Qiuwan Zhang

Skin-Derived Mesenchymal Stem Cells Help Restore Function to Ovaries in a Premature Ovarian Failure Mouse Model

Skin-derived mesenchymal stem cells (SMSCs) can differentiate into the three embryonic germ layers. For this reason, they are considered a powerful tool for therapeutic cloning and offer new possibilities for tissue therapy. Recent studies showed that skin-derived stem cells can differentiate into cells expressing germ-cell specific markers in vitro and form oocytes in vivo. The idea that SMSCs may be suitable for the treatment of intractable diseases or traumatic tissue damage has attracted attention. To determine the ability of SMSCs to reactivate injured ovaries, a mouse model with ovaries damaged by busulfan and cyclophosphamide was developed and is described here. Female skin-derived mesenchymal stem cells (F-SMSCs) and male skin-derived mesenchymal stem cells (M-SMSCs) from red fluorescence protein (RFP) transgenic adult mice were used to investigate the restorative effects of SMSCs on ovarian function. Significant increases in total body weight and the weight of reproductive organs were observed in the treated animals. Both F-SMSCs and M-SMSCs were shown to be capable of partially restoring fertility in chemotherapy-treated females. Immunostaining with RFP and anti-Müllerian hormone (AMH) antibodies demonstrated that the grafted SMSCs survived, migrated to the recipient ovaries. After SMSCs were administered to the treated mice, real-time PCR showed that the expression levels of pro-inflammatory cytokines TNF-α, TGF-β, IL-8, IL-6, IL-1β, and IFNγ were significantly lower in the ovaries than in the untreated controls. Consistent with this observation, expression of oogenesis marker genes Nobox, Nanos3, and Lhx8 increased in ovaries of SMSCs-treated mice. These findings suggest that SMSCs may play a role within the ovarian follicle microenvironment in restoring the function of damaged ovaries and could be useful in reproductive health.

The Paracrine Effect of Transplanted Human Amniotic Epithelial Cells on Ovarian Function Improvement in a Mouse Model of Chemotherapy-Induced Primary Ovarian Insufficiency.

Human amnion epithelial cells (hAECs) transplantation via tail vein has been reported to rescue ovarian function in mice with chemotherapy-induced primary ovarian insufficiency (POI). To test whether intraperitoneally transplanted hAECs could induce therapeutic effect and to characterize the paracrine effect of transplanted hAECs, we utilized a chemotherapy induced mice model of POI and investigated the ability of hAECs and conditioned medium collected from cultured hAECs (hAECs-CM) to restore ovarian function. We found that transplantation of hAECs or hAECs-CM either 24 hours or 7 days after chemotherapy could increase follicle numbers and partly restore fertility. By PCR analysis of recipient mice ovaries, the presence of SRY gene was only detected in mice transplanted with male hAECs 24 hours following chemotherapy. Further, the gene expression level of VEGFR1 and VEGFR2 in the ovaries decreased, although VEGFA increased 2 weeks after chemotherapy. After treatment with hAECs or hAEC-CM, the expression of both VEGFR1 and VEGFR2 increased, consistent with the immunohistochemical analysis. In addition, both hAECs and hAECs-CM treatment enhanced angiogenesis in the ovaries. The results suggested that hAECs-CM, like hAECs, could partly restore ovarian function, and the therapeutic function of intraperitoneally transplanted hAECs was mainly induced by paracrine-mediated ovarian protection and angiogenesis.

Human amniotic epithelial cells inhibit granulosa cell apoptosis induced by chemotherapy and restore the fertility

IntroductionPremature ovarian failure and insufficiency are significant long-term side-effects of chemotherapy for female cancer patients. Recently, stem cell transplantation has been identified as a promising treatment for premature ovarian failure and insufficiency. We have previously demonstrated that human amniotic epithelial cells (hAECs) migrate into injured tissue and promote the recovery of ovarian function in chemoablated mice. However, the molecular mechanism guiding this process remains unclear.MethodsTo further investigate the effect of hAECs on chemotherapy-induced apoptosis, cultured primary hAECs were injected intravenously into mice treated with cyclophosphamide and busulphan. Apoptosis of granulosa cells was observed by TUNEL staining, and apoptosis-related gene expression was performed on ovarian tissue by real-time PCR and Western blot 7 days after hAEC transplantation. Additionally, the ovarian function and fertility of mice were assessed via counts of follicles and mating experiments at 4 weeks after hAEC transplantation.ResultshAECs significantly inhibited tumor necrosis factor-alpha-mediated granulosa cell apoptosis induced by chemotherapeutics and reduced the inflammatory reaction in ovaries at 7 days after transplantation. In addition, 4 weeks after transplantation, hAECs promoted the development of follicles and increased the number of cumulus oocyte complexes in chemoablated mice. Furthermore, hAECs improved ovarian mass and increased the number of follicles compared to those of the chemoablated group, and hAEC transplantation partially rescued the fertility of chemoablated mice.ConclusionshAEC transplantation promotes ovarian function by inhibiting tumor necrosis factor-alpha-mediated cell apoptosis and reducing inflammation in chemotherapy-induced premature ovarian failure. These results suggest a potential molecular mechanism for the effective therapy of hAEC transplantation in chemotherapy-induced premature ovarian failure and insufficiency.

Melatonin ameliorates restraint stress-induced oxidative stress and apoptosis in testicular cells via NF-κB/iNOS and Nrf2/ HO-1 signaling pathway

Decline in semen quality has become a global public health concern. Psychological stress is common in the current modern society and is associated with semen decline. Increasing evidence demonstrated that melatonin has anti-apoptotic and antioxidant functions. Whether melatonin can ameliorate the damage in testes induced by psychological stress has never been investigated. Here, a mouse model of restraint stress demonstrated that melatonin normalized the sperm density decline, testicular cells apoptosis, and testicular oxidative stress in stressed male mice. Melatonin decreased reactive oxygen species (ROS) level, increased superoxide dismutase (SOD) and glutathione (GSH) activities, and downregulated inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) activities in stressed mice testes. Furthermore, melatonin reduced the stress-induced activation of the NF-κB signaling pathway by decreasing the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and p65 nuclear translocation. In addition, melatonin upregulated the expression of anti-oxidant proteins including nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Meanwhile, in vitro studies also demonstrated melatonin could reduce oxidative apoptosis of testicular cells. Collectively, melatonin mitigated psychological stress-induced spermatogenic damage, which provides evidence for melatonin as a therapy against sperm impairment associated with psychological stress.

Human endometrial mesenchymal stem cells exhibit intrinsic anti-tumor properties on human epithelial ovarian cancer cells

Epithelial ovarian cancer (EOC) is the most lethal tumor of all gynecologic tumors. There is no curative therapy for EOC thus far. The tumor-homing ability of adult mesenchymal stem cells (MSCs) provide the promising potential to use them as vehicles to transport therapeutic agents to the site of tumor. Meanwhile, studies have showed the intrinsic anti-tumor properties of MSCs against various kinds of cancer, including epithelial ovarian cancer. Human endometrial mesenchymal stem cells (EnSCs) derived from menstrual blood are a novel source for adult MSCs and exert restorative function in some diseases. Whether EnSCs endow innate anti-tumor properties on EOC cells has never been reported. By using tumor-bearing animal model and ex vivo experiments, we found that EnSCs attenuated tumor growth by inducing cell cycle arrest, promoting apoptosis, disturbing mitochondria membrane potential and decreasing pro-angiogenic ability in EOC cells in vitro and/or in vivo. Furthermore, EnSCs decreased AKT phosphorylation and promoted nuclear translocation of Forkhead box O-3a (FoxO3a) in EOC cells. Collectively, our findings elucidated the potential intrinsic anti-tumor properties of EnSCs on EOC cells in vivo and in vitro. This research provides a potential strategy for EnSC-based anti-cancer therapy against epithelial ovarian cancer.

Identification and characterization of epithelial cells derived from human ovarian follicular fluid

IntroductionFollicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program.MethodsFollicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography.ResultsEpithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues.ConclusionsThe epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine.

Chronic restraint stress induces excessive activation of primordial follicles in mice ovaries

Chronic stress is an important factor influencing people’s health. It usually causes endocrinal disorders and a decline in reproduction in females. Although studies of both human and animals suggest a detrimental effect of stress on reproduction, the influence of chronic stress on the ovarian reservation and follicular development is still not clear. In this study, a chronic restraint stress (CRS) mouse model was used to investigate the effect of stress on ovarian reservation and follicular development and explore the underlying mechanism. In this study, after 8 weeks of CRS, primordial follicles were excessively activated in the ovaries of the CRS group compared with the control group. Further results showed that the activation of primordial follicles induced by CRS was involved in the increasing expression level of Kit ligand and its receptor Kit and the activation of phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) pathway. The corticotropin-releasing hormone (CRH) is a neuropeptide released due to stress, which plays an important role in regulating follicle development. A high level of serum CRH was detected in the CRS mouse model, and the real-time polymerase chain reaction assay showed that the mRNA level of its main receptor CRHR1increased in the ovaries of the CRS mouse group. Moreover, 100nM CRH significantly improved the activation of primordial follicles in newborn mouse ovaries in vitro. These results demonstrated that CRS could induce immoderate activation of primordial follicles accompanied by the activation of Kit-PI3K signaling, in which CRH might be an important endocrine factor.

Chronic restraint stress disturbs meiotic resumption through APC/C-mediated cyclin B1 excessive degradation in mouse oocytes

ABSTRACT Psychological stress, which exerts detrimental effects on human reproduction, may compromise the meiotic competence of oocytes. Meiotic resumption, germinal vesicle breakdown (GVBD), is the first milestone to confer meiotic competence to oocytes. In the practice of assisted reproductive technology (ART), the timing for GVBD is associated with the rates of cleavage and blastocyst formation. However, whether chronic stress compromises oocyte competence by influencing GVBD and the underlying mechanisms are unclear. In the present study, a chronic restraint stress (CRS) mouse model was used to investigate the effects of stress on oocyte meiotic resumption, as well as the mechanisms. Following a 4-week chronic restraint stress in female mice, the percentage of abnormal bipolar spindles increased and indicated compromised oocyte competence in the CRS group. Furthermore, we identified a decreased percentage of GVBD and prolonged time of GVBD in the CRS mouse oocytes compared with the control group. CRS simultaneously reduced the expression of cyclin B1 (CCNB1), which represents a regulatory subunit of M-phase/mature promoting factor (MPF). However, MG132, an inhibitor of anaphase-promoting complex/cyclosome (APC/C), could rescue the prolonged time of GVBD and increase the expression level of CCNB1 of oocytes from the CRS mice. Collectively, our results demonstrated that stress disturbed meiotic resumption through APC/C-mediated CCNB1 degradation, thus providing a novel understanding for stress-related oocyte quality decline; moreover, it may provide a non-invasive approach to select high-quality gametes and novel targets for molecular therapy to treat stress-related female infertility.

Human amniotic epithelial cells inhibit growth of epithelial ovarian cancer cells via TGF‑β1-mediated cell cycle arrest

It is reported that human amniotic epithelial cells (hAECs) endow intrinsic antitumor effects on certain kinds of cancer. This research was designed to evaluate whether hAECs endowed potential anticancer properties on epithelial ovarian cancer (EOC) cells in vivo and in vitro, which has not been reported before. In this study, we established a xenografted BALB/c nude mouse model by subcutaneously co-injecting ovarian cancer cell line, SK-OV-3, and hAECs for 28 days. In ex vivo experiments, CCK-8 cell viability assay, real-time PCR, cell counting assay, cell cycle analysis and immunohistochemistry (IHC) assay were used to detect the effects of hAEC-secreted factors on the proliferation and cell cycle progression of EOC cells. A cytokine array was conducted to detect anticancer-related cytokines released from hAECs. Human recombinant TGF-β1 and TGF-β1 antibody were used to treat EOC cells and analyzed whether TGF-β1 contributed to the cell cycle arrest. Results from in vivo and ex vivo experiments showed that hAEC-secreted factors and rhTGF-β1 decreased proliferation of EOC cells and induced G0/G1 cell cycle arrest in cancer cells, which could be partially reversed by excess TGF-β1 antibody. These data indicate that hAECs endow potential anticancer properties on epithelial ovarian cancer in vivo and in vitro which is partially mediated by hAEC-secreted TGF-β1-induced cell cycle arrest. This study suggests a potential application of hAEC-based therapy against epithelial ovarian cancer.

Epithelial ovarian cancer stem-like cells expressing α-gal epitopes increase the immunogenicity of tumor associated antigens

BackgroundAs ovarian cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and chemo-resistance, new stratagems that selectively target ovarian CSCs are critically significant. Our previous work have demonstrated that ovarian cancer spheroid cells are tumorigenic and chemo-resistant, and have the properties of ovarian CSCs. Herein, we hypothesized that expressing α-gal epitopes on ovarian spheroid cells may help eliminate CSCs and improve the outcome of therapeutic intervention for ovarian cancer patients.MethodsLentivirus-mediated transfer of a pig α(1,3)galactosyltransferase [α1,3GT] enzyme gene into human ovarian cell line SKOV3 cells formed α-gal epitope-expressing cells (SKOV3-gal cells), and then these cells were maintained in a serum-free culture system to form SKOV3-gal spheroid cells. Efficacy of this cell vaccine was demonstrated in α1,3GT knockout mice (α1,3GT KO mice).ResultsThe antibody titers to α-gal epitopes measured by ELISA were significantly increased in α1,3GT KO mice after immunization with SKOV3-gal spheroid cells. Furthermore, compared with the non-immunized KO mice, the SKOV3 tumors grafted under renal capsules of KO mice immunized with SKOV3-gal spheroid cells grew slower and began to shrink on day 12. Western blot analysis also showed that immunized KO mice can produce effective antibody against certain tumor associated antigens (TAAs) derived from both SKOV3 cells and SKOV3 spheroid cells. The TAAs were further investigated by mass spectrometry and RNA interference (RNAi) technology. The results suggested that antibodies responding to protein c-erbB-2 may be raised in the sera of the mice after immunization with SKOV3-gal spheroid cells. Ultimately, vaccination with SKOV3-gal spheroid cells induced more CD3 + CD4 + T cells in the spleen of immunized mice than non-immunized KO mice.ConclusionsThe results suggest that vaccination using ovarian cancer stem-like cells engineered to express α-gal epitopes may be a novel strategy for treatment of ovarian cancer.