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.

Attenuation of Expression of Splenocytes Pro-Inflammatory Cytokines and Lung Alpha-Smooth Muscle Actin By Human Amniotic Mesenchymal Stem Cells-Conditioned Medium in Asthmatic Mice

Background Asthma is a chronic respiratory illness characterized by lung tissue remodeling, T helper cell imbalance, and the generation of inflammatory factors. The Human amniotic mesenchymal stem cells-conditioned medium (hAM-MSC-CM) contains various immunomodulatory components and has been utilized in certain studies as a source for anti-inflammatory factors. We investigated the impacts of CM on splenocytes pro-inflammatory cytokines and alpha-smooth muscle actin (α-SMA) expression in Balb/c mice with Ovalbumin (OVA)-induced asthma.Methods and results Forty mice were separated into four groups of ten: control (challenged and sensitized with normal saline solution), asthma (sensitized on days 1, 8, and 14 and challenged daily with OVA from days 21 to 28), OVA+CM (asthmatic mice treated with CM on days 29 and 30), and OVA+DMEM (DMEM-treated asthmatic mice on days 29 and 30). The spleen and lung tissues were removed 48 hours after the final challenge, and the expression of the inflammatory factors in the splenocyte culture supernatant was determined by ELISA, while the α-SMA expression in the lung was determined using western blotting. α-SMA protein expression was significantly greater in lung tissue. Also, inflammatory agents were significantly higher in the splenocytes supernatant in the DMEM and asthma groups compared to the control group. CM therapy has been shown to inhibit the production of the α-SMA protein and inflammatory cytokines. Conclusions Results showed that CM Treatment was able to decrease the α-SMA expression in lung and splenocytes pro-inflammatory cytokines.

S100A8/A9 Enhances Immunomodulatory and Tissue-Repairing Properties of Human Amniotic Mesenchymal Stem Cells in Myocardial Ischemia-Reperfusion Injury

Paracrine factors of human mesenchymal stem cells (hMSCs) have the potential of preventing adverse cardiac remodeling after myocardial infarction (MI). S100A8 and S100A9 are calcium-binding proteins playing essential roles in the regulation of inflammation and fibrous tissue formation, and they might modulate the paracrine effect of hMSCs. We isolated human amniotic mesenchymal stem cells (hAMSCs) and examined the changes in the expression level of regulatory genes of inflammation and fibrosis after hAMSCs were treated with S100A8/A9. The anti-inflammatory and anti-fibrotic effects of hAMSCs pretreated with S100A8/A9 were shown to be superior to those of hAMSCs without S100A8/A9 pretreatment in the cardiomyocyte hypoxia/reoxygenation experiment. We established a murine myocardial ischemia/reperfusion model to compare the therapeutic effects of the conditioned medium of hAMSCs with or without S100A8/A9 pretreatment. We found the hearts administered with a conditioned medium of hAMSCs with S100A8/A9 pretreatment had better left ventricular systolic function on day 7, 14, and 28 after MI. These results suggest S100A8/A9 enhances the paracrine therapeutic effects of hAMSCs in aspects of anti-inflammation, anti-fibrosis, and cardiac function preservation after MI.

Distribution of amniotic stem cells in human term amnion membrane

Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane–derived stem cells. The amnion can be divided into a site that is continuous with the umbilical cord (region A), a site that adheres to the placenta (region B), and a site that is located opposite the placenta (region C). We found that human amnion epithelial stem cells (HAECs) that strongly express stem cell markers were abundant in area A. HAEC not only expressesed stem cell-specific surface markers TRA-1-60, Tra-1-81, SSEA4, SSEA3, but was also OCT-3/4 positive and had alkaline phosphatase activity. Human amniotic mesenchymal stem cells expressed KLF-A, OCTA, Oct3/4, c-MYC and Sox2 which is transcription factor. Especially, in regions A and B they have expressed CD73, and the higher expression of BCRP which is drug excretion transporter protein than the other parts. These data suggest that different types of stem cells may have existed in different area. The understanding the relation with characteristics of the stem cells in each area and function would allow for the efficient harvest of suitable HAE and HAM stem cells as using tool for regenerative medicine.

Human amniotic mesenchymal stem cells-conditioned medium protects mice from high-fat diet-induced obesity

Background Obesity is a metabolic disorder syndrome characterized by excessive fat accumulation that is related to many diseases. Human amniotic mesenchymal stem cells (hAMSCs) have a great potential for cell-based therapy due to their characteristics such as pluripotency, low immunogenicity, no tumorigenicity, potent paracrine effects, and no ethical concern. Recently, we observed that both hAMSCs and their conditioned medium (hAMSCs-CM) efficiently repaired skin injury, inhibited hepatocellular carcinoma, and alleviated high-fat diet (HFD)-induced diabetes. However, the effects and the underlying mechanisms of hAMSCs-CM on high-fat diet (HFD)-induced obesity were not explored. Methods The characteristics of hAMSCs were confirmed by flow cytometry, RT-PCR, and immunofluorescence. Obese mice were induced by administrating HFD for 15 weeks and simultaneously, the mice were intraperitoneally injected with hAMSCs-CM weekly to evaluate the effects of hAMSCs-CM on HFD-induced obesity. GTT and ITT assays were used to assess the effects of hAMSCs-CM on HFD-induced glucose tolerance and insulin resistance. The lipid accumulation and adipocytes hypertrophy in mouse adipose tissues were determined by histological staining, in which the alterations of blood lipid, liver, and kidney function were also examined. The role of hAMSCs-CM in energy homeostasis was monitored by examining the oxygen consumption (VO2), carbon dioxide production (VCO2), and food and water intake in mice. Furthermore, the expressions of the genes related to glucose metabolism, fatty acid β oxidation, thermogenesis, adipogenesis, and inflammation were determined by western blot analysis, RT-PCR, and immunofluorescence staining. The roles of hAMSCs-CM in adipogenesis and M1/M2 macrophage polarization were investigated with 3T3-L1 preadipocytes or RAW264.7 cells in vitro. Results hAMSCs-CM significantly restrained HFD-induced obesity in mice by inhibiting adipogenesis and lipogenesis, promoting energy expenditure, and reducing inflammation. The underlying mechanisms of the anti-obesity of hAMSCs-CM might be involved in inhibiting PPARγ and C/EBPα-mediated lipid synthesis and adipogenesis, promoting GLUT4-mediated glucose metabolism, elevating UCP1/PPARα/PGC1α-regulated energy expenditure, and enhancing STAT3-ARG1-mediated M2-type macrophage polarization. Conclusion Our studies demonstrated that hAMSCs significantly alleviated HFD-induced obesity through their paracrine effects. Obviously, our results open up an attractive therapeutic modality for the prevention and treatment of obesity and other metabolic disorders clinically. Graphic Abstract The cytokines, exosomes, or micro-vesicles secreted from hAMSCs significantly inhibited HFD-induced obesity in mice by inhibiting lipid production and adipogenesis, promoting energy consumption, and reducing inflammation.