KLB dysregulation mediates disrupted muscle development in intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified ß-Klotho (KLB), a fibroblast growth factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co-receptor, ß-Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.

Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96-182 cluster miRNAs in endothelial cells.

Our previous studies showed that the miRNA clusters, miR-183-96-182 and miR-212-132, may be critical in promoting luteal cell survival and progesterone production in both bovine and humans. To further understand their involvement in luteal development, this study aimed to establish the expression of these miRNAs in different bovine luteal cell types, namely, endothelial and steroidogenic, isolated using fluorescence-activated cell sorting (FACS). We isolated each of the two cell populations based on the presence of the endothelia surface marker, CD144, and uptake of the lipophilic dye, Nile Red, respectively. Using quantitative Polymerase Chain Reaction (qPCR) in the sorted cell fractions we confirmed that CD144 and the endothelia-specific miRNA, miR-126, were predominantly expressed in endothelial cells (CD144+), whereas HSD3B1 was expressed predominantly in steroidogenic cells (Nile RedHI). Finally, we found that whereas the miR-212-132 cluster was expressed at similar levels in luteal endothelial and steroidogenic cells, miR-183-96-182 was expressed at > 4-fold higher levels in endothelial than in steroidogenic cells (P < 0.05), suggesting that these two miRNA clusters, and particularly miR-183-96-182, may be important in functionally regulating not only steroidogenic cells but also endothelial cells in the corpus luteum (CL).

Pericytes in Veterinary Species: Prospective Isolation, Characterization and Tissue Regeneration Potential.

Although pericytes have long been known for their roles in blood vessel regulation, it was not until a decade ago that their tissue regeneration potential began to be considered, after studies showed that pericytes were the in vivo counterparts of mesenchymal stem/stromal cells (MSCs). The prospective isolation and culture expansion of pericytes brought great excitement as it opened the way to the therapeutic use of well-defined cell populations with known regenerative potential to overcome concerns associated with the use of traditional MSC preparations. Studies first in humans and later in the horse and other domestic species showed that indeed cultured pericytes had key characteristics of MSCs, namely, their immunophenotype and the abilities to grow clonally and to differentiate into mature mesenchymal cells both in vitro and vivo. Several studies with human pericytes, and to a much lesser extent with animal pericytes, have also shown significant promise in tissue repair in different disease models. This review summarizes current knowledge on the tissue regeneration properties of pericytes from domestic animals and outlines future steps necessary for realizing their full potential both in clinical veterinary medicine and in preclinical testing of human therapies using large animal models, including the need for robust approaches for isolation, culture and appropriate in vivo testing of the tissue regenerative properties of pericytes in these species.

Transcriptome profiling of granulosa and theca cells during dominant follicle development in the horse.

Several aspects of equine ovarian physiology are unique among domestic species. Moreover, follicular growth patterns are very similar between horses and humans. This study aimed to characterize, for the first time, global gene expression profiles associated with growth and preovulatory (PO) maturation of equine dominant follicles. Granulosa cells (GCs) and theca interna cells (TCs) were harvested from follicles (n = 5) at different stages of an ovulatory wave in mares corresponding to early dominance (ED; diameter ≥22 mm), late dominance (LD; ≥33 mm) and PO stage (34 h after administration of crude equine gonadotropins at LD stage), and separately analyzed on a horse gene expression microarray, followed by validation using quantitative PCR and immunoblotting/immunohistochemistry. Numbers of differentially expressed transcripts (DETs; ≥2-fold; P < 0.05) during the ED-LD and LD-PO transitions were 546 and 2419 in GCs and 5 and 582 in TCs. The most prominent change in GCs was the down-regulation of transcripts associated with cell division during both ED-LD and LD-PO. In addition, DET sets during LD-PO in GCs were enriched for genes involved in cell communication/adhesion, antioxidation/detoxification, immunity/inflammation, and cholesterol biosynthesis. In contrast, the largest change in TCs during the LD-PO transition was an up-regulation of genes involved in immune activation, with other DET sets mapping to GPCR/cAMP signaling, lipid/amino acid metabolism, and cell proliferation/survival and differentiation. In conclusion, distinct expression profiles were identified between growing and PO follicles and, particularly, between GCs and TCs within each stage. Several DETs were identified that have not been associated with follicle development in other species.

Derivation and long-term maintenance of porcine skeletal muscle progenitor cells.

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat.

Characterization of canine adipose- and endometrium-derived Mesenchymal Stem/Stromal Cells and response to lipopolysaccharide.

Mesenchymal stem/stromal cells (MSCs) are used for regenerative therapy in companion animals. Their potential was initially attributed to multipotency, but subsequent studies in rodents, humans and veterinary species evidenced that MSCs produce factors that are key mediators of immune, anti-infective and angiogenic responses, which are essential in tissue repair. MSCs preparations have been classically obtained from bone marrow and adipose tissue (AT) in live animals, what requires the use of surgical procedures. In contrast, the uterus, which is naturally exposed to external insult and infection, can be accessed nonsurgically to obtain samples, or tissues can be taken after neutering. In this study, we explored the endometrium (EM) as an alternative source of MSCs, which we compared with AT obtained from canine paired samples. Canine AT- and EM-MSCs, formed CFUs when seeded at low density, underwent tri-lineage differentiation into adipocytes, osteocytes and chondrocytes, and expressed the CD markers CD73, CD90 and CD105, at equivalent levels. The immune genes IL8, CCL2 and CCL5 were equally expressed at basal levels by both cell types. However, in the presence of the inflammatory stimulus lipopolysaccharide (LPS), expression of IL8 was higher in EM- than in AT-MSCs (p < 0.04) while the other genes were equally elevated in both cell types (p < 0.03). This contrasted with the results for CD markers, where the expression was unaltered by exposing the MSCs to LPS. Overall, the results indicate that canine EM-MSCs could serve as an alternative cell source to AT-MSCs in therapeutic applications.

Differentiation Potential of Mesenchymal Stem/Stromal Cells Is Altered by Intrauterine Growth Restriction.

Consistency in clinical outcomes is key to the success of therapeutic Mesenchymal Stem/Stromal cells (MSCs) in regenerative medicine. MSCs are used to treat both humans and companion animals (horses, dogs, and cats). The properties of MSC preparations can vary significantly with factors including tissue of origin, donor age or health status. We studied the effects of developmental programming associated with intrauterine growth restriction (IUGR) on MSC properties, particularly related to multipotency. IUGR results from inadequate uterine capacity and placental insufficiency of multifactorial origin. Both companion animals (horses, dogs, cats) and livestock (pigs, sheep, cattle) can be affected by IUGR resulting in decreased body size and other associated changes that can include, alterations in musculoskeletal development and composition, and increased adiposity. Therefore, we hypothesized that this dysregulation occurs at the level of MSCs, with the cells from IUGR animals being more prone to differentiate into adipocytes and less to other lineages such as chondrocytes and osteocytes compared to those obtained from normal animals. IUGR has consequences on health and performance in adult life and in the case of farm animals, on meat quality. In humans, IUGR is linked to increased risk of metabolic (type 2 diabetes) and other diseases (cardiovascular), later in life. Here, we studied porcine MSCs where IUGR occurs spontaneously, and shows features that recapitulate human IUGR. We compared the properties of adipose-derived MSCs from IUGR (IUGR-MSCs) and Normal (Normal-MSCs) new-born pig littermates. Both MSC types grew clonally and expressed typical MSC markers (CD105, CD90, CD44) at similar levels. Importantly, tri-lineage differentiation capacity was significantly altered by IUGR. IUGR-MSCs had higher adipogenic capacity than Normal-MSCs as evidenced by higher adipocyte content and expression of the adipogenic transcripts, PPARγ and FABP4 (P < 0.05). A similar trend was observed for fibrogenesis, where, upon differentiation, IUGR-MSCs expressed significantly higher levels of COL1A1 (P < 0.03) than Normal-MSCs. In contrast, chondrogenic and osteogenic potential were decreased in IUGR-MSCs as shown by a smaller chondrocyte pellet and osteocyte staining, and lower expression of SOX9 (P < 0.05) and RUNX2 (P < 0.02), respectively. In conclusion, the regenerative potential of MSCs appears to be determined prenatally in IUGR and this should be taken into account when selecting cell donors in regenerative therapy programmes both in humans and companion animals.

Farmer and Veterinary Practices and Opinions Related to Fertility Testing and Pregnancy Diagnosis of UK Dairy Cows.

Dairy cow farming plays an important role in the UK and worldwide economies. Significant challenges are currently being faced regarding sustainability of the dairy industry. Dairy cow subfertility remains an important issue limiting herd productivity, resulting in annual losses of hundreds of millions of pounds in the UK alone. To address this, accurate monitoring of reproductive status and early detection of fertility issues in individual cows is essential. The aim of this study was to gather farmer and veterinarian opinions on current practices and perceived gaps related to diagnosis of fertility issues and pregnancy testing in UK dairy farms. Using online questionnaires, data were collected and analyzed from a total of 40 farmers and 59 veterinarians. The results showed that non-seen bulling checks and ultrasound were the most frequent tools to detect fertility issues, and that most farmers tested post-calving, and often again before or during mating. Most farmers believed that current tests did not meet their expectations, with half of those being willing to pay more than they were currently paying for fertility testing. In regard to pregnancy testing, ultrasound was most commonly used, at 30-50 days post-insemination either in individual or groups of cows. Again, most farmers believed that current tests did not meet their expectations, and a majority would consider paying a higher cost for a test that was better than those currently available. In addition, a majority of farmers would consider using a test that could detect pregnancy within 2 weeks post-insemination, if such test existed, because they believed it would help improve their herds' reproductive performance. Overall, the opinions of farmers and veterinarians indicate that there is significant scope for improving dairy herd fertility monitoring practices in the UK, through development of improved assays that can diagnose pregnancy and infertility earlier, are less disruptive to farm operations and are more cost effective than available tools. They also provide useful information to guide the future development and implementation of better diagnostics for improving reproductive performance of dairy cattle.

Farmer and Veterinary Practices and Opinions Related to the Diagnosis of Mastitis and Metabolic Disease in UK Dairy Cows.

Production diseases are highly prevalent in modern dairy herds, resulting in lost productivity and reduced animal welfare. Two important production diseases are mastitis and metabolic disorders. The availability of robust diagnostic tools that can detect animals at early stages of disease is crucial to prevent the high costs associated with lost productivity and the treatment of clinically and/or chronically diseased animals. Despite a variety of diagnostic methods being available to farmers and veterinarians, the incidence of these diseases in UK dairy herds has not changed over the last decade, underscoring the need for improved approaches for early disease detection. To this end, we administered a questionnaire to farmers and veterinarians to understand current diagnostic practices in the UK dairy cow sector, and to gather opinions on the suitability of currently available diagnostic tests in order to identify specific areas where improvement in diagnostic technologies and/or practices are needed. Data from a total of 34 farmers and 42 veterinarians were analyzed. Results indicated that most farmers surveyed used a combination of methods to diagnose mastitis and metabolic disorders, the most popular of which were visual inspection and milk recording somatic cell count data for mastitis, and body condition score and milk ketone testing for metabolic disorders. These preferences were not always in line with veterinarian recommendations of different diagnostic tools. Moreover, veterinarians indicated they were not satisfied with currently available diagnostic tools or how these were implemented by farmers. Both farmers and veterinarians recognized there was substantial room for improvement of current diagnostic tools, particularly in regard to the need to detect disease early. A majority of respondents preferred new diagnostic tests to be suitable for use with milk rather than blood or urine samples, and to yield results within 24 h. Finally, both groups surveyed identified economic cost as the most important barrier for the future uptake of new diagnostic technologies. The information obtained should guide the future development of diagnostic approaches that meet both the expectations of farmers and veterinarians, and help bring about a reduction in the incidence of production diseases in UK dairy herds.

Glucocorticoid regulation of the promoter of 11beta-hydroxysteroid dehydrogenase type 1 is indirect and requires CCAAT/enhancer-binding protein-beta.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inert 11keto-glucocorticoids to active 11beta-hydroxy forms, thereby amplifying intracellular glucocorticoid action. Up-regulation of 11beta-HSD1 in adipose tissue and liver is of pathogenic importance in metabolic syndrome. However, the mechanisms controlling 11beta-HSD1 transcription are poorly understood. Glucocorticoids themselves potently increase 11beta-HSD1 expression in many cells, providing a potential feed-forward system to pathology. We have investigated the molecular mechanisms by which glucocorticoids regulate transcription of 11beta-HSD1, exploiting an A549 cell model system in which endogenous 11beta-HSD1 is expressed and is induced by dexamethasone. We show that glucocorticoid induction of 11beta-HSD1 is indirect and requires new protein synthesis. A glucocorticoid-responsive region maps to between -196 and -88 with respect to the transcription start site. This region contains two binding sites for CCAAT/enhancer-binding protein (C/EBP) that together are essential for the glucocorticoid response and that bind predominantly C/EBPbeta, with C/EBPdelta present in a minority of the complexes. Both C/EBPbeta and C/EBPdelta are rapidly induced by glucocorticoids in A549 cells, but small interfering RNA-mediated knockdown shows that only C/EBPbeta reduction attenuates the glucocorticoid induction of 11beta-HSD1. Chromatin immunoprecipitation studies demonstrated increased binding of C/EBPbeta to the 11beta-HSD1 promoter in A549 cells after glucocorticoid treatment. A similar mechanism may apply in adipose tissue in vivo where increased C/EBPbeta mRNA levels after glucocorticoid treatment were associated with increased 11beta-HSD1 expression. C/EBPbeta is a key mediator of metabolic and inflammatory signaling. Positive regulation of 11beta-HSD1 by C/EBPbeta may link amplification of glucocorticoid action with metabolic and inflammatory pathways and may represent an endogenous innate host-defense mechanism.

Comparison of Antibacterial and Immunological Properties of Mesenchymal Stem/Stromal Cells from Equine Bone Marrow, Endometrium, and Adipose Tissue.

Equine mesenchymal stem/stromal cells (MSCs) are multipotent cells that are widely used for treatment of musculoskeletal injuries, and there is significant interest in expanding their application to nonorthopedic conditions. MSCs possess antibacterial and immunomodulatory properties that may be relevant for combating infection; however, comparative studies using MSCs from different origins have not been carried out in the horse, and this was the focus of this study. Our results showed that MSC-conditioned media attenuated the growth of Escherichia coli, and that this effect was, on average, more pronounced for endometrium (EM)-derived and adipose tissue (AT)-derived MSCs than for bone marrow (BM)-derived MSCs. In addition, the antimicrobial lipocalin-2 was expressed at mean higher levels in EM-MSCs than in AT-MSCs and BM-MSCs, and the bacterial component lipopolysaccharide (LPS) stimulated its production by all three MSC types. We also showed that MSCs express interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, chemokine ligand-5, and Toll-like receptor 4, and that, in general, these cytokines were induced in all cell types by LPS. Low expression levels of the macrophage marker colony-stimulating factor 1 receptor were detected in BM-MSCs and EM-MSCs but not in AT-MSCs. Altogether, these findings suggest that equine MSCs from EM, AT, and BM have both direct and indirect antimicrobial properties that may vary between MSCs from different origins and could be exploited toward improvement of regenerative therapies for horses.

Reproductive stage and sex steroid hormone levels influence the expression of mesenchymal stromal cell (MSC) markers in the equine endometrium.

Mesenchymal stem or stromal cells (MSCs) play key roles in tissue homeostasis. In the cyclic equine endometrium, this may be regulated by changes in serum concentrations of sex steroid hormones. This study was designed to investigate the changes in endometrial expression of MSC markers during reproductive cycles in mares and the influence of sex steroid hormones on endometrial MSC proliferation in vitro. Endometrial biopsies were collected from pony mares at different reproductive stages (estrus; day 5 and 13 after ovulation; seasonal anestrus; 20 h and 7days post-partum; n = 5 per stage) and were analyzed by RT-qPCR. MSC (CD29, CD44, CD73, CD90, CD105) and perivascular (CD146, NG2) markers were present in all samples irrespective of reproductive stage. Transcript levels of most markers were present at lowest levels on day 5 after ovulation and at 20 h post-partum. MSCs isolated from endometrial tissue (n = 6 mares) were cultured in the presence of progesterone (0.01-100 µM) and estradiol 17ß (0.1-1 µM), and cell proliferation was analyzed using alamarBlue® assay. Relative to cells incubated in steroid-depleted media, both progesterone and estradiol 17ß moderately increased cell proliferation (1.1- and 1.2-fold, respectively) independently of the concentration used. In conclusion, our results suggest that levels of MSC markers in equine endometrium dynamically change across reproductive cycles and that MSC populations are in part regulated by sex steroids.

Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.

Isolation and characterization of equine endometrial mesenchymal stromal cells.

BACKGROUND: Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. METHODS: The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. RESULTS: Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. CONCLUSIONS: We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

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 equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Isolation and characterization of equine native MSC populations.

BACKGROUND: In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell populations in that species. Moreover, a comparison between perivascular CD146+ and CD34+ cell populations has not been performed in any species. METHODS: Immunohistochemistry was used to identify adventitial cells (CD34+) and pericytes (CD146+) and to determine their localization in relation to MSCs in equine tissues. Isolation of CD34+ (CD34+/CD146-/CD144-/CD45-) and CD146+ (CD146+/CD34-/CD144-/CD45-) cell fractions from equine adipose tissue was achieved by fluorescence-activated cell sorting. The isolated cell fractions were cultured and analyzed for the expression of MSC markers, using qPCR and flow cytometry, and for the ability to undergo trilineage differentiation. Angiogenic properties were analyzed in vivo using a chorioallantoic membrane (CAM) assay. RESULTS: Both CD34+ and CD146+ cells displayed typical MSC features, namely growth in uncoated tissue culture dishes, clonal growth when seeded at low density, expression of typical MSC markers, and multipotency shown by the capacity for trilineage differentiation. Of note, CD146+ cells were distinctly angiogenic compared with CD34+ and non-sorted cells (conventional MSCs), demonstrated by the induction of blood vessels in a CAM assay, expression of elevated levels of VEGFA and ANGPT1, and association with vascular networks in cocultures with endothelial cells, indicating that CD146+ cells maintain a pericyte phenotype in culture. CONCLUSION: This study reports for the first time the successful isolation and culture of CD146+ and CD34+ cell populations from equine tissues. Characterization of these cells evidenced their distinct properties and MSC-like phenotype, and identified CD146+ cells as distinctly angiogenic, which may provide a novel source for enhanced regenerative therapies.

11ß-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice.

Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11ß-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11ß-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11ß-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b(+),Ly6G(+),7/4(+) cells) as the thioglycollate-recruited cells that most highly express 11ß-HSD1 and show dynamic regulation of 11ß-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11ß-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11ß-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11ß-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11ß-HSD1, acute inhibition of 11ß-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11ß-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11ß-HSD1 expression, suggesting the antiinflammatory effects of 11ß-HSD1 in neutrophils may be conserved in humans.

Prospects and Challenges of Induced Pluripotent Stem Cells in Equine Health.

Pluripotent stem cells (PSCs) hold, through the capacity to differentiate into virtually all body cell types, unprecedented promise for human and animal medicine. PSCs are naturally found in the early embryo, and in rodents and humans they can be robustly harvested and grown in culture in the form of embryonic stem cells (ESCs); however, the availability of ESCs from horses is limited. ES-like cells named induced pluripotent stem cells (iPSCs) can be derived in vitro by transcription factor-mediated reprogramming of adult cells. As such, iPSCs can be generated in a patient-specific manner providing unmatched potential for tissue transplantation and in vitro disease modeling. In humans, clinical trials using iPSC-derived cells are already taking place and the use of in vitro iPSC models has identified novel mechanisms of disease and therapeutic targets. Although to a more limited extent, iPSCs have also been generated from horses, a species in which, after humans, these cells are likely to hold the greatest potential in regenerative medicine. Before a clinical use can be envisioned, however, significant challenges will need to be addressed in relation to the robust derivation, long-term culture, differentiation, and clinical safety of equine iPSCs. Toward this objective, recent studies have reported significant improvement in culture conditions and the successful derivation for the first time of functional cell types from equine iPSCs. Given the wide range of exciting applications they could have, it is hoped future research will make the biomedical promise of iPSCs a reality not only for humans but also horses.

Expression of putative markers of pluripotency in equine embryonic and adult tissues.

Expression of several putative markers of pluripotency (OCT4, SOX2, NANOG, LIN28A, REX1, DNMT3B and TERT) was examined in a range of equine tissues, including early embryos, induced pluripotent stem cells (iPSCs), testis, adipose- and bone marrow-derived mesenchymal stromal cells (MSCs), and keratinocytes. Transcript levels of all markers were highest in embryos and iPSCs and, except for SOX2, were very low or undetectable in keratinocytes. Mean expression levels of all markers were lower in testis than in embryos or iPSCs and, except for DNMT3B, were higher in testis than in MSCs. Expression of OCT4, NANOG and DNMT3B, but not the other markers, was detected in MSCs. Of all markers analysed, only LIN28A, REX1 and TERT were associated exclusively with pluripotent cells in the horse.