Topic:Adipose Tissue
Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype. Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in ...
Minicircle Mediated Gene Delivery to Canine and Equine Mesenchymal Stem Cells. Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA v...
From skeletal muscle to stem cells: an innovative and minimally-invasive process for multiple species. Bone marrow and adipose tissue represent the two most commonly exploited sources of adult mesenchymal stem cells for musculoskeletal applications. Unfortunately the sampling of bone marrow and fat tissue is invasive and does not always lead to a sufficient number of cells. The present study describes a novel sampling method based on microbiopsy of skeletal muscle in man, pigs, dogs and horses. The process includes explant of the sample, Percoll density gradient for isolation and subsequent culture of the cells. We further characterized the cells and identified their clonogenic and immunomodula...
Degenerative Suspensory Ligament Desmitis (DSLD) in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts. Equine degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses typically presents at 7-15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding) we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs) from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05) in cells from DSL...
Evaluation of adipose-derived stromal vascular fraction from the lateral tailhead, inguinal region, and mesentery of horses. Use of mesenchymal stem cells (MSCs) found in the stromal vascular fraction (SVF) of equine adipose tissue has promising applications for regenerative therapies. The most commonly used source of equine adipose tissue is the subcutaneous tailhead. The objective of this study was to compare 3 adipose depot sites in horses and determine the viability and cellular yield, capillary density, gene expression for selected markers, and colony-forming unit fibroblasts (CFU-Fs) in adipose tissue taken from these sites. Adipose tissue was excised from the area lateral to the tailhead, the inguinal region,...
Allogeneic Mesenchymal Stem Cell Treatment Induces Specific Alloantibodies in Horses. Background. It is unknown whether horses that receive allogeneic mesenchymal stem cells (MSCs) injections develop specific humoral immune response. Our goal was to develop and validate a flow cytometric MSC crossmatch procedure and to determine if horses that received allogeneic MSCs in a clinical setting developed measurable antibodies following MSC administration. Methods. Serum was collected from a total of 19 horses enrolled in 3 different research projects. Horses in the 3 studies all received unmatched allogeneic MSCs. Bone marrow (BM) or adipose tissue derived MSCs (ad-MSCs) were admini...
Assessment of regeneration in meniscal lesions by use of mesenchymal stem cells derived from equine bone marrow and adipose tissue. OBJECTIVE To assess the ability to regenerate an equine meniscus by use of a collagen repair patch (scaffold) seeded with mesenchymal stem cells (MSCs) derived from bone marrow (BM) or adipose tissue (AT). SAMPLE 6 female Hispano-Breton horses between 4 and 7 years of age; MSCs from BM and AT were obtained for the in vitro experiment, and the horses were subsequently used for the in vivo experiment. PROCEDURES Similarities and differences between MSCs derived from BM or AT were investigated in vitro by use of cell culture. In vivo assessment involved use of a meniscus defect and implantation o...
Tracking of autologous adipose tissue-derived mesenchymal stromal cells with in vivo magnetic resonance imaging and histology after intralesional treatment of artificial equine tendon lesions–a pilot study. Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine tendinopathies. Up to now, knowledge about the fate of autologous AT-MSCs after intralesional injection into equine superficial digital flexor tendons (SDFTs) is very limited. The purpose of this study was to monitor the presence of intralesionally injected autologous AT-MSCs labelled with superparamagnetic iron oxide (SPIO) nanoparticles and green fluorescent protein (GFP) over a staggered period of 3 to 9 weeks with standing magnetic resonance imaging (MRI) and histology. Methods: Four adult warmbl...
Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues. Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. Objective: The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horse...
GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells. Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on exp...
The effect of metamizole and tolfenamic acid on canine and equine adipose-derived mesenchymal stem cells (ASCs) an in vitro research. The influences of NSAIDs (Nonsteroidal Anti-inflammatory Drugs)--non-selective metamizole and selectively-acting tolfenamic acid were estimated on morphology, ultrastructure, and cytophysiological activity of canine (Ca) and equine (Eq) adipose-derived mesenchymal stem cells (ASCs). The lowest concentration of metamizole (0.01 mg/mL) stimulated the viability and cytophysiological activity of Ca ASCs and did not affect cell morphology. Stimulated cells possessed a proper, fibroblastic shape, with large, eccentrically located nuclei. Similar effects to those observed in Ca ASCs were found in Eq ...
Carotenoids and fat-soluble vitamins in horse tissues: a comparison with cattle. Carotenoids are important for human health because of their provitamin A function among other biological actions. Their implication on consumer point of view of cattle products have been widely studied, but very little information is available for horse products. The aim of this study was to study the accumulation of carotenoids, retinoids and tocopherol by HPLC and HPLC-MS analysis in different horse tissues (plasma, milk, adipose tissue and liver) and compare it with that of cattle. Fat color was also studied. Four groups of animals were studied (15 animals within each group): lactating mare...
Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study. Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprisi...
Application of a novel sorting system for equine mesenchymal stem cells (MSCs). The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Al...
Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential. Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods: The BM-MS...
Expression of inflammation-related genes is associated with adipose tissue location in horses. In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine A...
Family of melanocortin receptor (MCR) genes in mammals-mutations, polymorphisms and phenotypic effects. The melanocortin receptor gene family consists of five single-exon members, which are located on autosomes. Three genes (MC2R, MC4R and MC5R) are syntenic in the human, mouse, cattle and dog genomes, while in the pig, the syntenic group comprises MC1R, MC2R and MC5R. Two genes (MC1R and MC4R) have been extensively studied due to their function in melanogenesis (MC1R) and energy control (MC4R). Conservative organisation of these genes in five mammalian species (human, mouse, cattle, pig and dog), in terms of the encoded amino acid sequence, is higher in the case of MC4R compared to MC1R. Polymo...
Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells. To characterize equine muscle tissue- and periosteal tissue-derived cells as mesenchymal stem cells (MSCs) and assess their proliferation capacity and osteogenic potential in comparison with bone marrow- and adipose tissue-derived MSCs. Methods: Tissues from 10 equine cadavers. Methods: Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and adipose tissue from the left subcutaneous region. Mesenchymal stem cells were characterized on the basis of morphology, ad...
Morphometric analyses of the body and the branches of the normal third interosseous muscle (suspensory ligament) in Standardbreds. The third interosseous muscle (suspensory ligament, TIOM) is composed of connective tissue (CT) with a variable proportion of muscle (MT) and adipose tissue (AT). The aim of our study is to quantify the CT, MT and AT within the body and the branches of right thoracic and pelvic limbs TIOM in sound horses to determine whether there are differences in CT, MT and AT between age, sex, limbs and levels. Right limbs from 11 sound horses were collected. Samples from 6 levels of the TIOM were embedded in paraffin or in Tissue-Tek(®) . Most of the paraffin sections were shredded. Using the cryosection...
Follicular fluid leptin concentrations and expression of leptin and leptin receptor in the equine ovary and in vitro-matured oocyte with reference to pubertal development and breeds. There is no published information about follicular-fluid leptin concentrations or the presence of leptin and leptin receptor in the equine ovary or oocyte. Three groups of mares - adult draft mares, draft fillies and adult Standardbred mares - were included in the study. Leptin and leptin receptor were detected in all immature oocytes by immunofluorescence with higher intensity in oocytes from draft mares compared with draft fillies and Standardbred mares. After in vitro maturation a higher proportion of oocytes reached metaphase II in draft mares than in draft fillies and Standardbred mares, ...
Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. Results: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxi...
Novel link between inflammation and impaired glucose transport during equine insulin resistance. Although insulin resistance (IR) has been increasingly recognized in horses, a clear understanding of its pathophysiology is lacking. The purpose of the present study was to determine the early pathologic changes in IR horses by characterizing alterations in proteins that play key roles in innate immunological responses and inflammatory pathways, and by identifying potential links with glucose transport and insulin signaling. Visceral (VIS) and subcutaneous (SC) adipose tissue and skeletal muscle (SM) biopsies were collected from horses, which were classified as insulin-sensitive (IS) or IR ba...
An explant based-method for differentiating adipocytes from equine adipose tissue. Culturing adipocytes enables fine control of experimental conditions and helps minimise animal use. This report describes an explant-based method for isolating stromal-vascular cells from equine adipose tissue that enables use of small amounts of tissue. Subcutaneous and mesenteric adipose tissues were harvested post mortem and stromal-vascular cells grown from explants, prior to testing the capacity of several differentiation media to induce lipid droplet formation and increase transcript abundance of adipocyte markers. Inclusion of rosiglitazone at 1 and 5 µmol/l concentrations, along with ...
Effects of the insulin sensitizing drug, pioglitazone, and lipopolysaccharide administration on markers of systemic inflammation and clinical parameters in horses. Equine metabolic syndrome (EMS) is a condition of obese horses characterized by insulin resistance, systemic inflammation, and an increased risk of laminitis. The pathogenesis of EMS is thought, in part, to be due to inflammatory proteins produced by adipose tissue. Reducing inflammation may decrease the incidence of laminitis in horses with EMS. Pioglitazone hydrochloride, a thiazolidinedione, has efficacy to reduce obesity associated inflammation in humans. Eight normal, adult, horses were administered 1mg/kg pioglitazone for 14 days, and eight horses served as controls. Physical examination...
Collection and propagation methods for mesenchymal stromal cells. Mesenchymal stromal cells (MSC) are derived from adult mesenchymal tissues and have the ability to undergo differentiation into bone, cartilage, and fat, and have therefore attracted great interest in regenerative medicine. Many isolation and culture methods have been described, making comparison between laboratories and quality-control protocols difficult. A uniform protocol to characterize equine MSC has recently been proposed, aiming to introduce consistency across the equine stem cell research field. This article reviews the published techniques for collection and propagation of equine MSC...
Adipose tissue-derived adiponectin expression is significantly associated with increased post operative mortality in horses undergoing emergency abdominal surgery. Adipose tissue is an important source of inflammatory cytokines (adipokines) and adiposity has been identified as having a significant effect on human morbidity and mortality. Obesity is also an emerging welfare problem in the UK horse population, but the role that it plays in secondary diseases is unclear. Objective: To examine the expression of inflammation-related adipokine genes in retroperitoneal adipose tissue of horses undergoing emergency abdominal surgery and to explore associations with adiposity and post operative survival. Methods: Retroperitoneal adipose tissue samples were obtain...
Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. 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 ...
Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells. Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic linea...
Evaluation of senescence in mesenchymal stem cells isolated from equine bone marrow, adipose tissue, and umbilical cord tissue. 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 prolife...
Effects of hyperinsulinemia on glucose and lipid transporter expression in insulin-sensitive horses. Plasma insulin concentrations are elevated (hyperinsulinemia) in horses with obesity-associated insulin resistance. In other species, insulin resistance is partly due to reduced levels of insulin receptor and the insulin-sensitive glucose transporter, and, in vitro, chronic hyperinsulinemic conditions reduce the expression of these proteins. Consumption of grain-based concentrate feeds results in postprandial hyperinsulinemia in horses, and adaptation to these diets is associated with insulin resistance. As such, it is possible that the repeated, chronic postprandial hyperinsulinemia associate...