas iNOS and IDO were not expressed under any condition. In conclusion, the replacement of FBS by allogeneic CPL as a supplement for ex vivo propagation of eBM-MSCs provides appropriate proliferation and cryopreservation, and mildly upregulates the gene expression of immunomodulatory markers, thus constituting a potentially suitable alternative to the use of FBS. Further studies are needed to clarify the composition and effects of CPL supplementation on equine MSCs immunological profile.
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Plaquetas/química , Células de la Médula Ósea/citología , Extractos Celulares/química , Medios de Cultivo/química , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/inmunología , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Femenino , Perfilación de la Expresión Génica , Caballos , Masculino , Células Madre Mesenquimatosas/inmunología , TranscriptomaRESUMEN
Domestic species such as cattle (Bos taurus taurus and B. t. indicus) represent attractive biological models to characterize the genetic basis of short-term evolutionary response to climate pressure induced by their post-domestication history. Here, using newly generated dense SNP genotyping data, we assessed the structuring of genetic diversity of 21 autochtonous cattle breeds from the whole Mediterranean basin and performed genome-wide association analyses with covariables discriminating the different Mediterranean climate subtypes. This provided insights into both the demographic and adaptive histories of Mediterranean cattle. In particular, a detailed functional annotation of genes surrounding variants associated with climate variations highlighted several biological functions involved in Mediterranean climate adaptation such as thermotolerance, UV protection, pathogen resistance or metabolism with strong candidate genes identified (e.g., NDUFB3, FBN1, METTL3, LEF1, ANTXR2 and TCF7). Accordingly, our results suggest that main selective pressures affecting cattle in Mediterranean area may have been related to variation in heat and UV exposure, in food resources availability and in exposure to pathogens, such as anthrax bacteria (Bacillus anthracis). Furthermore, the observed contribution of the three main bovine ancestries (indicine, European and African taurine) in these different populations suggested that adaptation to local climate conditions may have either relied on standing genomic variation of taurine origin, or adaptive introgression from indicine origin, depending on the local breed origins. Taken together, our results highlight the genetic uniqueness of local Mediterranean cattle breeds and strongly support conservation of these populations.
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Aclimatación/genética , Variación Genética , Genómica , Animales , Cruzamiento , Bovinos , Mapeo Cromosómico , Clima , Genética de Población , Genoma , Genotipo , Filogenia , Termotolerancia/genéticaRESUMEN
BACKGROUND: This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. RESULTS: Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p < 0.05). Cartilage histopathology did not reveal differences but histochemistry suggested delayed progression of proteoglycan loss in MSC-treated groups. Synovium histopathology indicated decreased inflammation (p < 0.01) in MSC-primed and MSC-naïve at two and six months, respectively. At two months, cartilage from MSC-primed group significantly (p < 0.05) upregulated collagen type II (COL2A1) and transforming growth factor (TGF)-ß1 and downregulated cyclooxygenase-2 and interleukin (IL)-1ß. At six months, MSC-treatments significantly downregulated TNFα (p < 0.05), plus MSC-primed upregulated (p < 0.05) COL2A1, aggrecan, cartilage oligomeric protein, tissue inhibitor of metalloproteinases-2 and TGF-ß1. In synovium, both MSC-treatments decreased (p < 0.01) matrix metalloproteinase-13 expression at two months and MSC-primed also downregulated TNFα (p < 0.05) and IL-1ß (p < 0.01). CONCLUSIONS: Both MSC-treatments provided beneficial effects, mostly observed at short-term. Despite no huge differences between MSC-treatments, the findings suggested enhanced anti-inflammatory and regulatory potential of MSC-primed. While further research is needed to better understand these effects and clarify immunogenicity implications, these findings contribute to enlarge the knowledge about MSC therapeutics and how they could be influenced.
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Enfermedades de los Caballos/terapia , Inflamación/veterinaria , Trasplante de Células Madre Mesenquimatosas , Osteoartritis/veterinaria , Anfotericina B/administración & dosificación , Animales , Enfermedades de los Caballos/inducido químicamente , Caballos , Interferón gamma/farmacología , Masculino , Osteoartritis/inducido químicamente , Osteoartritis/terapia , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Mesenchymal stem cells (MSCs) are a promising treatment for equine musculoskeletal injuries because of their ability to regulate the inflammation and to differentiate into other cell types. Since interest in allogeneic therapy is rising, concerns about MSC immunogenicity need to be addressed. Differentiated MSCs from several species increase their expression of immunogenic molecules and induce alloresponses, but equine MSC immunogenic profile after differentiation has not been reported. Therefore, the aim of this study was to assess the gene expression of immunogenic markers in tri-lineage differentiated equine bone marrow derived MSCs (eBM-MSCs). For this purpose, eBM-MSCs (nâ¯=â¯4) were differentiated into osteoblasts, adipocytes and chondrocytes. Differentiation was confirmed by specific staining and gene expression of lineage-related markers. Subsequently, gene expression of MHC-I, MHC-II, CD40 and CD80 was analyzed in undifferentiated (control) and tri-lineage differentiated eBM-MSCs. Osteogenesis and adipogenesis, but not chondrogenesis, significantly upregulated MHC-I; MHC-II expression significantly increased in the three lineages, while CD40 and CD80 expression did not change. Despite this, MHC-I and MHC-II upregulation after differentiation might lead to increased immunogenicity and risk of allorecognition, either eBM-MSCs differentiate in vivo after administration or they are differentiated prior to administration, with potential negative consequences for effectiveness and safety of allogeneic therapy.
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Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Genes MHC Clase II , Genes MHC Clase I , Células Madre Mesenquimatosas/metabolismo , Adipogénesis , Animales , Antígeno B7-1/metabolismo , Antígenos CD40/metabolismo , Condrogénesis , Expresión Génica/genética , Caballos , Masculino , Osteogénesis , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
A 1-month-old Purebred Spanish Horse (PSH) foal presented with progressive hepatic failure culminating in death. Hepatic lesions were consistent with congenital hepatic fibrosis (CHF). Genetic studies in the PKHD1 gene in the affected foal revealed that it was heterozygous for the 2 previously described single-nucleotide polymorphisms (SNPs) linked to CHF in Swiss Franches-Montagnes (SFM) horses. In addition, 2 novel mutations were detected, the foal being homozygous for one of them and heterozygous for the other. Genetic studies in a healthy PSH population ( n = 35) showed a 3-fold higher genotypic frequency for PKHD1 SNP g.49,630,834G>A and a 5-fold higher genotypic frequency for PKHD1 SNP g.49,597,760A>T compared with those reported for SFM horses. SNPs in the PKHD1 gene in CHF-affected SFM horses might not fully explain the CHF observed in the PSH. Other mutations in the PKHD1 gene could play a more important role in the PSH.
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Enfermedades Genéticas Congénitas/veterinaria , Enfermedades de los Caballos/congénito , Cirrosis Hepática/veterinaria , Receptores de Superficie Celular/metabolismo , Animales , Resultado Fatal , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Genotipo , Enfermedades de los Caballos/genética , Enfermedades de los Caballos/patología , Caballos , Hígado/patología , Cirrosis Hepática/congénito , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular/genéticaRESUMEN
The aim of this Regional Research Communication was to validate a panel of 30 microsatellite markers recommended by FAO/ISAG for studies of biodiversity in cattle to improve the characterisation of Cuban buffalo populations. The water buffalo (Bubalus bubalis) is an economically important livestock species. Therefore, research focused on the study of the genetic relationships among water buffalo populations is useful to support conservation decisions and to design breeding schemes. Twenty-eight of the 30 tested regions were amplified, one of which (ETH10) turned out to be monomorphic. A total of 143 alleles were observed in the Cuban water buffalo population. The average number of alleles per locus was 5·04. The number of alleles per polymorphic locus ranged from two (INRA 63 and MM12) to nine (ETH185). The observed and expected heterozygosity ranged from 0·108 (HAUT24) to 0·851 (CSSM66) and 0·104 (MM12) to 0·829(INRA32), respectively. The polymorphic information content (PIC) ranged from 0·097 (MM12) to 0·806 (INRA32), and the overall value for these markers was 0·482. Within the population, inbreeding estimates (F IS) was positive in 14 of the 30 loci analysed. This study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in Cuban water buffalo breeds. Furthermore, the results can be utilised for future breeding strategies and conservation.
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Cruzamiento/métodos , Búfalos/genética , Repeticiones de Microsatélite/genética , Alelos , Animales , Conservación de los Recursos Naturales , Cuba , ADN/análisis , Variación Genética/genética , Polimorfismo Genético/genéticaRESUMEN
Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy.
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Enfermedades de los Caballos/inmunología , Inflamación/veterinaria , Interferón gamma/metabolismo , Células Madre Mesenquimatosas/citología , Líquido Sinovial/citología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Enfermedades de los Caballos/metabolismo , Caballos , Inflamación/inmunología , Inflamación/metabolismo , Inyecciones Intraarticulares/veterinaria , MasculinoRESUMEN
Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.
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Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , Citocinas/farmacología , Inmunomodulación/efectos de los fármacos , Mediadores de Inflamación/farmacología , Células Madre Mesenquimatosas/citología , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , Regulación de la Expresión Génica/efectos de los fármacos , Caballos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genéticaRESUMEN
Acute phase proteins are useful inflammatory markers in horses. Haptoglobin (Hp) serum level is increased in horses undergoing different inflammatory processes, including arthritis. However, Hp concentration has not been assessed in inflammatory synovial fluid (SF). The aim of the present study was to investigate the Hp response in serum and SF in horses undergoing experimentally induced arthritis. For this purpose, serum and SF samples were collected from 12 animals before amphotericin B-induced arthritis was created (T0, healthy) and 15days after the lesion induction (T1, joint inflammation) and Hp was determined by single radial immunodiffusion. The Hp increase between T0 and T1 was significant in both serum and SF, and serum Hp concentration at T0 was significantly higher than in SF, but significant differences were not found at T1, indicating a higher Hp increase in SF. A significant positive correlation for Hp concentration between serum and SF samples was found. These results highlight the potential usefulness of Hp as inflammatory marker in horses, showing for the first time the increase of Hp in SF from joint inflammation in the horse.
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Artritis/veterinaria , Haptoglobinas/metabolismo , Enfermedades de los Caballos/sangre , Enfermedades de los Caballos/metabolismo , Animales , Artritis/sangre , Artritis/metabolismo , Artritis Experimental/sangre , Artritis Experimental/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Caballos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Masculino , Líquido Sinovial/metabolismo , Factores de TiempoRESUMEN
Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed as in vitro cell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such as CD29 were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential .The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPC was upregulated in MSCs. This characteristic could be useful for developing in vitro models for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and CreutzfeldtJakob disease patients, protein misfolding cyclic amplification did not detect PrPSc in BM- or PB-MSCs from scrapie-infected sheep, which limits their use for in vivo diagnosis for scrapie.
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Células Madre Mesenquimatosas/fisiología , Scrapie/patología , Animales , Diferenciación Celular , Extensiones de la Superficie Celular/genética , Extensiones de la Superficie Celular/metabolismo , Regulación de la Expresión Génica , OvinosRESUMEN
The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-ß1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-ß1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.