Proteomic profiling of human uterine extracellular vesicles reveal dynamic regulation of key players of embryo implantation and fertility during menstrual cycle.

Endometrial extracellular vesicles (EVs) are emerging as important players in reproductive biology. However, how their proteome is regulated throughout the menstrual cycle is not known. Such information can provide novel insights into biological processes critical for embryo development, implantation, and successful pregnancy. Using mass spectrometry-based quantitative proteomics, we show that small EVs (sEVs) isolated from uterine lavage of fertile women (UL-sEV), compared to infertile women, are laden with proteins implicated in antioxidant activity (SOD1, GSTO1, MPO, CAT). Functionally, sEVs derived from endometrial cells enhance antioxidant function in trophectoderm cells. Moreover, there was striking enrichment of invasion-related proteins (LGALS1/3, S100A4/11) in fertile UL-sEVs in the secretory (estrogen plus progesterone-driven, EP) versus proliferative (estrogen-driven, E) phase, with several players downregulated in infertile UL-sEVs. Consistent with this, sEVs from EP- versus E-primed endometrial epithelial cells promote invasion of trophectoderm cells. Interestingly, UL-sEVs from fertile versus infertile women carry known players/predictors of embryo implantation (PRDX2, IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU, ANXA2), and implantation success (CAT, YWHAE, PPIA), highlighting their potential to inform regarding endometrial status/pregnancy outcomes. Thus, this study provides novel insights into proteome reprograming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility.

The proteomes of endometrial stromal cell-derived extracellular vesicles following a decidualizing stimulus define the cells' potential for decidualization success.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics. On Day 2, in PD- versus WD-ESC-sEVs, 17 sEV- proteins were down-regulated (C5, C6; complement/coagulation cascades, and SERPING1, HRG; platelet degranulation and fibrinolysis) and 39 up-regulated (FLNA, COL1A1; focal adhesion, ENO1, PKM; glycolysis/gluconeogenesis, and RAP1B, MSN; leukocyte transendothelial migration). On Day 14, in PD- versus WD-ESC-sEVs, FLNA was down-regulated while 21 proteins were up-regulated involved in complement/coagulation cascades (C3, C6), platelet degranulation (SERPINA4, ITIH4), B-cell receptor signalling and innate immune response (immunoglobulins). Changes from Days 2 to 14 suggested a subsequent response in PD-ESC-sEVs with 89 differentially expressed proteins mostly involved in complement and coagulation cascades (C3, C6, C5), but no change in WD-ESC-sEVs ESC. Poor decidualization was also associated with loss of crucial sEV-proteins for cell adhesion and invasion (ITGA5, PFN1), glycolysis (ALDOA, PGK1) and cytoskeletal reorganization (VCL, RAC1). Overall, this study indicates varied ESC response even prior to decidualization and provides insight into sEVs-proteomes as a benchmark of well-decidualized ESC. It shows distinct variation in sEV-protein composition depending on the ESC decidual response that is critical for embryo implantation, enabling and limiting trophoblast invasion during placentation and sensing a healthy embryo.

Endometrial mesenchymal stem/stromal cell modulation of T cell proliferation.

Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2 and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-ß1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-ß receptors, despite a reduction in regulatory T cells. Thus, the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-ß1 and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSCs were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

Tissue engineering approaches for treating pelvic organ prolapse using a novel source of stem/stromal cells and new materials.

PURPOSE OF REVIEW: Nondegradable transvaginal polypropylene meshes for treating pelvic organ prolapse (POP) are now generally unavailable or banned. In this review, we summarize recent developments using tissue engineering approaches combining alternate degradable scaffolds with a novel source of mesenchymal stem/stromal cells from human endometrium (eMSC). RECENT FINDINGS: Tissue engineering constructs comprising immunomodulatory, reparative eMSC and biomimetic materials with nanoarchitecture are a promising approach for vaginal repair and improving outcomes of POP surgery. Culture expansion of eMSC that maintains them (and other MSC) in the undifferentiated state has been achieved using a small molecule transforming growth factor-ß receptor inhibitor, A83-01. The mechanism of action of A83-01 has been determined and its suitability for translation into the clinic explored. Novel blends of electrospun synthetic and natural polymers combined with eMSC shows this approach promotes host cell infiltration and slows biomaterial degradation that has potential to strengthen the vaginal wall during healing. Improving the preclinical ovine transvaginal surgical model by adapting the human clinical POP-Quantification system for selection of multiparous ewes with vaginal wall weakness enables assessment of this autologous eMSC/nanobiomaterial construct. SUMMARY: A tissue engineering approach using autologous eMSC with degradable nanobiomaterials offers a new approach for treating women with POP.

Posterior Segment Retained Intraocular Foreign Body: A Study from Western Nepal.

INTRODUCTION: Posterior segment retained Intraocular foreign body (IOFB) management is challenging. Facility of pars plana vitrectomy (PPV) and availability of well trained vitreo retina surgeons are the basic need to accomplish this work. Encircling band provide permanent 360° support to close the anterior retinal break and prevent traction on the retina. The objective of this study is to analyse the clinical characteristics and predictors of the final visual outcome and survival of the globe in cases of retained IOFB in the posterior eye segment. MATERIALS AND METHODS: A hospital based retrospective observational study was conducted. All the patients of retained IOFB in the posterior segment presented from January 2016 to June 2019 were enrolled. Patients presented with visual acuity of NPL were excluded. Statistical analysis was performed using a variety of tests using SPSS version 21. RESULTS: Forty eyes of 40 patients were included. The mean age was 27.08±10.68 years (range 5-66). 95% of our patients were male. Most of them (52.5%) worked on the farm. 26(65%) of 40 eyes had Zone I injury. The median time spent before presentation was 13.5 day. Retinal detachment, vitreous hemorrhage, and endophthalmitis were present in 15, 23 and 5 eyes, respectively, before IOFB removal. The mean LogMAR visual acuity was improved significantly from 2.50±0.87 to 1.33± 1.01 (p=0.003). Poor presenting visual acuity, retinal detachment and large diameter of IOFB were found as the predictor of poor final visual acuity. CONCLUSION: Pars plana vitrectomy by a vitreo retinal surgeon can give encouraging results in the cases of retained posterior segment IOFB. Poor presenting visual acuity, large diameter of IOFB and RD before IOFB removal are predictors of poor visual outcome.

Early phase Vogt-Koyanagi-Harada Disease in Nepalese Elderly woman: A case report.

INTRODUCTION: Vogt-Koyanagi-Harada (VKH) disease is defined as an autoimmune disorder characterized by bilateral granulomatous panuveitis with systemic manifestations, such as tinnitus, vertigo, and meningism caused by melanocyte antigen-reactive T-cells. Majority of VKH patients present at the age between 20 and 50 years. VKH is uncommon in elderly and challenging to manage. VKH is one of the important differential diagnosis of bilateral pan uveitis Case: A 65 year/ female brought with chief complaint of sudden loss of vision in both eyes, headache and hearing problem for 1 month. She didn't give any history of other systemic illness, ocular surgery, ocular trauma, chronic use of medicament. Her visual acuity was hand movement with accurate projection of rays (HM) in both eyes The intraocular pressure (IOP) was 12mmHg in both eye. Slit-lamp bio microscopy revealed features of Pan uveitis in both eye. Systemic work up revealed no any other abnormalities. A diagnosis of early phase VKH was made and treated with intravenous pulse steroid therapy followed by tapering dose of oral steroid along with immunemodulator resulting in a very good visual recovery. CONCLUSION: VKH can present in elderly. immunomodulator should be considered in elderly to prevent side effect of steroid along with recurrence of inflammation.

Comparing the Effect of TGF-ß Receptor Inhibition on Human Perivascular Mesenchymal Stromal Cells Derived from Endometrium, Bone Marrow and Adipose Tissues.

Rare perivascular mesenchymal stromal cells (MSCs) with therapeutic properties have been identified in many tissues. Their rarity necessitates extensive in vitro expansion, resulting in spontaneous differentiation, cellular senescence and apoptosis, producing therapeutic products with variable quality and decreased potency. We previously demonstrated that A83-01, a transforming growth factor beta (TGF-ß) receptor inhibitor, maintained clonogenicity and promoted the potency of culture-expanded premenopausal endometrial MSCs using functional assays and whole-transcriptome sequencing. Here, we compared the effects of A83-01 on MSCs derived from postmenopausal endometrium, menstrual blood, placenta decidua-basalis, bone marrow and adipose tissue. Sushi-domain-containing-2 (SUSD2+) and CD34+CD31-CD45- MSCs were isolated. Expanded MSCs were cultured with or without A83-01 for 7 days and assessed for MSC properties. SUSD2 identified perivascular cells in the placental decidua-basalis, and their maternal origin was validated. A83-01 promoted MSC proliferation from all sources except bone marrow and only increased SUSD2 expression and prevented apoptosis in MSCs from endometrial-derived tissues. A83-01 only improved the cloning efficiency of postmenopausal endometrial MSCs (eMSCs), and expanded adipose tissue MSCs (adMSCs) underwent significant senescence, which was mitigated by A83-01. MSCs derived from bone marrow (bmMSCs) were highly apoptotic, but A83-01 was without effect. A83-01 maintained the function and phenotype in MSCs cultured from endometrial, but not other, tissues. Our results also demonstrated that cellular SUSD2 expression directly correlates with the functional phenotype.

Endometrial and Menstrual Blood Mesenchymal Stem/Stromal Cells: Biological Properties and Clinical Application.

A highly proliferative mesenchymal stem/stromal cell (MSC) population was recently discovered in the dynamic, cyclically regenerating human endometrium as clonogenic stromal cells that fulfilled the International Society for Cellular Therapy (ISCT) criteria. Specific surface markers enriching for clonogenic endometrial MSC (eMSC), CD140b and CD146 co-expression, and the single marker SUSD2, showed their perivascular identity in the endometrium, including the layer which sheds during menstruation. Indeed, cells with MSC properties have been identified in menstrual fluid and commonly termed menstrual blood stem/stromal cells (MenSC). MenSC are generally retrieved from menstrual fluid as plastic adherent cells, similar to bone marrow MSC (bmMSC). While eMSC and MenSC share several biological features with bmMSC, they also show some differences in immunophenotype, proliferation and differentiation capacities. Here we review the phenotype and functions of eMSC and MenSC, with a focus on recent studies. Similar to other MSC, eMSC and MenSC exert immunomodulatory and anti-inflammatory impacts on key cells of the innate and adaptive immune system. These include macrophages, T cells and NK cells, both in vitro and in small and large animal models. These properties suggest eMSC and MenSC as additional sources of MSC for cell therapies in regenerative medicine as well as immune-mediated disorders and inflammatory diseases. Their easy acquisition via an office-based biopsy or collected from menstrual effluent makes eMSC and MenSC attractive sources of MSC for clinical applications. In preparation for clinical translation, a serum-free culture protocol was established for eMSC which includes a small molecule TGFß receptor inhibitor that prevents spontaneous differentiation, apoptosis, senescence, maintains the clonogenic SUSD2+ population and enhances their potency, suggesting potential for cell-therapies and regenerative medicine. However, standardization of MenSC isolation protocols and culture conditions are major issues requiring further research to maximize their potential for clinical application. Future research will also address crucial safety aspects of eMSC and MenSC to ensure these protocols produce cell products free from tumorigenicity and toxicity. Although a wealth of data on the biological properties of eMSC and MenSC has recently been published, it will be important to address their mechanism of action in preclinical models of human disease.

Impact of Sustained Transforming Growth Factor-ß Receptor Inhibition on Chromatin Accessibility and Gene Expression in Cultured Human Endometrial MSC.

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-ß receptor (TGFß-R) inhibitor, promotes expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFß-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFß-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RARß). Selective RARß inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture.

Lens induced glaucoma in a tertiary eye care centre in Western Nepal.

INTRODUCTION: Lens induced glaucoma due to hypermature cataracts is an important cause of secondary glaucoma in the developing world. The most common etiology of lens induced glaucoma is phacomorphic glaucoma from untreated senile cataract. OBJECTIVES: To study the demographics, clinical presentations and surgical outcome of lens induced glaucoma (LIG). MATERIALS AND METHODS: It is the prospective case series of fifty three patients eyes with LIG over a 6 months period from June 2015 to November 2015. All cases of lens induced glaucoma underwent cataract surgery. Data including demographics, clinical presentations, surgical outcome were analysed using Statistical Package for Social Studies 20.0. RESULTS: The mean age was 61.5 years (Standard deviation 8.9) with predominantly women (30, 56.6%) were affected. Phacomorphic glaucoma (38, 71.7%) was the main cause of lens induced glaucoma, followed by phacolytic glaucoma (15, 28.3%). The main clinical symptoms were reduced vision (100%), eye pain (96.2%) and redness of eyes (62.3%). All patients (100%) presented with visual acuity of <3/60 or worse and intraocular pressure (IOP) more than 40 mm Hg (34, 64.2%). All 53(100%) patients underwent cataract surgery and all of them had tremendously reduced intraocular pressure with a mean 13.9 mmHg and vision had improved from >6/18 as noted in 16(30.2%) cases. CONCLUSIONS: The main clinical presentations of LIG are triad of acute reduced vision, eye pain and redness. The better final BCVA is found when there is an early presentation and less IOP at the time of presentation Public awareness, early detection and early intervention aids in good visual recovery and control of intraocular pressure in LIG.

The Transcriptome of Human Endometrial Mesenchymal Stem Cells Under TGFßR Inhibition Reveals Improved Potential for Cell-Based Therapies.

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-ß 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-ß, 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.

In Vivo Survival of Human Endometrial Mesenchymal Stem Cells Transplanted Under the Kidney Capsule of Immunocompromised Mice.

Human endometrial mesenchymal stem cells (eMSCs) are a well-characterized adult stem cell type with potential for use in regenerative medicine or cell therapy. As a proof of principle, we demonstrated that eMSCs promoted wound healing by reducing the inflammatory response through a paracrine action in a subcutaneous rat model of wound repair. However, an efficient protocol for culturing eMSCs in the undifferentiated state and a reliable method of labeling them for cell tracking were lacking. In this study, we investigated the use of a lentiviral vector containing the mCherry fluorescent reporter gene to transduce and label eMSCs following in vitro culturing in A83-01 containing medium, and different methods of tracing the labeled cells following transplantation under the kidney capsule of immunocompromised NSG mice. Perivascular SUSD2+ eMSCs were isolated from human endometrium. Passage 1 eMSCs were transduced by lentiviruses with mCherry fluorescent reporter gene; mCherry+ cells were isolated by fluorescence-activated cell sorting and cultured until passage 6 in 5% O2 in serum-free medium with fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). The cells were subsequently divided into two flasks and treated with either dimethyl sulfoxide (0.01%) or A83-01 (1 µM) for 7 days. 5 × 105 control or A83-01 pretreated cells were encapsulated into a fibrin gel and transplanted under the subrenal capsules of NSG mice. Tissues were analyzed at 7, 14, and 30 days posttransplantation. Human eMSCs were efficiently transduced with mCherry gene. They proliferated and maintained high mCherry expression over five passages. Analyzing transplanted kidneys using polymerase chain reaction, flow cytometry, and immunofluorescence showed that both cell types survived at least 30 days. Efficient labeling of eMSCs using a lentiviral vector and culturing them in an environment maintaining them in an undifferentiated state enable reliable detection in preclinical animal models and highlight the need for generating a pure population of undifferentiated MSCs for long-term survival in vivo to prolong their treatment effect.

Preterm umbilical cord blood derived mesenchymal stem/stromal cells protect preterm white matter brain development against hypoxia-ischemia.

INTRODUCTION: Preterm infants are at high risk for white matter injury and subsequent neurodevelopmental impairments. Mesenchymal stem/stromal cells (MSC) have anti-inflammatory/immunomodulatory actions and are of interest for neural repair in adults and newborns. This study examined the neuroprotective effects of allogeneic MSC, derived from preterm umbilical cord blood (UCB), in a preterm sheep model of white matter injury. METHODS: Quad-lineage differentiation, clonogenicity and self-renewal ability of UCB-derived MSC were confirmed. Chronically instrumented fetal sheep (0.7 gestation) received either 25 min hypoxia-ischemia (HI) to induce preterm brain injury, or sham-HI. Ten million MSC, or saline, were administered iv to fetuses at 12 h after HI. Fetal brains were collected 10d after HI for histopathology and immunocytochemistry. RESULTS: HI induced white matter injury, as indicated by a reduction in CNPase-positive myelin fiber density. HI also induced microglial activation (Iba-1) in the periventricular white matter and internal capsule (P < .05 vs control). MSC administration following HI preserved myelination (P < .05), modified microglial activation, and promoted macrophage migration (CD163) and cell proliferation (Ki-67) within cerebral white matter (P < .05). Cerebral CXCL10 concentration was increased following MSC administration (P < .05), which was likely associated with macrophage migration and cell proliferation within the preterm brain. Additionally, MSC administration reduced systemic pro-inflammatory cytokine TNFα at 3d post-HI (P < .05). CONCLUSIONS: UCB-derived MSC therapy preserved white matter brain structure following preterm HI, mediated by a suppression of microglial activation, promotion of macrophage migration and acceleration of self-repair within the preterm brain. UCB-derived MSC are neuroprotective, acting via peripheral and cerebral anti-inflammatory and immunomodulatory mechanisms.

Demographic pattern and clinical characteristics of optic neuritis in a tertiary eye care centre.

PURPOSE: To study the demographic pattern and clinical characteristics of optic neuritis cases in a tertiary eye care centre in Nepal. DESIGN: Descriptive, cross-sectional study. METHODOLOGY: Complete ocular examination was done in all the newly diagnosed cases of optic neuritis presenting from January 1st 2012 to June 30th 2013. Demographic pattern, clinical features, visual acuity, colour vision, contrast sensitivity and visual field defects were studied. RESULTS: Sixty seven eyes of 50 patients (28 females and 22 males) with optic neuritis were included in the study. The mean age was 34.32 years ± 13.72 years. The male: female ratio was 1:1.27. All the cases presented with complaint of blurring of vision. Painful ocular movement was noted in 58%. On ophthalmoscopic examination around 2/3rd of eyes suffered from papillitis (72%) and 1/3rd from retrobulbar optic neuritis (27%). Only one case of neuroretinitis (1%) was seen in the study. The colour vision pattern was variable. Contrast sensitivity was reduced in 94%. Centrocaecal scotoma was seen in 10.5%. CONCLUSION: Females were predominantly affected. Unilateral involvement was the most common presenting as papillitis.

Inhibition of Transforming Growth Factor-ß Receptor signaling promotes culture expansion of undifferentiated human Endometrial Mesenchymal Stem/stromal Cells.

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-ß 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 ß-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-ß receptors. Small molecules such as A83-01 may enable the expansion of undifferentiated MSC for use in tissue engineering and cell-based therapies.

Stem Cells in Endometrial Physiology.

Human endometrial mucosa is a dynamically remodeling tissue, undergoing cyclical morphologic and functional changes in response to fluctuating sex steroid hormones each menstrual cycle during a woman's reproductive life. Postmenopausal endometrium responds similarly to exogenous estrogen. Cyclical endometrial regeneration also occurs in nonmenstruating rodents, although to a lesser extent. The recent identification of rare populations of endogenous epithelial progenitor cells, mesenchymal stem/stromal cells (MSCs), the side population (SP) cells, and label-retaining cells (LRCs) suggests these stem/progenitor cell populations may play a key role in endometrial regeneration during menstrual and estrus cycles. This review summarizes the identification of epithelial progenitors, MSC, SP, and LRC, and discusses their contribution to endometrial tissue regeneration, maintaining tissue homeostasis, decidualization, and placentation. Markers for human endometrial MSC have been identified, revealing their perivascular location in both the functionalis and basalis layers. These markers also allow their purification from biopsy tissue and menstrual blood. These findings have advanced our understanding of normal endometrial physiology and will provide new insight into endometrial proliferative disorders (endometriosis, endometrial cancer). The ability to prospectively isolate endometrial MSC will enable their utilization in cell-based therapies for reproductive tract pathologies.

Isolation and characterisation of mesenchymal stem/stromal cells in the ovine endometrium.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.