Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.

Primary Culture of Cornea-Limbal Epithelial Cells In Vitro.

The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.

The Incidence of Dichorionic Diamniotic Twin Pregnancy After Single Blastocyst Embryo Transfer and Zygosity: 8 Years of Single-Center Experience.

Dichorionic diamniotic (DCDA) twin pregnancies after single blastocyst embryo transfer have been reported recently, although a blastocyst ovum is generally believed to divide into monochorionic twin pregnancy. We investigated the incidence of DCDA twin pregnancy after single blastocyst embryo transfer and their zygosity. This prospective cohort study included 655 consecutive twin pregnancies that were managed from 2006 to 2014 at our institution. Chorionicity and amnionicity were determined using first-trimester ultrasonography and/or placental pathology. Zygosity was analyzed if the cases were DCDA twins after single blastocyst embryo transfer. Among 655 twin pregnancies, there were 348 DCDA cases, 295 monochorionic diamniotic (MCDA) cases and 12 monochorionic monoamniotic cases. Single blastocyst embryo transfer was performed in 43 cases. Six out of the 43 (14%) cases involved DCDA twin pregnancies and the other 37 cases involved MCDA twin pregnancies. Three DCDA twins born after single blastocyst embryo transfer, wherein frozen embryo transfer (FET) was performed in the natural cycle, were dizygotic, and the other three cases, wherein FET with hormone replacement therapy was performed, were monozygotic. DCDA twin pregnancy occurred in 14% (7% for monozygotic and 7% for dizygotic) of twin pregnancies after single blastocyst embryo transfer cases.

Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells.

The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.

Genes upregulated in the amnion at labour are bivalently marked by activating and repressive histone modifications.

Inflammatory genes are expressed increasingly in the foetal membranes at late gestation triggering birth. Here we have examined whether epigenetic histone modifications contribute to the upregulation of proinflammatory genes in the amnion in late pregnancy and at labour. Amnion samples were collected from early pregnancy, at term in the absence of labour and after spontaneous birth. The expression of the labour-associated proinflammatory genes PTGS2, BMP2 and NAMPT was determined by reverse transcription-coupled quantitative real-time PCR (qRT-PCR). Chromatin immunoprecipitation (ChIP) and sequential double ChIP were performed to determine the levels and co-occurrence of activating histone-3, lysine-4 trimethylation (H3K4me3) and repressive histone-3, lysine-27 trimethylation (H3K27me3) at the gene promoters. H3K4 methyltransferase, H3K27me3 demethylase and H3K27 methyltransferase expression was determined by qRT-PCR and immunofluorescence confocal microscopy. PTGS2, BMP2 and NAMPT expression was upregulated robustly between early pregnancy and term (P < 0.05). The promoters were marked bivalently by both the H3K4me3 and H3K27me3 modifications. Bivalence was reduced at term by the decrease of the H3K27me3-modified fraction of promoter copies marked by H3K4me3 indicating epigenetic activation. Messenger RNAs encoding the H3K4-specific methyl transferases MLL1,-2,-3,-4, SETD1A,-B and the H3K27me3-specific demethylases KDM6A,-B were expressed increasingly while the H3K27 methyl transferase EZH2 was expressed decreasingly at term. Histone modifying enzyme proteins were detected in amnion epithelial and mesenchymal cells. These results with prototypical proinflammatory genes suggest that nucleosomes at labour-promoting genes are marked bivalently in the amnion, which is shifted towards monovalent H3K4me3 modification at term when the genes are upregulated. Bivalent epigenetic regulation by histone modifying enzymes may control the timing of labour.

Pluripotency markers in tissue and cultivated cells in vitro of different regions of human amniotic epithelium.

Studies have described the presence of pluripotent markers in vivo and in vitro in human amnion. However, the amnion can be divided into reflected, placental and umbilical regions that are anatomically and functionally heterogeneous. Here, we evaluated the expression of pluripotency markers in tissue and cultivated cells in vitro of different regions of human amnion. To this end, we determined the presence of the core pluripotency factors OCT-4, NANOG and SOX-2 by immunofluorescence and RT-PCR and also performed transcriptome analysis of the different regions of amnion tissue. We identified the mRNA and protein of the pluripotency factors in the different regions of human amnion tissue. However, the OCT-4 and NANOG immunolocalization was cytoplasmic, whereas SOX-2 immunolocalization was nuclear regardless of the region analyzed. Moreover, we found three subpopulations of cells in the in vitro cultures of reflected and placental amnion: cells with immunostaining only in the nucleus, only in the cytoplasm, or in both compartments. Yet no statistically significant differences were found between the reflected and placental amnion. These results suggest a homogeneous distribution of the pluripotency transcription factors of the different regions of human amnion to isolate stem cells that can be used in regenerative medicine.

Non-Ionizing Radiation for Cardiac Human Amniotic Mesenchymal Stromal Cell Commitment: A Physical Strategy in Regenerative Medicine.

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for "in vitro" stem cell commitment, preparatory to the "in vivo" stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 µT), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

Comparison of Human Denuded Amniotic Membrane and Porcine Small Intestine Submucosa as Scaffolds for Limbal Mesenchymal Stem Cells.

Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.

Two consecutive microtubule-based epithelial seaming events mediate dorsal closure in the scuttle fly Megaselia abdita.

Evolution of morphogenesis is generally associated with changes in genetic regulation. Here, we report evidence indicating that dorsal closure, a conserved morphogenetic process in dipterans, evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation. In Drosophila melanogaster, dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis. We find that dorsal closure in Megaselia abdita, a three-tissue system comprising serosa, amnion and epidermis, differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling. In addition to an actomyosin cable, M. abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis. Our study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression.

Human amniotic epithelial cells regulate osteoblast differentiation through the secretion of TGFß1 and microRNA-34a-5p.

Since the beginning of the use of stem cells in tissue regenerative medicine, there has been a search for optimal sources of stem cells. Human amniotic epithelial cells (hAECs) are derived from human amnions, which are typically discarded as medical waste, but were recently found to include cells with trilineage differentiation potential in vitro. Previous study has focused on the osteogenic differentiation ability of hAECs as seed cells in bone regeneration; however, their paracrine effects on osteoblasts (OBs) are yet to be elucidated. In the present study, conditioned medium (CM) derived from hAECs was used to determine their paracrine effects on the human fetal OB cell line (hFOB1.19), and the potential bioactive factors involved in this process were investigated. The results suggested that hAEC-CM markedly promoted the proliferation, migration and osteogenic differentiation of hFOB1.19 cells. Expression of transforming growth factor ß1 (TGFß1) and microRNA 34a-5p (miR-34a-5p) were detected in hAECs. Furthermore, it was demonstrated that TGFß1 and miR-34a-5p stimulated the differentiation of hFOB1.19 cells, and that TGFß1 promoted cell migration. Moreover, the effects of hAEC-CM were downregulated following the depletion of either TGFß1 or miR-34a-5p. These results demonstrated that hAECs promote OB differentiation through the secretion of TGFß1 and miR-34a-5p, and that hAECs may be an optimal cell source in bone regenerative medicine.

The Amniotic Membrane: Development and Potential Applications - A Review.

Foetal membranes are essential tissues for embryonic development, playing important roles related to protection, breathing, nutrition and excretion. The amnion is the innermost extraembryonic membrane, which surrounds the foetus, forming an amniotic sac that contains the amniotic fluid (AF). In recent years, the amniotic membrane has emerged as a potential tool for clinical applications and has been primarily used in medicine in order to stimulate the healing of skin and corneal diseases. It has also been used in vaginal reconstructive surgery, repair of abdominal hernia, prevention of surgical adhesions and pericardium closure. More recently, it has been used in regenerative medicine because the amniotic-derived stem cells as well as AF-derived cells exhibit cellular plasticity, angiogenic, cytoprotective, immunosuppressive properties, antitumoural potential and the ability to generate induced pluripotent stem cells. These features make them a promising source of stem cells for cell therapy and tissue engineering. In this review, we discussed the development of the amnion, AF and amniotic cavity in different species, as well as the applicability of stem cells from the amnion and AF in cellular therapy.

DNA Methylation Landscapes of Human Fetal Development.

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.

High aminopeptidase N/CD13 levels characterize human amniotic mesenchymal stem cells and drive their increased adipogenic potential in obese women.

Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.

Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils--a promising candidate formulation for amnioinfusion fluid.

Oligohydramnios is often caused by the premature rupturing of membranes and subsequent intrauterine infections, such as chorioamnionitis, in which event oxidative stress is hypothesized to be closely associated with the damage to the fetal organs. The clinical efficiency of amnioinfusion using warmed saline in cases of premature rupture of membranes is still controversial, especially concerning the prognosis for the fetus. In the present study, we found that human amniotic fluid per se suppresses the release of superoxide from cultured human neutrophils, suggesting an acute or chronic shortage of amniotic fluid in cases of premature rupture of membranes can affect the shielding of intrauterine organs from oxidative stress. The aim of this study was to propose a formula of zinc and magnesium ions in saline for amnioinfusion, by assessing antioxidative activities. A combination of 5 microM zinc and 5mM magnesium in saline synergistically inhibited superoxide production by cultured human neutrophils, equivalent to human amniotic fluid. The intraperitoneal administration of this formula significantly improved the survival rate in a rat model of peritonitis compared to the saline control (46.7% vs. 10%). The combination of these metals with saline may thus be a promising formula for an amnioinfusion fluid with the capacity to protect fetal organs from oxidative stress.

Initial development of bovine placentation (Bos indicus) from the point of view of the allantois and amnion.

The aim of this study was to perform a morphological characterization of the initial bovine placental development, between 20 and 70 days post-insemination (p.i.), with emphasis on the differentiation of the allantois and amnion. After collection, the conceptuses were dissected, macroscopically measured and photographically documented. The extraembryonic membranes were cut into fragments measuring 5 cm(2), and then fixed in 4% paraformaldehyde for analysis using light microscopy, and in 2.5% glutaraldehyde for use in scanning and transmission electron microscopy. The extraembryonic and fetal membranes presented variable degrees of development throughout the periods analysed. The macroscopic appearance of vascularization of the allantois, its attempt to merge with the chorium and the effective appearance of the first cotyledons in development were the events observed from 30 to 40 days of pregnancy. The measurements of the amnion increased gradually as gestation developed. The allantoic epithelia presented cellular dimorphism from 20 to 25 days of pregnancy, but was shown to be immature from 60 to 70 days of pregnancy.

Chronic stretching of amniotic epithelial cells increases pre-B cell colony-enhancing factor (PBEF/visfatin) expression and protects them from apoptosis.

In normal pregnancy, the fetal membranes become increasingly distended towards term and in multifetal gestations they become over-distended. Apoptosis of the amniotic epithelium increases with advancing gestation and may contribute to fetal membrane weakening and rupture. The effects of chronic static stretching for 36h have been investigated using primary amniotic epithelial cells. Pre-B cell colony-enhancing factor (PBEF) is a stretch-responsive cytokine and expression of its gene, intracellular and secreted protein were all significantly increased by 4h and its secretion sustained over 36h, contrasting with the rapid increase and decline in expression of IL-8. Increased expression of SIRT1 and decreased p53 paralleled the changes in PBEF, are known to be responsive to PBEF, and contribute to cell survival. Distension had no effects on proliferation or necrosis but protected the cells from apoptosis, knocking-down PBEF with antisense probes abrogated this protective effect. There was increased immunostaining of PBEF in the compact layer of the amnion in multifetal tissues and significantly fewer apoptotic amniotic epithelial cells. These results show that chronic stretching of the amniotic epithelial cells increases PBEF expression, which protects them from apoptosis.

El saco vitelino y su contribución al desarrollo del saco amniótico antes de las 6 semanas de gestación: a propósito de un caso de aborto retenido / The yolk sac and its contribution to the development of the amniotic sac before the sixth week of pregnancy: a case of missed abortion

La expansión del saco amniótico entre las semanas 5 y 14 de gestación se correlaciona con los cambios de la superficie embrionaria. En este trabajo describimos un embarazo de 6 semanas en el que las imágenes ecográficas demostraron los sacos amniótico y vitelino sin polo embrionario. Se especula sobre el mecanismo por el que puede acumularse líquido amniótico en ausencia de embrión (AU)

Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo.

Conversion of a solid primordium to a hollow tube of cells is a morphogenetic process used frequently during vertebrate embryogenesis. In the early mouse embryo, this process of cavitation transforms the solid embryonic ectoderm into a columnar epithelium surrounding a cavity. Using both established cell lines and normal embryos, we provide evidence that cavitation in the early mouse embryo is the result of the interplay of two signals, one from an outer layer of endoderm cells that acts over short distances to create a cavity by inducing apoptosis of the inner ectodermal cells, and the other a rescue signal mediated by contact with the basement membrane that is required for the survival of the columnar cells that line the cavity. This simple model provides a paradigm for investigating tube morphogenesis in diverse developmental settings.

Roles of prostaglandins and intracellular free calcium mobilisation in epidermal growth factor-induced proliferation of human amnion cells.

OBJECTIVE: To investigate the mechanisms which regulate the growth of human amnion cells. DESIGN: A prospective descriptive study. SUBJECTS: Women undergoing caesarean section at term before the onset of labour. INTERVENTIONS: Amnion cells were cultured in monolayer. MAIN OUTCOME MEASURES: Cell cycle analysis, intracellular calcium levels, prostaglandin (PG) production rates. RESULTS: Epidermal growth factor (EGF) stimulated intracellular Ca2+ mobilisation and PGE2 production in cultured amnion cells. The addition of a Ca2+ channel blocker (cobalt) or a Ca2+ chelator (EGTA) into the culture medium inhibited intracellular Ca2+ mobilisation and PGE2 production induced by EGF. The analysis of cell cycles showed that EGF induced the initiation of DNA synthesis and that the addition of cobalt or EGTA into the culture medium inhibited EGF-induced DNA synthesis. The addition of a cyclo-oxygenase inhibitor (indomethacin) inhibited PGE2 production and DNA synthesis induced by EGF without the effect on intracellular Ca2+ mobilisation. Moreover, the inhibitory effect of indomethacin on EGF-induced DNA synthesis was attenuated by the addition of exogenous PGE2 or PGF2 alpha. CONCLUSIONS: These data suggest that EGF induces an increase in intracellular Ca2+ levels and the rate of prostaglandin production which leads to proliferation of human amnion cells.