Menstrual Stem Cells Alleviate Endometrial Fibrosis And Promote Endometrial Repair By Paracrine Mechanisms

PART ONE:Menstrual stem cells promote endometrial repairBackground and objective:Essential to embryo implantation and development, the endometrium is a key factor for successful pregnancy. A thin endometrium, resulting from endometrial injury, adhesions or absence, often results in implantation failure. Endometrial stem cells are responsible for the cyclic regeneration of the endometrial functionalis. A quantity researches have conferred that menstrual stem cells have enormous proliferation and differentiation ability, could restore liver functions for injured liver. However, the contribution of menstrual stem cells to endometrial repair has not been elucidated yet. In this work, we aimed to examine whether the menstrual stem cells are able to promote the proliferation and migration of damaged endometrial stromal cells, and alleviate the apoptosis of damaged stromal cells.Methods:Mifepristone was added to damage the endometrial stromal cells. The damaged endometrial stromal cells were cocultured with menstrual stem cells. The proliferation of endometrial stromal cells was investigated by Cell Counting Kit 8, and the apoptosis by annexin-V-fluorescein isothiocyanate binding. The protein expression of VEGF was determined by western blot.Results:The proliferation of endometrial stromal cells decreased after exposure to mifepristone in a dose-and time-dependent manner. And 25mg/1 concentration and 48 hours of mifepristone treatment with endometrial stromal cells were the best concentration and duration. After cocultured the damaged endometrial stromal cells with menstrual stem cells, the proliferation of damaged endometrial stromal cells was significantly increased and the apoptosis decreased. In addition, the protein level of VEGF increased after cocultured with menstrual stem cells.Conclusions:Using coculture system, it indicated that menstrual stem cells may serve as a promising treatment approach to ameliorate endometrial damage by paracrine mechanismsPART TWO:Menstrual stem cells alleviate endometrial fibrosis by paracrine effectBackground and objective:Intrauterine adhesions refer to trauma to the basal layer of the endometrium, which leads to partial or total adherence of endometrial surfaces with largely fibrous tissue and results in clinical manifestations such as reduced volume of menstrual fluid, amenorrhea and infertility. Although the chance of recurrent intrauterine adhesions has reduced after the use of hormone-therapy intrauterine devices and antibiotics, the management of moderate to severe intrauterine adhesions is still a challenge. Repeated surgery may be necessary and may not always obtain a desired outcome in some cases. Research in stem cell regenerative medicine has produced promising clinical trials, and indeed there are reports that transplantation of autologous bone marrow cells in restoration of the endometrial function in human intrauterine adhesions. Menstrual stem cells which have enormous proliferation and differentiation ability as bone marrow mesenchymal stem cells, have been conferred that could restore injured myocardial cells and ameliorate the fibrosis after myocardial infarction. However, the role and the mechanism of menstrual stem cells responsible for the prevention and reversal of intrauterine adhesions remain largely unknown. In this work, we aimed to examine whether the menstrual stem cells were able to mitigate the endometrial fibrosis, and hippo/TAZ signaling pathway involved in this progress.Methods:The endometrial stromal cells were cocultured with menstrual stem cells in transwell plates. Conditioned media from menstrual stem cells (MenSCs-CM) was prepared. The proliferation of endometrial stromal cells was investigated by Cell Counting Kit 8. The migration and motility of endometrial stromal cells were accessed by in vitro wound healing assay. The mRNA and protein expressions of aSMA and collagen Ⅰ were determined by real-time PCR and western blot. The protein expressions of phos-LATS1(1059), phos-MOB1 and phos-TAZ were determined by western blot. The localization of TAZ in endometrial stromal cells was detected by immunofluorescence.Results:Both MenSCs-CM and MenSCs coculture down-regulated the mRNA and protein expressions of aSMA and collagen Ⅰ in endometrial stromal cells, and promoted endometrial wound-healing. MenSCs-CM significantly promoted the proliferation of human stromal cells. Both MenSCs-CM and MenSCs coculture stimulated Hippo/TAZ signaling activity in stromal cells, up-regulated the protein expressions of phos-LATS1(1059), phos-MOB1 and phos-TAZ. Moreover, MenSCs-CM induced the TAZ translocate from nucleus to cytoplasm, and cytoplasm accumulation.Conclusions:We uncovered a paracrine role of menstrual stem cells in impeding endometrial stroma-myofibroblast transiton, and promoting stromal cells proliferation and wound healing. Hippo/TAZ signal pathway may be the one in those pathways activated by menstrual stem cells.PART THREE:Menstrual stem cells promote endometrial repair by alleviating TGFβ-innduced endometrial fibrosisBackground and objective:Intrauterine adhesions have become the second major cause of female secondary infertility. It is reported that 13% of infertility patients have intrauterine adhesions, and 43% of patients with intrauterine adhesions were affected by infertility. There are insufficient endometrial stem cells, which may or may not be dysfunctional, in intrauterine adhesion patients. Thus, stem cell regenerative medicine may be a promising treatment for intrauterine adhesions. Our previous research had found that menstrual stem cells could impede endometrial stroma-myofibroblast transition, and down-regulate the mRNA and protein expressions of aSMA and collagen Ⅰ in endometrial stromal cells, which manifested that menstrual stem cells may alleviate endometrial fibrosis. However, the mechanism of menstrual stem cells regulating stroma-myofibroblast transition and inhibiting endometrial fibrosis still needs further research. The TGFβ family is a multifunctional signaling pathway which regulates many different physiological processes. It has been reported that the expression of TGFβ in endometrium of intrauterine adhesions was up-regulated, and was positive related to the adhesion degree. In this work, we aimed to verify the role of TGFP in inducing endometrial stroma-myofibroblast transition, and explicit whether menstrual stem cells were able to attenuate TGFβ-induced endometrial fibrosis.Methods:The effects of TGFβ on proliferation and motility of endometrial stromal cells were analyzed, as well as on the expression of aSMA, collagen Ⅰ, CTGF and fibronectin. The endometrial stromal cells were cocultured with menstrual stem cells in transwell plates. Conditioned media from menstrual stem cells (MenSCs-CM) was prepared. The proliferation of endometrial stromal cells was investigated by Cell Counting Kit 8. The migration and motility of endometrial stromal cells were accessed by in vitro wound healing assay. The mRNA expressions of aSMA, collagen Ⅰ, CTGF and fibronectin were determined by real-time PCR.Results:TGFβ significantly up-regulated the mRNA expression levels of aSMA, collagen Ⅰ, CTGF and fibronectin of endometrial stromal cells. Moreover, TGFP inhibited the motility of endometrial stromal cells and impeded the wound healing. However, TGFβ had no influence on the proliferation of endometrial stromal cells. Both MenSCs-CM and MenSCs coculture could reverse the effect of TGFp. Both MenSCs-CM and MenSCs coculture down-regulated the expression of aSMA, collagen Ⅰ, CTGF and fibronectin induced by TGFβ, and recovered the motility of endometrial stromal cells and promoted wound healing.Conclusions:TGFβ may be involved in the injured endometrial fibrosis by regulating endometrial stromal cell function. Paracrine mechanisms activated by menstrual stem cells may be associated with inhibiting TGFβ-induced fibrosis through the mechanisms of activated hippo/TAZ and suppressed TGFβ pathways.