Human Endometrial Mesenchymal Stem Cells Research Articles

P-796 Single-cell transcriptomics unveils a role for leptin receptor in human endometrial mesenchymal stromal/stem cells

Abstract Study question What are the characteristics of leptin receptor (LepR) expressing cells within the endometrial mesenchymal stromal/stem cells (eMSCs) population? Summary answer LepR+ eMSCs display better stem-cell capacities as well as distinctively quiescent properties compared to eMSCs and LepR- eMSCs. What is known already Single-cell RNA sequencing (scRNA-seq) reveals that cultured eMSCs are heterogeneous. LepR is highly expressed in bone marrow MSCs, and LepR+ cells contribute towards the formation of fibroblast colony-forming units (CFU-F). Mouse LepR+ MSCs are quiescent and activate upon injuries to form osteoblasts and adipocytes in the bone marrow. In this study, we hypothesized that LepR+ cells play a critical role in the stem cell population in human endometrium. Study design, size, duration We performed scRNA-seq on magnetic bead selected eMSCs (CD140b+CD146+ cells). Functional stem cell assays such as clonogenicity and serial subcloning were performed on the different stromal subsets (eMSC, LepR- eMSC, and LepR+ eMSC) isolated by fluorescence-activated cell sorting (FACS) cultured endometrial stromal cells. The cell cycle analysis was evaluated by multicolor flow cytometry on freshly isolated endometrial stromal cells. Participants/materials, setting, methods The endometrial samples were collected from women aged 37–53 years with regular menstrual cycles undergoing hysterectomy. The expression and localization of LepR in human endometrium were determined by flow cytometry and immunohistochemistry, respectively. The in vitro colony-forming and self-renewal abilities were assessed on FACS sorted endometrial stromal populations. The cell cycle status of the stromal subsets was determined by flow cytometry and the quiescent property was determined by qPCR (RB1, RBL2, CDKN1A, CDKN1B, and E2F4). Main results and the role of chance Clustering analysis of the scRNA-seq data revealed 5 subpopulations (SP0-4) of cultured eMSCs. A high proportion (>90%) of the cells in SP3 and SP4 were at the G1 phase. The majority of the SP3 cells (92%) were located at the root of the cell trajectory and pseudo time inference analysis. The expression of LepR was highly specific in SP3 suggesting LepR eMSCs are potentially the origin progenitors.The expression of LepR in endometrial stromal cells was 2.61 ± 1.43%, and in eMSCs was 25.59 ± 19.04%. In vitro experiments demonstrated that LepR+ eMSCs exhibit better stem-cell properties, including clonogenic and self-renewal ability, than its negative counterparts. QPCR validation of cell-quiescence associated genes revealed higher expression of RB1, RBL2, and CDKN1B in the LepR+ eMSCs than eMSCs and LepR- eMSCs. In addition, multicolor flow analysis on freshly isolated endometrial cells revealed significantly higher proportion of LepR+ eMSCs are in the G0 phase of the cell cycle when compared to eMSCs and LepR- eMSCs. The above results indicate in vivo LepR+ eMSCs are relatively more quiescent. Limitations, reasons for caution The mechanism involved in maintaining or activating quiescent LepR+ eMSCs remains unknown. The role of LepR+ is likely to be different in human compared to mouse. We also lack an in vivo model for studying LepR+ eMSCs during endometrial repair. Wider implications of the findings Our findings extend the understanding of heterogeneous human endometrial mesenchymal stem cells and unveil a new role for LepR in endometrial regeneration. Trial registration number not applicatble

Differentiation of human endometrial stem cells encapsulated in alginate hydrogel into oocyte-like cells.

Human endometrial mesenchymal stem cells (hEnMSCs) are a rich source of mesenchymal stem cells (MSCs) with multi-lineage differentiation potential, making them an intriguing tool in regenerative medicine, particularly for the treatment of reproductive and infertility issues. The specific process of germline cell-derived stem cell differentiation remains unknown, the aim is to study novel ways to achieve an effective differentiation method that produces adequate and functioning human gamete cells. We adjusted the optimum retinoic acid (RA) concentration for enhancement of germ cell-derived hEnSCs generation in 2D cell culture after 7 days in this study. Subsequently, we developed a suitable oocyte-like cell induction media including RA and bone morphogenetic protein 4 (BMP4), and studied their effects on oocyte-like cell differentiation in 2D and 3D cell culture media utilizing cells encapsulated in alginate hydrogel. Our results from microscopy analysis, real-time PCR, and immunofluorescence tests revealed that 10 µM RA concentration was the optimal dose for inducing germ-like cells after 7 days. We examined the alginate hydrogel structural characteristics and integrity by rheology analysis and SEM microscope. We also demonstrated encapsulated cell viability and adhesion in the manufactured hydrogel. We propose that in 3D cell cultures in alginate hydrogel, an induction medium containing 10 µM RA and 50 ng/mL BMP4 can enhance hEnSC differentiation into oocyte-like cells. The production of oocyte-like cells using 3D alginate hydrogel may be viable in vitro approach for replacing gonad tissues and cells.

Optimizing human endometrial mesenchymal stem cells for maximal induction of angiogenesis.

Human endometrial mesenchymal stem cells (hEMSCs) have been shown to promote neo-vascularization; however, its angiogenic function lessens with age. To determine the optimal conditions for maximizing hEMSC angiogenic capacity, we examined the effects of serial passaging on hEMSC activity. hEMSCs were cultured from passages (P) 3, 6, 9, and 12, and analyzed for proliferation, migration, differentiation and senescence, as well as their capacity to induce angiogenesis. The results showed that hEMSC proliferation and migration significantly decreased after P12. Furthermore, hEMSC differentiation into adipogenic and osteogenic lineages, as well as their proangiogenic capacity, gradually decreased from P9-12, while senescence only occurred after P12. Evaluation of angiogenic-related protein levels showed that both transforming growth factor β2 and Tie-2 was significantly reduced in hEMSCs at P12, compared to P3, possibly serving as the basis behind their lowered angiogenic capacity. Furthermore, in vivo angiogenesis evaluation with Matrigel plug assay showed that the optimal hEMSC to HUVEC ratio, for maximizing vessel formation, was 1:4. This study showed that hEMSC passaging was associated with lowered cellular functioning, bringing them closer to a senescent phenotype, especially after P12, thereby defining the optimal time period for cultivating fully functional hEMSCs for therapeutic applications.

The anti-angiogenic effect of atorvastatin loaded exosomes on glioblastoma tumor cells: An in vitro 3D culture model

Exosomes are endogenous nanoparticles with a lipid bilayer membrane whose natural function as carriers of biological materials has attracted much attention. The ability of exosomes to cross biological barriers, especially the blood-brain barrier, has highlighted them as tools of drug delivery to brain tumors. In a previous study, we isolated and characterized exosomes derived from human endometrial mesenchymal stem cells (hEnMSCs exosomes). In the present study, we used hEnMSCs exosomes as carriers for atorvastatin and investigated its pro-apoptotic and anti-angiogenic effects on U87 glioblastoma spheroids 3D co-cultured with Human Umbilical Vein Endothelial cells (HUVECs). In the study of HUVEC proliferation by using MTT assay, cell treatments with concentrations of 5 and 10 μM of free atorvastatin and atorvastatin-loaded hEnMSCs exosomes (AtoEXOs) showed significant differences in inhibition of proliferation compared to other concentrations. Also, 5 and 10 μM of AtoEXOs inhibited HUVEC migration in both scratch closure and transwell migration assays significantly more than that of free atorvastatin. In addition, in vitro HUVEC capillary tube network formation was inhibited by 5 and 10 μM treatment of AtoEXOs significantly more that of free atorvastatin. Moreover, a significant decrease in VEGF secretion and a significant increase in Bax/Bcl2 expression ratio were observed in U87 spheroids 3D co-cultured with HUVECs, especially for 10 μM AtoEXOs compared to other treated cell groups. Our results showed that hEnMSCs exosomes loaded with atorvastatin not only mimicked the anti-tumor effects of free atorvastatin but also potentiated its anti-tumor effects on glioblastoma cells. The enhanced pro-apoptotic and anti-angiogenic capabilities of atorvastatin loaded in hEnMSCs exosomes offer promising new perspectives for the treatment of glioblastoma.

Stability of the Human Endometrial Mesenchymal Stem Cells Karyotype In Vitro

Stability of the Human Endometrial Mesenchymal Stem Cells Karyotype In Vitro

Functional Activity of Sodium Channels ENaC in Human Endometrial Mesenchymal Stem Cells

Functional Activity of Sodium Channels ENaC in Human Endometrial Mesenchymal Stem Cells

Tropism of Extracellular Vesicles and Cell-Derived Nanovesicles to Normal and Cancer Cells: New Perspectives in Tumor-Targeted Nucleic Acid Delivery.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

O-065 The naughty cells of the endometriumxx

Abstract Stem/progenitor cells are the naughty cells of the endometrium! The term “naughty” has a number of connotations, one being immaturity which I will apply to the rare stem/progenitor cell populations hiding in the endometrium, where they have eluded scientists for so long. Despite their rarity, these immature cells have the capability of growing up and differentiating into the functional cells of the endometrium, producing their progenies in the process. The self-willed human endometrial epithelial progenitor cells (eEPC) and mesenchymal stem cells (eMSC) first revealed themselves through their clonogenic activity, shunning their mates and setting up clones of cells on their own. Their risqué production of identical copies of themselves ensures their continuity, much to the chagrin of their mature counterparts. They are sneaky and can produce large numbers of mature progeny, but rarely proliferate themselves preferring to take life easy and do little. They also spit out viability dyes (Hoechst) at a greater rate than mature endometrial cells to become Side Population (SP) cells. A number of approaches have been used to tame these naughty endometrial stem/progenitors. In order to determine the identity and location of these elusive cells, specific markers had to be found. The immature endometrial epithelial progenitor cells play tricks with the specific markers they express. For example, clonogenic eEPC are N-cadherin+, an epithelial mesenchymal transition marker, found by unbiassed gene profiling, revealed their hiding place in the bases of glands deep in the endometrial basalis. Similarly, SSEA-1+ basalis epithelial progenitors pirated their marker from mature neutrophils and differentiating human pluripotent stem cells. In mice the stem/progenitor cells like to play chase, with lineage tracing of individual genetically marked cells revealing their location in the intersection zone of the glands and luminal epithelium, and also in the gland bases (Axin2+ and Lgr5+). The identity of eMSCs has also been determined by discovery of specific markers, but even here the eMSC play games in human endometrium where sometimes they are pericytes (CD140b and CD146 double positive cells), sometimes perivascular cells (SUSD2+) and sometimes CD34+ cells in the adventitia of blood vessels. They are also adventitial perivascular cells in ovine endometrium, but this time they are CD271+. Mature endometrial stromal cell progeny are also naughty, often pretending to be eMSC, particularly when shed into menstrual fluid, confusing many of their status. Adding further to their misbehaviour, they express the same official MSC surface markers. To get even immature endometrial MSC strike back, claiming immunomodulatory properties in attempt to upstage their mature stromal progeny, also endowed with these properties. Finally, other endometrial cells such as macrophages may also be naughty as their mischievousness in evading detection can trick us to consider them as stem cells from the bone marrow, masquerading as endometrial epithelial or stromal cells. Naughty implies behaving badly and I will show data suggesting that stem/progenitor cells may escape the endometrium to cause a nasty disease, endometriosis. They may also become wayward and unruly, invading the myometrium to form adenomyosis. Some naughty epithelial progenitors defiantly pick up mutations to become cancer stem cells and initiate endometrial cancer. They may also malfunction because they do not obey estrogen signalling instructions, failing to proliferate and causing thin unresponsive endometrium. In their naughtiness, they may run away or get totally lost, thereby resulting in Asherman’s syndrome. Therefore, for numerous reasons, stem/progenitor cells are the naughty cells of the endometrium. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

In Vitro Implantation Model Using Human Endometrial SUSD2+ Mesenchymal Stem Cells and Myometrial Smooth Muscle Cells.

Objective This study evaluated a novel in vitro implantation model using human endometrial mesenchymal stem cells (EMSCs), SUSD2+, and myometrial smooth muscle cells (SMCs) that were co-cultured with mouse blastocysts as the surrogate embryo. Materials and Methods In this experimental study, SUSD2+ MSCs were isolated from human endometrial cell suspensions (ECS) at the fourth passage by magnetic-activated cell sorting. The ECS and SUSD2+ cells were separately co-cultured with human myometrial muscle cells for five days. After collection of mouse blastocysts, the embryos were placed on top of the co-cultured cells for 48 hours. The interaction between the embryo and the cultured cells was assessed morphologically at the histological and ultrastructural levels, and by expression profiles of genes related to implantation. Results Photomicrographs showed that trophoblastic cells grew around the embryonic cells and attached to theECS and SUSD2+ cells. Ultrastructural observations revealed pinopode and microvilli-like structures on the surfaces of both the ECS and SUSD2+ cells. Morphologically, the embryos developed to the egg-cylinder stage in both groups. Gene expression analysis showed no significant differences between the two groups in the presence of an embryo, but an increased expression of αV was detected in SUSD2+ cells compared to ECS cells in the absence of an embryo. ConclusionThis study showed that SUSD2+ cells co-cultured with SMCs could interact with mouse embryos. The co-cultured cells could potentially be used as an implantation model.

Human endometrial mesenchymal stem cells: cellular and exosome phenotype, and clinical prospects

Human endometrial mesenchymal stem cells: cellular and exosome phenotype, and clinical prospects

Presence of Clustered GM1 Ganglioside in the Membrane of Endometrial Mesenchymal Stem Cells is Dependent on Cell Cycle Stage

Gangliosides, representing the group of sialic glycosphingolipids, are typical components of lipid rafts of cell membranes and play an important role in the processes of reception and signal transduction. Gangliosides attract specific attention as possible regulators of directed differentiation of mesenchymal stem cells (MSCs). GM1 ganglioside is considered as a marker of lipid microdomains, and fluorescence labeling of clusters of GM1 molecules is one of the main methods for assessing the integrity or destruction of rafts in various types of cells. At the same time, data on the presence and possibility of determining GM1 ganglioside in the plasma membrane of MSCs are extremely limited. The objective of this work was to identify the ganglioside GM1 in the membrane of human endometrial MSC (eMSC) using an experimental approach based on interaction with the pentamer of the beta subunits of cholera toxin (CTB). Fluorescence staining of cells using FITC-CTB conjugate revealed the heterogeneity of the eMSC culture by the presence of GM1 membrane clusters. Experiments on synchronized culture have shown that the amount of clustered GM1 in the eMSC plasma membrane depends on the phase of the cell cycle: it is maximum when the cells are stopped at the G0/G1 phase boundary and decreases when proliferation starts, reaching a minimum in the G2/M phases. The data suggest the functional role of GM1-containing lipid rafts in cell cycle of eMSCs and possible changes in the mechanical properties of the membrane before cell division.

Chondrogenic differentiation followed IGFBP3 loss in human endometrial mesenchymal stem cells

Insulin-like growth factor binding protein 3 (IGFBP3) is a multifunctional protein, able either to stimulate the cell growth or to promote apoptosis. In particular, IGFBP3 plays significant role in propagation of stress-induced senescence in human endometrium-derived mesenchymal stem cells (MESCs) (Vassilieva et al., 2020). We undertook CRISPR/Cas9-mediated IGFBP3 knockout in an effort to decelerate stress-induced senescence in MESCs, but, unexpectedly, IGFBP3-knockout MESCs culture acquired chondrocyte-like features, such as cell condensation and aggregation. We revealed that IGFBP3-knockout MESCs completely lost CD73 and CD90 MESCs positive surface markers, and significantly decreased expression of CD105 and CD146 MESCs positive surface markers. In addition, we found IGFBP3-knockout MESCs aggregates positively stained for Alcian Blue. We also detected expression of collagen type II in IGFBP3-knockout MESCs. The obtained results indicate that MESCs lost stemness after IGFBP3-knockout and underwent differentiation toward chondrogenic lineage. Our findings can enlighten IGFBP3 role in regulation of MESCs chondrogenesis.

Increased Migration Ability of Adenomyosis-Derived Endometrial Mesenchymal Stem Cells

Adenomyosis is a form of endometriosis—a gynecological disease associated with abnormal functional activity of endometrial cells. Endometrial stem cells can play a key role in the pathogenesis of this disease. Despite the numerous studies that have been conducted of cultures of endometrial mesenchymal stem cells (eMSCs) obtained from patients with adenomyosis, information on their phenotypic and functional properties is very contradictory. In this work, a comparative study of morphological and migratory characteristics of human endometrial mesenchymal stem cells isolated from the menstrual blood of healthy donors (eMSCs) and a donor with adenomyosis (eMSCs-A) was performed. eMSC migration was evaluated by the method of “wound healing” using live-cell microscopy. It was established that the rate of wound healing of eMSCs-A is significantly higher compared to normal cells, which indicates an increased migration potential of eMSCs-A in adenomyosis. However, when transferring cells to a serum-free medium, eMSC-A migrated more slowly than normal cells. As a result of the assessment of morphological characteristics, it was found that eMSCs-A are smaller (in area and perimeter) than normal eMSCs, while the remaining morphometric parameters reflecting cell polarization did not differ. The data obtained allow the use of eMSCs in culture as a model for elucidating the membrane and intracellular mechanisms that underlie changes in cellular mechanics, motility, and invasive activity in various pathologies, including adenomyosis.

Paracrine senescence of human endometrial mesenchymal stem cells: a role for the insulin-like growth factor binding protein 3.

Stress-induced premature cell senescence is well recognized to be accompanied by emerging the senescence-associated secretory phenotype (SASP). Secreted SASP factors can promote the senescence of normal neighboring cells through autocrine/paracrine pathways and regulate the senescence response, as well. Regarding human endometrium-derived mesenchymal stem cells (MESCs), the SASP regulation mechanisms as well as paracrine activity of senescent cells have not been studied yet. Here, we examined the role of insulin-like growth factor binding protein 3 (IGFBP3) in the paracrine senescence induction in young MESCs. The H2O2-induced premature senescence of MESCs led to increased IGFBP3 in conditioned media (CM). The inhibitory analysis of both MAPK and PI3K signaling pathways showed that IGFBP3 releasing from senescent cells is mainly regulated by PI3K/Akt pathway activity. IGFBP3 appears to be an important senescence-mediating factor as its immunodepletion from the senescent CM weakened the pro-senescent effect of CM on young MESCs and promoted their growth. In contrast, young MESCs acquired the senescence phenotype in response to simultaneous addition of recombinant IGFBP3 (rIGFBP3). The mechanism of extracellular IGFBP3 internalization was also revealed. The present study is the first to demonstrate a significant role of extracellular IGFBP3 in paracrine senescence induction of young MESCs.

61: Derivation of Extracellular Vesicles In Vitro from Human Endometrial Mesenchymal Stem Cells with Potential for the Treatment Neurodegenertive Diseases

61: Derivation of Extracellular Vesicles In Vitro from Human Endometrial Mesenchymal Stem Cells with Potential for the Treatment Neurodegenertive Diseases

Human amniotic epithelial cells improve fertility in an intrauterine adhesion mouse model

BackgroundIntrauterine adhesion (IUA) is an adhesion of the uterine cavity or cervical canal resulting from damage to the basal layer of the endometrium; this condition is usually accompanied by fibrosis of the endometrium. Previous studies have demonstrated that human amniotic epithelial cells (hAECs) have stem cell characteristics; however, it is unclear whether hAECs have the therapeutic potential to restore fertility after IUA.MethodsA murine IUA model was established by mechanical injury to the uterus. Then, 106 hAECs were transplanted by intraperitoneal injection. The endometrium thickness, number of glands, and fibrosis area were measured by hematoxylin and eosin (H&E) staining and Masson staining. Molecules (including vWF, VEGF, PCNA, ER, PR, LC3, and p62) related to endometrial angiogenesis, cell proliferation, and autophagy were assayed by IHC staining. Pregnancy outcomes were also evaluated. Finally, hAECs were cocultured with human endometrial mesenchymal stem cells (hEnSCs) damaged by H2O2 to verify the paracrine effect on endometrial stromal cells in vitro.ResultsThe IUA uterine cavity presented with adhesion and even atresia, accompanied by a thinner endometrium, fewer glands, increased fibrosis area, and fewer microvessels. However, hAECs significantly improved the uterine structure after IUA. After hAEC treatment, the endometrium was thicker, the number of endometrial glands was increased, fibrosis was reduced, and more microvessels were generated. The expression levels of VEGF, PCNA, and ER were increased in the hAEC-treated endometrium, indicating improvements in angiogenesis and stromal cell proliferation. hAECs also increased pregnancy outcomes in IUA mice, and the pregnancy rate and fetus number increased. Furthermore, we observed altered autophagy in the IUA uterine model, and hAEC transplantation upregulated autophagy. An in vitro study showed that hAECs activated autophagy in (hEnSCs) treated with H2O2 in a paracrine manner.ConclusionsOur results demonstrated that hAECs have the potential to repair the uterus after injury, providing a new strategy for the prevention and treatment of Asherman syndrome.

Optimization of lentiviral transduction parameters and its application for CRISPR-based secretome modification of human endometrial mesenchymal stem cells

ABSTRACTMesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.

The effects of human menstrual blood stem cells-derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure.

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy-induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty-four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF-induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI-labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI-labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.

The Transcriptome of Human Endometrial Mesenchymal Stem Cells Under TGFβR 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-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.

Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice

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-IL2Rgammanull) 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.