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.     

Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4)) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6)) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4) treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4) administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4) demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4) treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4) alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4) treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis.     

Changes in culture expanded human amniotic epithelial cells: implications for potential therapeutic applications

Human amniotic epithelial cells (hAEC) isolated from term placenta have stem cell-like properties, differentiate into tissue specific cells and reduce lung and liver inflammation and fibrosis following transplantation into disease models established in mice. These features together with their low immunogenicity and immunosuppressive properties make hAEC an attractive source of cells for potential therapeutic applications. However, generation of large cell numbers required for therapies through serial expansion in xenobiotic-free media may be a limiting factor. We investigated if hAEC could be expanded in xenobiotic-free media and if expansion altered their differentiation capacity, immunophenotype, immunosuppressive properties and production of immunomodulatory factors. Serial expansion in xenobiotic-free media was limited with cumulative cell numbers and population doubling times significantly lower than controls maintained in fetal calf serum. The epithelial morphology of primary hAEC changed into mesenchymal-stromal like cells by passage 4-5 (P4-P5) with down regulation of epithelial markers CK7, CD49f, EpCAM and E-cadherin and elevation of mesenchymal-stromal markers CD44, CD105, CD146 and vimentin. The P5 hAEC expanded in xenobiotic-free medium differentiated into osteocyte and alveolar epithelium-like cells, but not chondrocyte, hepatocyte, α- and β-pancreatic-like cells. Expression of HLA Class IA, Class II and co-stimulatory molecules CD80, CD86 and CD40 remained unaltered. The P5 hAEC suppressed mitogen stimulated T cell proliferation, but were less suppressive compared with primary hAEC at higher splenocyte ratios. Primary and P5 hAEC did not secrete the immunosuppressive factors IL-10 and HGF, whereas TGF-β1 and HLA-G were reduced and IL-6 elevated in P5 hAEC. These findings suggest that primary and expanded hAEC may be suitable for different cellular therapeutic applications.     

Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.     

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.     

CD117+ amniotic fluid stem cells

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

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.     

CD117+ amniotic fluid stem cells: State of the art and future perspectives

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

In vitro differentiation of human amniotic fluid‐derived cells: Augmentation towards a neuronal dopaminergic phenotype

Amniotic fluid is known to yield a number of cell types which are multipotent, ethically derived, genetically stable, easily grown, expanded and possess favourable immunogenicity, which has resulted in an increasing interest for use in various neuronal disorders such as Parkinson's disease. The neuronal potential of cells derived from the adherent fraction of amniotic fluid, routinely taken by amniocentesis, are least explored. The aim of the present study was to investigate the capacity of these cells for neuronal and dopaminergic differentiation using in vitro differentiation protocols with canonical inductive factors not previously tested. To do this, samples derived from multiple donors were grown under four conditions: standard serum‐containing media, NB (neurobasal) media designed specifically for propagation and maintenance of neuronal cells, NB media with addition of retinoic acid and BDNF (brain‐derived neurotrophic factor) for NI (neuronal induction), and NB media with addition of FGF8 (fibroblast growth factor‐8) and Shh (sonic hedgehog) after NI. Our results showed the presence of multiple neuronal markers after growth in serum‐containing medium [TUJ1, MAP2, NF‐M, TH (tyrosine hydroxylase)], which was significantly up‐regulated after serum withdrawal in NB medium alone with induction of NeuN (neuronal nuclei) and NSE (neuron‐specific enolase). NI and DA.I (dopaminergic induction) was accompanied by further increases in expression and a distinct transition to a sustained neuronal morphology. Western blot analysis confirmed increasing TH expression and NURR1, expressed in base serum‐containing media, found to be down‐regulated after induction. In conclusion, these cells possess a highly favourable base neuronal and dopaminergic prepotential, which may easily be accentuated by standard induction protocols.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

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。     

人羊水祖细胞的分离和基因修饰

探讨从孕中期羊水中分离出人羊水祖细胞的有效方法和FIX基因修饰后的效果,为血友病B的产前治疗提供可行的基础。从镜下分离出呈致密克隆生长的梭形细胞集落,经培养传代后,通过第3代慢病毒载体将hFIX导入该细胞,经酶联免疫反应(ELISA)等方法检测hFIX的表达并检测凝血活性。用这种方法得到的羊水祖细胞呈成纤维细胞样,倍增时间为39.05 h,该细胞在不仅在蛋白水平表达干细胞表面分子SSEA4,TRA1-60,在基因水平还可检测到NANOG,OCT4,SOX2mRNA的表达。羊水祖细胞导入hFIX cDNA后,能合成并分泌hFIX蛋白,传代后48 h在上清液中的浓度为20.37%±2.77%,凝血活性16.42%±1.78%。上清液中的浓度在第4天达到平台期,为50.35%±5.42%,凝血活性可达45.34%±4.67%。ELISA检测显示转染后的羊水细胞表达的hFIX蛋白的水平呈现基本稳定趋势,波动幅度较小;同时检测FIX凝血活性也与蛋白浓度呈正相关。从羊水中可以分离得到具有多能性祖细胞,转染了hFIX的羊水祖细胞在体外能持续合成有凝血功能的hFIX蛋白,为血友病B产前治疗的新方法提供了实验依据。     

Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function

MSCs are kind of cultured cells that reside in different tissues as inducers or regulators of physiological and pathological processes. Here, we derived MSCs from amniotic fluid and compared their differentiation ability and immunosuppression effect on PHA-activated PBMC with those of MSCs isolated from umbilical cords. Amniotic fluid MSCs were isolated and cultured on commercial AFC medium and classic MSC medium, and the number and size of colonies were used to evaluate differences in primary and passaged culture. Rate of proliferation, population doubling time, cell morphology, cell surface markers and mRNA expression were measured in subcultured cells. Furthermore, a comparative study was performed with umbilical cord MSCs to assess the ability of differentiation and immunosuppressive effect of PHA-stimulated PBMCs. Amniotic fluid MSCs were isolated and expanded by three methods, and exhibited nearly all the characteristics of umbilical cord MSCs. Compared with umbilical cord MSCs, amniotic fluid MSCs had an enhanced osteogenic and chrondrogenic differentiation capability, and stronger immunosuppression effect of inhibition of PHA-activated PBMC division. Culture with commercial AFCs medium yielded the highest percentage of CD105 expression and showed some advantages in primary cell isolation, cell source-specific marker retention and cell proliferation. We demonstrated that amniotic fluid MSCs exhibited some advantages over umbilical cord MSCs, and different culture media caused cell proliferation, cell surface marker and cell morphology change, but were not associated with varying differentiation capability and immune effects.     

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.     

人羊膜间充质细胞具有向心肌样细胞分化的特性

摘 要 探讨人羊膜间充质细胞（human amniotic mesenchymal cells,hAMCs）向心肌细胞分化的能力。采用胶原酶消化法分离hAMCs,用流式细胞仪进行表型鉴定;用5-氮杂胞苷和碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）诱导hAMCs向心肌细胞分化,免疫荧光染色法检测诱导后细胞中特异蛋白结蛋白和α-辅肌动蛋白的表达,RT-PCR检测心肌特异性转录因子Nkx2.5 、GATA-4和心肌特异性收缩蛋白α-肌球蛋白重链（α-myosin heavy chain,α-MHC）mRNA的表达。结果显示：①hAMCs原代培养至第6 d,贴壁细胞汇合度可达80%,呈漩涡状生长。传代后hAMCs增殖迅速,3~4 d细胞汇合度可达100%,细胞呈梭形或多角形。②hAMCs表达CD44和波形蛋白,不表达CK19。③hAMCs经诱导分化8~10 d后细胞排列紧密,多为长梭形。③hAMCs诱导2 w和4 w表达α-辅肌动蛋白和心肌特异性转录因子Nkx2.5。④诱导前后的hAMCs均表达结蛋白和GATA-4,但均未见α-MHC表达。说明hAMCs具有向心肌样细胞分化的能力,可望成为细胞心肌成形术（cellular cardiomyoplasty,CCM）的候选细胞。