Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells.

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC-AF), adipose tissue (MSC-AT) and fibroblasts (FIB-control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT-PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC-AF were less hydroxymethylated for both animals. MSC-AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC-AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC-AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.

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

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial-mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF-ß, the main fibrogenic growth factor, through αvß6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvß6 integrin expression, with a consequent decrease in TGF-ß activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury-induced and fibrosis-promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

Gene Expression in Amnion-Derived Cells Cultured on Recombinant Laminin 332-A Preliminary Study.

Human amniotic cells (hAC) exhibit characteristics of undifferentiated cells and immunomodulatory properties. Recognition of the relationship between amniotic cells and components of the extracellular matrix is an important condition for their ex vivo preparation and further successful clinical application in regenerative medicine and transplantology. Laminin 332 (LN-332), as a natural component of the basement membrane of amniotic epithelial cells and a ligand for integrin receptors, may strongly influence the phenotype and fate of amniotic cells. We investigated the impact of recombinant LN-332 on hAC viability and expression of markers for pluripotency, early differentiation, adhesion, and immunomodulatory properties. During 14 days of culture, hAC were quantified and qualified by light microscopy, immunohistochemistry, immunocytochemistry, and flow cytometry. Gene expression was assessed with real-time polymerase chain reaction (RT-PCR) arrays and compared with differentiated cells originated from the three germ layers. LN-332 caused an over 2-fold increase in the total number of hAC, accompanied by a 75% reduction of SSEA-4-positive cells and an increase in HLA-ABC-positive cells. In particular, we observed that the presence of laminin 332 in the medium of a short-time culture modifies the effect of culture duration on hAC, enhancing time-dependent inhibition of expression of certain genes, including pluripotency and differentiation markers, laminin 332 subunits (which may be part of self-regulation of LN-332 synthesis by amniotic cells), and integrins. The changes observed in hAC were more distinct with respect to differentiated mesenchymal cells, resulting in more comparable phenotypes than those represented by differentiated endo- and ectodermal cells. We concluded that laminin 332 present in the culture medium influences to a certain extent proliferation, adhesion, and differentiation of amniotic cells in culture.

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.

Increased immunomodulatory capacity of human amniotic cells after activation by pro-inflammatory chemokines.

Human amniotic cells (hAC) possess multiple unique immunomodulatory properties. They are believed to be a very appealing and safe material for clinical applications. Primary hAC have been proposed as an efficient source of immunomodulatory factors that could be used as alternative or supporting to classical drug immunosuppression. The aim of this study was to evaluate hAC immunomodulatory properties post-activation by inflammatory cytokines as Interleukin 1ß and Interferon γ. hAC were isolated and characterized by the expression of pluripotency marker SSEA4, epithelial marker CK7, HLA-G antigen, mRNA for PTGS2, NOS2 and HLA-G gene, and secretion of soluble mediators as HLA-G and PGE2 in the culture medium in presence or absence of INF-γ and IL-1ß. Heterogeneity of the cultured hAC was proved, with 50 ±â€¯8% of cells positive for epithelial marker (CK7), and 73 ±â€¯3% expressing SSEA4 pluripotency marker. Priming effect by in vitro exposure to INF-γ and IL-1ß resulted in a significant increase in expression of PTGS2, NOS2 and HLA-G gene, with a peak between 32 and 64 h. The highest PGE2 concentration was measured in the culture medium at 48 h. At 96 h, a significant difference in the percentage of SSEA4+ hAC between activated and non-activated cells, as well as the highest expression of HLA-G - especially in SSEA4+ cells, and highly elevated concentration of soluble HLA-G (sHLA-G) in the medium of activated cells, were found. The prolonged exposure of primary human amnion-derived cells to inflammatory cytokines INF-γ and IL-1ß may result in enhanced expression and secretion of immunomodulatory molecules important in allogenic therapies.

NPs-TiO2 and Lincomycin Coexposure Induces DNA Damage in Cultured Human Amniotic Cells.

Titanium dioxide nanoparticles (NPs-TiO2 or TiO2-NPs) have been employed in many commercial products such as medicines, foods and cosmetics. TiO2-NPs are able to carry antibiotics to target cells enhancing the antimicrobial efficiency; so that these nanoparticles are generally used in antibiotic capsules, like lincomycin, added as a dye. Lincomycin is usually used to treat pregnancy bacterial vaginosis and its combination with TiO2-NPs arises questions on the potential effects on fetus health. This study investigated the potential impact of TiO2-NPs and lincomycin co-exposure on human amniocytes in vitro. Cytotoxicity was evaluated with trypan blue vitality test, while genotoxic damage was performed by Comet Test, Diffusion Assay and RAPD-PCR for 48 and 72 exposure hours. Lincomycin exposure produced no genotoxic effects on amniotic cells, instead, the TiO2-NPs exposure induced genotoxicity. TiO2-NPs and lincomycin co-exposure caused significant increase of DNA fragmentation, apoptosis and DNA damage in amniocytes starting from 48 exposure hours. These results contribute to monitor the use of TiO2-NPs combined with drugs in medical application. The potential impact of antibiotics with TiO2-NPs during pregnancy could be associated with adverse effects on embryo DNA. The use of nanomaterials in drugs formulation should be strictly controlled in order to minimize risks.

Different Culture Times Affect MicroRNA Cargo in Equine Amniotic Mesenchymal Cells and Their Microvesicles.

Conditioned medium (CM) and microvesicles (MVs) are produced using different protocols: CM is collected following 12-96 h of cell culture without renewal of tissue culture medium, while MVs are collected after overnight cell culture. For future comparative studies in regenerative medicine looking at the efficacy of CM and MVs, it is important to understand how the quality of cell secretions is affected by culture. The aim of this study was to evaluate whether the duration of culturing influences the micro-RNAs (miRNAs) cargo of equine amniotic mesenchymal cells (AMCs) and their MVs. The analysis identified 990 miRNAs. After one night, there were 347 differently expressed (DE)-miRNAs between MVs and cells, whereas after four nights there were 359. About 58.3% of the DE-miRNAs were shared between samples produced under the two conditions. The comparison between miRNA content in AMC cells cultured for one night versus four nights showed eight DE-Equus caballus (eca)-miRNAs, which target genes were involved in immune response to external stimulus, inflammatory response, and production of reactive oxygen species. Comparing MVs isolated from one or four nights, four DE-miRNAs that target genes regulating cell cycle progression and production of reactive oxygen species were found, but only eca-miR-214 was enriched in the MVs after four nights. In conclusion, after 4 days of cell culture, the profile of AMC miRNAs was altered, indicating a probable phenotypic transition versus a new cell culture environment and aging. After this time, MVs accumulated eca-miR-214, which may help cells survive or adapt to new culture conditions.

Amniotic Epithelial Cells: A New Tool to Combat Aging and Age-Related Diseases?

The number of elderly people is growing at an unprecedented rate and this increase of the aging population is expected to have a direct impact on the incidence of age-related diseases and healthcare-associated costs. Thus, it is imperative that new tools are developed to fight and slow age-related diseases. Regenerative medicine is a promising strategy for the maintenance of health and function late in life; however, stem cell-based therapies face several challenges including rejection and tumor transformation. As an alternative, the placenta offers an extraordinary source of fetal stem cells, including the amniotic epithelial cells (AECs), which retain some of the characteristics of embryonic stem cells, but show low immunogenicity, together with immunomodulatory and anti-inflammatory activities. Because of these characteristics, AECs have been widely utilized in regenerative medicine. This perspective highlights different mechanisms triggered by transplanted AECs that could be potentially useful for anti-aging therapies, which include: Graft and differentiation for tissue regeneration in age-related settings, anti-inflammatory behavior to combat "inflammaging," anti-tumor activity, direct lifespan and healthspan extension properties, and possibly rejuvenation in a manner reminiscent of heterochronic parabiosis. Here, we critically discuss benefits and limitation of AECs-based therapies in age-related diseases.