Human Amniotic Epithelial Cell Research Articles

Human amniotic epithelial cell transplantation is safe and well tolerated in patients with compensated cirrhosis: a first-in-human trial.

Placenta-derived human amniotic epithelial cells (hAEC) exhibit anti-inflammatory and anti-fibrotic effects in cirrhosis models. We conducted a first-in-human phase I clinical trial to assess the safety and tolerability of hAEC in adults with compensated cirrhosis. We examined increasing and repeated doses of hAEC in 9 patients in 3 cohorts. Cohort 1 patients received 0.5 × 106/kg hAEC in one IV infusion. Cohort 2 patients received 1 × 106/kg hAEC in one IV infusion. The patients in cohort 3 received 1 × 106/kg hAEC on days 0 and 28. Here, we report follow-up to post-infusion day 56 (D56), during which no serious adverse events occurred. Six patients experienced no study-related adverse events, while 3 patients reported mild (grade 1) headaches that were possibly infusion-related. A transient decrease in serum platelet count occurred in all patients, which returned to baseline screening values by day 5. FIB-4 values to assess fibrosis were significantly lower at D56. Although not statistically significant, serum AST levels and liver stiffness measurements at D56 were lower than those at baseline. The hepatic venous pressure gradient, a measure of portal hypertension, declined in 4 patients, did not change in 3 patients, and increased in 2 patients. In conclusion, intravenous infusion of allogeneic hAEC in patients with compensated cirrhosis at the doses used in this study was safe and well tolerated, with no difference observed between 1 and 2 doses. Decreased hepatic inflammation, liver stiffness, and portal hypertension support larger studies aimed at identifying patients who may benefit from this therapy. Clinical Trial registration: The trial was prospectively entered on the Australian Clinical Trials Registry (ANZCTR12616000437460).

Hyaluronan delays human amniotic epithelial stem cell senescence by regulating CD44 isoform switch to activate AKT/mTOR signals

The replicative senescence of human amniotic epithelial stem cells (hAECs) is a major concern towards its clinical application. This study found that a 300-kDa hyaluronic acid (HA) could effectively delay the replicative senescence of hAECs, as indicated by the downregulation of cellular senescence markers and alteration of the cell cycle, and substantially improve the differentiation capacities of hAECs. HA was confirmed to regulate the CD44 isoform switch by upregulating the CD44s and downregulating the CD44v, thus exerting an anti-aging effect. We further found that HA induced the upregulation of hyaluronan synthase (HAS) 2, resulting in the activation of epithelial splicing regulatory protein 1 (ESRP1) and alternative splicing of CD44 mRNA, thereby promoting CD44s expression and inhibiting CD44v expression. Knockdown of HAS2 blocked ESRP1 expression and attenuated the anti-aging effects of HA. Hermes-1, a specific blocker of CD44, caused partial loss of the anti-aging effect of HA, upregulated senescence markers, and downregulated stemness markers. Furthermore, CD44s receptor activation was shown to initiate the AKT/mTOR downstream signaling. Conclusively, the study suggested that HA delayed hAEC senescence by regulating CD44 isoform switch to activate the AKT/mTOR signaling pathway, and there is potential for the clinical application of hAECs in combination with HA.

NETs promote ROS production to induce human amniotic epithelial cell apoptosis via ERK1/2 signaling in spontaneous preterm birth.

Premature birth is a common obstetric complication but its pathogenesis is unclear.Inflammation at the maternal-fetal interface in preterm labor leads to the infiltration of neutrophils, which promotes inflammatory responses and induces the degradation of extracellular matrix and cell apoptosis, thus contributing to preterm labor.It is unclear whether neutrophil extracellular traps (NETs), a functional form of neutrophils, are involved in preterm labor. After collecting amniotic membranes from research objects, we localized NETs by immunofluorescence and evaluated the expression of matrix metalloproteinase (MMP)-9 and MMP-2 by western blotting. Primary human amniotic epithelial cells (hAECs) subjected to treatment with NETs, 5-ethynyl-20-deoxyuridine cell proliferation assay, lactate dehydrogenase (LDH) assay, western blotting, and flow cytometry apoptosis assay were used to determine the effects of NETs on hAECs. We also elucidated possible mechanisms underlying the effects. Compared with normal term women, NETs infiltration and MMP-9 expression in the amniotic membrane from preterm women had increased.Thereafter, NETs might suppress the proliferation and promote the apoptosis of hAECs.Furthermore, after NETs treatment, the mitochondrial membrane potential was significantly decreased, ERK1/2 phosphorylation expression was upregulated and reactive oxygen species (ROS) production was increased in hAECs.Changes in cell proliferation, LDH release, and cell apoptosis level due to NETs could be reversed by ROS inhibitor or ERK phosphorylation inhibitors. NETs can promote the apoptosis of hAECs via ERK1/2 pathways dependent on ROS release.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro

Objectives: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. Design: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. Materials: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. Settings: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. Methods: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. Results: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 μm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 μm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). Limitations: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. Conclusions: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

Repair of Retinal Degeneration by Human Amniotic Epithelial Stem Cell-Derived Photoreceptor-like Cells.

The loss of photoreceptors is a major event of retinal degeneration that accounts for most cases of untreatable blindness globally. To date, there are no efficient therapeutic approaches to treat this condition. In the present study, we aimed to investigate whether human amniotic epithelial stem cells (hAESCs) could serve as a novel seed cell source of photoreceptors for therapy. Here, a two–step treatment with combined Wnt, Nodal, and BMP inhibitors, followed by another cocktail of retinoic acid, taurine, and noggin induced photoreceptor–like cell differentiation of hAESCs. The differentiated cells demonstrated the morphology and signature marker expression of native photoreceptor cells and, intriguingly, bore very low levels of major histocompatibility complex (MHC) class II molecules and a high level of non–classical MHC class I molecule HLA–G. Importantly, subretinal transplantation of the hAESCs–derived PR–like cells leads to partial restoration of visual function and retinal structure in Royal College of Surgeon (RCS) rats, the classic preclinical model of retinal degeneration. Together, our results reveal hAESCs as a potential source of functional photoreceptor cells; the hAESCs–derived photoreceptor–like cells could be a promising cell–replacement candidate for therapy of retinal degeneration diseases.

Fetal Lung-Derived Exosomes in Term Labor Amniotic Fluid Induce Amniotic Membrane Senescence.

The mechanism of parturition is still unclear. Evidence has shown that delivery is associated with cellular senescence of the amniotic membrane. We isolated fetal lung-associated exosomes from the amniotic fluid from term labor (TL-exos) and verified that the exosomes can cause primary human amniotic epithelial cell (hAEC) senescence and apoptosis and can release higher levels of senescence-associated secretory phenotype (SASP)-related molecules and proinflammatory damage-associated molecular patterns (DAMPs) than exosomes isolated from the amniotic fluid from term not in labor (TNIL-exos). The human lung carcinoma cell lines (A549) can be used as an alternative to alveolar type 2 epithelial cells producing pulmonary surfactant. Therefore, we isolated A549 cell-derived exosomes (A549-exos) and found that they can trigger hAEC to undergo the same aging process. Finally, the animal experiments suggested that A549-exos induced vaginal bleeding and preterm labor in pregnant mice. Therefore, we conclude that exosomes derived from fetal lungs in term labor amniotic fluid induce amniotic membrane senescence, which may provide new insight into the mechanism of delivery.

MiR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor.

Labor is a process of inflammation and hormonal changes involving both fetal and maternal compartments. MicroRNA-132-3p (miR-132-3p) has been reported to be involved in the development of inflammation-related diseases. However, little is known about its potential role in labor onset. This study aimed to explore the mechanism of miR-132-3p in amnion for labor initiation. In the mouse amnion membranes, the expression of miR-132-3p was found to increase gradually during late gestation. In human amniotic epithelial cell line (WISH), upregulation of miR-132-3p was found to increase proinflammatory cytokines and cyclooxygenase 2 (COX2) as well as prostaglandin E2 (PGE2), which was suppressed by miR-132-3p inhibitor. Dual-specificity phosphatase 9 (DUSP9) was identified as a novel target gene of miR-132-3p, which could be negatively regulated by miR-132-3p. DUSP9 was present in the mouse amnion epithelial cells, with a decrease in its abundance at 18.5 days post coitum (dpc) relative to 15.5 dpc. Silencing DUSP9 was found to facilitate the expression of proinflammatory cytokines and COX2 as well as PGE2 secretion in WISH cells, which could be attenuated by p38 inhibitor SB203580 or JNK inhibitor SP600125. Additionally, intraperitoneal injection of pregnant mice with miR-132-3p agomir not only caused preterm birth, but also promoted the abundance of COX2 as well as phosphorylated JNK and p38 levels, and decreased DUSP9 level in mouse amnion membranes. Collectively, miR-132-3p might participate in inflammation and PGE2 release via targeting DUSP9-dependent p38 and JNK signaling pathways to cause preterm birth.

Human amniotic epithelial cell transplantation improves scar remodeling in a rabbit model of acute vocal fold injury: a pilot study

ObjectiveTo gain insight into the molecular mechanisms underlying the early stages of vocal fold extracellular matrix (ECM) remodeling after a mid-membranous injury resulting from the use of human amniotic epithelial cells (hAEC), as a novel regenerative medicine cell-based therapy.MethodsVocal folds of six female, New Zealand White rabbits were bilaterally injured. Three rabbits had immediate bilateral direct injection of 1 × 106 hAEC in 100 µl of saline solution (hAEC) and three with 100 µl of saline solution (controls, CTR). Rabbits were euthanized 6 weeks after injury. Proteomic analyses (in-gel trypsin protein digestion, LC–MS/MS, protein identification using Proteome Discoverer and the Uniprot Oryctolagus cuniculus (Rabbit) proteome) and histological analyses were performed.ResultshAEC treatment significantly increased the expression of ECM proteins, elastin microfibril interface-located protein 1 (EMILIN-1) and myocilin that are primarily involved in elastogenesis of blood vessels and granulation tissue. A reactome pathway analysis showed increased activity of the anchoring fibril formation by collagen I and laminin, providing mechanical stability and activation of cell signaling pathways regulating cell function. hAEC increased the abundance of keratin 1 indicating accelerated induction of the differentiation programming of the basal epithelial cells and, thereby, improved barrier function. Lastly, upregulation of Rab GDP dissociation inhibitor indicates that hAEC activate the vesicle endocytic and exocytic pathways, supporting the exosome-mediated activation of cell–matrix and cell-to-cell interactions.ConclusionsThis pilot study suggests that injection of hAEC into an injured rabbit vocal fold favorably alters ECM composition creating a microenvironment that accelerates differentiation of regenerated epithelium and promotes stabilization of new blood vessels indicative of accelerated and improved repair.

Effects of histone H4 hyperacetylation on inhibiting MMP2 and MMP9 in human amniotic epithelial cells and in premature rupture of fetal membranes.

Histone modification is closely associated with several diseases. The aim of the current study was to investigate the associations among histone acetylation, matrix metalloproteinases (MMPs) and premature rupture of membranes (PROM) during pregnancy. A total of 180 puerperants were divided into three groups: i) Preterm-PROM (PPROM), ii) term-PROM (TPROM) and iii) full-term labor (FTL). Enzyme-linked immunosorbent assay (ELISA) kits and western blotting were used to determine the protein concentrations of MMP2, MMP9, histone deacetylase (HDAC)1, HDAC2 and HDAC6, and the protein levels of histone H4 lysine (H4K)5 and H4K8 acetylation, respectively, in three types of fetal membranes. Additionally, human amniotic epithelial cells were used to determine the effects of the HDAC inhibitors droxinostat and chidamide on cell viability, histone acetylation and the levels of MMP2, MMP9, HDAC1, HDAC2 and HDAC6 in vitro, using the Cell Counting Kit-8 assay, western blotting and ELISA, respectively. Furthermore, the effects of droxinostat and chidamide on the invasion and migration abilities of human amniotic epithelial cells were investigated using transwell assays. In fetal membranes, the activities of MMP2 and MMP9 increased in PPROM, but decreased in TPROM. Further, the expression of HDAC1 was decreased and histone hyperacetylation was increased in both PPROM and TRPOM. In vitro experiments revealed that 5 µM droxinostat and 0.5 µM chidamide selectively decreased the level of HDAC and induced acetylation of H4K5 and H4K8. Additionally, the aforementioned HDAC inhibitors reduced human amniotic epithelial cell viability, invasion and migration, and decreased the expression levels of MMP2 and MMP9. The current study revealed a high expression level of MMP2 and MMP9 in PPROM compared with TPROM and FL tissue, which was in accordance with previously published studies. Furthermore, the in vitro tests performed in the current study revealed the effect of histone H4 hyperacetylation on inhibiting MMP2 and MMP9 levels in vitro was similar to that observed in TPROM. The results obtained in the current study may be used as a theoretical guide for clinical treatment of premature rupture of membranes.

Clinical perspective on the use of human amniotic epithelial cells to treat congenital metabolic diseases with a focus on maple syrup urine disease

Congenital metabolic diseases are a group of hereditary disorders caused by the deficiency of a single specific enzyme activity. Without appropriate therapy, affected patients suffer severe neurologic disability and eventual death. The current mainstays of management attempt to slow disease progression, but are not curative. Several of these diseases have demonstrated significant benefits from liver transplantation; however, this approach is limited by the morbidity associated with this invasive procedure and a shortage of donor organs. Therefore, there is a need to establish a new strategy for improving the quality of a life for these patients. One potential solution is regenerative therapy using hepatocytes generated from stem cells. Herein, we discuss pertinent issues necessary for clinical application of the human amniotic epithelial cell, a type of placental stem cell. Focusing on maple syrup urine disease as an example, where liver replacement is an effective therapy, we explore this approach from a clinician's perspective.

IL-37 Exerts Anti-Inflammatory Effects in Fetal Membranes of Spontaneous Preterm Birth via the NF-κB and IL-6/STAT3 Signaling Pathway.

Spontaneous preterm birth (sPTB), defined as delivery before 37 weeks of gestation, is thought to be a multifactorial syndrome. However, the inflammatory imbalance at the maternal-fetal interface promotes excessive secretion of inflammatory factors and induces apoptosis and degradation of the extracellular matrix (ECM), which can subsequently lead to preterm birth. As an anti-inflammatory molecule in the IL-1 family, interleukin-37 (IL-37) mainly plays an inhibiting role in a variety of inflammatory diseases. However, as a typical inflammatory disease, no previous studies have been carried out to explore the role of IL-37 in sPTB. In this study, a series of molecular biological experiments were performed in clinical samples and human amniotic epithelial cell line (Wistar Institute Susan Hayflick (WISH)) to investigate the deficiency role of IL-37 and the potential mechanism. Firstly, the results indicated that the expression of IL-37 in human peripheral plasma and fetal membranes was significantly decreased in the sPTB group. Afterward, it is proved that IL-37 could significantly suppress the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in WISH cells. Simultaneously, once silence IL-37, LPS-induced apoptosis and activity of matrix metalloproteinases (MMPs) 2 and 9 were significantly increased. In addition, the western blot data showed that IL-37 performed its biological effects by inhibiting the NF-κB and IL-6/STAT3 pathway. In conclusion, our results suggest that IL-37 limits excessive inflammation and subsequently inhibits ECM remodeling and apoptosis through the NF-κB and IL-6/STAT3 signaling pathway in the fetal membranes.

The effect of human amniotic epithelial cell on dendritic cell differentiation of peripheral blood monocytes: An experimental study.

Background The amniotic membrane plays an important role in maintaining a healthy pregnancy. The main population cells from amniotic membrane include human amnion epithelial cells (hAECs) which have been shown to possess immunomodulatory properties.ObjectiveThe proximity of hAECs with monocyte leads to the generation of tollerogenic dendritic cells.Materials and MethodshAECs were obtained from normal pregnancy. Peripheral blood monocytes were isolated by anti-CD14 MACS method. Co-cultures of monocytes and hAECs were established in Transwell chambers supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the absence and presence of lipopolysaccharide (LPS) to produce immature and mature DCs, respectively. Immunophenotyping of the obtained DCs was done through flow cytometry and the production of cytokines was measured by ELISA. Mixed leukocyte Reaction (MLR) was also performed for the functional assessment of DCs.Results Immunophenotyping of [hAECs - Immature DC (iDC)] and [hAECs - iDC] + LPS cells revealed that the expression of CD1a, CD80, CD86, CD40, HLA-DR, and CD83 markers showed no significant difference as compared with the control group (iDCs and mDCs alone). In the [hAECs-iDCs] + LPS cells, the percentage of CD14 cells at the ratio of 1:2.5 showed significant differences compared to the control group. The production of IL-10 and IL-12 showed no significant difference in any of the cultures as compared to the control groups. Also, co-cultured DCs did not inhibit proliferation of lymphocyte.ConclusionOur findings show that factors secreted from cultured hAECs are unable to generate of tollerogenic dendritic cells. To achieve a better understanding of other mechanisms more investigations are needed.

Histologic Evaluations of Xenotransplanted Rabbit Knees by In Vitro-Propagated Human Amniotic Epithelial Cells: A Preclinical Study.

Human amniotic epithelial cells have multipotent differentiation capacity and are considered as potential therapeutic cells for clinical use. This study represents the first published report on the evaluation of the safety and clinical feasibility of human amniotic epithelial cells for transplant into knee joints, serving as an initial step for subsequent therapeutic evaluations within arthritis clinics. Our experimental design was based on subjecting groups of rabbits as a recipient model for human amniotic epithelial cell transplant into knee joints. Twenty rabbits received 200 μL sterile 0.9% sodium chloride solution containing 1 × 10⁹ human amniotic epithelial cells/knee joint by intra-articular injection. Control groups received cell-free saline into knees, and some animals were not treated. After 10 days of xenotransplant, radiology scans and histologic sections of transplanted and nontrans planted knees were examined and compared. Immunohistochemistry staining was also applied to detect tumor necrosis factor-alpha and interleukin 17 (as inflammatory and immuno-rejection markers) in knee sections. Similar to results shown in noninjected and saline-injected knees, all treated knees appeared normal, with no signs of acute immunorejection, no microbial colonization, no pain, no allergic reactions, no inflammation, and normal motion. Use of human amniotic epithelial cells appeared safe without risk of immunorejection or tumor formation in the transplanted knee joint. Human amniotic epithelial cells can be safely transplanted into knee joints, encouraging a need for complementary research for further therapeutic evaluations of human amniotic epithelial cells for curing arthritis.

Human amniotic epithelial cell transplantation promotes neurogenesis and ameliorates social deficits in BTBR mice

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interactions and communication and stereotypical patterns of behaviors, interests, or activities. Even with the increased prevalence of ASD, there is no defined standard drug treatment for ASD patients. Currently, stem cells, including human amniotic epithelial cell (hAEC) transplantation, seem to be a promising treatment for ASD, but the effectiveness needs to be verified, and the mechanism has not been clarified.MethodsWe intraventricularly transplanted hAECs into a 2-month-old BTBR T+tf/J (BTBR) mouse model of ASD. Behavior tests were detected 1 month later; hippocampal neurogenesis, neuroprogenitor cell (NPC) pool, and microglia activation were analyzed with immunohistochemistry and immunofluorescence; the levels of pro-inflammatory cytokines, brain-derived neurotrophic factor (BDNF), and TrkB in the hippocampus were determined by real-time PCR or western blotting.ResultsAfter intraventricular injection of hAECs into adult males, social deficits in BTBR mice were significantly ameliorated. In addition, hAEC transplantation restored the decline of neurogenesis and NPCs in the hippocampus of BTBR mice by expanding the stem cell pool, and the decreased levels of BDNF and TrkB were also rescued in the hippocampus of the hAEC-injected BTBR mice. Meanwhile, the transplantation of hAECs did not induce microglial overactivation or excessive production of pro-inflammatory cytokines in the hippocampus of BTBR mice.ConclusionsBased on these results, we found that hAEC transplantation ameliorated social deficits and promoted hippocampal neurogenesis in BTBR mice. Our study indicates a promising therapeutic option that could be applied to ASD patients in the future.

Human Amniotic Epithelial Cell-Derived Exosomes Restore Ovarian Function by Transferring MicroRNAs against Apoptosis

Premature ovarian failure (POF) is one of the most common complications among female patients with tumors treated with chemotherapy and requires advanced treatment strategies. Human amniotic epithelial cell (hAEC)-based therapy mediates tissue regeneration in a variety of diseases, and increasing evidence suggests that the therapeutic efficacy of hAECs mainly depends on paracrine action. This study aimed to identify exosomes derived from hAECs and explored the therapeutic potential in ovaries damaged by chemotherapy and the underlying molecular mechanism. hAEC-derived exosomes exhibited a cup- or sphere-shaped morphology with a mean diameter of 100 nm and were positive for Alix, CD63, and CD9. hAEC exosomes increased the number of follicles and improved ovarian function in POF mice. During the early stage of transplantation, hAEC exosomes significantly inhibited granulosa cell apoptosis, protected the ovarian vasculature from damage, and were involved in maintaining the number of primordial follicles in the injured ovaries. Enriched microRNAs (miRNAs) existed in hAEC exosomes, and target genes were enriched in phosphatidylinositol signaling and apoptosis pathways. Studies in vitro demonstrated that hAEC exosomes inhibited chemotherapy-induced granulosa cell apoptosis via transferring functional miRNAs, such as miR-1246. Our findings demonstrate that hAEC-derived exosomes have the potential to restore ovarian function in chemotherapy-induced POF mice by transferring miRNAs.

Involvement of calcium in 50-Hz magnetic field-induced activation of sphingosine kinase 1 signaling pathway.

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce human amniotic epithelial (FL) cell proliferation and sphingosine kinase 1 (SK1) activation, but the mechanism was not clearly understood. In the present study, the possible signaling pathways which were involved in SK1 activation induced by 50-Hz MF exposure were investigated. Results showed that MF exposure increased intracellular Ca2+ which was dependent on the L-type calcium channel, and induced Ca2+ -dependent phosphorylation of extracellular regulated protein kinase (ERK), SK1, and protein kinase C α (PKCα). Also, treatment with U0126, an inhibitor of ERK, could block MF-induced SK1 phosphorylation, but had no effect on PKCα phosphorylation. Also, the inhibitor of PKCα, Gö6976, had no effect on MF-induced SK1 activation in FL cells. In addition, the activation of ERK and PKCα could be abolished by SKI II, the inhibitor of SK1. In conclusion, the intracellular Ca2+ mediated the 50-Hz MF-induced SK1 activation which enhanced PKCα phosphorylation, and there might be a feedback mechanism between SK1 and ERK activation in responding to MF exposure in FL cells. Bioelectromagnetics. 9999:XX-XX, 2019. © 2019 Bioelectromagnetics Society.

Effects of Δ(9)-tetrahydrocannabinol (THC) on human amniotic epithelial cell proliferation and migration.

BackgroundThe deleterious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) is able to cross the placenta barrier and cause alterations in fetal growth, low birth weight and preterm labor. However, the effects of THC on the human placenta amnion are still unknown. MethodsThe distributions of CB1R and CB2R in human amnion tissues were observed by immunohistochemistry (IHC). Human amniotic epithelial cell proliferation and migration in response to THC treatment were measured by MTS and transwell assays, respectively. The PCR array was performed to study the key regulators involved in the cell migration. The protein levels of CB1R, CB2R in amnion tissues and MMP2, MMP9 in cells were detected by western blotting. Small interfering RNAs (siRNAs) were used to knockdown MMP2 and MMP9 in WISH cells. ResultsOur results indicated that both CB1R and CB2R primarily identified in the epithelial layer of human placental amnion tissue. The CB1R expression in the amnion tissue was higher in the preterm group than normal control. High-dose of THC (30uM, but not 20 and 10uM) significantly inhibited (p<0.01) human amniotic epithelial cell lines (WISH) proliferation. Meanwhile, THC at both 10uM and 20uM (p<0.05) significantly suppressed cells migration in both WISH and primary human amniotic epithelial cells. The PCR array data and siRNA experiments demonstrated that MMP2/9 were tightly involved in the regulation of THC-inhibited cell migration in WISH cells. ConclusionThese results suggested that THC inhibited the migration of human amniotic epithelial cell through the regulation of MMP2 and MMP9, which in turn altered the development of the amnion during the gestation and partially resulted in preterm labor and other adverse pregnancy outcomes.

Liver-Directed Human Amniotic Epithelial Cell Transplantation Improves Systemic Disease Phenotype in Hurler Syndrome Mouse Model.

Mucopolysaccharidosis type 1 (MPS1) is an inherited lysosomal storage disorder caused by a deficiency in the glycosaminoglycan (GAG)‐degrading enzyme α‐l‐iduronidase (IDUA). In affected patients, the systemic accumulation of GAGs results in skeletal dysplasia, neurological degeneration, multiple organ dysfunction, and early death. Current therapies, including enzyme replacement and bone marrow transplant, improve life expectancy but the benefits to skeletal and neurological phenotypes are limited. In this study, we tested the therapeutic efficacy of liver‐directed transplantation of a placental stem cell, which possesses multilineage differentiation potential, low immunogenicity, and high lysosomal enzyme activity. Unfractionated human amniotic epithelial cells (hAECs) were transplanted directly into the liver of immunodeficient Idua knockout mouse neonates. The hAECs engraftment was immunohistochemically confirmed with anti‐human mitochondria staining. Enzyme activity assays indicated that hAECs transplantation restored IDUA function in the liver and significantly decreased urinary GAG excretion. Histochemical and micro‐computed tomography analyses revealed reduced GAG deposition in the phalanges joints and composition/morphology improvement of cranial and facial bones. Neurological assessment in the hAEC treated mice showed significant improvement of sensorimotor coordination in the hAEC treated mice compared to untreated mice. Results confirm that partial liver cell replacement with placental stem cells can provide long‐term (>20 weeks) and systemic restoration of enzyme function, and lead to significant phenotypic improvement in the MPS1 mouse model. This preclinical data indicate that liver‐directed placental stem cell transplantation may improve skeletal and neurological phenotypes of MPS1 patients. Stem Cells Translational Medicine 2017;6:1583–1594

Effects of an early intervention using human amniotic epithelial cells in a COPD rat model

The study aimed to investigate the effect of an early intervention using human amniotic epithelial cell (hAEC) in a rat model of chronic obstructive pulmonary disease (COPD). Twenty-four specific pathogen-free Wistar rats were randomized to the control, COPD, and COPD+hAEC groups. COPD was established by intratracheal LPS injection combined with smoke fumigation over 30days. On the first day of model establishment rats in the AEC group also received intratracheal instillation of 500,000 hAECs isolated from the placenta of healthy donors. The mean linear intercept (MLI) and mean alveolar number (MAN) were used to assess the degree of lung emphysema. IL-8 was measured using a radioimmunoassay, surfactant protein D (SP-D) was measured by ELISA, and matrix metalloproteinase (MMP)2 and MMP8 expression was assessed by PCR. Smoke fumigation combined to LPS injection successfully established a COPD rat model with significant emphysema and airway inflammation, elevated MLI and MAN, elevated systemic and lung tissue levels of IL-8 and SP-D (P<0.05), and high expression of MMP2 and MMP8. Rats in the COPD+hAEC group exhibited alleviated lung damage, MLI and MAN (P<0.05), reduced systemic and lung tissue levels of IL-8 and SP-D (P<0.05) and MMP2 and MMP8 expression (P<0.05). Early intervention using hAECs could delay disease progression in rats with COPD.

THC suppresses human amniotic epithelial cell migration via the inhibition of MMP2 and MMP9

THC suppresses human amniotic epithelial cell migration via the inhibition of MMP2 and MMP9