Human endometrial MSC (eMSC) are a novel source of MSC easily harvested from the highly regenerative uterine lining. We have developed protocols for eMSC isolation from single cell suspensions using magnetic bead-sorting using a perivascular marker antibody to SUSD2 and culture expansion in serum free medium (SFM). Similar to other MSC, eMSC spontaneously differentiate into fibroblasts during culture expansion decreasing their purity and efficacy. The aim of this study was to determine if A83-01, a TGF-β receptor inhibitor prevents eMSC differentiation in culture. SUSD2⁺ eMSC were cultured in SFM with bFGF/EGF in 5% O₂/5% CO₂. At passage 6, eMSC were incubated with or without A83-01 for 7 days, then analysed for MSC properties. A83-01 dose dependently promoted SUSD2⁺ eMSC proliferation and blocked apoptosis via the SMAD 2/3 pathway. Fewer A83-01 treated cells were autofluorescent or stained with β-galactosidase, indicating reduced senescence. A83-01-treated cells had higher cloning efficiency, differentiated into mesodermal lineages and expressed MSC phenotypic markers. These data suggest that A83-01 maintains SUSD2⁺ eMSC stemness, promoting proliferation by blocking senescence and apoptosis in late passage cultures through binding to TGF-β receptors. Small molecules such as A83-01 may enable the expansion of undifferentiated MSC for use in tissue engineering and cell-based therapies.

A successful pregnancy requires a synchronous dialogue between endometrium and embryo within the endometrial milieu. The aim of this study was to assess the role in the implantation of mediators in the endometrial milieu. Total secretome (TS), soluble secretome (SS) and small extracellular vesicles (containing exosomes) were generated from hormonally primed human endometrial epithelial cell culture medium. Human trophectoderm stem cell-derived spheroids were cultured with TS, SS or exosomes (30 µg/ml) on hormonally primed epithelial cells, with exosomes significantly increasing cell adhesion and outgrowth. Furthermore, F1 mouse 2-cell embryos were cultured in groups for 48 h followed by culture with each secretome fraction (30 µg/ml) for 48 h. Blastocyst cell number and hatching were quantified. In addition, blastocysts were further cultured on a fibronectin matrix for 72 h or transferred to recipient mice (with corresponding secretomes) with embryo implantation assessed after 6 days. Exosomes significantly increased total cell number in mouse embryos and complete hatching from zona pellucida, with both exosomes and SS significantly enhancing mouse embryo outgrowth. Importantly, exosomes increased the embryo implantation rate in comparison to other secretome fractions (normalized based on treatment amount) from the endometrial epithelia. These data indicate that endometrial epithelial exosomes support embryo growth, development and implantation while the SS has selective involvement specifically on mouse embryo outgrowth. This finding provides new insights into the molecular differences of endometrial secretome components in implantation and early embryo development and may implicate endometrial exosomes in the pathophysiology of implantation failure in infertility.

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgamma ^null ) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1β, Tnfα) secretion, and in C57BL6 mice reduced CCR7⁺ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. There was a small population CD271⁺ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271⁺CD49f^- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271^-CD49f^- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271⁺ cells. This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.

Mesenchymal stem/stromal cells (MSCs) are multipotent cells with favorable properties for cell therapies and regenerative medicine. Human endometrium harbors a small population of perivascular, clonogenic MSCs (eMSCs) identified by the SUSD2 marker. As for other MSCs, eMSCs require extensive in vitro expansion to generate clinically relevant numbers of cells, resulting in spontaneous differentiation, replicative senescence and cell death, decreasing therapeutic potency. We previously demonstrated that A83-01, a TGF-β receptor inhibitor, maintained eMSC clonogenicity, promoted proliferation, prevented apoptosis and maintained MSC function in vitro. Here we compare the transcriptome of passaged eMSCs from six women cultured with and without A83-01 for 7 days. We identified 1206 differentially expressed genes (DEG) using a false discovery rate cut-off at 0.01 and fold change >2. Significant enrichment of genes involved in anti-inflammatory responses, angiogenesis, cell migration and proliferation, and collagen fibril and extracellular matrix organization were revealed. TGF-β, Wnt and Akt signaling pathways were decreased. Anti-fibrotic and anti-apoptotic genes were induced, and fibroblast proliferation and myofibroblast related genes were downregulated. We found increased MSC potency genes (TWIST1, TWIST2, JAG1, LIFR, and SLIT2) validating the enhanced potency of A83-01-treated eMSCs, and importantly no pluripotency gene expression. We also identified eMSCs’ potential for secreting exosomes, possibly explaining their paracrine properties. Angiogenic and cytokine protein arrays confirmed the angiogenic, anti-fibrotic and immunomodulatory phenotype of A83-01-treated eMSCs, and increased angiogenic activity was functionally demonstrated in vitro. eMSCs culture expanded with A83-01 have enhanced clinically relevant properties, suggesting their potential for cell-therapies and regenerative medicine applications.