Human amniotic mesenchymal stem cells combined with PPCNg facilitate injured endometrial regeneration

Background Caused by the injury to the endometrial basal layer, intrauterine adhesions (IUA) are characterized by uterine cavity obliteration, leading to impaired fertility. Human amniotic mesenchymal stem cells (hAMSCs) have the potential to promote endometrial regeneration mainly through paracrine ability. PPCNg is a thermoresponsive biomaterial consisted of Poly (polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin, which has been reported as a scaffold for stem cell transplantation. This study aims to investigate the therapeutic effect of hAMSCs combined with PPCNg transplantation in promoting the regeneration of injured endometrium. Methods hAMSCs were cultured in different concentrates of PPCNg in vitro, and their proliferation, apoptosis and cell cycle were examined by CCK-8 assay and flow cytometry. Immunofluorescence was used to determine the MSCs specific surface markers. The expression of pluripotent genes was analyzed by qRT-PCR. The multiple-lineage differentiation potential was further evaluated by detecting the differentiation-related genes using qRT-PCR and specific staining. The Sprague–Dawley (SD) rat IUA model was established with 95% ethanol. hAMSCs combined with PPCNg were transplanted through intrauterine injection. The retention of DiR-labeled hAMSCs was observed by vivo fluorescence imaging. The endometrium morphology was assessed using hematoxylin and eosin (H&E) and Masson staining. Immunohistochemistry staining was performed to detect biomarkers related to endometrial proliferation, re-epithelialization, angiogenesis and endometrial receptivity. The function of regenerated endometrium was evaluated by pregnancy tests. Results hAMSCs maintained normal cell proliferation, apoptosis and cell cycle in PPCNg. Immunofluorescence and qRT-PCR showed that hAMSCs cultured in PPCNg and hAMSCs cultured alone expressed the same surface markers and pluripotent genes. hAMSCs exhibited normal multilineage differentiation potential in PPCNg. Vivo fluorescence imaging results revealed that the fluorescence intensity of hAMSCs combined with PPCNg intrauterine transplantation was stronger than that of direct hAMSCs intrauterine transplantation. Histological assays showed the increase in the thickness of endometrial and the number of endometrial glands, and the remarkably decrease in the fibrosis area in the PPCNg/hAMSCs group. The expressions of Ki-67, CK7, CK19, VEGF, ER and PR were significantly increased in the PPCNg/hAMSCs group. Moreover, the number of implanted embryos and pregnancy rate were significantly higher in the PPCNg/hAMSCs group than in the hAMSCs group. Conclusions PPCNg is suitable for growth, phenotype maintenance and multilineage differentiation of hAMSCs. hAMSCs combined with PPCNg intrauterine transplantation can facilitate the regeneration of injured endometrium by improving utilization rates of hAMSCs, and eventually restore reproductive capacity.

BMSCs and Pectin-Based E2-Loaded Microcapsules with Injectable Pectin-Pluronic® F-127 Scaffolds for Mouse Endometrial Regeneration Application

Background Uterine endometrium is a highly dynamic tissue which consists of a basal layer and a functional layer. Bone marrow-derived mesenchymal stem cells (BMSCs) have been recognized as new candidates for the treatment of serious endometrial injuries. However, due to the local microenvironment of damaged endometrium, transplantation of BMSCs yielded disappointing results with respect to survival, attachment, differentiation, and proliferation. Methods Pectin-Pluronic® F-127 scaffolds were fabricated. E2 was encapsulated into the W/O/W microspheres to construct pectin-based E2-loaded microcapsules (E2 MPs). The BMSCs/E2 MPs/scaffolds system was then injected into the uterine cavity of mouse endometrial injury model. Furthermore, the mechanism of E2 in promoting the repair of endometrial injury was also investigated. Result Pectin-Pluronic® F-127 scaffolds could provide three-dimensional architecture for the attachment, growth, and migration of BMSCs. E2 MPs has the potential to serve as a long-term reliable source of E2 for endometrial regeneration. At four weeks after transplantation, it was demonstrated that the system increased proliferative abilities of uterine endometrial cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive an embryo, suggesting that the BMSCs/E2 MPs/scaffolds system is a promising treatment option for endometrial regeneration. Exosomes are critical paracrine mediators that act as biochemical cues to direct stem cell differentiation. In this study, it was found that the expression of endometrial epithelial cells (EECs) markers was up-regulated in BMSCs treated by exosomes secreted from endometrial stromal cells (ESCs-Exos). Exosomes derived from E2-stimulated ESCs further promoted the expression level of EECs markers in BMSCs, suggesting exosomes released from ESCs by E2 stimulation could enhance the differentiation efficiency of BMSCs. Conclusion The BMSCs/E2 MPs/scaffolds therapeutic strategy may be beneficial in the treatment of severely damaged endometrium. Exosomes derived from ESCs play paracrine roles in endometrial regeneration stimulated by E2, potentially modulating the differentiation of BMSCs.

A Robotic System with Robust Remote Center Motion Constraint for Endometrial Regeneration Surgery

Surgical robots have been widely used in various surgeries in order to improve and facilitate the surgeries. However, there is no robot intended for endometrial regeneration surgery which is a new therapy to restore women’s fertility by using stem cells. Endometrial regeneration surgery requires processing endometrium and transplanting stem cells with the minimal trauma to uterus. In this paper, we introduce a surgical robotic system which consists of a dexterous hysteroscope, a supporting arm and additional novel instruments to facilitate the operations and decrease trauma to the uterus. To protect the cervix of the uterus, remote center of motion (RCM) constraint is required. First, the supporting arm and the hysteroscope are controlled separately in kinematics in order to ensure that the RCM constraint and the hysteroscope’s shape and posture are predictable. Then, a task decoupled method is used to improve the robustness of the RCM constraint. The experiments show that our method is more robust and achieves higher accuracy of RCM. Besides, the master slave control of robot with RCM constraint is also verified.

Stromal Heterogeneity in the Human Proliferative Endometrium—A Single-Cell RNA Sequencing Study

The endometrium undergoes regular regeneration and stromal proliferation as part of the normal menstrual cycle. To better understand cellular interactions driving the mechanisms in endometrial regeneration we employed single-cell RNA sequencing. Endometrial biopsies were obtained during the proliferative phase of the menstrual cycle from healthy fertile women and processed to single-cell suspensions which were submitted for sequencing. In addition to known endometrial cell types, bioinformatic analysis revealed multiple stromal populations suggestive of specific stromal niches with the ability to control inflammation and extracellular matrix composition. Ten different stromal cells and two pericyte subsets were identified. Applying different R packages (Seurat, SingleR, Velocyto) we established cell cluster diversity and cell lineage/trajectory, while using external data to validate our findings. By understanding healthy regeneration in the described stromal compartments, we aim to identify points of further investigation and possible targets for novel therapy development for benign gynecological disorders affecting endometrial regeneration and proliferation such as endometriosis and Asherman’s syndrome.