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Dori L. Borjesson - One of the best experts on this subject based on the ideXlab platform.
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Equine Bone marrow volume reduction, red blood cell depletion, and mononuclear cell recovery using the PrepaCyte-CB processing system
Veterinary Clinical Pathology, 2015Co-Authors: Andrew G. Burton, Julie Burges, Danielle D. Carrade, Dori L. Borjesson, Kaitlin C. Clark, Sean D. OwensAbstract:Background Volume reduction and RBC depletion of Equine Bone marrow specimens are necessary processing steps for the immediate therapeutic use of Bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. Objectives The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from Equine BM specimens. Methods One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduction, and total nucleated cell (TNC) and MNC recoveries were determined. Results Bone marrow volume was reduced from 120 mL to 21 mL with a median RBC depletion of 90.1% (range, 62.0–96.7%). The median preprocessing total TNC count was 2.2 × 109 (range, 0.46–7.9 × 109) and the median postprocessing TNC count was 1.7 × 109 (range, 0.3–4.4 × 109; P
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evaluation of senescence in mesenchymal stem cells isolated from Equine Bone marrow adipose tissue and umbilical cord tissue
Stem Cells and Development, 2012Co-Authors: Martin A Vidal, Naomi J Walker, Eleonora Napoli, Dori L. BorjessonAbstract:Mesenchymal stem cells (MSCs) from adult and neonatal tissues are intensively investigated for their use in regenerative medicine. The purpose of this study was to compare the onset of replicative senescence in MSCs isolated from Equine Bone marrow (BMSC), adipose tissue (ASC), and umbilical cord tissue (UCMSC). MSC proliferation (cell doubling), senescence-associated β-galactosidase staining, telomere length, Sox-2, and lineage-specific marker expression were assessed for MSCs harvested from tissues of 4 different donors. The results show that before senescence ensued, all cell types proliferated at ∼1 day/cell doubling. BMSCs significantly increased population doubling rate by passage 10 and ceased proliferation after a little >30 total population doublings, whereas UCMSCs and ASCs achieved about 60 to 80 total population doublings. UCMSC and ASCs showed marked β-galactosidase staining after ∼70 population doublings, whereas BMSCs stained positive by ∼30 population doublings. The onset of senescence was...
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Processing of Equine Bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery.
Veterinary Clinical Pathology, 2011Co-Authors: Sean D. Owens, Julie Burges, Jennifer L. Johns, Danielle D. Carrade, Larry D. Galuppo, Fred Librach, Dori L. BorjessonAbstract:Background The therapeutic use of Bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in Equine patients is expanding. After collection, Bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human Bone marrow samples. Objectives The purpose of this study was to evaluate the capacity of the MXP System to process Equine Bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. Methods Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. Results For Equine Bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 107 and after processing was significantly higher at 7.8 ± 3.3 × 107 (P
B Ranera - One of the best experts on this subject based on the ideXlab platform.
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Primary Cilia in Chondrogenic Differentiation of Equine Bone Marrow Mesenchymal Stem Cells: Ultrastructural Study
Journal of Equine Veterinary Science, 2016Co-Authors: María José Luesma, Ana Rosa Remacha, Antonio Romero, Clementina Rodellar, B Ranera, Irene Cantarero, Tomás Castiella, Inmaculada Martín, C. JunqueraAbstract:Locomotor system disorders in Equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and phenotyping. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from Bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes.
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effect of inflammatory environment on Equine Bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties
Veterinary Immunology and Immunopathology, 2016Co-Authors: Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Pilar Zaragoza, Francisco José Vázquez, B Ranera, Jorge Albareda, M Prades, Inmaculada MartinburrielAbstract:Mesenchymal stem cells (MSCs) are being investigated for the treatment of Equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of Equine Bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for Equine joint diseases.
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comparative study of Equine Bone marrow and adipose tissue derived mesenchymal stromal cells
Equine Veterinary Journal, 2012Co-Authors: B Ranera, Ana Rosa Remacha, Antonio Romero, Francisco José Vázquez, Jaber Lyahyai, M L Bernal, L Ordovas, F Fernandes, Rosario Osta, C ConsAbstract:REASONS FOR PERFORMING STUDY: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. OBJECTIVES: To compare and better understand the behaviour of Equine MSCs obtained from Bone marrow (BM) and adipose tissue (AT) in culture. METHODS: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. RESULTS: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. CONCLUSIONS: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. POTENTIAL RELEVANCE: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.
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immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from Equine Bone marrow and adipose tissue
Veterinary Immunology and Immunopathology, 2011Co-Authors: B Ranera, Ana Rosa Remacha, Antonio Romero, Pilar Zaragoza, Francisco José Vázquez, Clementina Rodellar, Jaber Lyahyai, M L Bernal, Inmaculada MartinburrielAbstract:Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in Equine Bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by reverse transcriptase real-time quantitative PCR along with the expression of 5 other MSC markers. Both populations of cells expressed CD13, CD29, CD44, CD49d, CD73, CD90, CD105, CD106, CD146 and CD166 transcripts. Significant differences in gene expression levels between BM- and AT-MSCs were observed for CD44, CD90, CD29 and CD34. Both cell types were negative for CD45 and CD31. The surface antigens tested revealed a similar phenotypic profile between horse and human MSCs, although specific differences in some surface antigens were noticed.
Naoki Sasaki - One of the best experts on this subject based on the ideXlab platform.
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enhanced chondrogenic differentiation of Equine Bone marrow derived mesenchymal stem cells in zirconia microwell substrata
Research in Veterinary Science, 2019Co-Authors: Tomohiro Inui, Shingo Haneda, Hidefumi Furuoka, Yasuhiko Tabata, Motoki Sasaki, Masashi Yanagawa, Masato Hiyama, Naoki SasakiAbstract:Abstract In human cartilage tissue engineering, three-dimensional zirconia substrata have the potential advantage of producing many uniform cell clusters of controlled size without xenobiotic material, allowing easy clinical application. The objective of this study was to evaluate the possibility of using zirconia porous three-dimensional microwell substrata for chondrogenic differentiation of Equine Bone marrow-derived mesenchymal stem cells (BMMSCs) in vitro. In regular medium, 8 × 105, 2 × 106, and 5 × 106 Equine BMMSCs from five thoroughbred horses were cultured on zirconia microwell substrata for 4 days to allow formation of clusters. The medium was replaced by chondrogenic culture medium. After chondrogenic culture for 7, 14 and 21 days, analysis of collagen type II alpha 1 gene (COL2A1) gene expression and observation of chondrogenic aggregates by scanning electron microscopy (SEM) were performed. SEM showed size-controlled cell clusters and increasing extracellular matrix over time when using 5 × 106 cells. The expression of COL2A1 on day 7 and 14 with 5 × 106 cells was significantly higher than that of conventional pellet culture with 2 × 106 cells. Histological evaluation by immunohistochemical staining for type II collagen (ColII) was performed after chondrogenic culture for 7 days. The clusters showed wide distribution of ColII. The results suggest that the zirconia substrata have the potential to enhance the chondrogenic differentiation of Equine BMMSCs, allowing effective Equine cartilage tissue engineering without xenobiotic materials.
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Effect of Biodegradable Gelatin β-Tri Calcium Phosphate Sponges Containing Mesenchymal Stem Cells and Bone Morphogenetic Protein-2 on Equine Bone Defect
Journal of Equine Veterinary Science, 2014Co-Authors: Nao Tsuzuki, Kazutaka Yamada, Shingo Haneda, Hidefumi Furuoka, Yasuhiko Tabata, Shougo Nakao, Naoki SasakiAbstract:Abstract This study evaluated the osteoanagenetic effects of administering biodegradable gelatin β-tri calcium phosphate sponges containing mesenchymal stem cells (MSCs) and Bone morphogenetic protein-2 (BMP-2) on Equine Bone defect. Six healthy Thoroughbred horses were used in this study. Horses were anesthetized, and skin incisions were made on all the limbs. Splint Bones were exposed and a 1-cm Bone defect was created in each exposed Bone. Gelatin β-tri calcium phosphate sponges containing MSC and BMP-2 (MSC + BMP-2 sponge), MSC only (MSC sponge), BMP-2 only (BMP-2 sponge), or saline (saline sponge) were implanted into each Bone defect at random. Defects were monitored for 16 weeks by radiography followed by computed tomography (CT) and histologic analyses. At 16 weeks, radiographic scores of MSC + BMP-2 sponge-treated defects were significantly higher than those of saline-treated defects ( P = .027). Moreover, the CT value of the MSC + BMP-2 sponge group was significantly higher than that of the other groups ( P = .027; P = .046; and P = .027, respectively), and the histologic score of the MSC + BMP-2 sponge group was significantly greater than that of the saline sponge group ( P = .041). We conclude that MSC + BMP-2 sponge administration to Bone defects accelerates Bone regeneration in Equines.
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Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and Bone morphogenetic protein-2 in an Equine Bone defect model
Veterinary Research Communications, 2014Co-Authors: Nao Tsuzuki, Kazutaka Yamada, Shingo Haneda, Hidefumi Furuoka, Yasuhiko Tabata, Naoki SasakiAbstract:Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and Bone morphogenetic protein-2 (BMP-2) in an Equine Bone defect model. Seven thoroughbred horses were used in this study. Eight Bone defects were created in the third metatarsal Bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2 × 10^5 MSCs; M-6: 2 × 10^6 MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of Bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact Bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted Bone regeneration in an Equine Bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with Bone injuries.
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osteoinductivity of gelatin β tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and Bone morphogenetic protein 2 in an Equine Bone defect model
Veterinary Research Communications, 2014Co-Authors: Nao Tsuzuki, Kazutaka Yamada, Shingo Haneda, Hidefumi Furuoka, Yasuhiko Tabata, Naoki SasakiAbstract:Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and Bone morphogenetic protein-2 (BMP-2) in an Equine Bone defect model. Seven thoroughbred horses were used in this study. Eight Bone defects were created in the third metatarsal Bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2 × 105 MSCs; M-6: 2 × 106 MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of Bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact Bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted Bone regeneration in an Equine Bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with Bone injuries.
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comparison of allogeneic platelet lysate and fetal bovine serum for in vitro expansion of Equine Bone marrow derived mesenchymal stem cells
Research in Veterinary Science, 2013Co-Authors: Nao Tsuzuki, Kazutaka Yamada, Shingo Haneda, Hidefumi Furuoka, Yasuhiko Tabata, Naoki SasakiAbstract:Abstract Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause Equine Bone marrow-derived MSC expansion. MSCs were isolated from Bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences in MSC yield, colony-forming unit-fibroblast (CFU-F) assay, and population doubling time between PL and FBS cultures. In addition, both PL-MSCs and FBS-MSCs showed similar results in term of ALP staining, osteogenic differentiation, and RT-PCR, although there were subtle differences in morphology, growth pattern, and adhesive properties. These results suggest that PL is a suitable alternative to FBS for use in Equine MSC expansion, without the problems related to FBS use.
Antonio Romero - One of the best experts on this subject based on the ideXlab platform.
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Effect of allogeneic platelet lysate on Equine Bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.
Veterinary Immunology and Immunopathology, 2019Co-Authors: Kevin Yaneselli, Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Francisco José Vázquez, Agustina Algorta, Arantza Vitoria, Alina Cequier, Jacqueline Maisonnave, Clementina RodellarAbstract:Abstract 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; 591 × 103 platelets/μL) or basal PL (BPL; 177 × 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 (P
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Differentiation of Equine Bone marrow derived mesenchymal stem cells increases the expression of immunogenic genes.
Veterinary Immunology and Immunopathology, 2018Co-Authors: Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Pilar Zaragoza, Francisco José Vázquez, Clementina RodellarAbstract:Abstract 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 (n = 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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Primary Cilia in Chondrogenic Differentiation of Equine Bone Marrow Mesenchymal Stem Cells: Ultrastructural Study
Journal of Equine Veterinary Science, 2016Co-Authors: María José Luesma, Ana Rosa Remacha, Antonio Romero, Clementina Rodellar, B Ranera, Irene Cantarero, Tomás Castiella, Inmaculada Martín, C. JunqueraAbstract:Locomotor system disorders in Equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and phenotyping. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from Bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes.
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effect of inflammatory environment on Equine Bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties
Veterinary Immunology and Immunopathology, 2016Co-Authors: Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Pilar Zaragoza, Francisco José Vázquez, B Ranera, Jorge Albareda, M Prades, Inmaculada MartinburrielAbstract:Mesenchymal stem cells (MSCs) are being investigated for the treatment of Equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of Equine Bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for Equine joint diseases.
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comparative study of Equine Bone marrow and adipose tissue derived mesenchymal stromal cells
Equine Veterinary Journal, 2012Co-Authors: B Ranera, Ana Rosa Remacha, Antonio Romero, Francisco José Vázquez, Jaber Lyahyai, M L Bernal, L Ordovas, F Fernandes, Rosario Osta, C ConsAbstract:REASONS FOR PERFORMING STUDY: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. OBJECTIVES: To compare and better understand the behaviour of Equine MSCs obtained from Bone marrow (BM) and adipose tissue (AT) in culture. METHODS: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. RESULTS: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. CONCLUSIONS: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. POTENTIAL RELEVANCE: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.
Ana Rosa Remacha - One of the best experts on this subject based on the ideXlab platform.
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Effect of allogeneic platelet lysate on Equine Bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.
Veterinary Immunology and Immunopathology, 2019Co-Authors: Kevin Yaneselli, Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Francisco José Vázquez, Agustina Algorta, Arantza Vitoria, Alina Cequier, Jacqueline Maisonnave, Clementina RodellarAbstract:Abstract 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; 591 × 103 platelets/μL) or basal PL (BPL; 177 × 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 (P
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Differentiation of Equine Bone marrow derived mesenchymal stem cells increases the expression of immunogenic genes.
Veterinary Immunology and Immunopathology, 2018Co-Authors: Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Pilar Zaragoza, Francisco José Vázquez, Clementina RodellarAbstract:Abstract 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 (n = 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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Primary Cilia in Chondrogenic Differentiation of Equine Bone Marrow Mesenchymal Stem Cells: Ultrastructural Study
Journal of Equine Veterinary Science, 2016Co-Authors: María José Luesma, Ana Rosa Remacha, Antonio Romero, Clementina Rodellar, B Ranera, Irene Cantarero, Tomás Castiella, Inmaculada Martín, C. JunqueraAbstract:Locomotor system disorders in Equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and phenotyping. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from Bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes.
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effect of inflammatory environment on Equine Bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties
Veterinary Immunology and Immunopathology, 2016Co-Authors: Laura Barrachina, Ana Rosa Remacha, Antonio Romero, Pilar Zaragoza, Francisco José Vázquez, B Ranera, Jorge Albareda, M Prades, Inmaculada MartinburrielAbstract:Mesenchymal stem cells (MSCs) are being investigated for the treatment of Equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of Equine Bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for Equine joint diseases.
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comparative study of Equine Bone marrow and adipose tissue derived mesenchymal stromal cells
Equine Veterinary Journal, 2012Co-Authors: B Ranera, Ana Rosa Remacha, Antonio Romero, Francisco José Vázquez, Jaber Lyahyai, M L Bernal, L Ordovas, F Fernandes, Rosario Osta, C ConsAbstract:REASONS FOR PERFORMING STUDY: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. OBJECTIVES: To compare and better understand the behaviour of Equine MSCs obtained from Bone marrow (BM) and adipose tissue (AT) in culture. METHODS: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. RESULTS: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. CONCLUSIONS: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. POTENTIAL RELEVANCE: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.