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

Abstract

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.

Amniotic stem cells as a source of regenerative medicine to treat female infertility

“…MSCs originating from hAM share many similarities with adult tissue-derived MSC and meet all the international standards for declaring them as "mesenchymal stem cells" [12]. The differentiation potential of hAMSCs has been discussed recently [8,13]. Although the potential of adult MSCs in restoring ovarian function has been explored extensively [14][15][16], very limited data are available for AMderived MSCs.…”

“…The studies that explored the role of hAMSCs to promote ovarian function in natural or premature ovarian aging (NOA or POA) or premature ovarian failure/inefficacy (POF/POI) mouse models are listed in Table 1 . In these investigations, the hAMSCs demonstrated their potential to reinstate normal ovarian functions by improving the local microenvironment of the ovaries [ 17 ], maintaining endometrial regeneration through paracrine actions [ 13 , 18 – 20 ], and/or regulating cytokines [ 21 , 22 ] (Table 1 , Fig. 1 ).…”

“…In vivo studies demonstrated that hAMSCs harbor the great ability to colonize uterine tissue and ovarian stroma which helps achieve the utilization rate of hAMSCs thus the desired outcomes. However, various strategies were adopted and compared to improve the homing of stem cells, e.g., repeated vs. single transplantation of hAMSCs [ 24 ], direct injection of cells into the ovary vs tail injection [ 25 ], or use of polymers for efficient transportation of transplanted cells [ 13 ]. Direct vs. tail injection via tail did not result in any significant differences in restoring ovarian function [ 25 ].…”

“…However, the long-term implications on fertility cannot be drawn from this study as these outcomes were not found persistent throughout the study time frame. Recently, Haung et al [ 13 ] proposed a polymer-based transportation system PPCNg (polyethylene glycol citrate-co–N-isopropyl acrylamide + gelatin) for hAMSCs transplantation to regenerate endometrial tissue in the Sprague–Dawley rat IUA model. The utilization of PPCNg-based transportation improved the retention of hAMSCs in ovarian tissue leading to the enhance regeneration capacity of endometrial tissue.…”

Applications of Hydrogels in Premature Ovarian Failure and Intrauterine Adhesion

“…The application of hydrogels in IUA is mainly focused on drug delivery and stem cell delivery therapy (Mettler et al, 2008;Xue et al, 2019). Hydrogels are capable of controlling drug release and extending stem cell retention Huang et al, 2022). In this review, we discuss the physicochemical properties of hydrogels and the latest applications of hydrogels in POF and IUA.…”