Human Amniotic Mesenchymal Cells Research Articles

Cocktail of Hyaluronic Acid and Human Amniotic Mesenchymal Cells Effectively Repairs Cartilage Injuries in Sodium Iodoacetate-Induced Osteoarthritis Rats.

Osteoarthritis (OA) is one of the most common refractory degenerative articular cartilage diseases. Human amniotic mesenchymal cells (hAMSCs) have emerged as a promising stem cell source for cartilage repair, and hyaluronic acid (HA) has proven to be a versatile regulator for stem cell transplantation. Herein, an effective and straightforward intra-articular injection therapy using a cocktail of hAMSCs and HA was developed to treat knee OA in a rat model. The injured cartilage was remarkably regenerated, yielding results comparable to normal cartilage levels after 56 days of treatment. Both hAMSCs and HA were indispensable organic components in this therapy, in which HA could synergistically enhance the effects of hAMSCs on cartilage repair. The regenerative mechanism was attributed to the fact that the addition of HA comprehensively enhances the activities of hAMSCs, including chondrogenic differentiation, proliferation, colonization, and regenerative modulation. This cocktail paves a new avenue for injection therapy to treat OA, holding the potential to realize rapid clinical translation.

The Human Amniotic Mesenchymal Stem Cells (hAMSCs) Improve the Implant Osseointegration and Bone Regeneration in Maxillary Sinus Floor Elevation in Rabbits.

Insufficient bone height in the posterior maxilla is a challenging problem in dental implantation. Bio-Oss, though routinely used in maxillary sinus floor elevation (MSFE), is not osteoinductive. Human amniotic mesenchymal cells (hAMSCs) isolated from placental tissues have potential for multidifferentiation and immunomodulatory properties and can be easily obtained without the need for invasive procedures and without ethical concerns. This is the first study to use hAMSCs to improve implant osseointegration and bone regeneration after MSFE. Human AMSCs were loaded into a fibrin gel and injected into rabbit MSFE models. The rabbits were assigned to four groups (n = 3 per group), i.e., the control group, the hAMSC group, the Bio-Oss group, and the hAMSC/Bio-Oss group. The animals were sacrificed at postsurgery for four and twelve weeks and evaluated by histology and immunohistochemistry. Bone volume, bone volume/tissue volume, bone-to-implant contact ratio, and vessel-like structures in the hAMSC/Bio-Oss group were significantly better than those in other groups in the peri-implant and augmented areas. Immunofluorescence staining showed that alkaline phosphatase (ALP) activities of two hAMSC groups were higher than those of the other two groups. Sequential fluorescent labeling was performed in all of the 12-week groups. Observations showed that hAMSCs accelerated mineralized deposition rates on implant surfaces and in bone-augmented areas. These data demonstrated that hAMSCs could enhance implant osseointegration and bone regeneration after MSFE and might be used to optimize dental implantation in the future.

Human Amnion Epithelial Cell Therapy for Chronic Liver Disease.

Liver fibrosis is a common consequence of chronic liver disease. Over time, liver fibrosis can develop into liver cirrhosis. Current therapies for liver fibrosis are limited, and liver transplant is the only curative therapy for patients who progress to end-stage disease. A potential approach to treat chronic liver disease with increasing interest is cell-based therapy. Among the multiple cell types which have been proposed for therapeutic uses, human amnion epithelial cells and amniotic fluid-derived mesenchymal cells are promising. These cells are highly abundant, and their use poses no ethical concern. Furthermore, they exert potent anti-inflammatory and antifibrotic effects in animal models of liver injury. This review highlights the therapeutic characteristics and discusses how human amnion epithelial cells can be utilised as a therapeutic tool for chronic liver disease.

Differential response of immortalized human amnion mesenchymal and epithelial cells against oxidative stress

Cells are equipped with various antioxidant defense factors to antagonize insults from reactive oxygen species (ROS), thus the antioxidant capacity has been characterized by a variety of cellular responses during the pathophysiological processes. Amniotic cells have been extensively applied in clinical practice for burn treatment, corneal repair, and tissue regeneration. However, the antioxidative properties of amniotic cells have not yet been fully understood. Therefore, the current study was aimed to observe the response of amniotic cells against ROS stimuli, and to investigate the underlying molecular mechanisms. The immortalized human amniotic mesenchymal cells (iHAMs) and immortalized human amniotic epithelial cells (iHAEs) were used. The human skin fibroblast (HSF) was used as a control cell line. Changes in intracellular ROS generation, cell viability, and cellular morphology were investigated to reveal the response of amniotic cells against oxidative stresses induced by x-rays and hydrogen peroxide. In addition, expression of apoptosis-related proteins and response to antioxidative stress was also examined. The intracellular ROS level and cell apoptosis in iHAMs was remarkably increased. iHAEs showed relatively high resistance to ROS stimulation, which can be attributed to the high SOD2 expression and up-regulation of Nrf2, HO-1 after x-rays exposure. In contrast, iHAMs were found sensitive to oxidative damage. Expression of caspase-3, caspase-8 and BAX was increased, whereas down-regulation of Bcl-xL, Nrf2, HO-1, and TrxR-1. Taken together, findings have highlighted the characterization of response of amniotic derived epithelial and mesenchymal cells to oxidative stress. In physiological processes, iHAMs may play an important role to maintain the homeostasis of the pregnancy environment. However, under oxidative stimulations, iHAEs provides protection against oxidative damage in amnion tissue.

Myogenic differentiation of human amniotic mesenchymal cells and its tissue repair capacity on volumetric muscle loss

Stem cell–based tissue engineering therapy is the most promising method for treating volumetric muscle loss. Human amniotic mesenchymal cells possess characteristics similar to those of embryonic stem cells. In this study, we verified the stem cell characteristics of human amniotic mesenchymal cells by the flow cytometry analysis, and osteogenic and adipogenic differentiation. Through induction with the DNA demethylating agent 5-azacytidine, human amniotic mesenchymal cells can undergo myogenic differentiation and express skeletal muscle cell–specific markers such as desmin and MyoD. The Wnt/β-catenin signaling pathway also plays an important role. After 5-azacytidine-induced human amniotic mesenchymal cells were implanted into rat tibialis anterior muscle with volumetric muscle loss, we observed increased angiogenesis and improved local tissue repair. We believe that human amniotic mesenchymal cells can serve as a potential source of cells for skeletal muscle tissue engineering.

Effects from transplantation of human amniotic mesenchymal stem cells on survival of random skin flap in rats

Objective To investigate the feasibility of human amniotic mesenchymal stem cells (hAMSCs) transplantation to improve the survival of ischemic ultra-long random skin flap vascularization, so as to promote the survival of skin flap. Methods The hAMSCs were isolated from human amnion, cultured in vitro, and identified by immunocytochemistry. The phenotype of hAMSCs was analyzed by flow cytometry. CellTracker™-CM-Dil was used to label before hAMSCs transplantation into skin flap. Twenty SD adult rats were selected and the 2 cm×8 cm ischemic ultra-long random skin flap models were constructed in the left and right sides of the rat back. The pedicles of flaps were on the lliac crest level. The flaps were divided into left group (injection with 0.5 ml LG-DMEM) and right group (0.5 ml 1×106/ml hAMSCs) after the flap was lifted. The survival rate of flap was observed 7 d after surgery. The blood perfusion values, namely blood perfusion unit at pedicle and in the middle, were monitored by laser Doppler flow monitor at the immediate time, 24 h, 48 h, 4 d and 7 d of the skin flap after surgery. The capillary density of the skin flap was observed through histological observation of the tissue (0.5 cm from adult and necrotic junction). The distribution and survival rate of CM-Dil labeled hAMSCs were observed by fluorescent microscope. Results In term of survival rate of the flap, left group was (50.6±2.2)%, and right group was (70.9±2.1)%. The survival rate of the flap in right group was greater than that of left group (P<0.05). Blood perfusion unit detected in the pedicle of left group at days 4 and 7 after surgery was higher than that of the right group (P<0.05). Blood perfusion unit in middle of flap of left group at 24 h, 48 h, 4 d and 7 d were lower than that of right group (P<0.05). The flap capillary density at 7 d after surgery were (8.8±1.2)/ mm2 in left group and (23.5±1.6)/mm2 in right group (P<0.05). The tissue of flap was made frozen section, and the fluorescence microscope showed there were CM-Dil labeled hAMSCs in skip flap in right group, which could manifest the survival and distribution of hAMSCs in skip flap. Conclusion The application of hAMSCs in the middle and distal parts of ultra-long random skin flap can significantly improve the survival rate of skin flap, and increase the density of microvascular reconstruction in the flap. Key words: Mesenchymal stem cell transplantation; Amnion; Surgical flaps

Mechanisms underlying intravenous transplantation of human amniotic mesenchymal stem cells for Alzheimer's disease in transgenic APP+ mice

Mechanisms underlying intravenous transplantation of human amniotic mesenchymal stem cells for Alzheimer's disease in transgenic APP+ mice

Electrospun Poly(l-lactide)/Poly(ethylene glycol) Scaffolds Seeded with Human Amniotic Mesenchymal Stem Cells for Urethral Epithelium Repair.

Tissue engineering-based urethral replacement holds potential for repairing large segmental urethral defects, which remains a great challenge at present. This study aims to explore the potential of combining biodegradable poly(l-lactide) (PLLA)/poly(ethylene glycol) (PEG) scaffolds and human amniotic mesenchymal cells (hAMSCs) for repairing urethral defects. PLLA/PEG fibrous scaffolds with various PEG fractions were fabricated via electrospinning. The scaffolds were then seeded with hAMSCs prior to implantation in New Zealand male rabbits that had 2.0 cm-long defects in the urethras. The rabbits were randomly divided into three groups. In group A, hAMSCs were grown on PLLA/PEG scaffolds for two days and then implanted to the urethral defects. In group B, only the PLLA/PEG scaffolds were used to rebuild the rabbit urethral defect. In group C, the urethral defect was reconstructed using a regular urethral reparation technique. The repair efficacy was compared among the three groups by examining the urethral morphology, tissue reconstruction, luminal patency, and complication incidence (including calculus formation, urinary fistula, and urethral stricture) using histological evaluation and urethral radiography methods. Findings from this study indicate that hAMSCs-loaded PLLA/PEG scaffolds resulted in the best urethral defect repair in rabbits, which predicts the promising application of a tissue engineering approach for urethral repair.

Human amnion favours tissue repair by inducing the M1-to-M2 switch and enhancing M2 macrophage features.

Human amniotic mesenchymal cells (hAMTCs) possess interesting immunomodulatory properties, making them attractive candidates for regenerative medicine applications. Recent in vivo reports argue in favour of an important role for macrophages as targets of hAMTC‐mediated suppression of inflammation and the enhancement of tissue repair. However, a comprehensive study of the effects of hAMTCs and their conditioned medium (CM) on human macrophage differentiation and function is unavailable. In the present study we found that hAMTCs and CM induce the differentiation of myeloid cells (U937 and monocytes) towards macrophages. We then investigated their effects on monocytes differentiated toward pro‐inflammatory M1 and anti‐inflammatory M2 macrophages. Monocytes treated under M1 conditions in the presence of hAMTCs or CMs shifted towards M2‐like macrophages, which expressed CD14, CD209, CD23, CD163 and PM‐2 K, possessed higher phagocytic activity and produced higher IL‐10 and lower pro‐inflammatory cytokines. They were also poor T cell stimulators and Th1 inducers, while they were able to increase activated and naïve suppressive Treg subsets. We show that prostaglandins, and not IL‐6, play a role in determining the M2 activation status. Instead, monocytes treated under M2 conditions in the presence of hAMTCs or CM retained M2‐like features, but with an enhanced anti‐inflammatory profile, having a reduced expression of the co‐stimulatory molecule CD80, reduced phagocytosis activity and decreased the secretion of inflammatory chemokines. Importantly, we provide evidence that macrophages re‐educated by CM improve tissue regeneration/repair in wound‐healing models. In conclusion, we identified new cell targets of hAMTCs and their bioactive factors and here provide insight into the beneficial effects observed when these cells are used in therapeutic approaches in vivo. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

N-BENZYLCINNAMIDE INDUCES CHOLINERGIC NEURONAL DIFFERENTIATION FROM HUMAN AMNIOTIC FLUID MESENCHYMAL STEM CELLS

N-BENZYLCINNAMIDE INDUCES CHOLINERGIC NEURONAL DIFFERENTIATION FROM HUMAN AMNIOTIC FLUID MESENCHYMAL STEM CELLS

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

Construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells in vitro

Background Corneal transplantation is an effective treatment to severe corneal diseases, but the shortage of cornea donor limits its application.Tissue-engineered cornea is being a new approach to corneal diseases. Objective This study was to investigate the possibility of construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells (hAMSCs) in vitro. Methods Fresh human amniotic membranes were obtained under the approval of Ethic Committee of Affiliated First Hospital of Jinan University and informed consent of maternal women.The 6 cm×6 cm amniotic membrane tissue explant was digested using trypsin+ EDTA, and then the amniotic epithelial cells (AECs) were scraped before putting into collagenase Ⅱ digestion medium to isolate hAMSCs.hAMSCs of passage 3 were cultured to achive 80%-90% confluence, and then the cells were incubated on rabbit deepithelial corneal stroma at a 1×105/ml density.The corneal stroma was co-cultured with hAMSCs at an air-liquid interface till 14 days.Rabbit deepithelial corneal stroma with and without hAMSCs (experimental group and control group) were fixed in 4% para formaldehyde, and sections were prepared for histopathological examination.Immunochemistry and immunofluorescence were empoyed to detect the expressions of cytokeratin3 (CK3) and CK12 in hAMSCs. Results hAMSCs grew well and formed a stratified epidermal structure resembling native corneal epithelium on rabbit corneal stroma in cultured 14 days in the experimental group, with the oval nucleus at basement and fusiform nucleus on the surface of corneal stroma.There was no cell structure in the control group.Immunochemistry revealed brown staining for CK3, CK12 in cytoplasm of hAMSCs on the rabbits corneal stroma, and the green fluorescence for CK3 and CK12 was also seen in the hAMSCs.However, the response for CK3 and CK12 was absent in the control sections either immunochemistry or immunofluorescence test. Conclusions hAMSCs can be induced to differentiate into corneal epithelioid cells at an air-liquid interface on the rabbit corneal stroma. Key words: Amnion; Mesenchymal stem cells; Cell culture; Tissue engineering; Epithelium, corneal; Differentiation; Humans

Human Amniotic Membrane-Derived Mesenchymal and Epithelial Cells Exert Different Effects on Monocyte-Derived Dendritic Cell Differentiation and Function.

We previously demonstrated that mesenchymal cells from human amniotic membrane (hAMTCs) inhibit the generation and maturation of monocyte-derived dendritic cells (DCs) in vitro. Considering the crucial role of DCs in the immune response and that epithelial cells of the human amniotic membrane (hAECs) share some of the immunoregulatory properties of hAMTCs, we investigated whether hAECs also modulate monocyte-derived DCs. We compared hAECs with hAMTCs in a cell-to-cell contact setting and their secreted factors in modulating DC differentiation and function. First, we demonstrated that primary and expanded hAMTCs strongly inhibited the differentiation of DCs and induced a shift toward M2-like macrophages. This was observed when hAMTCs were cultured in contact (hAMTC-DC(cont)) or in Transwells (hAMTC-DC(tw)) with monocytes and even when medium conditioned by hAMTCs was used instead of hAMTCs. hAECs also prevented DC development, but to a lesser extent than hAMTCs. hAECs were more effective when cultured in contact with monocytes (hAEC-DC(cont)) rather than in Transwells (hAEC-DC(tw)). The modulatory capacity of hAECs changed during passaging unlike the hAMSCs. The ability to stimulate CD4(+) and CD8(+) T-cell proliferation was almost completely abolished by hAMTC-DC(cont), whereas hAMTC-DC(tw) and hAEC-DC(cont) displayed only a reduced ability to stimulate CD8(+) T cells. Furthermore, monocytes cocultured with hAMTCs and hAECs showed some similarities, but also differences in cytokine/chemokine secretion. Similarities were observed in the inhibition of IL-12p70 and TNF-α and the increase in IL-10 in supernatants taken from monocyte-DCs cocultured with hAMTCs and hAECs in contact and Transwell settings. The inflammatory factors IL-8, CXCL9, and MIP-1α were significantly lower in hAMTC-DC(cont), hAMTC-DC(tw), and hAEC-DC(cont) conditions. In contrast, only hAMTCs (in both contact and Transwell conditions) were able to significantly increase IL-1β and CCL2. Altogether, we demonstrated that hAMTCs and hAECs affect DC differentiation, but that hAMTCs exerted a stronger inhibitory effect, abolished T-cell proliferation, and also induced more changes in cytokine/chemokine production.

Effects of conditioned medium from human amniotic mesenchymal tissue cell cultures on prostate cancer cells

It has been recently demonstrated that human amniotic mesenchymal tissue cells (hAMTC) derived from term placenta inhibit lymphocyte proliferation and significantly reduce the growth of haemopoietic and non haemopoietic cancer cell lines (HeLa and Saos cells) in vitro (1). The aim of our study was to evaluate the effects of hAMTC-conditioned medium (CM) on two human prostate cancer cells lines: LNCaP, androgen responsive and well differentiated, and PC-3, androgen unresponsive and less differentiated. Cells were grown in their standard culture conditions in the absence or in the presence of various concentrations (0.001–50%) of hAMTC-CM or their own exhausted medium. Cell numbers were determined by using a haemocytometer, after three days. Moreover, E- and N-cadherin expression was evaluated in PC-3 cells cultured in medium with 0.01, 1 or 25% hAMTC-CM by Immunocytochemistry and Western blot analysis. Our findings indicate that hAMTC-CM reduces the growth of both PC-3 and LNCaP cells. The effect is more pronounced in PC-3 cells in which inhibition is about 25% vs control (p<0.001) at a very low concentration (0.001%) and reaches the maximum (about 55% vs control, p<0.001) with the highest concentration used (50%). In LNCaP cells only the highest concentration of hAMTC-CM (50%) inhibits cell proliferation (about 40% vs control, p<0.001). Interestingly, growth of LNCaP cells is reduced by their own exhausted medium, while proliferation of PC-3 cells is not affected by their spent medium. Both E- and N-cadherin expression have been detected at the membrane level in untreated PC-3 cells and the localization does not change in hAMTC-CM-treated cells. Preliminary data obtained by Western blot analysis seem to indicate an increase in both E- and N-cadherin levels. Our findings show that hAMTC-CM reduces prostate cancer cell proliferation in relationship to their androgen sensitivity and modifies the expression levels of adhesion molecules. Experiments are in progress to determine the mechanisms which underlie the observed effects and assess if hAMTC-CM can determine any variation in the differentiation status of prostate cancer cells.

Therapeutic effect and immunologic regulation of human amniotic mesenchymal stem cells in rats with experimental type 1 diabetes mellitus

Objective To study the therapeutic effect and immunologic regulation of human amniotic mesenchymal stem cells (hAMSCs) in rats with experimental type 1 diabetes mellitus (T1DM).Methods The hAMSCs from human amnion were isolated and cultured in vitro,then phenotype was analyzed by flow cytometry.T1DM were produced by administering streptozocin to rats.The rats were divided into normal control group (n =6),T1 DM model group (n =6),medium treated group (n =6),hAMSCs transplanted group(n =6),and insulin treated group(n =6).5 × 106of hAMSCs or vehicle were administered to rats via sublingual vein.Blood glucose levels of rats were recorded weekly in the groups for six weeks by Blood Sugar Meter.At the end of 6 weeks after hAMSCs transplantation,concentrations of plasma insulin were detected by ELISA; histopathological changes of pancreas,surviwl and differentiation of transplanted hAMSCs in pancreatic tissue were studied with HE staining,and immunofluorescence staining; percentages of lymphocyte subsets in peripheral blood mononuclear cells were determined by flow cytometry ; concentrations of plasma cytokines were determined by cytometric bead array.Results After hAMSCs transplantation,blood glucose levels in rats with T1DM were decreased (P ＜ 0.01),while concentrations of plasma insulin were increased significantly (P＜0.01).At 6 weeks,cell-treated animals showed an improvement in pancreas damage ; the percentages of CD4 + IFN-γ+ (Th 1) and C D4 + interleukin (IL)-17 + (Th 17) cells were reduced (all P＜0.05),while the percentages of FoxP3-positive regulatory T cells (FoxP3 +Treg) and CD4+ IL-4+(Th2) cells were increased (all P＜0.01) ; plasma concentrations of interferon-γ,IL-2,and tumor necrosis factor-αwere decreased (all P＜0.01),but IL-4 level was increased (P＜0.05).No histological evidence of insulin producing cells from hAMSCs was seen within pancreas.Conclusions hAMSCs may reduce blood glucose and alleviate the islet damage in rats with T1 DM,which is related to their potential to up-regulate FoxP3 +Treg cells. Key words: Diabetes mellitus, type 1 ; Human amniotic mesenchymal stem cells; Immunologic regulation; Rat

Human Amniotic Fluid-Derived Mesenchymal Cells from Fetuses with a Neural Tube Defect Do Not Deposit Collagen Type I Protein After TGF-β1 Stimulation In Vitro

In spina bifida, the neural tube fails to close during the embryonic period. Exposure of the neural tube to the amniotic fluid during pregnancy causes additional neural damage. Intrauterine tissue engineering using a biomaterial seeded with stem cells might prevent this additional damage. For this purpose, autologous cells from the amniotic fluid are an attractive source. To close the defect, it is important that these cells deposit an extracellular matrix. However, it is not known if amniotic fluid mesenchymal cells (AFMCs) from a fetus with a neural tube defect (NTD) share the same characteristics as AFMCs from a healthy fetus. We found that cells derived from fetuses with a NTD, in contrast to healthy human amniotic fluid cells, did not deposit collagen type I. Furthermore, the NTD cells showed, compared with both healthy amniotic fluid cells and fetal fibroblasts, much lower mRNA expression levels of genes that are involved in collagen biosynthesis [procollagen C-endopeptidase enhancer proteins (PCOLCE), PCOLCE2, ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2), ADAMTS14]. This indicates that NTD-AFMCs have different characteristics compared with healthy AFMCs and might not be suitable for fetal therapy to close the defect in spina bifida patients.

Human Amnion Mesenchymal Cells Negative Co-stimulatory Molecules PD-L1 Expression and Its Capacity of Modulating Microglial Activation of CNS

The objective of this study is to investigate the negative immunomodulatory capacity of human amniotic mesenchymal cells (AMSCs) and their possible intrinsic mechanism, by which we can confirm that they modulate microglial activation of central nervous system from multiple perspectives at the molecular level. The identification of the immune phenotype of AMSCs and microglial cells was executed by immunohistochemical methods and flow cytometry. Meanwhile, the influence and mechanism of amniotic mesenchymal cells in vitro on proliferation, cell cycle, and cytokine release of activated microglia (MI) would be detected by ELISA, β-liquid scintillation counting method, and flow cytometry. Human amnion mesenchymal cells highly expressed negative co-stimulatory molecules PD-L1, while its ligand PD1 was expressed with high level by activated MI. When adding the PD-L1mAb to the mixed culture system composed of AMSCs and activated MI, the proliferation inhibitory effect and the cycle-blocking effect produced by the former on the latter would be partially reversed; at the same time, the impact of the latter cytokine secretion would be adjusted. As a conclusion, AMSCs play inhibitory effects on microglial activation, proliferation, and immune effects partially through the PD-L1-PD1 signaling pathways.

Effect of Exogenous Oct4 Overexpression on Cardiomyocyte Differentiation of Human Amniotic Mesenchymal Cells

Regenerative therapy is a new strategy for the end-stage heart failure; however, the ideal cell source has not yet been established for this therapy. We expected that the amnion might be an ideal cell source for cardiac regenerative therapy and that the differentiation potency of the human amnion mesenchymal cells (hAMCs) could be improved by overexpression of Oct4, a key factor that maintains the undifferentiated state. A plasmid vector was made by insertion of the Oct4 open reading frame (ORF) under control of a cytomegalovirus (CMV) promoter (pCMV-hOct4) and transfected into hAMCs by electroporation. The optimum induction time was investigated by comparing the quantity of stem cell-specific mRNAs, cardiac-specific mRNAs, and cardiac-specific proteins with time. hAMCs already expressed cardiac-specific proteins such as Nkx2.5 and Connexin43. After pCMV-hOct4 transfection, endogenous Oct4 mRNA and other stem cell markers showed a transient increase. With 5-azacytidine treatment, quantities of the cardiac-specific mRNAs, such as GATA4 and myosin light-chain-2v (Mlc-2v), were increased significantly. After Oct4 overexpression, the highest expression of cardiac-specific mRNAs and stem cell makers was seen at almost the same time. Furthermore, more mature myocardial contraction proteins were observed when hAMCs were induced at specific optimal times after gene transfection. In conclusion, hAMCs were activated to an undifferentiated state by overexpression of Oct4, and their cardiac differentiation potency was improved. Thus, the single-time transfection of the Oct4 expression vector may be a useful strategy for effective cell therapy. The use of cryopreserved hAMCs in cell therapy still requires more investigation.

Hypoxia enhances the induction of human amniotic mesenchymal side population cells into vascular endothelial lineage

Human amniotic mesenchymal side population (hAM-SP) cells have pluripotency and weak immunogenicity, and have promising roles in the field GAPDH of regenerative medicine. The aim of the present study was to determine whether hypoxic conditions induce the differentiation of hAM-SP cells into the vascular endothelial lineage. Mesenchymal cells were isolated from enzyme-treated amniotic membranes and stained with Hoechst 33342. The hAM-SP cells were negatively sorted by FACS and cultured in induction medium containing vascular endothelial growth factor (VEGF) under normoxic (20% O2) or hypoxic (1% O2) conditions for 1 or 2 weeks. The expression of endothelial markers such as kinase domain region (KDR), fms-like tyrosine kinase (Flt)-1, von Willebrand factor (vWF), vascular endothelial (VE)-cadherin and human vascular cell adhesion molecule (VCAM) at the gene and protein level was evaluated by real-time PCR and fluorescent immunostaining, respectively. The gene expression of KDR, Flt-1, VE-cadherin and vWF peaked after 2 weeks of culture. The protein expression of KDR and VE-cadherin was also enhanced after 2 weeks of culture under hypoxic conditions. To confirm the involvement of hypoxia-inducible factor (HIF) in the induction under hypoxic conditions, the expression of genes which are known to be upregulated by HIF was analyzed by DNA microarray. The expression of these genes increased under hypoxic conditions. hAM-SP cells cultured under hypoxic conditions differentiated into the vascular endothelial lineage, probably due to upregulation of the gene expression associated with angiogenesis through activation of the HIF system.

Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells

Human amniotic mesenchymal cells (HAM cells) are known to contain somatic stem cells possessing the characteristics of pluripotency. However, little is known about the biology of these somatic cells because isolated HAM cells from amniotic membrane have a limited lifespan. To overcome this problem, we attempted to prolong the lifespan of HAM cells by infecting retrovirus encoding human papillomavirus type16E6 and E7 (HPV16E6E7), bmi-1, and/or human telomerase reverse transcriptase (hTERT) genes and investigated their characteristics as stem cells. We confirmed the immortalization of the four lines of cultured HAM cells for about 1 year. Immortalized human amnion mesenchymal cells (iHAM cells) have continued to proliferate over 200 population doublings (PDs). iHAM cells were positive for CD73, CD90, CD105, and CD44 and negative for CD34, CD14, CD45, and HLA-DR. They expressed stem cell markers such as Oct3/4, Sox2, Nanog, Klf4, SSEA4, c-myc, vimentin, and nestin. They showed adipogenic, osteogenic, and chondrogenic differentiation abilities after induction. These results suggested that immortalized cell lines with characteristics of stem cells can be established. iHAM cells with an extended lifespan can be used to produce good experimental models both in vitro and in vivo.