| Objective:The current advances in assisted reproductive technology have made it possible to overcome many causes of female infertility.However,pregnancy is still notably difficult for patients with severe endometrial injury,which is characterized by intrauterine adhesions or fibrosis resulting as a consequence of damage to the basal layer of endometrium.Regeneration of the endometrial cells is essential for patients with endometrial damage.However,severe damage to the basal layer may not be amenably repairable due to loss of the basal endometrial progenitor stem cells.Using bone marrow-derived mesenchymal stem cells（BMSCs）to repair endometrium has become promising in the clinical research.BMSCs can differentiate into endometrial epithelium and accelerate the regeneration of injured endometrium.A major challenge of using stem cell,however,is the low percentage of cell retention after the transplantation to the wounded site.Previous reports showed that BMSCs were surrounded by endometrial stromal cell（ESCs）providing a local environment for donor BMSCs differentiation,we investigated the effect of ESCs on differentiation conditions of BMSCs into endometrial epithelial cells and to confirm the effect of estrogen in this process.In addition,considering that insulin-like Growth Factor-1（IGF-1）can effectively promote endometrial regeneration,transduction of BMSCs with IGF-1 was performed with adenovirus infection.Furthermore,we keep E2 into the culture to induce the IGF-1 release of BMSCs and the restoration and regeneration of injured endometrium.We investigated the potential therapeutic effect of IGF-1-overexpressed BMSC with E2 transplantation in mouse uterus injury model by histological structures of the regenerative uterine horns.Our goal is to provide a novel and multidisciplinary approach to restore the endometrial function.Methods:1.BMSCs and ESCs were co-cultured in Transwell system with gradient concentration of E2(0 mol/L,1×10-9mol/L,1×10-8mol/L,1×10-7mol/L,1×10-6mol/L)in conditioned media（DMEM/F12,2%CSF,1%P/S）.After 6 weeks in culture,the expressions of epithelial marker（Pan-CKs,CK13,CK18,CK19）and endometrium marker（PR,ER-α）in BMSCs during differentiation were examined.2.Transduction of BMSCs with IGF-1 was performed with adenovirus infection.The expression of IGF-1 in BMSCs was validated by RT-PCR and western-blot.In addition,IGF-1 transduced BMSCs were co-cultured for 7 days with E2 at equivalent concentration 1*10-8 mol/L.The media were harvested daily for 7 consecutive days to measure the IGF-1 release of BMSCs using ELISA.Furthermore,we investigated the potential therapeutic effect of IGF-1-overexpressed BMSC with E2 transplantation in mouse uterus injury model by histological structures of the regenerative uterine horns.Results:1.After 6 weeks in culture,CK13,CK18,CK19 and PR expression was up-regulated in BMSCs during differentiation.The expressions of endometrial epithelial marker（CK13,CK18,CK19 and PR）were higher in BMSCs co-cultured with ESCs in conditioned media containing gradient concentration of E2 than that in conditioned media without E2.Notably,the expression levels of those markers were highest in cells cultured with 1×10-8mol/L E2.2.The IGF-1 expression in E2-treated BMSCs cell increased in a sustainable manner and reached the peak level at day 4.Notably,the IGF-1 overexpressing BMSCs cells with E2 displayed the remarkable improvement in this setting while in comparison with regular BMSCs transplantation.The angiogenesis indicated by vWF labelling was significantly induced post-BMSCs transplantation,which was further enhanced by overexpression of IGF-1 with E2.Similarly,the smooth muscle regeneration,which was greatly impaired in model group,was greatly improved by IGF-1 in BMSCs with E2 transplantation.Conclusion:1.Bone marrow mesenchymal stem cells can differentiate in the direction of endometrial epithelial cells in a certain microenvironment and appropriate concentration of E2 can facilitate this differentiation.2.E2 could induce IGF-1 expression of BMSCs.Our in vivo data supported the beneficial effect of IGF-1 with E2 in promotion recovery of injured uterus. |
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