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Stem cell therapy in cat patients needs a high quantity of mesenchymal stromal/stem cells (MSCs) requiring in vitro propagation under culture conditions which may potentially impact cellular characteristics and genetic stability. This study aimed to assess the in vitro characteristics and cytogenetic stability of cat adipose tissue-derived MSCs (cAT-MSCs). For this purpose, morphological features, clonogenic potential, and proliferative capacity of cAT-MSCs were assessed at passages 2 (P2), P4, and P6. Multipotency and immunophenotype were evaluated. Cytogenetic analyses were conducted up to P6. The cAT-MSCs exhibited a spindle-shaped morphology in early passages. The doubling time increased from 2.5 days at P2 to 9.4 at P4 and 10.5 at P6, accompanied by the observation of nuclear abnormalities such as cluster formation, karyorrhexis, karyolysis, and a decline in the mitotic index at P4. Cells demonstrated multipotency capacity and were CD45-, CD90+, and CD44+. Metaphase analysis at P2 and P4 revealed some indications of structural instability such as gaps, breaks, deletions, duplications, and early chromatid segregation, but these alterations did not show an increase across passages. In conclusion, cAT-MSCs decreased their proliferative capacity after P4, accompanied by morphological alterations and signs of structural instability.
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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
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
Mesenchymal stem cells (MSCs) have desirable characteristics for use in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. The objective of this study was to evaluate the differences between canine adipose tissue-derived MSCs (AD-MSCs) extracted from subcutaneous (Sc) and visceral (Vs) sites. Surface antigenic markers, in vitro differentiation, and mineralized matrix quantification of AD-MSCs at different passages (P4, P6, and P8) were studied. Immunophenotypic analysis showed that AD-MSCs from both sites were CD44+, CD90+, and CD45-. Moreover, they were able, in vitro, to differentiate into fat, cartilage, and bone. Sc-AD-MSCs preserve in vitro multipotentiality up to P8, but Vs-AD-MSCs only tri-differentiated up to P4. In addition, compared to Vs-AD-MSCs, Sc-AD-MSCs had greater capacity for in vitro mineralized matrix synthesis. In conclusion, Sc-AD-MSCs have advantages over Vs-AD-MSCs, as Sc AD-MSCs preserve multipotentiality during a greater number of passages, have more osteogenic potential, and require less invasive extraction.
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Diferenciación Celular , Grasa Intraabdominal/citología , Células Madre Mesenquimatosas/citología , Grasa Subcutánea/citología , Animales , Perros , Femenino , Inmunofenotipificación/veterinariaRESUMEN
Bovine herpesviruses (BoHVs) types 1 (BoHV-1) and 5 (BoHV-5) are alphaherpesviruses of major importance to the bovine production chain. Such viruses are capable of establishing latent infections in neuronal tissues. Infected animals tend to develop a serological response to infection; however, such response-usually investigated by antibody assays in serum-may eventually not be detected in laboratory assays. Nevertheless, serological tests such as virus neutralization (VN) and various enzyme-linked immunosorbent assays (ELISAs) are widely employed to check individual or herd status of BoHV infections. The correlation between detection of antibodies and the presence of viral nucleic acids as indicatives of infection in infected cattle has not been deeply examined. In order to investigate such correlation, 248 bovine serum samples were tested by VN to BoHV-1 and BoHV-5, as well as in a widely employed (though not type-differential) gB ELISA (IDEXX IBR gB X2 Ab Test) in search for antibodies to BoHVs. Immediately after blood withdrawal, cattle were slaughtered and trigeminal ganglia (TG) excised for DNA extraction and viral nucleic acid detection (NAD) by nested PCR. Neutralizing antibodies to BoHV-1 and/or BoHV-5 were detected in 44.8% (111/248) of sera, whereas the gB ELISA detected antibodies in 51.2% (127/248) of the samples. However, genomes of either BoHV-1, BoHV-5, or both, were detected in TGs of 85.9% (213/248) of the animals. These findings reveal that the assays designed to detect antibodies to BoHV-1 and/or BoHV-5 employed here may fail to detect a significant number of latently infected animals (in this study, 35.7%). From such data, it is clear that antibody assays are poorly correlated with detection of viral genomes in BoHV-1 and BoHV-5-infected animals.