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Transplantation of Human Adipose Stem Cells Using Acellular Human Amniotic Membrane Improves Angiogenesis in Injured Endometrial Tissue in a Rat Intrauterine Adhesion Model.

ABSTRACT: Endometrial injury resulting in intrauterine adhesion is associated with extensive damage to the regenerative basal layer of the endometrium and represents a major therapeutic challenge. Human adipose stem cells (hASCs) hold promise for future clinical use in the individualized therapy of injured endometrial tissue. Here, we observed that the use of the acellular human amniotic membrane (AHAM) significantly increased the expression of angiogenic factors, including angiogenin (ANG) and vascular endothelial growth factor (VEGF), in hASCs in vitro. The three-dimensional engineered hASC-AHAM grafts significantly increased the endometrial receptivity, as increased endometrial thickness, greater numbers of endometrial glands, and higher protein levels of leukemia inhibitory factor were observed in injured endometrial tissue that was treated with these grafts compared to those detected in injured endometrial tissue that was treated with AHAM alone. In addition, the hASC-AHAM grafts significantly increased the vascular density in the injured endometrial tissue in rats, when transplanted into an injured uterine cavity. Using the EGFP⁺-hASC-AHAM grafts for transplantation, we confirmed that the hASCs maintained higher protein levels of ANG and VEGF in the injured uterine cavity in vivo. The results of this study suggest that the ability of the engineered hASC-AHAM grafts to repair injured endometrial tissue may be associated with their ability to promote angiogenesis through the upregulated expression of angiogenic factors in hASCs. These findings may support individualized stem cell-based therapy for endometrial disease using bioartificial grafts.

SUBMITTER: Han X

PROVIDER: S-EPMC7784510 | biostudies-literature | 2020 Jan-Dec

REPOSITORIES: biostudies-literature

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Transplantation of Human Adipose Stem Cells Using Acellular Human Amniotic Membrane Improves Angiogenesis in Injured Endometrial Tissue in a Rat Intrauterine Adhesion Model.

Han Xiao X Ma Yuejiao Y Lu Xin X Li Weihong W Xia Enlan E Li Tin-Chiu TC Zhang Haiyan H Huang Xiaowu X

Cell transplantation 20200101

Endometrial injury resulting in intrauterine adhesion is associated with extensive damage to the regenerative basal layer of the endometrium and represents a major therapeutic challenge. Human adipose stem cells (hASCs) hold promise for future clinical use in the individualized therapy of injured endometrial tissue. Here, we observed that the use of the acellular human amniotic membrane (AHAM) significantly increased the expression of angiogenic factors, including angiogenin (ANG) and vascular e ...[more]

PMID: 32838542

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Project description:BackgroundCaused 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.MethodshAMSCs 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.ResultshAMSCs 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.ConclusionsPPCNg 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.

Project description:Intrauterine adhesions (IUAs) have caused serious harm to women's reproductive health. Although emerging evidence has linked intrauterine microbiome to gynecological diseases, the association of intrauterine microbiome with IUA, remains unknown. We performed metagenome-wide association, metabolomics, and transcriptomics studies on IUA and non-IUA uteri of adult rats to identify IUA-associated microbial species, which affected uterine metabolites and endometrial transcriptions. A rat model was used with one side of the duplex uterus undergoing IUA and the other remaining as a non-IUA control. Both 16S rRNA sequencing and metagenome-wide association analysis revealed that instead of Mycoplasmopsis specie in genital tract, murine lung pathogen Mycoplasmopsispulmonis markedly increased in IUA samples and displayed a distinct positive interaction with the host immune system. Moreover, most of the IUA-enriched 58 metabolites positively correlate with M.pulmonis, which inversely correlates with a mitotic progression inhibitor named 3-hydroxycapric acid. A comparison of metabolic profiles of intrauterine flushing fluids from human patients with IUA, endometritis, and fallopian tube obstruction suggested that rat IUA shared much similarity to human IUA. The endometrial gene Tenascin-N, which is responsible for extracellular matrix of wounds, was highly up-regulated, while the key genes encoding parvalbumin, trophectoderm Dkkl1 and telomerase involved in leydig cells, trophectoderm cells, activated T cells and monocytes were dramatically down-regulated in rat IUA endometria. Treatment for rat IUA with estrogen (E2), oxytetracycline (OTC), and a traditional Chinese patent medicine GongXueNing (GXN) did not reduce the incidence of IUA, though inflammatory factor IL-6 was dramatically down-regulated (96-86%) with all three. Instead, in both the E2 and OTC treated groups, IUA became worse with a highly up-regulated B cell receptor signaling pathway, which may be associated with the significantly increased proportions of Ulvibacter or Staphylococcus. Our results suggest an association between intrauterine microbiota alterations, certain uterine metabolites, characteristic changes in endometrial transcription, and IUA and the possibility to intervene in IUA formation by targeting the causal factors, microbial infection, and Tenascin-like proteins.

Project description:As a refractory fibrosis disease, intrauterine adhesions (IUAs) is defined as fibrosis of the physiological endometrium. Although hysteroscopic adhesiolysis is widely recommended as an effective treatment, prognosis and recurrence remain poor in severe cases. Recently, stem cell therapy has been promoted as a promising treatment for IUAs. The ability of human amniotic epithelial cells (hAECs), emerging as a new candidate for stem cell therapy, to treat IUAs has not been demonstrated. To study the potential effects of hAECs on IUAs, we created an IUA rat model using mechanical injury and injected cultured primary hAECs into the rats' uteri. Next, we observed the morphological structure of endometrial thickness and glands using hematoxylin and eosin staining, and we detected extracellular-matrix collagen deposition using Masson staining. In addition, we performed immunohistochemical staining and reverse-transcription polymerase chain reaction (RT-PCR) to investigate potential fibrosis molecules and angiogenesis factors 7 d after hAECs transplantation. Finally, we detected estrogen receptor (ER) and growth factors via RT-PCR to verify the molecular mechanism underlying cell therapy. In the IUA rat models, endometrial thickness and endometrial glands proliferated and collagen deposition decreased significantly after hAEC transplantation. We found that during the recovery of injured endometrium, the crucial fibrosis marker transforming growth factor-β (TGF-β) was regulated and angiogenesis occurred in the endometrial tissue with the up-regulation of vascular endothelial growth factor. Furthermore, hAECs were shown to promote ER expression in the endometrium and regulate the inflammatory reaction in the uterine microenvironment. In conclusion, these results demonstrated that hAEC transplantation could inhibit the progression of fibrosis and promote proliferation and angiogenesis in IUA rat models. The current study suggests hAECs as a novel stem cell candidate in the treatment of severe IUA.

Project description:Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with ?-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin-eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve; the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.

Dataset Information

Transplantation of Human Adipose Stem Cells Using Acellular Human Amniotic Membrane Improves Angiogenesis in Injured Endometrial Tissue in a Rat Intrauterine Adhesion Model.

Publications

Transplantation of Human Adipose Stem Cells Using Acellular Human Amniotic Membrane Improves Angiogenesis in Injured Endometrial Tissue in a Rat Intrauterine Adhesion Model.

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