Anti-inflammatory effects of dexmedetomidine on human amnion-derived WISH cells.

Background: To maintain the normal pregnancy, suppression of inflammatory signaling pathway is a crucial physiologic response. Dexmedetomidine has been used for labor analgesia or supplement of inadequate regional analgesia during delivery. And it has been reported that dexmedetomidine has an anti-inflammatory effect. In this study, we examined the influence of dexmedetomidine on the expression of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human amnion-derived WISH cells. In addition, we evaluated the association of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathway in anti-inflammatory effect of dexmedetomidine. Methods: Human amnion-derived WISH cells were pretreated with various concentrations of dexmedetomidine (0.001-1 µg/ml) for 1 h and after then treated with LPS (1 µg/ml) for 24 h. MTT assay was conducted to evaluate the cytotoxicity. Nitric oxide (NO) production was analyzed using Griess-reaction microassay. RT-PCR was performed for analysis of mRNA expressions of COX-2, PGE2, tumor necrosis factor (TNF)-α and interlukin (IL)-1ß. Protein expressions of COX-2, PGE2, p38 and NF-κB were analyzed by western blotting. Results: LPS and dexmedetomidine had no cytotoxic effect on WISH cells. There was no difference in NO production after dexmedetomidine pretreatment. The mRNA and protein expressions of COX-2 and PGE2 were decreased by dexmedetomidine pretreatment in LPS-treated WISH cells. Dexmedetomidine also attenuated the LPS-induced mRNA expression of TNF-α and IL-1ß. The activation of p38 and NF-κB was suppressed by dexmedetomidine pretreatment in LPS-treated WISH cells. Conclusion: We demonstrated that dexmedetomidine pretreatment suppressed the expressions of inflammatory mediators increased by LPS. In addition, this study suggests that anti-inflammatory effect of dexmedetomidine on WISH cells was mediated by the inhibitions of p38 and NF-κB activation.

Carbon nanodot decorated acellular dermal matrix hydrogel augments chronic wound closure.

Impaired skin regeneration in chronic wounds like in diabetes corresponds to high oxidative stress, poor angiogenesis and insufficient collagen hyperplasia. Therefore, a multifaceted strategy for treatment is required to address critical issues associated with chronic wound healing. Fascinating application of nanomaterials in chronic wounds is still limited; hence, in the present work bioactive solubilized decellularized dermal matrix (sADM) was employed to form a hydrogel with chitosan (CTS) at physiological pH/temperature and modified with reactive oxygen species (ROS) scavenging carbon nanodots (ND). A detailed in vitro investigation found that the ND modified bioactive hydrogel (CsADMND) is suitable for human amniotic membrane derived stem cell (hAMSC) delivery. Also, CsADMND was observed to possess a good ROS scavenging property, hemocompatibility and pro-angiogenic potential as demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), haemolysis and chick chorioallantoic membrane (CAM) assay, respectively. The hybrid hydrogel promoted migration of cells in vitro in scratch assay owing to its antioxidant potential and the presence of bioactive moieties. Further, its efficacy in healing full thickness (FT) chronic wounds was evaluated in a streptozotocin (STZ) induced diabetic model. The CsADMND hydrogel after association with hAMSCs led to stimulation of early angiogenesis, superior collagen deposition, rapid wound closure, complete reepithelialisation, and formation of distinct organized dermal epidermal junctions (DEJ) post 21 days of healing. These results suggest that the hAMSC laden CsADMND hydrogel may serve as a promising therapeutic strategy for the management of chronic wounds.

The Relative Permittivity Changes of EGF by 50 Hz MF Exposure Neither Affect the Interaction of EGF With EGFR Nor Its Biological Effects.

The biophysical mechanism of magnetic fields (MFs) acting on living systems is not clear. Previous research showed that, similar to epidermal growth factor (EGF), MF exposure induced EGF receptor (EGFR) clustering and activated EGFR signaling. In this study, we investigated whether MF exposure induced the changes in physical characteristics of EGF and downstream effects of EGF and EGFR interaction. The phase-interrogation surface plasmon resonance (SPR) sensing analyses showed that 50 Hz MF exposure at 4.0 mT for 1 h induced reversible relative permittivity changes of EGF solution. However, compared with sham-exposed EGF solution, the MF-exposed EGF solution did not affect the binding of EGF to EGFR, nor the cell viability and EGFR clustering in human amniotic epithelial cells (FL cells). Our data suggest that cellular EGFR clustering response to MF exposure might not be a result of changes in relative permittivity of EGF in cell culture solution. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.

Differential Effector Response of Amnion- and Adipose-Derived Mesenchymal Stem Cells to Inflammation; Implications for Intradiscal Therapy.

Intervertebral disc degeneration (IVDD) is a progressive condition marked by tissue destruction and inflammation. The therapeutic effector functions of mesenchymal stem cells (MSCs) makes them an attractive therapy for patients with IVDD. While several sources of MSCs exist, the optimal choice for use in the inflamed IVD remains a significant question. Adipose (AD)- and amnion (AM)-derived MSCs have several advantages compared with other sources, however, no study has directly compared the impact of IVDD inflammation on their effector functions. Human MSCs were cultured in media with or without supplementation of interleukin-1ß (IL-1ß) and tumor necrosis factor-α at concentrations reportedly produced by IVDD cells. MSC proliferation and production of pro- and anti-inflammatory cytokines were quantified following 24 and 48 h of culture. Additionally, the osteogenic and chondrogenic potential of AD- and AM-MSCs was characterized via histology and biochemical analysis following 28 days of culture. In inflammatory culture, AM-MSCs produced significantly more anti-inflammatory IL-10 (14.47 ± 2.39 pg/ml; p = 0.004) and larger chondrogenic pellets (5.67 ± 0.26 mm2 ; p = 0.04) with greater percent area staining positively for glycosaminoglycan (82.03 ± 3.26%; p < 0.001) compared with AD-MSCs (0.00 ± 0.00 pg/ml; 2.76 ± 0.18 mm2 ; 34.75 ± 2.49%; respectively). Conversely, AD-MSCs proliferated more resulting in higher cell numbers (221,000 ± 8,021 cells; p = 0.048) and produced higher concentrations of pro-inflammatory cytokines prostaglandin E2 (1,118.30 ± 115.56 pg/ml; p = 0.030) and IL-1ß (185.40 ± 7.63 pg/ml; p = 0.010) compared with AM-MSCs (109,667 ± 5,696 cells; 1,291.40 ± 78.47 pg/ml; 144.10 ± 4.57 pg/ml; respectively). AD-MSCs produced more mineralized extracellular matrix (3.34 ± 0.05 relative absorbance units [RAU]; p < 0.001) compared with AM-MSCs (1.08 ± 0.06 RAU). Under identical inflammatory conditions, a different effector response was observed with AM-MSCs producing more anti-inflammatories and demonstrating enhanced chondrogenesis compared with AD-MSCs, which produced more pro-inflammatory cytokines and demonstrated enhanced osteogenesis. These findings may begin to help inform researchers which MSC source may be optimal for IVD regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2445-2456, 2019.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

Insulin-Transferrin-Selenium as a Novel Serum-free Media Supplement for the Culture of Human Amnion Mesenchymal Stem Cells

This study aimed to evaluate the use of Insulin-Transferrin-Selenium (ITS) medium in place of fetal bovine serum (FBS) to culture human amnion mesenchymal stem cells (hAMSCs). Cell morphology, ultrastructure, proliferation, migration and MSC related markers were assessed accordingly. The hAMSCs were induced to osteocyte, chondrocyte, adipocyte and keratinocyte by culturing in appropriate induction medium. hAMSCs mRNA expression was detected for the matrix metalloproteinases 2 (MMP2), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), Platelet-derived Growth Factor (PDGF), and transforming growth factor beta 1 (TGF-ß) by real-time quantitative RT-PCR. Our results showed that hAMSCs cultured in ITS medium exhibited similar proliferation rates, demonstrated a statistically significant increased migration and expressed similar levels of MSC markers(CD73+, CD90+, CD105+, CD45-, CD34-) compared with those cultured in FBS. Osteoblasts, chondrocytes, adipocytes and keratinocytes were differentiated. Results of transmission electron microscope (TEM) revealed that hAMSCs cultured in ITS medium underwent active metabolism. The mRNA expression of MMP2, VEGF, KGF, TGF-ß, IGF-I and PDGF upregulated in ITS medium. In conclusion, ITS medium has the potential to be used for the expansion of hAMSCs before clinical application.

Immunological and Differentiation Properties of Amniotic Cells Are Retained After Immobilization in Pectin Gel.

Mesenchymal stromal cells from the human amniotic membrane (i.e., human amniotic mesenchymal stromal cells [hAMSCs]) of term placenta are increasingly attracting attention for their applications in regenerative medicine. Osteochondral defects represent a major clinical problem with lifelong chronic pain and compromised quality of life. Great promise for osteochondral regeneration is held in hydrogel-based constructs that have a flexible composition and mimic the physiological structure of cartilage. Cell loading within a hydrogel represents an advantage for regenerative purposes, but the encapsulation steps can modify cell properties. As pectin gels have also been explored as cell vehicles on 3D scaffolds, the aim of this study was to explore the possibility to include hAMSCs in pectin gel. Immobilization of hAMSCs into pectin gels could expand their application in cell-based bioengineering strategies. hAMSCs were analyzed for their viability and recovery from the pectin gel and for their ability to differentiate toward the osteogenic lineage and to maintain their immunological characteristics. When treated with a purposely designed pectin/hydroxyapatite gel biocomposite, hAMSCs retained their ability to differentiate toward the osteogenic lineage, did not induce an immune response, and retained their ability to reduce T cell proliferation. Taken together, these results suggest that hAMSCs could be used in combination to pectin gels for the study of novel osteochondral regeneration strategies.

Danshen extract regulates the expression of aquaporin 3 in human amniotic epithelial cells.

Danshen extract has been used in the treatment of oligohydramnios, however, the mechanism of its action has not been elucidated. Previously, we demonstrated that down-regulation of AQP3 in fetal membranes may contribute to the development of oligohydramnios. In this study, we investigated the effects of Danshen extract on AQP3 expression in human amniotic epithelial cells from term pregnancies with oligohydramnios or those with those with (those with) normovolemic amniotic fluid. Human amniotic epithelial cells from the oligohydramnios group expressed a lower level of AQP3 mRNA and protein than those with normovolemia. Tweleve hour (Twelvehours) of treatment with Danshen extract, in a dose dependent manner, significantly increased the expression of AQP3 in the two groups. However, human amniotic epithelial cells from the oligohydramnios patients showed a greater sensitivity to the treatment of Danshen extract. These data provide a molecular basis for the treatment of patients with oligohydraminos.

Clinical Application of Pluripotent Stem Cells: An Alternative Cell-Based Therapy for Treating Liver Diseases?

The worldwide shortage of donor livers for organ and hepatocyte transplantation has prompted the search for alternative therapies for intractable liver diseases. Cell-based therapy is envisaged as a useful therapeutic option to recover and stabilize the lost metabolic function for acute liver failure, end-stage and congenital liver diseases, or for those patients who are not considered eligible for organ transplantation. In recent years, research to identify alternative and reliable cell sources for transplantation that can be derived by reproducible methods has been encouraged. Human pluripotent stem cells (PSCs), which comprise both embryonic and induced PSCs, may offer many advantages as an alternative to hepatocytes for liver cell therapy. Their capacity for expansion, hepatic differentiation and self-renewal make them a promising source of unlimited numbers of hepatocyte-like cells for treating and repairing damaged livers. Immunogenicity and tumorigenicity of human PSCs remain the bottleneck for successful clinical application. However, recent advances made to develop disease-corrected hepatocyte-like cells from patients' human-induced PSCs by gene editing have opened up many potential gateways for the autologous treatment of hereditary liver diseases, which may likely reduce the risk of rejection and the need for lifelong immunosuppression. Well-defined methods to reduce the expression of oncogenic genes in induced PSCs, including protocols for their complete and safe hepatic differentiation, should be established to minimize the tumorigenicity of transplanted cells. On top of this, such new strategies are currently being rigorously tested and validated in preclinical studies before they can be safely transferred to clinical practice with patients.

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRß, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.

[Compound danshen injection regulated the expression of AQP3 in the human amnion epithelium cells through JNK signal pathway].

OBJECTIVE: To explore the role of Compound Danshen Injection (CDI) in regulating the expression of aquaporin 3 (AQP3) in human amnion epithelium cells (hAECs), and to study the relation between c-Jun N-terminal kinase (JNK) signal pathway and AQP3. METHODS: hAECs were isolated and primarily cultured from term pregnancy with normal amniotic fluid volume and from term pregnancy with oligohydramnios, and then hAECs were further divided into four groups, i.e., the blank control group (A), the SP600125 group (B), the CDI group (C), and the SP600125 +CDI group (D). The cell viability was measured by cell counting kit-8 assay (CCK-8). The expression of total JNK, phosphorylated JNK, and AQP3 were determined by Western blot. RESULTS: (1) In hAECs with normal AFV or with oligohydramnios: There was no statistical difference in the cell viability or the expression of total JNK among the 4 groups (P > 0.05). But there was statistical difference in the expression of p-JNK (P < 0.05). Compared with A group, the expression of p-JNK was obviously down-regulated in B group, but obviously up-regulated in C group (P < 0.05). The expression of p-JNK was significantly lower in D group than in C group, but higher than that in A group or B group (P < 0.05).The AQP3 expression in the hAECs with normal amniotic fluid volume of C group and D group were higher than that in the A group (P < 0.05). However, there was no statistical difference in the AQP3 expression between C group and D group (P > 0.05). In hAECs with oligohydramnios, the expression of AQP3 obviously decreased in B group, but up-regulated in C group (both P < 0.05). The expression of AQP3 was lower in D group than in C group, but higher than in B group (P < 0.05). CONCLUSION: CDI could regulate the AQP3 expression in hAECs with oligohydramnios via activating the JNK signal pathway.

[The role of MAPK signal pathway in the regulation of AQP3 expression induced by compound danshen injection in human amniotic epithelial cells].

OBJECTIVE: To investigate the role of mitogen-activated protein kinases (MAPKs)-extracellular signal regulated kinase1/2 (ERK1/2) signal pathway in the regulation of Compound Danshen Injection (CDI) induced AQP3 expression in the human amniotic epithelial cells (hAECs). METHODS: hAECs of term pregnancy with normal amniotic fluid volume (AFV) or isolated oligohydramnios were primarily cultured. And the cells were equally divided into four groups, i.e., the vehicle control group, the U0126 group, the CDI group, the CDI + U0126 group. The expressions of phosphorylated ERK1/2 (p-ERK1/2) and AQP3 in hAECs were detected using Western blot analysis. RESULTS: (1) When compared with the control group, the expression level of p-ERK1/2 in hAECs in those with normal AFV and oligohydramnios obviously decreased in the U0126 group (P < 0.05). The expression level of p-ERK1/2 could be elevated in the CDI group (P < 0.05). The expression level of p-ERK1/2 in hAECs was higher in the CDI +U0126 group than in the U0126 group, but lower in the CDI + U0126 group than in the CDI group (P < 0.05). (2) There was no obvious change in AQP3 expression in hAECs with normal AFV between the U0126 group and the vehicle control group (P > 0.05). There was no statistical difference in the expression level of AQP3 between the CDI group and the U0126 +CDI group (P > 0.05), but they were higher than those in the vehicle control group (P < 0.05). (3) Compared with the vehicle control group, the expression level of AQP3 in hAECs with oligohydramnios significantly decreased in the U0126 group and increased in the CDI group (P < 0.05). The expression level of AQP3 was lower in the U0126 + CDI group than in the CDI group, but higher in the U0126 +CDI group than in the U0126 group (P < 0.05). CONCLUSION: CDI could regulate AQP3 expression level in hAECs with oligohydramnios via activating the MAPK-ERK1/2 signal transduction pathway.

In vitro cytotoxic activity of seed oil of fenugreek against various cancer cell lines.

In the present study, investigations were carried out to screen the anticancer activities of fenugreek seed oil against cancer cell lines (HEp-2, MCF-7, WISH cells), and a normal cell line (Vero cells). Cytotoxicity was assessed with MTT and NRU assays, and cellular morphological alterations were studied using phase contrast light microscopy. All cells were exposed toi 10-1000 µg/ml of fenugreek seed oil for 24 h. The results show that fenugreek seed oil significantly reduced the cell viability, and altered the cellular morphology in a dose dependent manner. Among the cell lines, HEp-2 cells showed the highest decrease in cell viability, followed by MCF-7, WISH, and Vero cells by MTT and NRU assays. Cell viability at 1000 µg/ml was recorded as 55% in HEp-2 cells, 67% in MCF-7 cells, 75% in WISH cells, and 86% in Vero cells. The present study provides preliminary screening data for fenugreek seed oil pointing to potent cytotoxicity against cancer cells.

Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice.

BACKGROUND AIMS: Human amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury. METHODS: Newborn mouse pups were randomized to either normoxia (inspired O2 content (FiO2) = 0.21, n = 60) or hyperoxia (FiO2 = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14. RESULTS: Hyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-ß and platelet-derived growth factor-ß). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia. CONCLUSIONS: Intraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.

[Effects of compound salvia miltiorrhiza injection on aquaporin 3 expression in human amniotic epithelial cells].

OBJECTIVE: To study the effects of Compound Salvia miltiorrhiza Injection (CSI) on aquaporin 3 (AQP3) expression in human amniotic epithelial cells (hAECs), and to explore its mechanisms for treating oligohydramnios. METHODS: The hAECs selected from 8 human term pregnancies with oligohydramnios and no other complications (as the test group)and 8 human term pregnancies with normal amniotic fluid volume (as the control group) were primarily cultured. The mRNA and protein expressions of AQP3 in hAECs were detected using reverse transcription-polymerase chain reaction and Western blot with various concentrations of CSI (0.000, 0.001, 0.010, 0.020, 0.060, and 0.100 mg/mL, respectively) at different time points (0, 6, 12,24, and 48 h, respectively). RESULTS: (1) Compared with the control group, the AQP3 expression was down-regulated in the test group (P < 0.05). (2) The AQP3 expression in the two groups reached the peak when the concentration of CSI was 0.010 mg/mL, showing statistical difference when compared with other concentrations (P < 0.05). (3) The AQP3 expression reached the peak when 0.010 mg/mL CSI acted for 12 h, showing statistical difference when compared with other concentrations (P < 0.05). (4) The AQP3 expression was up-regulated in the two groups when 0.010 mg/mL CSI acted for 12 h. But the up-regulated AQP3 expression was more obvious in the test group than in the control group (P < 0.05). CONCLUSIONS: CSI could regulate the AQP3 expression in hAECs. CSI showed more obvious effects on the AQP3 expression in hAECs of oligohydramnios human term pregnancies.

Improved amino acid, bioenergetic metabolite and neurotransmitter profiles following human amnion epithelial cell transplant in intermediate maple syrup urine disease mice.

Orthotopic liver transplant (OLT) significantly improves patient outcomes in maple syrup urine disease (MSUD; OMIM: 248600), yet organ shortages point to the need for alternative therapies. Hepatocyte transplantation has shown both clinical and preclinical efficacy as an intervention for metabolic liver diseases, yet the availability of suitable livers for hepatocyte isolation is also limited. Conversely, human amnion epithelial cells (hAEC) may have utility as a hepatocyte substitute, and they share many of the characteristics of pluripotent embryonic stem cells while lacking their safety and ethical concerns. We reported that like hepatocytes, transplantation of hAEC significantly improved survival and lifespan, normalized body weight, and significantly improved branched-chain amino acid (BCAA) levels in sera and brain in a transgenic murine model of intermediate maple syrup urine disease (imsud). In the current report, we detail the neural and peripheral metabolic improvements associated with hAEC transplant in imsud mice, including amino acids associated with bioenergetics, the urea cycle, as well as the neurotransmitter systems for serotonin, dopamine, and gamma-aminobutyric acid (GABA). This stem cell therapy results in significant global correction of the metabolic profile that characterizes the disease, both in the periphery and the central nervous system, the target organ for toxicity in iMSUD. The significant correction of the disease phenotype, coupled with the theoretical benefits of hAEC, particularly their lack of immunogenicity and tumorigenicity, suggests that human amnion epithelial cells deserve serious consideration for clinical application to treat metabolic liver diseases.

Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries.

Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1ß induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture.

Equine bone marrow mesenchymal or amniotic epithelial stem cells as feeder in a model for the in vitro culture of bovine embryos.

Various studies have shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In the present study we investigated the effects of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSCs) or equine amniotic epithelial stem cells (AE-SCs) on in vitro blastocysts development. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and the isolated cells were resuspended in Dulbecco Modified Earle's Medium supplemented with 10 ng/ml of basic fibroblast growth factor (bFGF). Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fragments were incubated with 0.05% trypsin for 45 min. Separated AE cells were plated in standard culture medium containing 10 ng/ml epidermal growth factor (EGF). Seven hundred and five cumulus-oocyte complexes were used and, after IVM and IVF, cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured up to day 7: (1) co-culture with cumulus cells (control); (2) co-culture with BM-MSCs; and (3) co-culture with AE-SCs. Statistical analyses were performed by ANOVA. Blastocyst developmental rates were significantly different (p < 0.001) between control, AE-SCs and BM-MSCs (respectively 35.45, 41.84 and 30.09%). In conclusion, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSCs and cumulus cells. It can be suggested that these cells secrete biologically active substances, including signalling molecules and growth factors of epithelial nature, different to those of the BM cells of mesenchymal origin.

Cultured epithelial grafting using human amniotic membrane: the potential for using human amniotic epithelial cells as a cultured oral epithelium sheet.

OBJECTIVE: Human amniotic cells are a valuable source of functional cells that can be used in various fields, including regenerative medicine and tissue engineering. The aim of this study was to investigate the utility of human amniotic epithelial (hAE) cells as a new cell source for culturing stratified epithelium sheets for intraoral grafting. METHODS: Enzymatically isolated hAE cells were submerged in a serum-free, low-calcium-supplemented MCDB 153 medium without a feeder layer. The hAE cells were seeded onto a Millicell cell culture plate insert and cultured while submerged in a high-calcium medium for 4 days. Then, they were cultured at an air-liquid interface for 3 weeks. Cultures of hAE cells proliferated at the air-liquid interface. RESULTS: After 3 weeks, the hAE cells cultivated using the air-liquid interface method lead to almost 10 continuous layers of stratified epithelium without parakeratinization or keratinization. It confirmed immunohistochemically that the presence of CK10/13 and Ki-67 positive cells were spread throughout almost all the epithelial layer, and that CK19 positive cells were expressed throughout the entire epithelial layer in the cultured hAE cell sheets. Cultured hAE cells sheets showed a staining pattern similar to that of uncultured oral mucosa: ZO-1 and occludin were located in the intercellular junctions throughout all the epithelial layers. It was suggested that the hAE sheets consisted of highly-active proliferating cells and undifferentiated cells, and had a barrier function. CONCLUSIONS: These results suggested that hAE cells may be a promising cell source for the development of stratified epithelium allograft sheets using a human cell strain.

Decrease in expression of histamine H2 receptors by human amniotic epithelial cells during differentiation into pancreatic beta-like cells.

OBJECTIVE AND DESIGN: Considering the role of histaminergic pathway in the differentiation of stem cells, we compared expression patterns of H(1) and H(2) receptors in the human amniotic epithelial cells (HAEC) culture at different stages of nicotinamide-induced differentiation into PBLC with the control HAEC. MATERIAL AND METHODS: HAEC isolated after term pregnancies (N = 12) were cultured in vitro. Altogether, 72 cultures were established. The culture medium in the studied group was supplemented on Day 5 with nicotinamide (10 mM). C-peptide concentration in the medium collected every 3 days for 15 days was determined immunoenzymatically as a marker of differentiation. At the same intervals the cultures were formalin-fixed and paraffinembedded for H(1) and H(2) receptors immunostaining. Quantitative immunohistochemistry was applied for evaluation of H(1) and H(2) expression. RESULTS: C-peptide was detected on Day 6 and the levels were kept gradually increased until Day 12, then stayed at almost the same level, 3.7-fold higher than initially. Expression of H(2) was unchanged until Day 9 after nicotinamide addition, then was significantly (p < 0.05) decreased and amounted (mean % value for the measurements performed on Day 12 and Day 15, +/-SEM) 49.73 +/- 11.03 of the reference value obtained in control HAEC. CONCLUSION: Variable expression of H(2) during nicotinamide-induced differentiation of HAEC into PBLC may define a time-point, indicating involvement of histamine at the earlier stages.