体外诱导人羊膜上皮细胞分化为胰岛素分泌细胞的研究

目的:通过体外诱导分化实验,探讨人羊膜上皮细胞(hAECs)向胰岛素分泌细胞(ISCs)分化的能力。方法:采用胰蛋白酶消化法从人羊膜组织分离提取hAECs,用流式细胞仪和免疫细胞化学法进行鉴定。取第3代hAECs在含尼克酰胺和N2补充物的无血清培养基中诱导培养,分别于诱导不同时间采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子-1(PDX-1)mRNA的表达。结果:①hAECs高表达CD29、CD73、CD166和CK19;②hAECs诱导组第7、142、1天胰岛素阳性细胞百分率分别为74.00%±1.73%、75.33%±1.15%和75.67%±0.58%,而对照组未见胰岛素阳性细胞;③hAECs诱导组第7、14、21天培养物上清液中胰岛素含量分别达(328.47±3.22)μIU/ml、(332.26±1.22)μIU/ml和(329.68±2.57)μIU/ml,均显著高于对照组(P均<0.01);④hAECs诱导前后均有PDX-1 mRNA和β2微球蛋白表达,胰岛素mRNA表达仅见于诱导组。结论:hAECs能分化为ISCs,在Ⅰ型糖尿病细胞移植治疗方面具有潜在应用前景。     

Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.     

人羊膜上皮细胞和胚胎干细胞

人胚胎干细胞有着巨大的医学应用前景,但人胚胎干细胞要求的生长条件很高,体外很难模拟其生长的体内环境,因此控制人胚胎干细胞的生长常不理想,而使用鼠胚胎成纤维细胞(MEF)作为滋养层则存在动物源性污染的问题。该文阐述人羊膜上皮细胞(HAEC)的特点及其作为滋养层培养胚胎干细胞的现状,并探讨基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用,为建立更优化的培养系统提供依据。     

Bactericidal effects and stability of LL-37 and CAMA in the presence of human lung epithelial cells

Cationic antimicrobial peptides (CAMPs) are important actors in host innate immunity and represent a promising alternative to combat antibiotic resistance. Here, the bactericidal activity of two CAMPs (LL-37 and CAMA) was evaluated against Pseudomonas aeruginosa (PA) in the presence of IB3-1 cells, a cell line derived from patients with cystic fibrosis. The two CAMPs exerted different effects on PA survival depending on the timing of their administration. We observed a greater bactericidal effect when IB3-1 cells were pretreated with sub-minimum bactericidal concentrations (Sub-MBCs) of the CAMPs prior to infection. These findings suggest that CAMPs induce the production of factors by IB3-1 cells that improve their bactericidal action. However, we observed no bactericidal effect when supra-minimum bactericidal concentrations (Supra-MBCs) of the CAMPs were added to IB3-1 cells at the same time or after infection. Western-blot analysis showed a large decrease in LL-37 levels in supernatants of infected IB3-1 cells and an increase in LL-37 binding to these cells after LL-37 administration. LL-37 induced a weak inflammatory response in the cells without being toxic. In conclusion, our findings suggest a potential prophylactic action of CAMPs. The bactericidal effects were low when the CAMPs were added after cell infection, likely due to degradation of CAMPs by bacterial or epithelial cell proteases and/or due to adherence of CAMPs to cells becoming less available for direct bacterial killing.     

Human amniotic epithelial cells inhibit activation and pro-inflammatory cytokines production of naive CD4+ T cells from women with unexplained recurrent spontaneous abortion

Unexplained recurrent spontaneous abortion (URSA) has been assumed to be caused by a defect in maternal immunological tolerance to the fetus. Human amniotic epithelial cells (hAECs) have stem cell-like features and the ability to modulate the innate and adoptive immune responses. This study aimed to investigate whether hAECs have immunomodulatory effects on naive CD4+ T cells from URSA patients. hAECs were obtained from 15 healthy pregnant women and phenotypic profile of hAECs was determined by flow cytometry. Naive CD4+ T cells were isolated from 25 URSA patients using an immunomagnetic separation method. Naive T cells were stimulated with anti-CD3/anti-CD28 antibodies and co-cultured with different numbers of hAECs for 3 and 6 days. Immunomodulatory effect of hAECs on activation of stimulated T cell was assessed by flow cytometry and Enzyme-linked immunoasorbent assay (ELISA). The hAECs effect on pro-inflammatory cytokines production of activated T cells was also measured by ELISA. Our results indicated that hAECs significantly inhibited the activation of naive T cells in a dose-dependent manner (p?<?0.0001–0.05). They significantly reduced the production of transforming growth factor-beta1 (TGF-β1) of stimulated CD4+T cells (p?<?0.0001–0.05). Moreover, hAECs had potent immunomodulatory effects on the production of interferon-gamma (IFN-γ) and interleukin-17A (IL-17A) of activated T cells (p?<?0.0001–0.01). These findings suggest that hAECs may be a suitable cell source to modulate abnormal immune responses in women with URSA.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Therapeutic effect of human amniotic epithelial cells in murine models of Hashimoto's thyroiditis and Systemic lupus erythematosus

Background aims

The chronic inflammation of autoimmune diseases develops repetitive localized destruction or systemic disorders, represented by Hashimoto's thyroiditis (HT) and Systemic lupus erythematosus (SLE) respectively. Currently, there are no efficient ways to treat these autoimmune diseases. Therefore, it is critically important to explore new therapeutic strategies. The aim of this study was to investigate the therapeutic efficacy of human amniotic epithelial cells (hAECs) in murine models of HT and SLE.

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

Treatment of intracerebral haemorrhage in rats with intraventricular transplantation of human amniotic epithelial cells

We explored the effects on brain oedema and neurological functional recovery after transplantation of hAECs (human amniotic epithelial cells) into the lateral ventricle of rats with ICH (intracerebral haemorrhage). hAECs were isolated from human term placenta and seeded for primary culture. We delivered hAECs labelled with Hoechst33258 and transfected with EGFP (enhanced green fluorescent protein) gene using lentiviral vectors into ICH rat models. The behaviour of the animals and brain oedema were evaluated after 28 days, and brain sections were made for morphological and immunohistochemical analyses with fluorescence microscopy. Our results were as follows. Transplanted hAECs were observed along the lateral wall and survived for at least 4 weeks. Some of the cells were stained with human specific antibody to vimentin and nestin. Around the injury site, activated microglia stained with OX42 were reduced. The water content of ICH rats decreased in the treatment group. The behaviour test scores were improved in the treatment group compared with those in the control groups. In conclusion, hAECs cannot only survive in the lateral ventricle of ICH rats after transplantation, but also express vimentin and nestin. hAEC transplantation reduced brain oedema and improved the motor deficits of ICH rats.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

TLR4 signaling promotes the expression of VEGF and TGFbeta1 in human prostate epithelial PC3 cells induced by lipopolysaccharide

Chronic inflammation promotes tumor development and progression, and Toll-like receptors (TLRs) may play an important role in this process. In this study, we found that human prostate epithelial PC3 cells constitutively express TLR4 in mRNA and protein level. lipopolysaccharide (LPS) promotes the expression and secretion of immunosuppressive cytokine TGFβ₁ and proangiogenic factor VEGF in human prostate epithelial PC3 cells. We further elucidated that functionally activation of TLR4 is essential for the increased VEGF and TGFβ₁ mRNA expression in the cells. In addition, after LPS stimulation, the increased expression of NF-_KB p65 protein was also detected in human PC3 cells. Our results demonstrate that TLR4 expressed on human PC3 cells is functionally active, and may play important roles in promoting prostate cancer immune escape, survival, progression, and metastasis by inducing immunosuppressive and proangiogenic cytokines.     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的：研究人羊膜间充质细胞（Humanamnioticmesenchymalcells,HAMCs）的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法：无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果：来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论：羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

The effect of pro-inflammatory cytokines on immunophenotype,differentiation capacity and immunomodulatory functions of human mesenchymal stem cells

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders.     

The fate of lipopolysaccharide in cultured rat Kupffer cells

Cultured rat Kupffer cells were incubated in presence of biologically tritiated Salmonella abortus equi lipopolysaccharide. Uptake of lipopolysaccharide increased rapidly during the first 2 h of incubation and then levelled off. Within the first h of incubation 10(6) Kupffer cells were able to ingest up to 18 micrograms lipopolysaccharide. Kupffer cells metabolised lipopolysaccharide and released lipopolysaccharide-related substances, but neither the cell-associated lipopolysaccharide nor the released lipopolysaccharide products were detoxified, as measured by the mouse lethality test.     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的:研究人羊膜间充质细胞(Humanamnioticmesenchymalcells,HAMCs)的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法:无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果:来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论:羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

Human amniotic epithelial cells suppress relapse of corticosteroid-remitted experimental autoimmune disease

Background aimsMultiple sclerosis (MS) is considered to be a T-cell–mediated disease. Although MS remits with corticosteroid treatment, the disease relapses on discontinuation of therapy. Human amniotic epithelial cells (hAEC) from the placenta are readily accessible in large quantities and have anti-inflammatory properties. Previously we reported that hAEC given near disease onset ameliorated clinical signs and decreased myelin oligodendrocyte glycoprotein (MOG)-specific immune responses in MOG-induced experimental autoimmune encephalomyelitis (EAE), an experimental MS model.MethodsTo examine the therapeutic effect of hAEC in a clinically relevant setting, we first treated MOG peptide–induced EAE mice with a corticosteroid, prednisolone, in drinking water to induce remission. hAEC were then infused intravenously into the remitted mice. Anti-MOG antibodies in serum were detected by enzyme-linked immunoassay. Splenocyte proliferation was assessed by ³H-thymidine incorporation. Immune cell subpopulations in spleens and lymph nodes and secreted cytokines in splenocyte culture were quantified by flow cytometry. Central nervous system histology was examined with the use of hematoxylin and eosin, Luxol fast blue and immunostaining.ResultsWith cessation of prednisolone treatment, hAEC delayed EAE relapse for 7 days, and, after another 7 days, largely remitted disease in six of eight responder mice. Splenocyte proliferation was suppressed, anti-MOG_35–55 antibodies in serum were decreased and interleukin-2 and interleukin-5 production by splenocytes were elevated after hAEC treatment. In the central nervous system, hAEC-treated mice had decreased demyelination and fewer macrophages in the inflammatory infiltrates. hAEC treatment also increased CD4⁺CD25⁺FoxP3⁺ regulatory T cells in inguinal lymph nodes.ConclusionsThese data demonstrate that the therapeutic effects of hAEC after corticosteroid treatment in an MS model probably are the consequence of peripheral immunoregulation. We suggest that hAEC may have potential as a cell therapy for remitted MS.