An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Association of plasma miRNAs with early life performance and aging in dairy cattle.

Early life performance traits in dairy cattle can have important influences on lifetime productivity. Poor health and fertility are of great economical and animal welfare concern. Circulating miRNAs have been linked to several livestock traits, including resistance to infection, fertility, and muscle development. This study aimed to identify circulating miRNAs associated with early life performance traits and aging in dairy cattle. Plasma samples from female calves (n = 12) identified retrospectively as differing in health, growth, and fertility outcomes prior to first calving were analyzed using PCR arrays detecting 378 miRNAs. Levels of 6 miRNAs differed significantly in calves with poor growth/fertility relative to controls (t-test: P<0.05). Additionally, general(ized) (non)linear mixed models identified 1 miRNA associated with average daily gain until weaning, 22 with live bodyweight at one year of age, 47 with age at first service, and 19 with number of infections before first calving. Out of 85 distinct miRNAs that were associated with at least one animal trait, 9 miRNAs were validated by RT-qPCR in a larger cohort (n = 91 animals), which included longitudinal plasma samples (calf, heifer, first lactation cow). Significant associations (P<0.05) involving individual miRNAs or ratios between miRNAs and early-life performance traits were identified, but did not retain significance after multiple testing adjustment. However, levels of 8 plasma miRNAs (miR-126-3p, miR-127, miR-142-5p, miR-154b, miR-27b, miR-30c-5p, miR-34a, miR-363) changed significantly with age, most prominently during the calf-to-heifer transition. Comparative RT-qPCR analyses of these miRNAs across 19 calf tissues showed that most were ubiquitously expressed. Online database mining identified several pathways involved in metabolism and cell signaling as putative biological targets of these miRNAs. These results suggest that miR-126-3p, miR-127, miR-142-5p, miR-154b, miR-27b, miR-30c-5p, miR-34a, miR-363 are involved in regulating growth and development from birth to first lactation (~2 years old) and could provide useful biomarkers of aging in cattle.

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.

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.

Generation of functional neurons from feeder-free, keratinocyte-derived equine induced pluripotent stem cells.

Pluripotent stem cells (PSCs) offer unprecedented biomedical potential not only in relation to humans but also companion animals, particularly the horse. Despite this, attempts to generate bona fide equine embryonic stem cells have been unsuccessful. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but their potential for directed differentiation into clinically relevant tissues has not been explored. In this study, we used retroviral vectors to generate induced pluripotent stem cells (iPSCs) with comparatively high efficiency from equine keratinocytes. Expression of endogenous PSC markers (OCT4, SOX2, LIN28, NANOG, DNMT3B, and REX1) was effectively restored in these cells, which could also form in vivo several tissue derivatives of the three germ layers, including functional neurons, keratinized epithelium, cartilage, bone, muscle, and respiratory and gastric epithelia. Comparative analysis of different reprogrammed cell lines revealed an association between the ability of iPSCs to form well-differentiated teratomas and the distinct endogenous expression of OCT4 and REX1 and reduced expression of viral transgenes. Importantly, unlike in previous studies, equine iPSCs were successfully expanded using simplified feeder-free culture conditions, constituting significant progress toward future biomedical applications. Further, under appropriate conditions equine iPSCs generated cells with features of cholinergic motor neurons including the ability to generate action potentials, providing the first report of functional cells derived from equine iPSCs. The ability to derive electrically active neurons in vitro from a large animal reveals highly conserved pathways of differentiation across species and opens the way for new and exciting applications in veterinary regenerative medicine.

Derivation and characterization of induced pluripotent stem cells from equine fibroblasts.

Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine.

The differential effects of the gonadotropin receptors on aromatase expression in primary cultures of immature rat granulosa cells are highly dependent on the density of receptors expressed and the activation of the inositol phosphate cascade.

Signaling pathways mediating the divergent effects of FSH and LH on aromatase in immature rat granulosa cells were studied by infecting cells with increasing amounts of adenoviral vectors for the human LH receptor (hLHR) or FSH receptor (hFSHR). Increasing amounts of Ad-hLHR, used at a multiplicity of infection (MOI) of 20 or 200 viable viral particles/cell, increased human chorionic gonadotropin (hCG) binding and hCG-induced cAMP and Akt phosphorylation, but inositol phosphates only increased in response to hCG in cells infected with 200 MOI Ad-hLHR. In contrast, hCG increased aromatase expression in cells infected with 20, but not in cells infected with 200, MOI Ad-hLHR. Cells infected with 20 or 200 MOI Ad-hFSHR showed increased hFSH binding and hFSH-induced Akt phosphorylation, but the hFSH-induced cAMP response was unchanged relative to control cells. However, hFSH was able to stimulate the inositol phosphate cascade in the Ad-hFSHR-infected cells, and the hFSH induction of aromatase was abolished. We also found that activation of C kinase or expression of a constitutively active form of Galphaq inhibited the induction of aromatase by hFSH or 8Br-cAMP. We conclude that the differential effects of FSH and LH on aromatase in immature granulosa cells are highly dependent on gonadotropin receptor density and on the signaling pathways activated. We propose that aromatase is induced by common signals generated by activation of the FSHR and LHR (possibly cAMP and Akt) and that the activation of the inositol phosphate cascade in cells expressing a high density of LHR or FSHR antagonizes this induction.