Therapeutic effect of human umbilical cord-derived mesenchymal stem cells on injured rat endometrium during its chronic phase.
ABSTRACT: Repair deficiency after endometrial injury is an important reason for intra-uterine adhesions, amenorrhea, and infertility in females. Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is effective in repairing the damaged endometrium. However, the possibility of using umbilical cord-derived MSCs (UC-MSCs) to treat endometrial injury is rarely reported.Ethanol (95%) was injected into rat uterus to establish a model of endometrial injury. UC-MSCs were injected through the tail vein, either as a single, twice, or thrice administration. Functional restoration of the uterus was assessed by testing embryo implantation rates. Endometrial morphological alteration was observed by hematoxylin and eosin staining. Endometrial fibrosis, markers of epithelial and stromal cells of endometrium, cell proliferation and angiogenesis, and inflammatory factors were detected using immunohistochemistry, Western blotting, and quantitative reverse-transcription polymerase chain reaction.Endometrial morphology and embryo implantation rates were significantly improved on day 8 of transplantation among single-, twice-, or thrice-administered rats. Moreover, UC-MSCs could alleviate fibrosis in general, and reduced the expression of fibrosis markers, ?-smooth muscle actin (?-SMA) and transforming growth factor (TGF)-?. The cell proliferation marker Ki-67 had a positive expression in the injured endometrium after UC-MSC transplantation. The endometrial stromal marker vimentin and epithelial marker cytokeratin-19 (CK-19) expressions were visibly increased. The expression of vascular markers CD31, vascular endothelial growth factor (VEGF)A, and matrix metalloprotein (MMP)9 was generally upregulated. Proinflammatory factors interferon (IFN)-?, tumor necrosis factor (TNF)-?, and interleukin (IL)-2 were significantly downregulated in the rats administered UC-MSCs twice and thrice.UC-MSC transplantation contributed to the repair of endometrial injury and restoration of fertility, likely through the suppression of excessive fibrosis and inflammation, and enhancement of endometrial cell proliferation and vascular remodeling.
Project description:Asherman's Syndrome is characterized by intrauterine adhesions or fibrosis resulting as a consequence of damage to the basal layer of endometrium and is associated with infertility due to loss of normal endometrium. We have previously shown that bone marrow derived stem cells (BMDSCs) engraft the endometrium in mice and humans and Ischemia/reperfusion injury of uterus promoted BMDSCs migration to the endometrium; however, the role of BMDSCs in Asherman's syndrome has not been characterized. Here a murine model of Asherman's syndrome was created by traumatizing the uterus. We evaluate stem cell recruitment and pregnancy after BMDSCs transplantation in a model of Asherman's syndrome. In the Asheman's syndrome model, after BMDSC transplant, the Y chromosome bearing CD45-cells represented less than 0.1% of total endometrial cells. Twice the number of Y+CD45- cells was identified in the damaged uterus compared to the uninjured controls. There was no significant difference between the damaged and undamaged uterine horns in mice that received injury to a single horn. In the BMDSC transplant group, 9 of the 10 mice conceived, while only 3 of 10 in the non-transplanted group conceived (Chi-Square p?=?0.0225); all mice in an uninjured control group conceived. The time to conception and mean litter size were not different between groups. Taken together, BMDSCs are recruited to endometrium in response to injury. Fertility improves after BMDSC transplant in Asherman's Syndrome mice, demonstrating a functional role for these cells in uterine repair. BMDSC transplantation is a potential novel treatment for Asherman's Syndrome and may also be useful to prevent Asherman's syndrome after uterine injury.
Project description:BACKGROUND:Intrauterine adhesion (IUA) is a major cause of female secondary infertility. We previously demonstrated that menstrual blood-derived stromal cell (MenSC) transplantation helped severe IUA patients have pregnancy and endometrium regeneration. We also initiated platelet-rich plasma (PRP) acted as a beneficial supplement in MenSC culturing and a potential endometrial receptivity regulator. Here, we investigated the therapeutic effect of combined transplantation of MenSCs with PRP in rat IUA models and the mechanisms of MenSCs in endometrium regeneration. METHODS:Rat IUA models were established by intrauterine mechanical injured. Nine days later, all rats were randomly assigned to four groups received different treatment: placebo, MenSC transplantation, PRP transplantation, and MenSCs + PRP transplantation. The traces of MenSCs were tracked with GFP label. Endometrial morphology and pathology, tissue proliferation, inflammation, pregnancy outcomes, and mechanism of MenSCs in the regeneration of endometrium were investigated. RESULTS:Notably, at days 9 and 18 post-treatment, MenSC transplantation significantly improved endometrial proliferation, angiogenesis, and morphology recovery and decreased collagen fibrosis and inflammation in the uterus. MenSCs had lesion chemotaxis, colonized around the endometrial glands. Gene expression of human-derived secretory protein IGF-1, SDF-1, and TSP-1 was detected in the uterus received MenSCs at day 18. The three treatments can all improve fertility in IUA rats. Moreover, gene expressions of cell proliferation, developmental processes, and other biological processes were induced in MenSC transplantation group. Hippo signaling pathway was the most significantly changed pathway, and the downstream factors CTGF, Wnt5a, and Gdf5 were significantly regulated in treatment groups. PRP enhanced these parameters through a synergistic effect. CONCLUSIONS:In summary, MenSCs could effectively improve uterine proliferation, markedly accelerate endometrial damage repairment and promote fertility restoration in IUA rats, suggesting a paracrine restorative effect and Hippo signaling pathway stimulation. Our results indicate MenSCs, a valuable source of cells for transplantation in the treatment intrauterine adhesion. Combined with PRP, this cell therapy was more effective.
Project description:BACKGROUND:Tissue regeneration disorder after endometrial injury is an important cause of intrauterine adhesions, amenorrhea, and infertility in women. Both bone marrow mesenchymal stem cell (BMSC) transplantation and electroacupuncture (EA) are promising therapeutic applications for endometrial injury. This study examined their combined effects on thin endometrium in rats and the possible mechanisms underlying these effects. METHODS:A thin endometrial model was established in Sprague-Dawley (SD) rats by perfusing 95% ethanol into the right side of the uterus. The wounds were randomly treated with PBS (model group), BMSCs only (BMSC group), EA only (EA group), and BMSCs combined with EA (BMSC + EA group). Endometrial morphological alterations were observed by hematoxylin and eosin (H&E) staining. Changes in markers of epithelial and stromal endometrium cells, endometrial receptivity-related chemokines, and paracrine factors were detected using immunohistochemistry, western blotting, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Finally, the functional recovery of the uterus was evaluated by determining the rate of embryo implantation. RESULTS:As shown by endometrial morphology, the damaged uteri in all the treatment groups recovered to some extent, with the best effects observed in the BMSC + EA group. Further studies showed that EA promoted the migration of transplanted BMSCs to damaged uteri by activating the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) axis. As compared with the other groups, upregulated expression of endometrial cytokeratin and vimentin, increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in endometrial lesions, and improved embryo implantation rates on the 8th day of pregnancy were found in the BMSC + EA group. CONCLUSIONS:EA plays an important role in supporting BMSCs in the repair of thin endometrium, most likely by promoting the migration of BMSCs and enhancing the paracrine effect of BMSCs.
Project description:Severe injuries of the uterus may trigger uterine scar formation, ultimately leading to infertility or obstetrical complications. To date, few methods have adequately solved the problem of collagen deposition in uterine scars. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have shown great promise in clinical applications. The objective of this study was to investigate the effect of a scaffold/UC-MSCs construct on collagen degradation and functional regeneration in rat uterine scars following full-thickness excision of uterine walls.In order to establish a rat model of uterine scars, the uterine wall of approximately 1.0 cm in length and 0.5 cm in width (one-third of the uterine circumference) was excised from each uterine horn. A total of 128 scarred uterine horns from 64 rats were randomly assigned to four groups, including a PBS group (n?=?32 uterine horns), scaffold group (n?=?32 uterine horns), UC-MSCs group (n?=?32 uterine horns) and scaffold/UC-MSCs group (n?=?32 uterine horns) to investigate the effect of different treatments on the structure and function of uterine scars. PBS, degradable collagen fibres, UC-MSCs or UC-MSCs mixed with gelatinous degradable collagen fibres were injected into four pre-marked points surrounding each uterine scar, respectively. At days 30 and 60 post-transplantation, a subset of rats (n?=?8 uterine horns) from each group was euthanized and serial sections of uterine tissues containing the operative region were prepared. Haematoxylin-eosin staining, Masson's trichrome staining, and immunohistochemical staining for MMP-2, MMP-9, ?-SMA and vWF were performed. Finally, another subset of rats (n?=?16 uterine horns) from each group was mated with male rats at day 60 post-transplantation and euthanized 18 days after the presence of vaginal plugs to check numbers, sizes and weights of fetuses, as well as sites of implantation.The scaffold/UC-MSCs group exhibited obvious collagen degradation compared with the other three groups. At day 60 post-transplantation, the number of MMP-9-positive cells in the scaffold/UC-MSCs group (25.96?±?3.63) was significantly higher than that in the PBS group (8.19?±?1.61, P?<?0.01), the scaffold group (7.25?±?2.17, P?<?0.01) and the UC-MSCs group (8.31?±?2.77, P?<?0.01). The pregnancy rate in the scaffold/UC-MSCs group (10/16) was also significantly higher than that in the PBS group (2/16, P?<?0.017), the scaffold group (1/16, P?<?0.017) and the UC-MSCs group (3/16, P?<?0.017).The scaffold/UC-MSCs system facilitated collagen degradation in uterine scars via upregulation of MMP-9, which was secreted by transplanted UC-MSCs, and promoted regeneration of the endometrium, myometrium and blood vessels in uterine scars. Furthermore, the scaffold/UC-MSCs-treated uterine scars showed nearly complete restoration of receptive fertility.
Project description:As a refractory fibrosis disease, intrauterine adhesions (IUAs) is defined as fibrosis of the physiological endometrium. Although hysteroscopic adhesiolysis is widely recommended as an effective treatment, prognosis and recurrence remain poor in severe cases. Recently, stem cell therapy has been promoted as a promising treatment for IUAs. The ability of human amniotic epithelial cells (hAECs), emerging as a new candidate for stem cell therapy, to treat IUAs has not been demonstrated. To study the potential effects of hAECs on IUAs, we created an IUA rat model using mechanical injury and injected cultured primary hAECs into the rats' uteri. Next, we observed the morphological structure of endometrial thickness and glands using hematoxylin and eosin staining, and we detected extracellular-matrix collagen deposition using Masson staining. In addition, we performed immunohistochemical staining and reverse-transcription polymerase chain reaction (RT-PCR) to investigate potential fibrosis molecules and angiogenesis factors 7 d after hAECs transplantation. Finally, we detected estrogen receptor (ER) and growth factors via RT-PCR to verify the molecular mechanism underlying cell therapy. In the IUA rat models, endometrial thickness and endometrial glands proliferated and collagen deposition decreased significantly after hAEC transplantation. We found that during the recovery of injured endometrium, the crucial fibrosis marker transforming growth factor-? (TGF-?) was regulated and angiogenesis occurred in the endometrial tissue with the up-regulation of vascular endothelial growth factor. Furthermore, hAECs were shown to promote ER expression in the endometrium and regulate the inflammatory reaction in the uterine microenvironment. In conclusion, these results demonstrated that hAEC transplantation could inhibit the progression of fibrosis and promote proliferation and angiogenesis in IUA rat models. The current study suggests hAECs as a novel stem cell candidate in the treatment of severe IUA.
Project description: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.
Project description:Transplantation of umbilical cord mesenchymal stem cells (UC-MSCs) is currently considered a novel therapeutic strategy for diabetic nephropathy (DN). However, the mechanisms by which UC-MSCs ameliorate renal fibrosis in DN are not well understood. Herein, we firstly investigated the therapeutic effects of mouse UC-MSC infusion on kidney structural and functional impairment in streptozotocin- (STZ-) induced diabetic mice. We found that the repeated injection with mUC-MSCs alleviates albuminuria, glomerulus injury, and fibrosis in DN mouse models. Next, mesangial cells were exposed to 5.6?mM glucose, 30?mM glucose, or mUC-MSC-conditioned medium, and then we performed western blotting, immunofluorescence, wound healing assay, and cell proliferation assay to measure extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs), myofibroblast transdifferentiation (MFT), and cell proliferation. We demonstrated that mUC-MSC paracrine decreased the deposition of fibronectin and collagen I by inhibiting TGF-?1-triggered MFT and cell proliferation mediated by PI3K/Akt and MAPK signaling pathways, and elevating the levels of MMP2 and MMP9. Importantly, we provided evidence that the antifibrosis role of mUC-MSC paracrine in DN might be determined by exosomes shed by MSCs. Together, these findings reveal the mechanisms underlying the therapeutic effects of UC-MSCs on renal fibrosis in DN and provide the evidence for DN cell-free therapy based on UC-MSCs in the future.
Project description:BACKGROUND:Intrauterine adhesion (IUA) is characterized by progressive intrauterine fibrosis as a consequence of trauma to the basal layer of the endometrium. In an attempt to relieve IUA, many approaches have been applied in the clinic but show limited effects. In this study, we investigated the effect of autologous oral mucosal epithelial cells (OMECs) seeded on decellularized and lyophilized amniotic membrane (DL-AM) on preventing the development of IUA in a rat model. METHODS:IUA model was established by surgical scraping of the endometrium in the left uteri (the right uteri were kept as control) of SD rats. Wounds were randomly treated as unrepaired (IUA group), repaired with DL-AM alone (DL-AM group), and DL-AM seeded with autologous OMECs (DL-AM+OMECs group), respectively, in a total of 54 rats (n = 18 each). Uterus samples were harvested for histological and immunohistochemical evaluation after 3, 7, 14, and 28 days (n = 3 in each time point) of operations. RESULTS:After surgery, the uterine cavity was observed to be filled with extensive fibrosis in the IUA and DL-AM groups, respectively, while a lower ratio of the fibrotic area was identified in the DL-AM transplantation group. Transplantation of OMECs seeded on DL-AM significantly reduced fibrosis of IUA with recovered uterine cavity and regenerated epithelium and endometrial glands as determined by CK-18 immunostaining. OMECs transplantation resulted in extensive cellular proliferation as revealed by the Ki-67 immunofluorescent staining exhibited. Meanwhile, microvessel density was significantly increased in uteri that received OMECs transplantation, which was concomitant with elevated expression of vascular endothelial growth factor. The pregnancy test (n = 6 in each group) showed successful conception in the OMEC-transplanted uteri, but not in the IUA and DL-AM groups. CONCLUSIONS:Engineered epithelium developed from DL-AM seeded with OMECs showed great potential in preventing progression of intrauterine adhesion by improved endometrial epithelium regeneration.
Project description:BACKGROUND:Endometriosis, characterized by the presence of functional endometrial tissues outside the uterus, is one of the most common gynecological disorders. Endometrial mesenchymal stem cells (MSCs) are crucial for the occurrence and development of endometriosis. Ectopic endometrial MSCs exist in the peritoneal cavity. Thus, the bioactive factors in endometriotic peritoneal fluid may regulate the biological behaviors of endometrial MSCs. METHODS:In this study, after assessing the concentration of Activin A in peritoneal fluid using ELISA, we isolated and cultured endometrial MSCs and investigated whether Activin A stimulated endometrial MSCs to differentiate into myofibroblasts and clarified the underlying mechanisms by quantitative real-time PCR, Western blot analysis, immunofluorescent staining, RNA interference and Chromatin immunoprecipitation. We also employed the inhibitors of Activin A to explore the possibility of suppressing the development of fibrosis in endometriosis using primary endometrial MSCs cultures and a mouse model of endometriosis. RESULTS:Here, we revealed that Activin A significantly elevated in endometriotic peritoneal fluid and activin receptor-like kinase (ALK4), the specific receptor for Activin A, obviously enhanced in ectopic endometrial MSCs compared with eutopic endometrial MSCs from women with or without endometriosis. Next, we found that Activin A drived myofibroblast differentiation of endometrial MSCs, with extremely enhanced expression of connective tissue growth factor (CTGF). CTGF was shown to be required for Activin A-induced expression of ACTA2, COL1A1 and FN1 in endometrial MSCs. CTGF induction by Activin A in endometrial MSCs involved the activation of Smad2/3, as evidenced by the phosphorylation and nuclear translocation of Smad2/3 as well as the binding of Smad2/3 to CTGF promoter. Furthermore, Smad/CTGF pathway in endometrial MSCs required activation of STAT3 while independent of PI3K, JNK and p-38 pathways. In addition, we also demonstrated that inhibition of Activin A pathway impeded myofibroblast differentiation of endometrial MSCs and ameliorated fibrosis in endometriosis mice. CONCLUSIONS:Activin A promotes myofibroblast differentiation of endometrial mesenchymal stem cells via STAT3-dependent Smad/CTGF pathway. The results provided the first evidence that STAT3 acted as a crucial Activin A downstream mediator to regulate CTGF production. Our data may supplement the stem cell theory of endometriosis and provide the experimental basis to treat endometriosis-associated fibrosis by manipulating Activin A signaling.
Project description:BACKGROUND:Intrauterine adhesions (IUA) are the most common cause of uterine infertility and are caused by endometrium fibrotic regeneration following severe damage to the endometrium. Although current stem cell treatment options using different types of autologous stem cells have exhibited some beneficial outcomes in IUA patients, the reported drawbacks include variable therapeutic efficacies, invasiveness and treatment unavailability. Therefore, the development of new therapeutic stem cell treatments is critical to improving clinical outcomes. METHODS:Twenty-six patients who suffered from infertility caused by recurrent IUA were enrolled in this prospective, non-controlled, phase I clinical trial with a 30-month follow-up. During the procedure, 1?×?107 umbilical cord-derived mesenchymal stromal cells (UC-MSCs), loaded onto a collagen scaffold, were transplanted into the uterine cavity following an adhesion separation procedure. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and the biological molecules related to endometrial proliferation and differentiation were assessed both before and 3 months after cell therapy. RESULTS:No treatment-related serious adverse events were found. Three months after the operation, the average maximum endometrial thickness in patients increased, and the intrauterine adhesion score decreased compared to those before the treatment. A histological study showed the upregulation of ER? (estrogen receptor ?), vimentin, Ki67 and vWF (von Willebrand factor) expression levels and the downregulation of ?NP63 expression level, which indicates an improvement in endometrial proliferation, differentiation and neovascularization following treatment. DNA short tandem repeat (STR) analysis showed that the regenerated endometrium contained patient DNA only. By the end of the 30-month follow-up period, ten of the 26 patients had become pregnant, and eight of them had delivered live babies with no obvious birth defects and without placental complications, one patient in the third trimester of pregnancy, and one had a spontaneous abortion at 7 weeks. CONCLUSIONS:Transplanting clinical-grade UC-MSCs loaded onto a degradable collagen scaffold into the uterine cavity of patients with recurrent IUA following adhesiolysis surgery is a safety and effective therapeutic method. TRIAL REGISTRATION:Clinicaltrials.gov . NCT02313415 , Registered December 6, 2014.