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.

BackgroundEquine 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.MethodsThe 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.ResultsTypical 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.ConclusionsWe 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.

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.

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Because of their distinct differentiation, immunomodulatory, and migratory capacities, endometrial mesenchymal stromal cells (MSCs) may provide an optimum source of therapeutic cells not only in relation to the uterus but also for regeneration of other tissues. This study reports the fate of endometrial MSCs following intrauterine application in mares. Stromal cell fractions were isolated from endometrial biopsies taken from seven reproductively healthy mares, expanded, and fluorescence labeled in culture. Phosphate-buffered saline (PBS) or MSCs (15 × 106) were autologously infused into each uterine horn during early diestrus and subsequently tracked by fluorescence microscopy and flow cytometry of endometrial biopsies and blood samples taken periodically after infusion. The inflammatory response to cell infusion was monitored in endometrial cytology samples. MSCs were detected in endometrial sections at 6, 12, and 24 h, but not later (7 or 14 days), after cell infusion. Cells were in all cases located in the uterine lumen, never within the endometrial tissue. No fluorescence signal was detected in blood samples at any time point after infusion. Cytology analyses showed an increase in % of polymorphonuclear neutrophils between 1 and 3 h after uterine infusion with either MSCs or PBS and a further increase by 6 h only in mares infused with PBS. In summary, endometrial MSCs were detected in the uterine lumen for up to 24 h after infusion, but did not migrate into the healthy endometrium. Moreover, MSCs effectively attenuated the inflammatory response to uterine infusion. We conclude that endometrial MSCs obtained from routine uterine biopsies could provide a safe and effective cell source for treatment of inflammatory conditions of the uterus and potentially other tissues.

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 17β (0.1-1 μM), and cell proliferation was analyzed using alamarBlue assay. Relative to cells incubated in steroid-depleted media, both progesterone and estradiol 17β 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.

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