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Additional file 2. Supplementary Table 1: Quantitative polymerase chain reaction calibration curve data.

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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.

BackgroundIn 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.MethodsImmunohistochemistry 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.ResultsBoth 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.ConclusionThis 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.

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.

Poor reproductive performance remains a major issue in the dairy industry, with low conception rates having a significant impact on milk production through extended calving intervals. A major limiting factor is the lack of reliable methods for early pregnancy diagnosis. Identification of animals within a herd that fail to conceive within 3 weeks after insemination would allow early re-insemination and shorten calving intervals. In a previous study, we found an increase in plasma miR-26a levels in Day 16-pregnant relative to non-pregnant heifers, however changes in miRNA levels that early during pregnancy were very small which likely prevented the identification of robust biomarkers. In this study, we extended our analyses to a wider interval during pregnancy (Days 8 to 60, n = 11 heifers) with the rationale that this may facilitate the identification of additional early pregnancy miRNA biomarkers. Using small RNA sequencing we identified a total of 77 miRNAs that were differentially expressed on Day 60 relative to Day 0 of pregnancy. We selected 14 miRNAs for validation by RT-qPCR and confirmed significant differences in the expression of let-7f, let-7c, miR-30c, miR-101, miR-26a, miR-205 and miR-143 between Days 0 and 60. RT-qPCR profiling throughout Days 0, 8, 16 and 60 of pregnancy showed a distinct increase in circulating levels of miR-26a (3.1-fold, P = 0.046) as early as Day 8 of pregnancy. In summary, in contrast to earlier stages of pregnancy (≤ Day 24), marked differences in the levels of multiple miRNAs can be detected in circulation by Day 60 in cattle. Retrospective analyses showed miR-26a levels to be increased in circulation as early as Day 8, sooner than previously reported in any species, suggesting a biological role for this miRNA in the very early events of pregnancy.

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 pre-ovulatory maturation of equine dominant follicles. Granulosa and theca interna cells (GCs, 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 pre-ovulatory stage (PO; 34h after administration of crude equine gonadotropins at LD stage), and separately analyzed on a horse gene expression microarray, followed by validation using qPCR and immunoblotting/immunohistochemistry. Numbers of differentially expressed transcripts (DETs; ≥2-fold; P