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Sample records for human cells amniotic

  1. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

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    H. Niknejad

    2013-04-01

    Full Text Available Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF, which is full of growth factors, as substitute for fetal bovine serum (FBS in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved in 24 different patterns for 12 months in -196 ?C (liquid nitrogen and viability of cells were determined before and after cryopreservation by trypan blue and MTT assay. Moreover, Oct-4 expression was studied to determine pluripotency before and after cryopreservation with immunocytochemistry. Results were compared between groups with ANOVA (Tukey Post-Test. P.value under 0.01 and 0.05 was considered statistically significant. Results: The presence of DMEM, FBS or AF is necessary for amniotic cell cryopreservation. Trypan-blue, MTT and immunocytochemistry showed that there isn’t significant difference between using AF and FBS in viability and pluripotency of cells. Moreover, results showed that DMSO is a better cryoprotectant compared to glycerol. Conclusion : Results showed that amniotic fluid can be a proper substitute for FBS in amniotic epithelial cells cryopreservation. (Sci J Hamadan Univ Med Sci 2013; 20 (1:15-24

  2. Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

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    Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

    2012-08-01

    As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

  3. Human amniotic fluid: a source of stem cells for possible therapeutic use.

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    Dziadosz, Margaret; Basch, Ross S; Young, Bruce K

    2016-03-01

    Stem cells are undifferentiated cells with the capacity for differentiation. Amniotic fluid cells have emerged only recently as a possible source of stem cells for clinical purposes. There are no ethical or sampling constraints for the use of amniocentesis as a standard clinical procedure for obtaining an abundant supply of amniotic fluid cells. Amniotic fluid cells of human origin proliferate rapidly and are multipotent with the potential for expansion in vitro to multiple cell lines. Tissue engineering technologies that use amniotic fluid cells are being explored. Amniotic fluid cells may be of clinical benefit for fetal therapies, degenerative disease, and regenerative medicine applications. We present a comprehensive review of the evolution of human amniotic fluid cells as a possible modality for therapeutic use.

  4. Vesicular uptake of macromolecules by human placental amniotic epithelial cells.

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    Sharshiner, Rita; Brace, Robert A; Cheung, Cecilia Y

    2017-09-01

    Studies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. Placental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%-50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1-30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris(®) software. Vesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. In human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

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    Hartmann-Fritsch, Fabienne; Hosper, Nynke; Luginbuehl, Joachim; Biedermann, Thomas; Reichmann, Ernst; Meuli, Martin

    Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis

  6. Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum

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    Zhiyuan Wu; Guozhen Hui; Yi Lu; Tianjin Liu; Qin Huang; Lihe Guo

    2012-01-01

    Human amniotic epithelial cells were isolated from a piece of fresh amnion. Using immunocytochemical methods, we investigated the expression of neuronal phenotypes (microtubule-associated protein-2, glial fibrillary acidic protein and nestin) in human amniotic epithelial cells. The conditioned medium of human amniotic epithelial cells promoted the growth and proliferation of rat glial cells cultured in vitro, and this effect was dose-dependent. Human amniotic epithelial cells were further transplanted into the corpus striatum of healthy adult rats and the grafted cells could integrate with the host and migrate 1-2 mm along the nerve fibers in corpus callosum. Our experimental findings indicate that human amniotic epithelial cells may be a new kind of seed cells for use in neurograft.

  7. Characteristics of human amniotic fluid mesenchymal stem cells and their tropism to human ovarian cancer.

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    Liru Li

    Full Text Available The mesenchymal stem cells (MSCs derived from amniotic fluid (AF have become an attractive stem cells source for cell-based therapy because they can be harvested at low cost and avoid ethical disputes. In human research, stem cells derived from AF gradually became a hot research direction for disease treatment, specifically for their plasticity, their reduced immunogenicity and their tumor tropism regardless of the tumor size, location and source. Our work aimed to obtain and characterize human amniotic fluid mesenchymal stem cells (AFMSCs and detect their ovarian cancer tropsim in nude mice model. Ten milliliters of twenty independent amniotic fluid samples were collected from 16-20 week pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis in the first affiliated hospital of Harbin medical university. We successfully isolated the AFMSCs from thirteen of twenty amniotic fluid samples. AFMSCs presented a fibroblastic-like morphology during the culture. Flow cytometry analyses showed that the cells were positive for specific stem cell markers CD73,CD90, CD105, CD166 and HLA-ABC (MHC class I, but negative for CD 45,CD40, CD34, CD14 and HLA-DR (MHC class II. RT-PCR results showed that the AFMSCs expressed stem cell marker OCT4. AFMSCs could differentiate into bone cells, fat cells and chondrocytes under certain conditions. AFMSCs had the high motility to migrate to ovarian cancer site but didn't have the tumorigenicity. This study enhances the possibility of AFMSCs as drug carrier in human cell-based therapy. Meanwhile, the research emphasis in the future can also put in targeting therapy of ovarian cancer.

  8. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

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    Hartmann-Fritsch, Fabienne; Hosper, Nynke; Luginbuehl, Joachim; Biedermann, Thomas; Reichmann, Ernst; Meuli, Martin

    2013-01-01

    Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formati

  9. Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

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    Tsuno, Hiroaki [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Yoshida, Toshiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nogami, Makiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Orthopedic Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Koike, Chika; Okabe, Motonori [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Noto, Zenko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Arai, Naoya; Noguchi, Makoto [Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nikaido, Toshio, E-mail: tnikaido@med.u-toyama.ac.jp [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan)

    2012-12-01

    Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAM{alpha} cells and induced to osteogenic status-their in vivo osteogenesis was subsequently investigated in rats. It was found that HAM{alpha} cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAM{alpha} cells. The expression of osteocalcin mRNA was increased in HAM{alpha} cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAM{alpha} cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy. - Highlights: Black-Right-Pointing-Pointer Human amniotic mesenchymal cells include cells (HAM{alpha} cells) that have the properties of MSCs. Black-Right-Pointing-Pointer HAM{alpha} cells have excellent osteogenic differentiation potential. Black-Right-Pointing-Pointer Osteogenic differentiation ability of HAM{alpha} was amplified by calcium phosphate scaffolds. Black-Right-Pointing-Pointer HAM{alpha} cells can be applicable to allogeneic cell transplantation in bone regenerative therapy.

  10. DNA methylation patterns of imprinting centers for H19, SNRPN, and KCNQ1OT1 in single-cell clones of human amniotic fluid mesenchymal stem cell

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    Hsiu-Huei Peng

    2012-09-01

    Conclusion: In conclusion, human amniotic fluid mesenchymal stem cells contain a unique epigenetic signature during in vitro cell culture. H19 and KCNQ1OT1 possessed a substantial degree of hypermethylation status, and variable DNA methylation patterns of SNRPN was observed during in vitro cell culture of human amniotic fluid mesenchymal stem cells. Our results urge further understanding of epigenetic status of human amniotic fluid mesenchymal stem cells before it is applied in cell replacement therapy.

  11. Conditioned Medium From Human Amniotic Mesenchymal Stromal Cells Limits Infarct Size and Enhances Angiogenesis

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    Danieli, Patrizia; Malpasso, Giuseppe; Cluffreda, Maria Chiara; Cervio, Elisabetta; Calvillo, Laura; Copes, Francesco; Pisano, Federica; Mura, Manuela; Kleijn, Lennaert; de Boer, Rudolf A.; Viarengo, Gianluca; Rosti, Vittorio; Spinillo, Arsenio; Roccio, Marianna; Gnecchi, Massimiliano

    2015-01-01

    The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiog

  12. Gram-Negative Bacterial Lipopolysaccharide Stimulates Activin A Secretion from Human Amniotic Epithelial Cells

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    Yumiko Abe

    2013-01-01

    Full Text Available Activin A is involved in inflammation. The present study was performed to clarify if lipopolysaccharide, a component of Gram-negative bacteria, stimulates activin A secretion from human amniotic epithelial cells and to determine if activin A plays a role in amnionitis. Fetal membranes were obtained during elective cesarean sections performed in full-term pregnancies of patients without systemic disease, signs of premature delivery, or fetal complications. Amniotic epithelial cells were isolated by trypsinization. The activin A concentrations in the culture media were measured by enzyme-linked immunosorbent assay, and cell proliferation was assessed by 5-bromo-2′-deoxyuridine incorporation. Amniotic epithelial cells secreted activin A in a cell density-dependent manner, and lipopolysaccharide (10 μg/mL enhanced the secretion at each cell density. Lipopolysaccharide (10–50 μg/mL also stimulated activin A secretion in a dose-dependent manner. Contrary to the effect of activin A secretion, lipopolysaccharide inhibited cell proliferation in amniotic epithelial cells. The present study suggests that lipopolysaccharide stimulation of activin A secretion may be a mechanism in the pathogenesis of amnionitis.

  13. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

    OpenAIRE

    2013-01-01

    PURPOSE: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. METHODS: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thicknes...

  14. Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

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    Singhal Shweta

    2005-01-01

    Full Text Available Purpose: Retinal pigment epithelial (RPE cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM as substrate. Materials and Methods: Human cadaver eyeballs (16 were obtained from the eye bank after corneal trephination. RPE cells were harvested by a mechanical dissection of the inner choroid surface (10, group 1 or by b enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2. The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students′ ′t′ test. Results: Eleven samples (68.75% showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days in the mechanical group, significantly earlier ( P Conclusions: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes . Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion

  15. Comparison of Characteristics of Human Amniotic Membrane and Human Adipose Tissue Derived Mesenchymal Stem Cells

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    Dizaji Asl, Khadijeh; Shafaei, Hajar; Soleimani Rad, Jafar; Nozad, Hojjat Ollah

    2017-01-01

    BACKGROUND Mesenchymal stem cells (MSCs) are ideal candidates for treatment of diseases. Amniotic membranes are an inexpensive source of MSCs (AM-MSC) without any donor site morbidity in cell therapy. Adipose tissue derived stem cells (ASCs) are also suitable cells for cell therapy. There is discrepancy in CD271 expression among MSCs from different sources. In this study, the characteristics of AM-MSC and ASCs and CD271 expression were compared. METHODS Adult adipose tissue samples were obtained from patients undergoing elective surgical procedure, and samples of amniotic membrane were collected immediately after caesarean operation. After isolation and expansion of MSCs, the proliferation rate and viability of cells were evaluated through calculating DT and MTT assay. Expression of routine mesenchymal specific surface antigens of MSCs and CD271 was evaluated by flow cytometry for both types of cells. RESULTS The growth rate and viability of the MSCs from the amniotic membrane was significantly higher compared with the ASCs. The low expression of CD14 and CD45 indicated that AM-MSC and ASCs are non hematopoietic cells, and both cell types expressed high percentages of CD44, CD105. The results revealed that AM-MSC and ASCs expressed no CD271 on their surfaces. CONCLUSION This study showed that amniotic membrane is a suitable cell source for cell therapy, and CD271 is a negative marker for MSCs identification from amniotic membrane and adipose tissue.

  16. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

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    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  17. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos

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    Daniela Ávila-González

    2015-09-01

    Full Text Available Data from the literature suggest that human embryonic stem cell (hESC lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1 from poor-quality (PQ embryos derived and maintained on human amniotic epithelial cells (hAEC. This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  18. [Human amniotic epithelium (HAE) as a possible source of stem cells (SC)].

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    García-López, Guadalupe; García-Castro, Irma Lydia; Avila-González, Daniela; Molina-Hernández, Anayansi; Flores-Herrera, Héctor; Merchant-Larios, Horacio; Díaz-Martínez, Fabián

    2015-01-01

    There have been major recent advances in the field of developmental biology due to the investigation on stem cells (SC). Stem cells are characterized by their capacity of auto-renewal and differentiation to different cellular phenotypes. Based on the developmental stage, they can be classified into two different types: embryonic SCs and adult SCs. It has been widely reported that several problems need to be resolved before their possible clinical applications. As a result, fetal membranes have been suggested as an alternative source of SCs. In the human amniotic epithelium, the presence of markers of pluripotent SC´s has been reported, and its capacity as a feeder layer for expansion of different SC types. Also, fetal membranes are a discarded product after delivery, and thus there are not any ethical issues related to its use. In conclusion, the human amniotic epithelium can be a strong candidate for regenerative medicine.

  19. Isolation of c-Kit+ human amniotic fluid stem cells from second trimester.

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    Pozzobon, Michela; Piccoli, Martina; Schiavo, Andrea Alex; Atala, Anthony; De Coppi, Paolo

    2013-01-01

    Amniotic fluid-derived stem (AFS) cells have been described as an appealing source of stem cells because of their (1) fetal, non-embryonic origin, (2) easy access during pregnancy overcoming the ethical issues related both to the use of human embryonic cells and to the postnatal tissue biopsy with donor site morbidity, and (3) their undemanding ability to be expanded. We and others have demonstrated the broad differentiation potential and here we describe the established protocol we developed to obtain c-Kit+ human AFS cells, starting from second trimester amniocentesis samples.

  20. Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases

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    Ivana Antonucci

    2016-04-01

    Full Text Available In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases.

  1. Human amniotic membrane-derived stromal cells (hAMSC) interact depending on breast cancer cell type through secreted molecules.

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    Kim, Sun-Hee; Bang, So Hee; Kang, So Yeong; Park, Ki Dae; Eom, Jun Ho; Oh, Il Ung; Yoo, Si Hyung; Kim, Chan-Wha; Baek, Sun Young

    2015-02-01

    Human amniotic membrane-derived stromal cells (hAMSC) are candidates for cell-based therapies. We examined the characteristics of hAMSC including the interaction between hAMSC and breast cancer cells, MCF-7, and MDA-MB-231. Human amniotic membrane-derived stromal cells showed typical MSC properties, including fibroblast-like morphology, surface antigen expression, and mesodermal differentiation. To investigate cell-cell interaction via secreted molecules, we cultured breast cancer cells in hAMSC-conditioned medium (hAMSC-CM) and analyzed their proliferation, migration, and secretome profiles. MCF-7 and MDA-MB-231 cells exposed to hAMSC-CM showed increased proliferation and migration. However, in hAMSC-CM, MCF-7 cells proliferated significantly faster than MDA-MB-231 cells. When cultured in hAMSC-CM, MCF-7 cells migrated faster than MDA-MB-231 cells. Two cell types showed different profiles of secreted factors. MCF-7 cells expressed much amounts of IL-8, GRO, and MCP-1 in hAMSC-CM. Human amniotic membrane-derived stromal cells interact with breast cancer cells through secreted molecules. Factors secreted by hAMSCs promote the proliferation and migration of MCF-7 breast cancer cells. For much safe cell-based therapies using hAMSC, it is necessary to study carefully about interaction between hAMSC and cancer cells.

  2. Therapeutic effect of human amniotic epithelial cell transplantation into the lateral ventricle of hemiparkinsonian rats

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    YANG Xin-xin; XUE Shou-ru; DONG Wan-li; Kong Yan

    2009-01-01

    Background Human amniotic epithelial cells (HAECs) are able to secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine neurons.Previous study showed that when human amniotic epithelial cells were transplanted into the striatum of 6-hydroxydopamine (6-OHDA)-induced Parkinson disease rats, the cells could survive and exert functional effects. The purpose of this study was to investigate the survival and the differentiation of human amniotic epithelial cells after being transplanted into the lateral ventricle of Parkinson's disease (PD) rats, and to investigate the effects of grafts on healing PD in models.Methods The Parkinson's model was made with stereotactic microinjection of 6-hydroxydopamine (6-OHDA) into the striatum of a rat. The PD models were divided into two groups: the HAECs group and the normal saline (NS) group.Some untreated rats were taken as the control. The rotational asymmetry induced by apomorphine of the HAECs group and the NS group were measured post cell transplantation. The expression of nestin and vimentin in grafts were determined by immunohistology. Ten weeks after transplantation the density of tyrosine hydroxylase positive cells in the substantia nigra of the HAECs group, NS group and the untreated group was determined. The differentiation of grafts was determined by TH immunohistology. High performance liquid chromatography (HPLC) was used to determine monoamine neurotransmitter levels in the striatum.Results The rotational asymmetry induced by apomorphine of the HAECs group was ameliorated significantly compared to the NS group two weeks after transplantation (P <0.01). The grafts expressed nestin and vimentin five weeks after transplantation. TH immunohistochemistry indicated that the TH positive cells in the substantia nigra of the HAECs group increased significantly compared to the NS group (P<0.01). Tyrosine hydroxylase (TH) positive

  3. Human amniotic epithelial cells combined with silk ifbroin scaffold in the repair of spinal cord injury

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    Ting-gang Wang; Jie Xu; Ai-hua Zhu; Hua Lu; Zong-ning Miao; Peng Zhao; Guo-zhen Hui; Wei-jiangWu

    2016-01-01

    Treatment and functional reconstruction atfer central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artiifcial scaffold materials, such as ifbroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithe-lial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk ifbroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk ifbroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inlfammatory cell inifltration at the trans-plant site, milder host-versus-gratf reaction, and a marked improvement in motor function. hTese ifndings conifrm that the transplantation of amniotic epithelial cells combined with silk ifbroin scaffold can promote the repair of spinal cord injury. Silk ifbroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

  4. Human and murine amniotic fluid c-Kit+Lin- cells display hematopoietic activity.

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    Ditadi, Andrea; de Coppi, Paolo; Picone, Olivier; Gautreau, Laetitia; Smati, Rim; Six, Emmanuelle; Bonhomme, Delphine; Ezine, Sophie; Frydman, René; Cavazzana-Calvo, Marina; André-Schmutz, Isabelle

    2009-04-23

    We have isolated c-Kit(+)Lin(-) cells from both human and murine amniotic fluid (AF) and investigated their hematopoietic potential. In vitro, the c-Kit(+)Lin(-) population in both species displayed a multilineage hematopoietic potential, as demonstrated by the generation of erythroid, myeloid, and lymphoid cells. In vivo, cells belonging to all 3 hematopoietic lineages were found after primary and secondary transplantation of murine c-Kit(+)Lin(-) cells into immunocompromised hosts, thus demonstrating the ability of these cells to self-renew. Gene expression analysis of c-Kit(+) cells isolated from murine AF confirmed these results. The presence of cells with similar characteristics in the surrounding amnion indicates the possible origin of AF c-Kit(+)Lin(-) cells. This is the first report showing that cells isolated from the AF do have hematopoietic potential; our results support the idea that AF may be a new source of stem cells for therapeutic applications.

  5. Therapeutic potential of amniotic fluid stem cells.

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    Abdulrazzak, Hassan; De Coppi, Paolo; Guillot, Pascale V

    2013-03-01

    Human amniotic fluid cells have been used traditionally as a diagnostic tool for genetic anomalies. More recently it has been recognized that amniotic fluid contains populations of stem cells. Mesenchymal stem cells (AFMSC) were first to be described. These cells are able to differentiate towards mesodermal lineages. More recently cells with broader potential, defined as amniotic fluid stem cells (AFSC), were also isolated. They have intermediate characteristics between embryonic and adult stem cells and are able to differentiate into lineages representative of all three germ layers but unlike ES cells they do not form tumours in vivo. Furthermore, AFSC have been reverted to functional pluripotency in a transgene-free approach using an epigenetics modifier. These characteristics, together with absence of ethical issues concerning their employment, have made stem cells from amniotic fluid a promising candidate for cell therapy and tissue engineering.

  6. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid

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    Sanie-Jahromi Fatemeh

    2012-04-01

    Full Text Available Abstract Background Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers during treatment of human retinal pigment epithelium (RPE cells with amniotic fluid (AF, RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age. Results Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1 confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation. Conclusion Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

  7. AFM studies of cellular mechanics during osteogenic differentiation of human amniotic fluid-derived stem cells.

    Science.gov (United States)

    Chen, Qian; Xiao, Pan; Chen, Jia-Nan; Cai, Ji-Ye; Cai, Xiao-Fang; Ding, Hui; Pan, Yun-Long

    2010-01-01

    Amniotic fluid-derived stem cells (AFSCs) are becoming an important source of cells for regenerative medicine given with apparent advantages of accessibility, renewal capacity and multipotentiality. In this study, the mechanical properties of human amniotic fluid-derived stem cells (hAFSCs), such as the average Young's modulus, were determined by atomic force microscopy (3.97 ± 0.53 kPa for hAFSCs vs. 1.52 ± 0.63 kPa for fully differentiated osteoblasts). These differences in cell elasticity result primarily from differential actin cytoskeleton organization in these two cell types. Furthermore, ultrastructures, nanostructural details on the surface of cell, were visualized by atomic force microscopy (AFM). It was clearly shown that surface of osteoblasts were covered by mineralized particles, and the histogram of particles size showed that most of the particles on the surface of osteoblasts distributed from 200 to 400 nm in diameter, while the diameter of hAFSCs particles ranged from 100 to 200 nm. In contrast, there were some dips on the surface of hAFSCs, and particles were smaller than that of osteoblasts. Additionally, as osteogenic differentiation of hAFSCs progressed, more and more stress fibers were replaced by a thinner actin network which is characteristic of mature osteoblasts. These results can improve our understanding of the mechanical properties of hAFSCs during osteogenic differentiation. AFM can be used as a powerful tool for detecting ultrastructures and mechanical properties.

  8. Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells.

    Science.gov (United States)

    Li, Chunliang; Zhou, Junmei; Shi, Guilai; Ma, Yu; Yang, Ying; Gu, Junjie; Yu, Hongyao; Jin, Shibo; Wei, Zhe; Chen, Fang; Jin, Ying

    2009-11-15

    Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific induced pluripotent stem (iPS) cells for disease modeling and cellular replacement therapies. However, the low efficiency and safety issues associated with generation of human iPS cells have limited their usage in clinical settings. Cell types can significantly influence reprogramming efficiency and kinetics. To date, human iPS cells have been obtained only from a few cell types. Here, we report for the first time rapid and efficient generation of iPS cells from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4/SOX2/KLF4/C-MYC. Significantly, typical single iPS cell colonies can be picked up 6 days after viral infection with high efficiency. Eight iPS cell lines have been derived. They can be continuously propagated in vitro and express pluripotency markers such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, maintaining the normal karyotype. Transgenes are completely inactivated and the endogenous OCT4 promoter is adequately demethylated in the established iPS cell lines. Moreover, various cells and tissues from all three germ layers are found in embryoid bodies and teratomas, respectively. In addition, microarray analysis demonstrates a high correlation coefficient between hAFDC-iPS cells and human embryonic stem cells, but a low correlation coefficient between hAFDCs and hAFDC-iPS cells. Taken together, these data identify an ideal human somatic cell resource for rapid and efficient generation of iPS cells, allowing us to establish human iPS cells using more advanced approaches and possibly to establish disease- or patient-specific iPS cells.

  9. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

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    Natascha Krömmelbein

    2016-02-01

    Full Text Available The human cytomegalovirus (HCMV replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.

  10. Atomized Human Amniotic Mesenchymal Stromal Cells for Direct Delivery to the Airway for Treatment of Lung Injury

    NARCIS (Netherlands)

    Kim, Sally Yunsun; Burgess, Janette K.; Wang, Yiwei; Kable, Eleanor P. W.; Weiss, Daniel J.; Chan, Hak-Kim; Chrzanowski, Wojciech

    2016-01-01

    Background: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were use

  11. Atomized human amniotic mesenchymal stromal cells for direct delivery to the airway for treatment of lung injury

    NARCIS (Netherlands)

    Kim, Sally Yunsun; Burgess, Janette K.; Wang, Yiwei; Kable, Eleanor P. W.; Weiss, Daniel J.; Chan, Hak-Kim; Chrzanowski, Wojciech

    2016-01-01

    Background: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were use

  12. EFFECT OF HUMAN AMNIOTIC MEMBRANE ON CORNEAL EPITHELIUM AND YAC-1 CELL

    Institute of Scientific and Technical Information of China (English)

    叶纹; 沈玺; 钟一声

    2003-01-01

    Objective To study the effect of the amniotic membrane on enhancing the proliferation of corneal epithelia and YAC 1 cell.MethodsAfter the primary culture of the rabbits corneal epithelia and YAC 1 cells, they were seeded on the upper surface or stromal matrix side of amniotic membrane respectively. The proliferation results were observed by MTT test.ResultsThe amniotic membrane was found significantly enhancing the proliferation of corneal epithelia on the d1,d3,and d5 after culture. The proliferation rate was 28.93%,23.32%,23.41%(P<0.05)respectively, but the d7 proliferation rate was 20.72%(P>0.05).On the d1,d3,d7 after culture,the YAC 1 cells proliferation rate was 34.87%,36.28%,33.86%(P<0.01)respectively.ConclusionOur results demonstrated that the amniotic membrane could enhance the prolifera tion of both corneal epithelia and YAC 1 cells significantly. Although amniotic membrane has been suggested as an ideal material for reconstruction of ocular surface, special attention should be paid during amniotic membrane transplantation for treating ocular surface lesion resulted from epibulbar tumors.

  13. Amniotic membrane and amniotic fluid-derived cells: potential tools for regenerative medicine?

    Science.gov (United States)

    Parolini, Ornella; Soncini, Maddalena; Evangelista, Marco; Schmidt, Dörthe

    2009-03-01

    Human amniotic membranes and amniotic fluid have attracted increasing attention in recent years as a possible reserve of stem cells that may be useful for clinical application in regenerative medicine. Many studies have been conducted to date in terms of the differentiation potential of these cells, with several reports demonstrating that cells from both the amniotic fluid and membrane display high plasticity. In addition, cells from the amniotic membrane have also been shown to display immunomodulatory characteristics both in vivo and in vitro, which could make them useful in an allotransplantation setting. Here, we provide an overview comparing the latest findings regarding the stem characteristics of cells from both the amniotic membrane and amniotic fluid, as well as on the potential utility of these cells for future clinical application in regenerative medicine.

  14. Human amniotic epithelial cell transplantation for the repair of injured brachial plexus nerve:evaluation of nerve viscoelastic properties

    Institute of Scientific and Technical Information of China (English)

    Hua Jin; Qi Yang; Feng Ji; Ya-jie Zhang; Yan Zhao; Min Luo

    2015-01-01

    The transplantation of embryonic stem cells can effectively improve the creeping strength of nerves near an injury site in animals. Amniotic epithelial cells have similar biological properties as em-bryonic stem cells; therefore, we hypothesized that transplantation of amniotic epithelial cells can repair peripheral nerve injury and recover the creeping strength of the brachial plexus nerve. In the present study, a brachial plexus injury model was established in rabbits using the C6root avulsion method. A suspension of human amniotic epithelial cells was repeatedly injected over an area 4.0 mm lateral to the cephal and caudal ends of the C6 brachial plexus injury site (1 × 106 cells/mL, 3μL/injection, 25 injections) immediately after the injury. The results showed that the decrease in stress and increase in strain at 7,200 seconds in the injured rabbit C6 brachial plexus nerve were mitigated by the cell transplantation, restoring the viscoelastic stress relaxation and creep properties of the brachial plexus nerve. The forepaw functions were also signiifcantly improved at 26 weeks after injury. These data indicate that transplantation of human amniotic epithelial cells can effec-tively restore the mechanical properties of the brachial plexus nerve after injury in rabbits and that viscoelasticity may be an important index for the evaluation of brachial plexus injury in animals.

  15. Human amniotic epithelial cell transplantation for the repair of injured brachial plexus nerve: evaluation of nerve viscoelastic properties

    Directory of Open Access Journals (Sweden)

    Hua Jin

    2015-01-01

    Full Text Available The transplantation of embryonic stem cells can effectively improve the creeping strength of nerves near an injury site in animals. Amniotic epithelial cells have similar biological properties as embryonic stem cells; therefore, we hypothesized that transplantation of amniotic epithelial cells can repair peripheral nerve injury and recover the creeping strength of the brachial plexus nerve. In the present study, a brachial plexus injury model was established in rabbits using the C 6 root avulsion method. A suspension of human amniotic epithelial cells was repeatedly injected over an area 4.0 mm lateral to the cephal and caudal ends of the C 6 brachial plexus injury site (1 × 10 6 cells/mL, 3 μL/injection, 25 injections immediately after the injury. The results showed that the decrease in stress and increase in strain at 7,200 seconds in the injured rabbit C 6 brachial plexus nerve were mitigated by the cell transplantation, restoring the viscoelastic stress relaxation and creep properties of the brachial plexus nerve. The forepaw functions were also significantly improved at 26 weeks after injury. These data indicate that transplantation of human amniotic epithelial cells can effectively restore the mechanical properties of the brachial plexus nerve after injury in rabbits and that viscoelasticity may be an important index for the evaluation of brachial plexus injury in animals.

  16. Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD-inducible genes in human amniotic epithelial cells

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    Kokame Koichi

    2006-05-01

    Full Text Available Abstract Background Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD treated primary cultures of human amniotic epithelial cells. Methods Human amniotic epithelial cells were dispersed by trypsin from amniotic membranes and cultured in DME/Ham's F12 medium supplemented with 10% FBS. Two weeks after plating, cells were treated with 50 nM TCDD or DMSO (control, further incubated for 48 hrs, and the gene expression was analyzed by DNA microarray technology and quantitative real-time PCR. Results Thirty eight TCDD-inducible genes, including cytochromeP4501A1 and cytochromeP4501B1, were identified. One of the remarkable profiles of the gene expression was the prominent up-regulation of interferon-inducible genes. The genes involved in the interferon gene expression and interferon signaling pathways were also up-regulated. Furthermore, the expression of genes related to collagen synthesis or degradation was enhanced by TCDD. Conclusion Using DNA microarray and quantitative real-time PCR analyses, we identified TCDD-inducible genes, including interferon-inducible genes and genes related to collagen synthesis or degradation, in human amniotic epithelial cells.

  17. Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation.

    Science.gov (United States)

    Zhao, Bin; Zhang, Yijie; Han, Shichao; Zhang, Wei; Zhou, Qin; Guan, Hao; Liu, Jiaqi; Shi, Jihong; Su, Linlin; Hu, Dahai

    2017-04-01

    Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.

  18. The use of human amniotic fluid mesenchymal stem cells as the feeder layer to establish human embryonic stem cell lines.

    Science.gov (United States)

    Soong, Yung-Kwei; Huang, Shang-Yu; Yeh, Chiu-Hsiang; Wang, Tzu-Hao; Chang, Kuo-Hsuan; Cheng, Po-Jen; Shaw, S W Steven

    2015-12-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into the three germ layers and possibly all tissues of the human body. To fulfil the clinical potentials for cell-based therapy, banks of hESC lines that express different combinations of the major histocompatibility genes should be established, preferably without exposing such cells to animal cells and proteins. In this study, we tested human amniotic fluid mesenchymal stem cells (AFMSCs) as feeder cells to support the growth of hESCs. Our results indicated that mitomycin-treated AFMSCs were able to support the newly established hESC lines CGLK-1 and CGLK-2. The hESC colonies cultured on AFMSCs expressed alkaline phosphatase (ALK-P), SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Nanog and Sox-2, which are markers for undifferentiated hESCs. Chromosomal analyses of both hESC lines, CGLK-1 and CGLK-2, which were cultured on AFMSC feeders for 22 and 14 passages, respectively, were confirmed to be normal karyotypes (46, XX). The ability of AFMSCs as feeder cells to maintain the undifferentiated growth and pluripotency of hESCs was confirmed by in vivo formation of teratomas derived on AFMSC hESCs in severe combined immune-compromised mice. The use of AFMSCs for feeder cells to culture hESCs has several advantages, in that AFMSCs are not tumourigenic and can be expanded extensively with a short doubling time.

  19. Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn

    Institute of Scientific and Technical Information of China (English)

    SONG E; YANG Wei; CUI Zhi-hua; DONG Yu; SUI Dong-ming; GUAN Xiao-kang; MA Yang-ling

    2005-01-01

    Background The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. Methods Human limbal cells were isolated and cultivated in vitro. Cytokertins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining.Results On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no

  20. Isolation and Partial Characterization of Human Amniotic Epithelial Cells: The Effect of Trypsin

    Science.gov (United States)

    Tabatabaei, Meraj; Mosaffa, Nariman; Nikoo, Shohreh; Bozorgmehr, Mahmood; Ghods, Roya; Kazemnejad, Somaieh; Rezania, Simin; Keshavarzi, Bahareh; Arefi, Soheila; Ramezani-Tehrani, Fahimeh; Mirzadegan, Ebrahim; Zarnani, Amir-Hassan

    2014-01-01

    Background Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells (hAECs) remains controversial. The aim of the present study was to characterize immunophenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion. Methods Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor (EGF). To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining. Results Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies. Conclusion Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC. PMID:24523953

  1. Human amniotic fluid stem cell injection therapy for urethral sphincter regeneration in an animal model

    Directory of Open Access Journals (Sweden)

    Kim Bum

    2012-08-01

    Full Text Available Abstract Background Stem cell injection therapies have been proposed to overcome the limited efficacy and adverse reactions of bulking agents. However, most have significant limitations, including painful procurement, requirement for anesthesia, donor site infection and a frequently low cell yield. Recently, human amniotic fluid stem cells (hAFSCs have been proposed as an ideal cell therapy source. In this study, we investigated whether periurethral injection of hAFSCs can restore urethral sphincter competency in a mouse model. Methods Amniotic fluids were collected and harvested cells were analyzed for stem cell characteristics and in vitro myogenic differentiation potency. Mice underwent bilateral pudendal nerve transection to generate a stress urinary incontinence (SUI model and received either periurethral injection of hAFSCs, periurethral injection of Plasma-Lyte (control group, or underwent a sham (normal control group. For in vivo cell tracking, cells were labeled with silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate (MNPs@SiO2 (RITC and were injected into the urethral sphincter region (n = 9. Signals were detected by optical imaging. Leak point pressure and closing pressure were recorded serially after injection. Tumorigenicity of hAFSCs was evaluated by implanting hAFSCs into the subcapsular space of the kidney, followed two weeks later by retrieval and histologic analysis. Results Flow activated cell sorting showed that hAFSCs expressed mesenchymal stem cell (MSC markers, but no hematopoietic stem cell markers. Induction of myogenic differentiation in the hAFSCs resulted in expression of PAX7 and MYOD at Day 3, and DYSTROPHIN at Day 7. The nanoparticle-labeled hAFSCs could be tracked in vivo with optical imaging for up to 10 days after injection. Four weeks after injection, the mean LPP and CP were significantly increased in the hAFSC-injected group compared with the control group. Nerve regeneration and

  2. Nuclear Nox4 Role in Stemness Power of Human Amniotic Fluid Stem Cells

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    Tullia Maraldi

    2015-01-01

    Full Text Available Human amniotic fluid stem cells (AFSC are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(PH oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4 depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

  3. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G. [Depto. Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510 (Mexico); Enríquez-Jiménez, Juana [Depto. Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), México City 14000 (Mexico); Alcántara-Quintana, Luz E. [Subd. de Investigación, Centro Nacional de la Transfusión Sanguínea, Secretaria de Salud, Mexico City 07370 (Mexico); Fuentes-Mera, Lizeth [Depto. Biología Molecular e Histocompatibilidad, Hospital General “Dr. Manuel Gea González”, México City 4800 (Mexico); Piña-Barba, María C. [Depto. Materiales Metálicos y Cerámicos, Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); Zepeda-Rodríguez, Armando [Depto. Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); and others

    2013-05-10

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

  4. Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis

    Directory of Open Access Journals (Sweden)

    Jurate Savickiene

    2015-01-01

    Full Text Available Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

  5. A new candidate substrate for cell-matrix adhesion study: the acellular human amniotic matrix.

    Science.gov (United States)

    Guo, Qianchen; Lu, Xuya; Xue, Yuan; Zheng, Hong; Zhao, Xiaotao; Zhao, Huajian

    2012-01-01

    In vivo adhesions between cells and the extracellular matrix play a crucial role in cell differentiation, proliferation, and migration as well as tissue remodeling. Natural three-dimensional (3D) matrices, such as self-assembling matrices and Matrigel, have limitations in terms of their biomechanical properties. Here, we present a simple method to produce an acellular human amniotic matrix (AHAM) with preserved biomechanical properties and a favorable adhesion potential. On the stromal side of the AHAM, human foreskin fibroblasts (HFFs) attached and extended with bipolar spindle-shaped morphology proliferated to multilayer networks, invaded into the AHAM, and migrated in a straight line. Moreover, αV integrin, paxillin, and fibronectin were observed to colocalize after 24 h of HFF culture on the stromal side of the AHAM. Our results indicate that the AHAM may be an ideal candidate as a cell-matrix adhesion substrate to study cell adhesion and invasion as well as other functions in vitro under a tensile force that mimics the in vivo environment.

  6. A New Candidate Substrate for Cell-Matrix Adhesion Study: The Acellular Human Amniotic Matrix

    Directory of Open Access Journals (Sweden)

    Qianchen Guo

    2012-01-01

    Full Text Available In vivo adhesions between cells and the extracellular matrix play a crucial role in cell differentiation, proliferation, and migration as well as tissue remodeling. Natural three-dimensional (3D matrices, such as self-assembling matrices and Matrigel, have limitations in terms of their biomechanical properties. Here, we present a simple method to produce an acellular human amniotic matrix (AHAM with preserved biomechanical properties and a favorable adhesion potential. On the stromal side of the AHAM, human foreskin fibroblasts (HFFs attached and extended with bipolar spindle-shaped morphology proliferated to multilayer networks, invaded into the AHAM, and migrated in a straight line. Moreover, αV integrin, paxillin, and fibronectin were observed to colocalize after 24 h of HFF culture on the stromal side of the AHAM. Our results indicate that the AHAM may be an ideal candidate as a cell-matrix adhesion substrate to study cell adhesion and invasion as well as other functions in vitro under a tensile force that mimics the in vivo environment.

  7. Genotoxic effects of cadmium chloride on human amniotic fluid cells cultured in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Fogu, G. [Sassari Univ., Sassari (Italy). Dept. of Physiological, Biochemical and Cellular sciences; Sassari Univ., Sassari (Italy). Centre of Clinical Genetics; Congiu, A. M.; Sini, M. C.; Ladu, R. [Sassari Univ., Sassari (Italy). Dept. of Physiological, Biochemical and Cellular sciences; Campus, P. M.; Sanna, R.; Soro, G. [Sassari Univ., Sassari (Italy). Centre of Clinical Genetics

    2000-12-01

    In this study it has been reported the results of cytogenetic tests, namely a search for chromosome aberrations (CA) and sister chromatid exchanges (SCEs), performed on human amniotic fluid cells cultured and treated with Cadmium chloride. The cells from primary cultures were exposed to CdCl{sub 2} at 1 {mu}M and 10 {mu}M for 24 h. At the higher dose, no metaphases were scored and at the lower dose (1 {mu}M) no effects were evident on cell proliferation, and no chromosome aberrations were found. In the subsequent experiments were used cells from subcultures exposed to 1 {mu}M and 5 {mu}M CdCl{sub 2}. At the 5 {mu}M dose was evident the induction of chromatid breaks, while the frequency of sister chromatid exchanges shows a small increase, not statistically significant at the dose of 1 {mu}M. In this study it was positively demonstrated that amniotic fluid cells grown in vitro are reliable for testing various mutagenic or teratogenic substances. With regard to cadmium treatment results, it is evident a clastogenic effect of cadmium chloride but not a significant induction of SCEs. [Italian] In questo studio abbiamo riportato i risultati dei tests citogenici di valutazione degli scambi fra cromatidi fratelli (SCEs) e di induzione di aberrazioni cromosomiche (CA) condotti su colture cellulari di liquido amniotico umano, trattate con cloruro di cadmio. Le cellule delle colture primarie venivano esposte al CdCl{sub 2} a concentrazioni di 1 {mu}M 10 {mu}M per 24 h. Alla dose piu' alta (10 {mu}M) non sono state osservate metafasi, mentre alla dose piu' bassa (1 {mu}M) non sono stati evidenziati ne' effetti sulla proliferazione cellulare ne' CA. Nei successivi esperimenti il CdC{sub 2} e' stato testato su subcolture cellulari alle dosi di 1 {mu}M e 5 {mu}M. Alla dose di 5 {mu}M sono state evidenziate rotture cromatidiche, mentre la frequenza di SCE alla dose di 1 {mu}M ha mostrato un piccolo incremento, statisticamente non significativo. In questo

  8. Human umbilical cord mesenchymal stem cell-loaded amniotic membrane for the repair of radial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Zhi Li; Hanjiao Qin; Zishan Feng; Wei Liu; Ye Zhou; Lifeng Yang; Wei Zhao; Youjun Li

    2013-01-01

    In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis, the injured radial nerve was enwrapped with the prepared nerve conduit, which was fixed to the epineurium by sutures, with the cellon the inner surface of the conduit. Simultaneously, a 1.0 mL aliquot of human umbilical cord mesenchymal stem cellsuspension was injected into the distal and proximal ends of the injured radial nerve with 1.0 cm intervals. A total of 1.75 × 107 cells were seeded on the amniotic membrane. In the control group, patients received only neurolysis. At 12 weeks after celltransplantation, more than 80%of patients exhibited obvious improvements in muscular strength, and touch and pain sensations. In contrast, these improve-ments were observed only in 55-65% of control patients. At 8 and 12 weeks, muscular electro-physiological function in the region dominated by the injured radial nerve was significantly better in the transplantation group than the control group. After celltransplantation, no immunological rejec-tions were observed. These findings suggest that human umbilical cord mesenchymal stem cel-loaded amniotic membrane can be used for the repair of radial nerve injury.

  9. Human Amniotic Fluid Cells Form Functional Gap Junctions with Cortical Cells

    Directory of Open Access Journals (Sweden)

    Anna Jezierski

    2012-01-01

    Full Text Available The usage of stem cells is a promising strategy for the repair of damaged tissue in the injured brain. Recently, amniotic fluid (AF cells have received a lot of attention as an alternative source of stem cells for cell-based therapies. However, the success of this approach relies significantly on proper interactions between graft and host tissue. In particular, the reestablishment of functional brain networks requires formation of gap junctions, as a key step to provide sufficient intercellular communication. In this study, we show that AF cells express high levels of CX43 (GJA1 and are able to establish functional gap junctions with cortical cultures. Furthermore, we report an induction of Cx43 expression in astrocytes following injury to the mouse motor cortex and demonstrate for the first time CX43 expression at the interface between implanted AF cells and host brain cells. These findings suggest that CX43-mediated intercellular communication between AF cells and cortical astrocytes may contribute to the reconstruction of damaged tissue by mediating modulatory, homeostatic, and protective factors in the injured brain and hence warrants further investigation.

  10. Value of human amniotic epithelial cells in tissue engineering for cornea.

    Science.gov (United States)

    Fatimah, Simat Siti; Ng, Sook Luan; Chua, Kien Hui; Hayati, Abdul Rahman; Tan, Ay Eeng; Tan, Geok Chin

    2010-11-01

    Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

  11. Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

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    Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

    2017-09-05

    Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

  12. Improvement of Heart Failure by Human Amniotic Mesenchymal Stromal Cell Transplantation in Rats

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    Seyed Mohammad Taghi Razavi Tousi

    2016-11-01

    Full Text Available Background: Recently, stem cells have been considered for the treatment of heart diseases, but no marked improvement has been recorded. This is the first study to examine the functional and histological effects of the transplantation of human amniotic mesenchymal stromal cells (hAMSCs in rats with heart failure (HF.Methods: This study was conducted in the years 2014 and 2015. 35 male Wistar rats were randomly assigned into 5 equal experimental groups (7 rats each as 1- Control 2- Heart Failure (HF 3- Sham 4- Culture media 5- Stem Cell Transplantation (SCT. Heart failure was induced using 170 mg/kg/d of isoproterenol subcutaneously injection in 4 consecutive days. The failure confirmed by the rat cardiac echocardiography on day 28. In SCT group, 3×106 cells in 150 µl of culture media were transplanted to the myocardium. At the end, echocardiographic and hemodynamic parameters together with histological evaluation were done.Results: Echocardiography results showed that cardiac ejection fraction in HF group increased from 58/73 ± 9% to 81/25 ± 6/05% in SCT group (p value < 0.001. Fraction shortening in HF group was increased from 27/53 ± 8/58% into 45/55 ± 6/91% in SCT group (p value < 0.001. Furthermore, hAMSCs therapy significantly improved mean diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, rate pressure product, and left ventricular end-diastolic pressure compared to those in the HF group, with the values reaching the normal levels in the control group. A marked reduction in fibrosis tissue was also found in the SCT group (p value < 0.001 compared with the animals in the HF group.Conclusion: The transplantation of hAMSCs in rats with heart failure not only decreased the level of fibrosis but also conferred significant improvement in heart performance in terms of echocardiographic and hemodynamic parameters.

  13. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.

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    Ursula Manuelpillai

    Full Text Available Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4 twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6 were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively. Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established

  14. Involvement of gene methylation changes in the differentiation of human amniotic epithelial cells into islet-like cell clusters.

    Science.gov (United States)

    Peng, Lin; Wang, Jian; Lu, Guangxiu

    2014-09-01

    Insulin-dependent diabetes results from destruction of the insulin-producing β-cells of the pancreas. Islet cell transplantation is a promising cure for diabetes. Here, we induced human amniotic epithelial cells (hAECs) to differentiate into islet-like cell clusters by nicotinamide plus betacellulin in vitro, and further investigated the DNA methylation status by a Nimble MeDIP microarray before and after cell differentiation to shed light on the molecular mechanisms of this differentiation. In addition, 5-Aza-2'-deoxycytidine was used to investigate whether the differentiation of hAECs into islet-like cells occurred through demethylation. Purified hAECs (CK18(+)/E-cadherin(+)/CD29(+)/CD90(-)/CD34(-)/CD45(-)) were isolated from human amnia. After induction, hAECs were found to be insulin positive and sensitive to glucose, indicating successful induction to islet-like cells. The methylation status of cell cytoskeleton-related genes was down-regulated and that of negative regulation of cell adhesion-related genes was up-regulated. The methylation status of pancreas development-related genes such as HNF1α and DGAT1 was decreased in hAECs after induction. After brief demethylation, INS gene expression was up-regulated in islet-like cell clusters, suggesting that DNA methylation changes were associated with the differentiation of hAECs into islet-like cell clusters.

  15. Transplantation of human amniotic epithelial cells improves hindlimb function in rats with spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    WU Zhi-yuan; HUI Guo-zhen; LU Yi; WU Xin; GUO Li-he

    2006-01-01

    Background Human amniotic epithelial cells (HAECs), which have several characteristics similar to stem cells,therefore could possibly be used in cell therapy without creating legal or ethical problems. In this study, we transplanted HEACs into the injured spinal cord of rats to investigate if the cells can improve the rats' hindlimb motor function.Methods HAECs were obtained from a piece of fresh amnion, labeled with Hoechst33342, and transplanted into the site of complete midthoracic spinal transections in adult rats. The rats (n=21) were randomly divided into three groups: Sham-operation group (n=7), cells-graft group (n=7), and PBS group (n=7). One rat of each group was killed for histological analysis at the second week after the transplantation. The other six rats of each group were killed for histological analysis after an 8-week behavioral testing. Hindlimb motor function was assessed by using the open-field BBB scoring system. Survival rate of the graft cells was observed at second and eighth weeks after the transplantation. We also detected the myelin sheath fibers around the lesions and the size of the axotomized red nucleus. A one-way ANOVA was used to compare the means among the groups. The significance level was set at P<0.05.Results The graft HAECs survived for a long time (8 weeks) and integrated into the host spinal cord without immune rejection. Compared with the control group, HAECs can promote the regeneration and sprouting of the axons, improve the hindlimb motor function of the rats (BBB score: cells-graft group 9.0± 0.89 vs PBS group 3.7± 1.03, P<0.01), and inhibit the atrophy of axotomized red nucleus [cells-graft group (526.47 ± 148.42) μm2 vs PBS group (473.69±164.73) μm2, P<0.01].Conclusion Transplantation of HAECs can improve the hindlimb motor function of rats with spinal cord injury.

  16. Comparison of human mesenchymal stromal cells from four neonatal tissues: Amniotic membrane, chorionic membrane, placental decidua and umbilical cord.

    Science.gov (United States)

    Araújo, Anelise Bergmann; Salton, Gabrielle Dias; Furlan, Juliana Monteiro; Schneider, Natália; Angeli, Melissa Helena; Laureano, Álvaro Macedo; Silla, Lúcia; Passos, Eduardo Pandolfi; Paz, Ana Helena

    2017-05-01

    Mesenchymal stromal cells (MSCs) are being investigated as a potential alternative for cellular therapy. This study was designed to compare the biological characteristics of MSCs isolated from amniotic membrane (A-MSCs), chorionic membrane (C-MSCs), placental decidua (D-MSCs) and umbilical cord (UC-MSCs) to ascertain whether any one of these sources is superior to the others for cellular therapy purposes. MSCs were isolated from amniotic membrane, chorionic membrane, umbilical cord and placental decidua. Immunophenotype, differentiation ability, cell size, cell complexity, polarity index and growth kinetics of MSCs isolated from these four sources were analyzed. MSCs were successfully isolated from all four sources. Surface marker profile and differentiation ability were consistent with human MSCs. C-MSCs in suspension were the smallest cells, whereas UC-MSCs presented the greatest length and least width. A-MSCs had the lowest polarity index and UC-MSCs, as more elongated cells, the highest. C-MSCs, D-MSCs and UC-MSCs exhibited similar growth capacity until passage 8 (P8); C-MSCs presented better lifespan, whereas insignificant proliferation was observed in A-MSCs. Neonatal and maternal tissues can serve as sources of multipotent stem cells. Some characteristics of MSCs obtained from four neonatal tissues were compared and differences were observed. Amniotic membrane was the least useful source of MSCs, whereas chorionic membrane and umbilical cord were considered good options for future use in cell therapy because of the known advantages of immature cells. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  17. Human amniotic fluid stem cells as a model for functional studies of genes involved in human genetic diseases or oncogenesis.

    Science.gov (United States)

    Rosner, Margit; Dolznig, Helmut; Schipany, Katharina; Mikula, Mario; Brandau, Oliver; Hengstschläger, Markus

    2011-09-01

    Besides their putative usage for therapies, stem cells are a promising tool for functional studies of genes involved in human genetic diseases or oncogenesis. For this purpose induced pluripotent stem (iPS) cells can be derived from patients harbouring specific mutations. In contrast to adult stem cells, iPS cells are pluripotent and can efficiently be grown in culture. However, iPS cells are modulated due to the ectopic induction of pluripotency, harbour other somatic mutations accumulated during the life span of the source cells, exhibit only imperfectly cleared epigenetic memory of the source cell, and are often genomically instable. In addition, iPS cells from patients only allow the investigation of mutations, which are not prenatally lethal. Embryonic stem (ES) cells have a high proliferation and differentiation potential, but raise ethical issues. Human embryos, which are not transferred in the course of in vitro fertilization, because of preimplantation genetic diagnosis of a genetic defect, are still rarely donated for the establishment of ES cell lines. In addition, their usage for studies on gene functions for oncogenesis is hampered by the fact the ES cells are already tumorigenic per se. In 2003 amniotic fluid stem (AFS) cells have been discovered, which meanwhile have been demonstrated to harbour the potential to differentiate into cells of all three germ layers. Monoclonal human AFS cell lines derived from amniocenteses have a high proliferative potential, are genomically stable and are not associated with ethical controversies. Worldwide amniocenteses are performed for routine human genetic diagnosis. We here discuss how generation and banking of monoclonal human AFS cell lines with specific chromosomal aberrations or monogenic disease mutations would allow to study the functional consequences of disease causing mutations. In addition, recently a protocol for efficient and highly reproducible siRNA-mediated long-term knockdown of endogenous gene

  18. Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response.

    Directory of Open Access Journals (Sweden)

    Emily C Moorefield

    Full Text Available Amniotic fluid stem (AFS cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO and monocyte chemotactic protein (MCP family members as well as interleukin-6 (IL-6. AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α, MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and

  19. Human amniotic fluid stem cells support undifferentiated propagation and pluripotency of human embryonic stem cell without b-FGF in a density dependent manner.

    Science.gov (United States)

    Ma, Xiaorong; Li, Huanqi; Xin, Shujia; Ma, Yueting; Ouyang, Tianxiang

    2014-01-01

    Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10(4)/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.

  20. Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

    Science.gov (United States)

    Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

    2012-03-01

    Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

  1. Cerebroside Sulfatase Activity in Cultivated Human Skin Fibroblasts and Amniotic Fluid Cells

    Science.gov (United States)

    Booth, Carol W.; And Others

    1975-01-01

    Prenatal monitoring for metachromatic leukodystrophy (a fatal inherited metabolic disorder) suggested that the determination of levels of cerebroside sulfatase in the amniotic fluid helped in the prenatal detection of this disorder. (DB)

  2. Neurogenic differentiation of amniotic fluid stem cells.

    Science.gov (United States)

    Rosner, M; Mikula, M; Preitschopf, A; Feichtinger, M; Schipany, K; Hengstschläger, M

    2012-05-01

    In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.

  3. Sphingosine 1-phosphate in amniotic fluid modulates cyclooxygenase-2 expression in human amnion-derived WISH cells.

    Science.gov (United States)

    Kim, Jung Im; Jo, Eun Jin; Lee, Ha-Young; Cha, Moon Seok; Min, Jung Kee; Choi, Chang Hwan; Lee, Yong Moon; Choi, Young-Ae; Baek, Suk-Hwan; Ryu, Sung Ho; Lee, Kyu Sup; Kwak, Jong-Young; Bae, Yoe-Sik

    2003-08-22

    The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several S1P receptors including S1P1, S1P2, and S1P3. When WISH cells were stimulated with S1P, PGE2 synthesis increased in a concentration-dependent manner, showing maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that S1P stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P1 or S1P3 but not of S1P2 antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE2 production in WISH cells, indicating the involvements of S1P1 and S1P3 in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells.

  4. Magnetic Resonance Imaging of Human-Derived Amniotic Membrane Stem Cells Using PEGylated Superparamagnetic Iron Oxide Nanoparticles

    Directory of Open Access Journals (Sweden)

    Maryam Naseroleslami

    2016-09-01

    Full Text Available Objective: The label and detection of cells injected into target tissues is an area of focus for researchers. Iron oxide nanoparticles can be used to label cells as they have special characteristics. The purpose of this study is to examine the effects of iron oxide nanoparticles on human-derived amniotic membrane stem cell (hAMCs survival and to investigate the magnetic properties of these nanoparticles with increased contrast in magnetic resonance imaging (MRI. Materials and Methods: In this experimental study, we initially isolated mesenchymal stem cells from amniotic membranes and analyzed them by flow cytometry. In addition, we synthesized superparamagnetic iron oxide nanoparticles (SPIONs and characterized them by various methods. The SPIONs were incubated with hAMCs at concentrations of 25-800 μg/mL. The cytotoxicity of nanoparticles on hAMCs was measured by the MTT assay. Next, we evaluated the effectiveness of the magnetic nanoparticles as MRI contrast agents. Solutions of SPION were prepared in water at different iron concentrations for relaxivity measurements by a 1.5 Tesla clinical MRI instrument. Results: The isolated cells showed an adherent spindle shaped morphology. Polyethylene glycol (PEG-coated SPIONs exhibited a spherical morphology. The average particle size was 20 nm and magnetic saturation was 60 emu/g. Data analysis showed no significant reduction in the percentage of viable cells (97.86 ± 0.41% after 72 hours at the 125 μg/ml concentration compared with the control. The relaxometry results of this SPION showed a transverse relaxivity of 6.966 (μg/ml.s-1 Conclusion: SPIONs coated with PEG used in this study at suitable concentrations had excellent labeling efficiency and biocompatibility for hAMCs.

  5. Magnetic Resonance Imaging of Human-Derived Amniotic Membrane Stem Cells Using PEGylated Superparamagnetic Iron Oxide Nanoparticles

    Science.gov (United States)

    Naseroleslami, Maryam; Parivar, Kazem; Khoei, Samideh; Aboutaleb, Nahid

    2016-01-01

    Objective The label and detection of cells injected into target tissues is an area of focus for researchers. Iron oxide nanoparticles can be used to label cells as they have special characteristics. The purpose of this study is to examine the effects of iron oxide nanoparticles on human-derived amniotic membrane stem cell (hAMCs) survival and to investigate the magnetic properties of these nanoparticles with increased contrast in magnetic resonance imaging (MRI). Materials and Methods In this experimental study, we initially isolated mesenchymal stem cells from amniotic membranes and analyzed them by flow cytometry. In addition, we synthesized superparamagnetic iron oxide nanoparticles (SPIONs) and characterized them by various methods. The SPIONs were incubated with hAMCs at concentrations of 25-800 μg/mL. The cytotoxicity of nanoparticles on hAMCs was measured by the MTT assay. Next, we evaluated the effectiveness of the magnetic nanoparticles as MRI contrast agents. Solutions of SPION were prepared in water at different iron concentrations for relaxivity measurements by a 1.5 Tesla clinical MRI instrument. Results The isolated cells showed an adherent spindle shaped morphology. Polyethylene glycol (PEG)-coated SPIONs exhibited a spherical morphology. The average particle size was 20 nm and magnetic saturation was 60 emu/g. Data analysis showed no significant reduction in the percentage of viable cells (97.86 ± 0.41%) after 72 hours at the 125 μg/ml concentration compared with the control. The relaxometry results of this SPION showed a transverse relaxivity of 6.966 (μg/ml.s)-1 Conclusion SPIONs coated with PEG used in this study at suitable concentrations had excellent labeling efficiency and biocompatibility for hAMCs. PMID:27602314

  6. Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.

    Science.gov (United States)

    Moschidou, Dafni; Mukherjee, Sayandip; Blundell, Michael P; Drews, Katharina; Jones, Gemma N; Abdulrazzak, Hassan; Nowakowska, Beata; Phoolchund, Anju; Lay, Kenneth; Ramasamy, T Selvee; Cananzi, Mara; Nettersheim, Daniel; Sullivan, Mark; Frost, Jennifer; Moore, Gudrun; Vermeesch, Joris R; Fisk, Nicholas M; Thrasher, Adrian J; Atala, Anthony; Adjaye, James; Schorle, Hubert; De Coppi, Paolo; Guillot, Pascale V

    2012-10-01

    Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.

  7. Ultracytochemical study on the permeability of the human amniotic epithelium.

    Science.gov (United States)

    Matsubara, S; Tamada, T

    1991-06-01

    In order to elucidate and characterize the transport pathway of the substances in the amniotic fluid, the permeability of the term human amnion was studied ultracytochemically, with lanthanum or horse radish peroxidase (HRP) as a tracer. Pieces of the term human amnion were exposed to the solutions containing lanthanum or HRP, and processed for electronmicroscopy. Precipitates indicating lanthanum or HRP were observed in the lateral intercellular spaces of the amniotic epithelial cells through the entire depth of the spaces. Generally, pinocytosis of HPR was not observed. In rare cases, however, diffuse uptake of HRP was noticed in the cells of the electron-lucent cytoplasm. These facts indicated that the human amniotic epithelium is quite permeable and that this particular intercellular pathway is important in the mechanism of the transfer of substances between the mother and the fetus.

  8. Study of p53 expression and post-transcriptional modifications after GSM-900 radiofrequency exposure of human amniotic cells.

    Science.gov (United States)

    Bourthoumieu, Sylvie; Magnaudeix, Amandine; Terro, Faraj; Leveque, Philippe; Collin, Alice; Yardin, Catherine

    2013-01-01

    The potential effects of radiofrequency (RF) exposure on the genetic material of cells are very important to determine since genome instability of somatic cells may be linked to cancer development. In response to genetic damage, the p53 protein is activated and can induce cell cycle arrest allowing more time for DNA repair or elimination of damaged cells through apoptosis. The objective of this study was to investigate whether the exposure to RF electromagnetic fields, similar to those emitted by mobile phones of the second generation standard, Global System for Mobile Communications (GSM), may induce expression of the p53 protein and its activation by post-translational modifications in cultured human cells. The potential induction of p53 expression and activation by GSM-900 was investigated after in vitro exposure of human amniotic cells for 24 h to average specific absorption rates (SARs) of 0.25, 1, 2, and 4 W/kg in the temperature range of 36.3-39.7 °C. The exposures were carried out using a wire-patch cell (WPC) under strictly controlled conditions of temperature. Expression and activation of p53 by phosphorylation at serine 15 and 37 were studied using Western blot assay immediately after three independent exposures of cell cultures provided from three different donors. Bleomycin-exposed cells were used as a positive control. According to our results, no significant changes in the expression and activation of the p53 protein by phosphorylation at serine 15 and 37 were found following exposure to GSM-900 for 24 h at average SARs up to 4 W/kg in human embryonic cells.

  9. Comparison of the neural differentiation potential of human mesenchymal stem cells from amniotic fluid and adult bone marrow.

    Science.gov (United States)

    Yan, Zhong-Jie; Hu, Yu-Qin; Zhang, Hong-Tian; Zhang, Peng; Xiao, Zong-Yu; Sun, Xin-Lin; Cai, Ying-Qian; Hu, Chang-Chen; Xu, Ru-Xiang

    2013-05-01

    Human mesenchymal stem cells (MSCs) are considered a promising tool for cell-based therapies of nervous system diseases. Bone marrow (BM) has been the traditional source of MSCs (BM-MSCs). However, there are some limitations for their clinical use, such as the decline in cell number and differentiation potential with age. Recently, amniotic fluid (AF)-derived MSCs (AF-MSCs) have been shown to express embryonic and adult stem cell markers, and can differentiate into cells of all three germ layers. In this study, we isolated AF-MSCs from second-trimester AF by limiting dilution and compared their proliferative capacity, multipotency, neural differentiation ability, and secretion of neurotrophins to those of BM-MSCs. AF-MSCs showed a higher proliferative capacity and more rapidly formed and expanded neurospheres compared to those of BM-MSCs. Both immunocytochemical and quantitative real-time PCR analyses demonstrated that AF-MSCs showed higher expression of neural stemness markers than those of BM-MSCs following neural stem cell (NSC) differentiation. Furthermore, the levels of brain-derived growth factor and nerve growth factor secreted by AF-MSCs in the culture medium were higher than those of BM-MSCs. In addition, AF-MSCs maintained a normal karyotype in long-term cultures after NSC differentiation and were not tumorigenic in vivo. Our findings suggest that AF-MSCs are a promising and safe alternative to BM-MSCs for therapy of nervous system diseases.

  10. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Te, E-mail: liute79@yahoo.com [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China); Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 (China); Cheng, Weiwei [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China); Huang, Yongyi [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France); Huang, Qin; Jiang, Lizhen [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo, Lihe, E-mail: liute79@yahoo.com [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder

  11. Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis.

    Directory of Open Access Journals (Sweden)

    Laura Perin

    Full Text Available Acute Tubular Necrosis (ATN causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit(pos stem cells, derived from human amniotic fluid (hAFSC can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

  12. Low microRNA-199a expression in human amniotic epithelial cell feeder layers maintains human-induced pluripotent stem cell pluripotency via increased leukemia inhibitory factor expression

    Institute of Scientific and Technical Information of China (English)

    Te Liu; Qing Chen; Yongyi Huang; Qin Huang; Lizhen Jiang; Lihe Guo

    2012-01-01

    Human-induced pluripotent stem (iPS) cells share the same key properties as embryonic stem cells,and may be generated from patient- or disease-specific sources,which makes them attractive for personalized medicine,drug screens,or cellular therapy.Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state is a major challenge.Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells,or spermatogonial stem cells,as they express endogenous leukemia inhibitory factor (LIF) at high levels.Here,we examined the effect of exogenous microRNA-199a regulation on endogenous LIF expression in HuAECs,and in torn on human iPS cell pluripotency.We found that HuAECs feeder cells transfected with microRNA-199a mutant expressed LIF at high levels,allowing iPS to maintain a high level of alkaline phosphatase activity in longterm culture and form teratomas in severe combined immunodeficient mice.The expression of stem cell markers was increased in iPS cultured on HuAECs feeder cells transfected with the microRNA-199a mutant,compared with iPS cultured on HuAECs transfected with microRNA-199a or mouse embryo fibroblasts.Taken together,these results suggested that LIF expression might be regulated by microRNA-199a,and LIF was a crucial component in feeder cells,and also was required for maintenance of human iPS cells in an undifferentiated,proliferative state capable of self-renewal.

  13. Endothelin-1 and macrophage colony-stimulating factor are co-localized in human amnion membrane cells and secreted into amniotic fluid.

    Science.gov (United States)

    Fried, Gabriel; Sand, Anna; Ostlund, Eva; Andersson, Eva; Byström, Birgitta; Ståbi, Berit

    2003-11-01

    We have examined the cellular localization and human amniotic fluid content of endothelin-1 (ET-1) and macrophage colony-stimulating factor (M-CSF). The study material consisted of amniotic fluid from 20 patients referred for amniocentesis, and placental samples from normal deliveries. ET-1 and M-CSF were analysed by radioimmunoassay and enzyme-linked immunosorbent assay respectively. The cellular localization of ET-1 and M-CSF in the amnion membranes was analysed by double-labelling immunocytochemistry using fluorescein isothiocyanate- and Cy3-labelled secondary antibodies. Release of ET-1 and M-CSF was studied in cultured amniocytes. We found that the mean +/- SD concentrations of ET-1 and M-CSF in fetal amniotic fluid were 45.6 +/- 17.3 pmol/l (range 16.8-85.5) and 7323 +/- 3415 ng/l (range 2640-12 110) respectively. Double-labelling immunocytochemistry showed that both M-CSF and ET-1 were co-localized in the same cells to a high extent. Further analysis revealed that levels of M-CSF, but not ET-1, were significantly correlated with pregnancy length. Both M-CSF and ET-1 were released from cultured amniocytes in response to interleukin-1. These findings show that ET-1 and M-CSF are partly co-localized to specific cells in the human amniotic membrane. As both M-CSF and ET-1 were released from cultured amniocytes in vitro, this suggests that they both may be secreted into fetal amniotic fluid in vivo as well.

  14. Feasibility of Human Amniotic Fluid Derived Stem Cells in Alleviation of Neuropathic Pain in Chronic Constrictive Injury Nerve Model.

    Directory of Open Access Journals (Sweden)

    Chien-Yi Chiang

    Full Text Available The neurobehavior of neuropathic pain by chronic constriction injury (CCI of sciatic nerve is very similar to that in humans, and it is accompanied by a profound local inflammation response. In this study, we assess the potentiality of human amniotic fluid derived mesenchymal stem cells (hAFMSCs for alleviating the neuropathic pain in a chronic constriction nerve injury model.This neuropathic pain animal model was conducted by four 3-0 chromic gut ligatures loosely ligated around the left sciatic nerve in Sprague-Dawley rats. The intravenous administration of hAFMSCs with 5x105 cells was conducted for three consecutive days.The expression IL-1β, TNF-α and synaptophysin in dorsal root ganglion cell culture was remarkably attenuated when co-cultured with hAFMSCs. The significant decrease of PGP 9.5 in the skin after CCI was restored by administration of hAFMSCs. Remarkably increased expression of CD 68 and TNF-α and decreased S-100 and neurofilament expression in injured nerve were rescued by hAFMSCs administration. Increases in synaptophysin and TNF-α over the dorsal root ganglion were attenuated by hAFMSCs. Significant expression of TNF-α and OX-42 over the dorsal spinal cord was substantially attenuated by hAFMSCs. The increased amplitude of sensory evoked potential as well as expression of synaptophysin and TNF-α expression was alleviated by hAFMSCs. Human AFMSCs significantly improved the threshold of mechanical allodynia and thermal hyperalgesia as well as various parameters of CatWalk XT gait analysis.Human AFMSCs administration could alleviate the neuropathic pain demonstrated in histomorphological alteration and neurobehavior possibly through the modulation of the inflammatory response.

  15. Effect of microcystin-LR on protein phosphatase 2A and its function in human amniotic epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Jing LIANG; Tan LI; Ya-li ZHANG; Zong-lou GUO; Li-hong XU

    2011-01-01

    Due to their toxicity,the increased distribution of microcystins (MCs) has become an important worldwide problem.MCs have been recognized as inhibitors of protein phosphatase 2A (PP2A) through their binding to the PP2A catalytic subunit.However,the exact mechanism of MC toxicity has not been elucidated,especially concerning the cellular response and its autoregulation.To further dissect the role of PP2A in MC-induced toxicity,the present study was undertaken to determine the response of PP2A in human amniotic epithelial (FL) cells treated with microcystin-LR (MCLR),one of the MC congeners.The results show that a low-dose treatment of MCLR in FL cells for 6 h induced an increase in PP2A activity,and a high-dose treatment of MCLR for 24 h decreased the activity of PP2A,as expected.The increased mRNA and protein levels of the PP2A C subunit may explain the increased activity of PP2A.Furthermore,MCLR altered microtubule post-translational modifications through PP2A.These results further clarify the underlying mechanism how MCLR affects PP2A and may be helpful for elucidating the complex toxicity of MCLR.

  16. Induction of micronuclei, hyperdiploidy and chromosomal breakage affecting the centric/pericentric regions of chromosomes 1 and 9 in human amniotic fluid cells after treatment with asbestos and ceramic fibers.

    Science.gov (United States)

    Dopp, E; Schuler, M; Schiffmann, D; Eastmond, D A

    1997-06-09

    This article describes the induction of micronuclei, hyperdiploidy and chromosome breakage in human amniotic cells in vitro by amosite, chrysotile and crocidolite asbestos, and ceramic fibers. The response of human (amniotic fluid cells) and rodent (Syrian hamster embryo fibroblasts, SHE) cells to fiber treatment was compared using the micronucleus assay. The data of the rodent studies were taken from a previous investigation (Dopp, E. et al. (1995) Environ. Health Perspect., 103, 268-271). All types of mineral fibers caused a significant increase of micronucleated cells. The kinetochore analysis revealed that all three types of asbestos and ceramic fibers yielded similar effects. Approximately 50% of the induced micronuclei were kinetochore-negative indicating formation through clastogenic events. Human amniotic cells were much less susceptible than SHE cells to the induction of micronuclei by mineral fibers. This again demonstrates that SHE cells are more susceptible to chromosomal changes than human amniotic fluid cells. The application of fluorescence in situ hybridization (FISH) with tandem DNA probes yielded more detailed information about specific structural chromosome aberrations in the 1 (cen-q12) and 9 (cen-q12) regions and about abnormal numbers of chromosomes in interphase human amniotic fluid cells. Using this FISH approach we found a statistically significant increase of chromosomal breakage in the pericentric heterochromatin regions of chromosomes 1 and 9 in interphase human amniotic cells after exposure to asbestos and ceramic fibers compared to control cells. The number of hyperdiploid cells was also significantly increased. Our results show that asbestos fibers as well as ceramic fibers are inducers of structural and numerical chromosomal aberrations in human amniotic fluid cells.

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

    Science.gov (United States)

    Lv, Xiaokui; Guo, Qianping; Han, Fengxuan; Chen, Chunyang; Ling, Christopher; Chen, Weiguo; Li, Bin

    2016-01-01

    Tissue engineering-based urethral replacement holds potential for repairing large segmental urethral defects, which remains a great challenge at present. This study aims to explore the potential of combining biodegradable poly(l-lactide) (PLLA)/poly(ethylene glycol) (PEG) scaffolds and human amniotic mesenchymal cells (hAMSCs) for repairing urethral defects. PLLA/PEG fibrous scaffolds with various PEG fractions were fabricated via electrospinning. The scaffolds were then seeded with hAMSCs prior to implantation in New Zealand male rabbits that had 2.0 cm-long defects in the urethras. The rabbits were randomly divided into three groups. In group A, hAMSCs were grown on PLLA/PEG scaffolds for two days and then implanted to the urethral defects. In group B, only the PLLA/PEG scaffolds were used to rebuild the rabbit urethral defect. In group C, the urethral defect was reconstructed using a regular urethral reparation technique. The repair efficacy was compared among the three groups by examining the urethral morphology, tissue reconstruction, luminal patency, and complication incidence (including calculus formation, urinary fistula, and urethral stricture) using histological evaluation and urethral radiography methods. Findings from this study indicate that hAMSCs-loaded PLLA/PEG scaffolds resulted in the best urethral defect repair in rabbits, which predicts the promising application of a tissue engineering approach for urethral repair. PMID:27517902

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

    Directory of Open Access Journals (Sweden)

    Xiaokui Lv

    2016-08-01

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

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

    Science.gov (United States)

    Lv, Xiaokui; Guo, Qianping; Han, Fengxuan; Chen, Chunyang; Ling, Christopher; Chen, Weiguo; Li, Bin

    2016-08-09

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

  20. Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo.

    Science.gov (United States)

    Ma, Xiaorong; Zhang, Shengli; Zhou, Junmei; Chen, Baisong; Shang, Yafeng; Gao, Tongbing; Wang, Xue; Xie, Hua; Chen, Fang

    2012-08-01

    Stem cell-based therapy may be the most promising method to cure skeletal muscle degenerative diseases such as Duchenne muscular dystrophy (DMD) and trauma in the future. Human amniotic fluid is enriched with early-stage stem cells from developing fetuses and these cells have cardiomyogenic potential both in vitro and in vivo. In the present study, we investigated the characteristics of human amniotic fluid-derived AF-type stem (HAF-AFS) cells by flow cytometry, immunofluorescence staining, reverse-transcription polymerase chain reaction, and osteogenic and adipogenic differentiation analysis. After confirming the stemness of HAF-AFS cells, we tested whether HAF-AFS cells could differentiate into skeletal myogenic cells in vitro and incorporate into regenerating skeletal muscle in vivo. By temporary exposure to the DNA demethylation agent 5-aza-2'-deoxycytidine (5-Aza dC) or co-cultured with C2C12 myoblasts, HAF-AFS cells differentiated into skeletal myogenic cells, expressing skeletal myogenic cell-specific markers such as Desmin, Troponin I (Tn I) and α-Actinin. Four weeks after transplantation into cardiotoxin-injured and X-ray-irradiated tibialis anterior (TA) muscles of NOD/SCID mice, HAF-AFS cells survived, differentiated into myogenic precursor cells and fused with host myofibres. The findings that HAF-AFS cells differentiate into myogenic cells in vitro and incorporate in skeletal muscle regeneration in vivo hold the promise of HAF-AFS cell-based therapy for skeletal muscle degenerative diseases.

  1. Investigation on biological characteristics of human amniotic fluid-derived stem cells%人羊水来源干细胞生物学性状的研究

    Institute of Scientific and Technical Information of China (English)

    张建芳; 顾潇; 陈必良

    2011-01-01

    Objective: Human amniotic fluid samples were isolated and cultured in vitro.To establish in vitro culture procedure of amniotic fluid-derived stem cells (AFS) and to study their biological characteristics.Methods: AFS were isolated from second-trimester amniotic fluid by adherence.The phenotypes of AFS were detected by flow cytometry and RT-PCR.Results: Primary cells grow slowly, The cells proliferated rapidly after passage and they were positive for CD29, CD44, CD105 and negative for CD45 and CD133 by flow cytometry.RT-PCR analysis showed that AFS were positive for Oct-4 and Nanog.Conclusion: Experiment has successfully isolated stem cells in amniotic fluid.The stem cells isolated from second-trimester amniotic fluid have a great potential of proliferation, which express mesenchymal stem cell markers.Their characteristics are in accord with mesenchymal stem cells.%目的 对人羊水标本进行体外分离培养,建立人羊水来源干细胞的体外培养体系,对其生物学形状进行研究.方法 贴壁法体外分离获得人羊水来源干细胞,多次传代扩增后,采用流式细胞仪和RT-PCR技术检测细胞表面抗原的表达.结果 羊水干细胞原代生长较慢,传代后生长迅速,体外倍增时间约36h,流式细胞仪检测证实细胞表达CD29、CD44、CD105等间充质干细胞标志,不表达造血干细胞标志CD45和CD133.RT-PCR检测显示羊水干细胞表达Oct-4、Nanog基因.结论 实验成功分离获得羊水中具有干细胞性质的细胞群,采用贴壁法分离获得的干细胞体外增殖能力强,表达间充质干细胞表面标志,符合间充质干细胞的特点.

  2. Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation.

    Science.gov (United States)

    Ab Hamid, Suzina Sheikh; Zahari, Nor Kamalia; Yusof, Norimah; Hassan, Asnah

    2014-03-01

    Human amniotic membrane that has been processed and sterilised by gamma irradiation is widely used as a biological dressing in surgical applications. The morphological structure of human amniotic membrane was studied under scanning electron microscopy (SEM) to assess effects of gamma radiation on human amniotic membrane following different preservation methods. The amniotic membrane was preserved by either air drying or submerged in glycerol before gamma irradiated at 15, 25 and 35 kGy. Fresh human amniotic membrane, neither preserved nor irradiated was used as the control. The surface morphology of glycerol preserved amnion was found comparable to the fresh amniotic membrane. The cells of the glycerol preserved was beautifully arranged, homogonous in size and tended to round up. The cell structure in the air dried preserved amnion seemed to be flattened and dehydrated. The effects of dehydration on intercellular channels and the microvilli on the cell surface were clearly seen at higher magnifications (10,000×). SEM revealed that the changes of the cell morphology of the glycerol preserved amnion were visible at 35 kGy while the air dried already changed at 25 kGy. Glycerol preservation method is recommended for human amniotic membrane as the cell morphological structure is maintained and radiation doses lower than 25 kGy for sterilization did not affect the appearance of the preserved amnion.

  3. 人类羊膜上皮细胞与间充质干细胞的免疫调节功能%The immune regulation functions of human amniotic epithelial cells and mesenchymal cells

    Institute of Scientific and Technical Information of China (English)

    王佳萍

    2010-01-01

    成体干细胞是一种具有自我更新和多向分化能力的细胞,在组织工程、基因治疗和细胞移植领域具有较好的应用前景.近年研究显示,人类羊膜中也存在两种干细胞:羊膜上皮细胞(HAEC)和羊膜间充质干细胞(hAMSC),由于具有来源广泛、取材方便、多向分化潜能以及免疫原性低等优点,在细胞移植中表现出免疫调节等作用,且可作为细胞组织工程种子,正逐渐成为干细胞研究领域的热点之一.现就两种羊膜细胞生物学特性、免疫调节机制以及临床运用的潜在价值进行综述.%Adult stem cells are a kind of cells which have the capability to differentiate into multiple cell types as well as self renew continuously. They have great therapeutic potential in tissue engineering,genetherapy and cell transplantation.In recent study, it has been found that there were two kinds of stem cells in human amniotic membrane,including human amniotic epithelial cells and human amniotic mesenchymal stem cells. As a new source of stem cells, these two amniotic cells have become the hot spot in stem cells research due to their advantages such as extensive resource, easy to acquire, multi-differentiation potential and negligible antigenicity. This article reviewed biological characteristics, immune regulation mechanism and prospect on amniotic epithelial cells and mesenchymal cells.

  4. Rat full term amniotic fluid harbors highly potent stem cells.

    Science.gov (United States)

    Mun-Fun, Hoo; Ferdaos, Nurfarhana; Hamzah, Siti Nurusaadah; Ridzuan, Noridzzaida; Hisham, Nurul Afiqah; Abdullah, Syahril; Ramasamy, Rajesh; Cheah, Pike See; Thilakavathy, Karrupiah; Yazid, Mohd Nazri; Nordin, Norshariza

    2015-10-01

    Amniotic fluid stem cells (AFSCs) are commonly isolated from mid-term amniotic fluid (AF) of animals and human collected via an invasive technique, amniocentesis. Alternatively, AFSCs could be collected at full-term. However, it is unclear whether AFSCs are present in the AF at full term. Here, we aimed to isolate and characterize stem cells isolated from AF of full term pregnant rats. Three stem cell lines have been established following immuno-selection against the stem cell marker, c-kit. Two of the new lines expressed multiple markers of pluripotency until more than passage 90. Further, they spontaneously differentiated into derivatives of the three primary germ layers through the formation of good quality embryoid bodies (EBs), and can be directly differentiated into neural lineage. Their strong stemness and potent neurogenic properties highlight the presence of highly potent stem cells in AF of full-term pregnancies, which could serve as a potential source of stem cells for regenerative medicine.

  5. Reprogramming human amniotic fluid stem cells to functional pluripotency by manipulation of culture conditions.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Dafni Moschidou & Pascale V Guillot ### Abstract Pluripotent stem cells have potential applications in regenerative medicine, disease modelling and drug screening. Induced pluripotent stem (iPS) cells have first been generated from fibroblasts using retroviral insertion of OCT4A, SOX2, c-MYC and KLF4. Since then, a number of methods have been developed to avoid the random integration of ectopic factors in the genome and the low efficiency of the process. Those include alt...

  6. Treatment of traumatic brain injury in rats with transplantation of human amniotic cells

    Institute of Scientific and Technical Information of China (English)

    LU Yi; HUI Guo-zhen; WU Zhi-yuan; GUO Li-he; JI Xun-he; WU Xin

    2006-01-01

    @@ Traumatic brain injury (TBI), which is an important reason of human mortality and morbidity in industrialized countries, still cannot be treated effectively. Since the self-repair capacity of brain is limited, cellular transplantation in TBI may be a therapeutic option.

  7. Synergic activation of toll-like receptor (TLR 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Martha Triantafilou

    Full Text Available Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM, preterm labour (PL pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR. In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA, trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

  8. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  9. Amniotic fluid may act as a transporting pathway for signaling molecules and stem cells during the embryonic development of amniotes.

    Science.gov (United States)

    Tong, Xinglong

    2013-11-01

    Amniotic fluid (AF) is formed at the very early stages of pregnancy, and is present throughout embryonic development of amniotes. It is well-known that AF provides a protective sac around the fetus that allows fetal movement and growth, and prevents mechanical and thermal shock. However, a growing body of evidence has shown that AF contains a number of proteins and peptides, including growth factors and cytokines, which potently affect cellular growth and proliferation. In addition, pluripotent stem cells have recently been identified in AF. Herein, this article reviews the biological properties of AF during embryonic development and speculates that AF may act as a transporting pathway for signaling molecules and stem cells during amniote embryonic development. Defining this novel function of AF is potentially significant for further understanding embryonic development and regenerative medicine, preventing genetic diseases, and developing therapeutic options for human malignancies.

  10. Phenotype and differentiation capacity of human amniotic epithelial cells cultured in vitro%体外培养人羊膜上皮细胞的表型及分化能力

    Institute of Scientific and Technical Information of China (English)

    连建春; 刘洋; 刘畅; 吕世杰; 郭昕; 南丰; 孙广炜; 贺欣; 马小军

    2014-01-01

    BACKGROUND:Human amniotic epithelial cells are an important source of cells in regenerative medicine as its multipotentation, but new studies mainly focused on differentiation features and there were little research oneffect of culture in vitro on biological property of amniotic epithelial cells. OBJECTIVE:To analyze the effects of in vitro culture on growth, cellphenotype and differentiation capacity of human amniotic epithelial cells into cardiomyocyte-like cells, and explore the correlation of primarily cultured human amniotic epithelial cells marker SSEA-4 expression level and the change of biological characteristics of human amniotic epithelial cells. METHODS:Primarily cultured human amniotic epithelial cells were obtained from amniotic tissues by using the same separation protocol. Human amniotic epithelial cells were cultured in vitro. The proliferation, cellphenotype and the differentiation capacity of human amniotic epithelial cells into cardiomyocyte-like cells were evaluated by means of cellcounting kit-8, flow cytometry and real-time PCR. RESULTS AND CONCLUSION:The SSEA-4 positive cells in primarily cultured human amniotic epithelial cells from different fetal tissues were between 26.7%-97%, which indicated that there was great individual difference among amniotic tissue samples. Moreover, with passage, the SSEA-4 expression in human amniotic epithelial cells decreased significantly, which did not correlate with the SSEA-4 expression in primarily cultured human amniotic epithelial cells. Results indicated that there was great individual difference in SSEA-4 expression level in primarily cultured human amniotic epithelial cells from different amniotic tissue samples. Thus, it is necessary to set up clinical screening indexes to get samples with higher SSEA-4 expression stably and to control the quality of human amniotic epithelial cells. In addition, during culture period, SSEA-4 expression level was affected by culture conditions. The culture

  11. Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

    Science.gov (United States)

    Lange, S; Viergutz, T; Simkó, M

    2004-10-01

    Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

  12. The targeted inhibitory effects of human amniotic fluid stem cells carrying CXCR4 promoter and DAL-1 on non-small cell lung carcinoma growth.

    Science.gov (United States)

    Li, L; Li, S; Cai, T; Wang, H; Xie, X; Liu, Z; Zhang, Y

    2016-02-01

    The differentially expressed in adenocarcinoma of the lung-1 (DAL-1) protein has been demonstrated to be suppressive to various types of tumors including lung cancer. This study aimed to determine the targeted effects of human amniotic fluid stem cells (hAFS cells) carrying CXCR4 promoter driven conditionally replicable adenovirus vector overexpressing DAL-1 (Ad-CXCR4-DAL-1) on non-small cell lung carcinoma (NSCLC) growth. The apoptotic effects of virus vectors were assessed using flow cytometry, and the cytotoxicity analyzed by CCK-8 assay. In vivo imaging system was used to determine the homing capability of hAFS cells. A549 cell xenograft mouse model was created to assess the in vivo effect of DAL-1 overexpression on NSCLC growth. We found that infection of Ad-CXCR4-DAL-1 increased the apoptosis of A549 NSCLC cells but not 16HBE normal human bronchial epithelial cells. Ad-CXCR4-DAL-1 administered via intratumoral injection led to significant reduced growth and greater necrosis of A549 xenograft tumors comparing to null vector treated animals. When infused via tail vein, hAFS cells carrying Ad-CXCR4-DAL-1 homed to lung cancer xenografts, caused virus replication and DAL-1 overexpression, and led to significant lower growth and greater necrosis of A549 cell xenografts comparing to non-treatment control. In conclusion, hAFS cells are capable of carrying Ad-CXCR4-DAL-1 vectors, specifically targeting to lung cancer, and causing oncolytic effects when administered in vivo.

  13. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta.

    Science.gov (United States)

    Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca

    2016-02-01

    Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications.

  14. Induction of E-cadherin+ human amniotic fluid cell differentiation into oocyte-like cells via culture in medium supplemented with follicular fluid.

    Science.gov (United States)

    Liu, Te; Huang, Yongyi; Bu, Yanzhen; Zhao, Yanhui; Zou, Gang; Liu, Zhixue

    2014-07-01

    Pluripotent human amniotic fluid cells (HuAFCs) can differentiate into various types of somatic cell in vitro. However, their differentiation into oocyte-like cells has never been described to the best of our knowledge. In the present study, differentiation of E-cadherin+ and E-cadherin- HuAFC sub-populations into oocyte-like cells was induced via culture in medium containing bovine follicular fluid and β-mercaptoethanol. The E-cadherin+ HuAFCs expressed DAZL highly. Post-induction, cells with an oocyte-like phenotype were found among the E-cadherin+ HuAFCs, expressing markers specific to germ cells and oocytes (VASA, ZP3 and GDF9) and meiosis (DMC1 and SCP3). When specific small interfering RNA (siRNA) was used to suppress E-cadherin in the E-cadherin+ HuAFCs, the levels of DAZL expression were reduced. Post-induction, the morphology of the siRNA‑E‑cadherin HuAFCs was poorer and the expression levels of germ cell-specific markers were lower compared with those of the siRNA-mock HuAFCs. Therefore, E-cadherin+ HuAFCs could be more easily induced to differentiate into oocyte-like cells by bovine follicular fluid and β-mercaptoethanol. In addition, the E-cadherin+ HuAFCs exhibited potential characteristics of DAZL protein expression, and thus it was conjectured that bovine follicular fluid acts on DAZL protein and promotes E-cadherin+ HuAFC differentiation into oocyte-like cells.

  15. Amniotic fluid-derived stem cells in regenerative medicine research.

    Science.gov (United States)

    Joo, Sunyoung; Ko, In Kap; Atala, Anthony; Yoo, James J; Lee, Sang Jin

    2012-02-01

    The stem cells isolated from amniotic fluid present an exciting possible contribution to the field of regenerative medicine and amniotic fluid-derived stem (AFS) cells have significant potential for research and therapeutic applications. AFS cells are multipotent, showing the ability to differentiate into cell types from all three embryonic germ layers. They express both embryonic and adult stem cell markers, expand extensively without feeder cells, double in 36 h, and are not tumorigenic. The AFS cells can be maintained for over 250 population doublings and preserve their telomere length and a normal karyotype. They differentiate easily into specific cell lineages and do not require human embryo tissue for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem (ES) cells. The discovery of the AFS cells has been recent, and a great deal of work remains to be performed on the characterization and use of these cells. This review describes the various differentiated lineages that AFS cells can form and the future of these promising new stem cells in regenerative medicine research.

  16. Comparative study of effects of magnesium and taurine on electrical parameters of natural and artificial membranes. VIII. Effect on the ultrastructure of human amniotic epithelial cells.

    Science.gov (United States)

    Guiet-Bara, A; Bara, M; Durlach, J

    1991-03-01

    The ultrastructure of human amniotic epithelial cells from normal pregnancies, at term, was studied using transmission electron microscopy. The results were analysed by a stereological method which indicates the ratio between the volume of the intercellular space (R1, the microvilli (R2), and the podocytes (R3) versus the cell volume. At low concentration (2 mM), MgCl2 decreased R1 and R3 and had no significant effect on R2. In contrast, taurine (2 mM) increased R1 and had no significant effect on R2 and R3. There is no vicarious action between Mg and taurine. These data are in contrast to the results obtained after electrophysiological studies, which indicates that the structural targets for Mg and taurine are different from the targets responsible for ionic transfer.

  17. Human beta-defensin 1, 2 and 3 production by amniotic epithelial cells with respect to human papillomavirus (HPV) infection, HPV oncogenic potential and the mode of delivery.

    Science.gov (United States)

    Szukiewicz, Dariusz; Alkhalayla, Habib; Pyzlak, Michal; Watroba, Mateusz; Szewczyk, Grzegorz; Wejman, Jaroslaw

    2016-08-01

    Human beta-defensins (HBD) produced by human amniotic epithelial cells (HAEC) co-create an innate antiviral immune response in the materno-placento-fetal unit. Oncogenic potential of HPV may reflect its ability to avoid immune recognition. In this study we assessed the risk of HAEC infection with human papillomavirus (HPV) in relation to the type of labor and the impact of the oncogenic potential of HPV on HBD production in HAEC. A comparative analysis [HPV(+) vs. HPV(-)HAEC] of the production of HBD were performed. HAEC were isolated from placentas of 116 HPV(+) and 36 HPV(-) parturients (groups I and II, respectively) using trypsin-based method. The cases of premature rupture of membranes (PROM), natural labors (NL) and cesarean sections (CS) were analysed in respective subgroups. High-risk (HR-HPV) and low-risk (LR-HPV) genotypes of HPV in cervical smears and HAEC were identified using the Roche Linear Array(®) HPV Genotyping Test. HBD-1,-2,-3 concentrations in the HAEC culture supernatant were assessed using ELISA. The highest percentage (42.1%) of HPV transmission to HAEC occurred in PROM, an intermediate value was observed after NL (38.5%), and the lowest (25.6%) after CS. The mean concentrations of HBD-2 and HBD-3 in group I were up to 3.1- and 2.8-fold higher (p infection compared with HR-HPV. The course of labor and the mode of delivery influence the risk of HPV transmission to the HAEC. HPV infection upregulates HBD-2 and HBD-3 production in HAEC. Smaller increases in HBD-2 level after HR-HPV infection as compared to LR-HPV may affect cancerogenesis. Therapeutic potential of HBD-2 for HR-HPV infection should be assessed in future studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    CHENJian-su; CHENRui; XUJin-tang; DINGYong; ZHAOSong-bin; LISui-lian

    2004-01-01

    Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.

  19. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    Science.gov (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  20. Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway.

    Science.gov (United States)

    Liu, Junfeng; Hua, Rong; Gong, Zhangbin; Shang, Bin; Huang, Yongyi; Guo, Lihe; Liu, Te; Xue, Jun

    2017-01-01

    In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. -Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3' UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the 'c-Rel-NFκB' complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Stem Cells in Amniotic Fluid - What are the Next Steps to Do?

    Directory of Open Access Journals (Sweden)

    Hengstschläger M

    2005-01-01

    Full Text Available It is the hope of patients and investigators that in future the characterisation and isolation of human stem cells will allow the establishment of new therapeutic concepts for a wide variety of diseases. Recently, we found a new source for stem cells. Human amniotic fluid contains cells, which express Oct-4, a marker for pluripotent stem cells. In addition, we described amniotic fluid cells expressing markers for neuronal stem cells. The latter harbour the potential to differentiate into neurogenic cells. This opened a new field in stem cell research. In this review I want to summarise the current knowledge about amniotic fluid cells focusing on the open questions, which need to be investigated in future.

  2. Amniotic Fluid Cells Proliferation in Normal and Down Syndrome Subjects

    Directory of Open Access Journals (Sweden)

    Honcea Adina

    2016-02-01

    Full Text Available Down Syndrome/Trisomy 21 is the most common chromosomal anomaly, and it represents the most common congenital cause of infants’ intellectual disability. Subjects with this syndrome are affected by degenerative processes caused by accelerated aging or unknown ethyologies. In recent years, accumulating evidence revealed increased potential of amniotic fluid-derived stem cells to be used in regenerative therapy. Our aim was to assess differences in immunophenotype, cell morphology and proliferation of amniotic fluid cells from normal and Down Syndrome pregnancies using a quantitative cytometry approach. Results revealed the emergence of a population of small sized cells in Down Syndrome derived amniotic fluid cells that are readily visible upon microscopic inspection. Hence, the fluorescence–based quantitative image cytometry determinations showed a tendency of decrease in both cell and nuclei size in trisomy, with no significant modification in nuclei circularity, as measured following actin cytoskeleton and nuclei labeling. The propensity of Ki67 positive cells was found to be increased in Down Syndrome derived cells (48.92% as compared to normal specimens (28.68%. However, cells in S and G2/M cell cycle phases decreased from 32.91% to 4.49% in diseased cells. Further studies are devoted to understanding the molecular basis of the observed differences in the proliferation ability of Down Syndrome amniotic cells, in order to evaluate the potential therapeutic effect of amniotic fluid stem cells for tissue regeneration in subjects with trisomy and to find correlations between amniotic cells phenotype and patient prognosis.

  3. Comparison of human amniotic fluid-derived and umbilical cord Wharton's Jelly-derived mesenchymal stromal cells: Characterization and myocardial differentiation capacity

    Institute of Scientific and Technical Information of China (English)

    Jing Bai; Yuan Hu; Yi-Ru Wang; Li-Feng Liu; Jie Chen; Shao-Ping Su; Yu Wang

    2012-01-01

    Objective To compare the characterization and myocardial differentiation capacity of amniotic fluid-derived mesenchymal stromal cells (AF MSCs) and umbilical cord Wharton's Jelly-derived mesenchymal stromal cells (WJ MSCs). Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton's Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes (GATA-4, c-TnT, α-actin, and Cx43) were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities of WJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility (MHC I) antigens (HLA-ABC), and were negative, or mildly positive, for MHC Class II (HLA-DR) antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, α-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.

  4. Ultrastructural study of the neovagina following the utilization of human amniotic membrane for treatment of congenital absence of the vagina

    Directory of Open Access Journals (Sweden)

    L.F. Bleggi-Torres

    1997-07-01

    Full Text Available We present an ultrastructural study of the utilization of human amniotic membrane in the treatment of congenital absence of the vagina in 10 patients. All patients were surgically treated with application of an amniotic membrane graft using the modified McIndoe and Bannister technique. Sixty days after surgery, samples of the vaginal neo-epithelium were collected for transmission electron microscopy analysis. The ultrastructural findings consisted of a lining of mature squamous epithelium indicating the occurrence of metaplasia of the amniotic epithelium into the vaginal epithelium. The cells were arranged in layers as in the normal vaginal epithelium, i.e., superficial, intermediate and deep layers. There were desmosomes and cytoplasmic intermediate cytokeratin filaments, as well as some remnant features of the previous amniotic epithelium. These findings suggest that human amniotic membrane is able to complete metaplasia into squamous cells but the mechanism of this cellular transformation is unknown

  5. Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways.

    Science.gov (United States)

    Zhao, Bin; Liu, Jia-Qi; Zheng, Zhao; Zhang, Jun; Wang, Shu-Yue; Han, Shi-Chao; Zhou, Qin; Guan, Hao; Li, Chao; Su, Lin-Lin; Hu, Da-Hai

    2016-07-01

    Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing.

  6. HUMAN AMNIOTIC MEMBRANE GRAFTING: A BOON IN OCULAR CHEMICAL INJURIES

    Directory of Open Access Journals (Sweden)

    Shashikala

    2013-03-01

    Full Text Available INTRODUCTION: Amniotic membrane is the innermost layer of the f etal membranes. It has a stromal matrix, a collagen layer, and an overlying basement membrane with a single layer of epithelium. (1 Amniotic membrane has unique properties including an ti-adhesive effects, bacterio-static properties, wound protection, pain redu ction, and epithelialisation effects. Another characteristic of amniotic membrane is the lack of imunogenicity. (2 Amniotic membranes have been used as a dressing to promote he aling of chronic ulcers of the leg and as a biological dressing for burned skin and skin woun ds. (3, 4 It has also been used in surgical reconstruction of artificial vagina, for repairing o mphaloceles, and to prevent tissue adhesion in surgeries of the abdomen, head, or pelvis. (5, 6 Amniotic membrane has been successfully used in ocular conditions like persistent epithelial defects (7, pterygium, (8 Symblepharon (9 and for ocular surface reconstruction. (10, 11 The purpose of this study was to evaluate the use of cryo- preserved Human amniotic membrane graft( HAMT, with or without limbal autograft transplantation (LAT in patients with previous and fr esh chemical eye injuries respectively. Institutional ethical committee approval was obtained .

  7. The effects of acellular amniotic membrane matrix on osteogenic differentiation and ERK1/2 signaling in human dental apical papilla cells.

    Science.gov (United States)

    Chen, Yi-Jane; Chung, Min-Chun; Jane Yao, Chung-Chen; Huang, Chien-Hsun; Chang, Hao-Hueng; Jeng, Jiiang-Huei; Young, Tai-Horng

    2012-01-01

    The amniotic membrane (AM) has been widely used in the field of tissue engineering because of the favorable biological properties for scaffolding material. However, little is known about the effects of an acellular AM matrix on the osteogenic differentiation of mesenchymal stem cells. In this study, it was found that both basement membrane side and collagenous stroma side of the acellular AM matrix were capable of providing a preferential environment for driving the osteogenic differentiation of human dental apical papilla cells (APCs) with proven stem cell characteristics. Acellular AM matrix potentiated the induction effect of osteogenic supplements (OS) such as ascorbic acid, β-glycerophosphate, and dexamethasone and enhanced the osteogenic differentiation of APCs, as seen by increased core-binding factor alpha 1 (Cbfa-1) phosphorylation, alkaline phosphatase activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Even in the absence of soluble OS, acellular AM matrix also could exert the substrate-induced effect on initiating APCs' differentiation. Especially, the collagenous stroma side was more effective than the basement membrane side. Moreover, the AM-induced effect was significantly inhibited by U0126, an inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. Taken together, the osteogenic differentiation promoting effect on APCs is AM-specific, which provides potential applications of acellular AM matrix in bone/tooth tissue engineering.

  8. Stem cells from amniotic fluid--Potential for regenerative medicine.

    Science.gov (United States)

    Loukogeorgakis, Stavros P; De Coppi, Paolo

    2016-02-01

    Regenerative medicine has recently been established as an emerging field focussing on repair, replacement or regeneration of cells, tissues and whole organs. The significant recent advances in the field have intensified the search for novel sources of stem cells with potential for therapy. Recently, researchers have identified the amniotic fluid as an untapped source of stem cells that are multipotent, possess immunomodulatory properties and do not have the ethical and legal limitations of embryonic stem cells. Stem cells from the amniotic fluid have been shown to differentiate into cell lineages representing all three embryonic germ layers without generating tumours, which make them an ideal candidate for tissue engineering applications. In addition, their ability to engraft in injured organs and modulate immune and repair responses of host tissues suggest that transplantation of such cells may be useful for the treatment of various degenerative and inflammatory diseases affecting major tissues/organs. This review summarises the evidence on amniotic fluid cells over the past 15 years and explores the potential therapeutic applications of amniotic fluid stem cells and amniotic fluid mesenchymal stem cells.

  9. Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

    Directory of Open Access Journals (Sweden)

    Salini Vincenzo

    2009-02-01

    Full Text Available Abstract Background Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells. Results The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2 within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium. Electron microscopy analysis evidenced the best cell growth on this latter surface. Conclusion The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

  10. Hair Follicle Morphogenesis in the Treatment of Mouse Full-Thickness Skin Defects Using Composite Human Acellular Amniotic Membrane and Adipose Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Wu Minjuan

    2016-01-01

    Full Text Available Early repair of skin injury and maximal restoration of the function and appearance have become important targets of clinical treatment. In the present study, we observed the healing process of skin defects in nude mice and structural characteristics of the new skin after transplantation of isolated and cultured adipose derived mesenchymal stem cells (ADMSCs onto the human acellular amniotic membrane (AAM. The result showed that ADMSCs were closely attached to the surface of AAM and grew well 24 h after seeding. Comparison of the wound healing rate at days 7, 14, and 28 after transplantation showed that ADMSCs seeded on AAM facilitated the healing of full-thickness skin wounds more effectively as compared with either hAM or AAM alone, indicating that ADMSCs participated in skin regeneration. More importantly, we noticed a phenomenon of hair follicle development during the process of skin repair. Composite ADMSCs and AAM not only promoted the healing of the mouse full-thickness defects but also facilitated generation of the appendages of the affected skin, thus promoting restoration of the skin function. Our results provide a new possible therapy idea for the treatment of skin wounds with respect to both anatomical regeneration and functional restoration.

  11. Hair Follicle Morphogenesis in the Treatment of Mouse Full-Thickness Skin Defects Using Composite Human Acellular Amniotic Membrane and Adipose Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Minjuan, Wu; Jun, Xiong; Shiyun, Shao; Sha, Xu; Haitao, Ni

    2016-01-01

    Early repair of skin injury and maximal restoration of the function and appearance have become important targets of clinical treatment. In the present study, we observed the healing process of skin defects in nude mice and structural characteristics of the new skin after transplantation of isolated and cultured adipose derived mesenchymal stem cells (ADMSCs) onto the human acellular amniotic membrane (AAM). The result showed that ADMSCs were closely attached to the surface of AAM and grew well 24 h after seeding. Comparison of the wound healing rate at days 7, 14, and 28 after transplantation showed that ADMSCs seeded on AAM facilitated the healing of full-thickness skin wounds more effectively as compared with either hAM or AAM alone, indicating that ADMSCs participated in skin regeneration. More importantly, we noticed a phenomenon of hair follicle development during the process of skin repair. Composite ADMSCs and AAM not only promoted the healing of the mouse full-thickness defects but also facilitated generation of the appendages of the affected skin, thus promoting restoration of the skin function. Our results provide a new possible therapy idea for the treatment of skin wounds with respect to both anatomical regeneration and functional restoration. PMID:27597871

  12. Dielectric properties of human placenta, umbilical cord and amniotic fluid

    Energy Technology Data Exchange (ETDEWEB)

    Peyman, A [Physical Dosimetry Department, Health Protection Agency, Chilton, Didcot OX11 0RQ (United Kingdom); Gabriel, C [MCL-P, Newbury RG14 5PY, Berkshire (United Kingdom); Benedickter, H R; Froehlich, J, E-mail: Azadeh.peyman@hpa.org.uk [Electromagnetic Fields and Microwave Electronics Laboratory, Swiss Federal Institute of Technology, Zurich (Switzerland)

    2011-04-07

    The dielectric properties of freshly delivered human placenta, umbilical cord and amniotic fluid have been acquired at 37 deg. C and in the frequency range of 200 MHz-10 GHz. The experimental data were fitted to a Cole-Cole expression. The results show that dielectric properties of the umbilical cord are significantly higher than placenta due to the presence of high water content Wharton's jelly. The results also demonstrate large differences in the dielectric properties of amniotic and cerebrospinal fluids. The data presented can be used in numerical simulations of the exposure of pregnant women to electromagnetic fields. (note)

  13. Dielectric properties of human placenta, umbilical cord and amniotic fluid

    Science.gov (United States)

    Peyman, A.; Gabriel, C.; Benedickter, H. R.; Fröhlich, J.

    2011-04-01

    The dielectric properties of freshly delivered human placenta, umbilical cord and amniotic fluid have been acquired at 37 °C and in the frequency range of 200 MHz-10 GHz. The experimental data were fitted to a Cole-Cole expression. The results show that dielectric properties of the umbilical cord are significantly higher than placenta due to the presence of high water content Wharton's jelly. The results also demonstrate large differences in the dielectric properties of amniotic and cerebrospinal fluids. The data presented can be used in numerical simulations of the exposure of pregnant women to electromagnetic fields.

  14. In vivo differentiation of human amniotic epithelial cells into cardiomyocyte-like cells and cell transplantation effect on myocardial infarction in rats: comparison with cord blood and adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Fang, Cheng-Hu; Jin, Jiyong; Joe, Jun-Ho; Song, Yi-Sun; So, Byung-Im; Lim, Sang Moo; Cheon, Gi Jeong; Woo, Sang-Keun; Ra, Jeong-Chan; Lee, Young-Yiul; Kim, Kyung-Soo

    2012-01-01

    Human amniotic epithelial cells (h-AECs), which have various merits as a cell source for cell therapy, are known to differentiate into cardiomyocytes in vitro. However, the ability of h-AECs to differentiate into cardiomyocytes in vivo and their cell transplantation effects on myocardial infarction are still unknown. In this study, we assessed whether h-AECs could differentiate into cardiomyocytes in vivo and whether h-AECs transplantation can decrease infarct size and improve cardiac function, in comparison to transplantation of cord blood-derived mesenchymal stem cells (MSCs) or adipose tissue-derived MSCs. For our study, we injected h-AECs, cord blood-derived MSCs, adipose tissue-derived MSCs, and saline into areas of myocardial infarction in athymic nude rats. After 4 weeks, 3% of the surviving h-AECs expressed myosin heavy chain, a marker specific to the myocardium. Compared with the saline group, all cell-implanted groups showed a higher ejection fraction, lower infarct area by positron emission tomography and histology, and more abundant myocardial gene and protein expression in the infarct area. We showed that h-AECs can differentiate into cardiomyocyte-like cells, decrease infarct size, and improve cardiac function in vivo. The beneficial effects of h-AECs were comparable to those of cord blood and adipose tissue-derived MSCs. These results support the need for further studies of h-AECs as a cell source for myocardial regeneration due to their plentiful availability, low immunity, and lack of ethical issues related to their use.

  15. Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

    Science.gov (United States)

    Sipahi, Mesut; Şahin, Sevinç; Arslan, Ergin; Börekci, Hasan; Metin, Bayram; Cantürk, Nuh Zafer

    2015-01-01

    Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations. PMID:26457000

  16. Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

    Directory of Open Access Journals (Sweden)

    Mesut Sipahi

    2015-01-01

    Full Text Available Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations.

  17. Human amniotic epithelial cells culture in vitro and the research of Stem cell characteristics.%羊膜上皮细胞的体外培养及干细胞特性研究

    Institute of Scientific and Technical Information of China (English)

    耿娟娟; 崔勇; 邱书奇

    2012-01-01

    Objective To establish in vitro culture procedure of human amniotic epithelial cells (hAECS) and to explore the biological characteristics. Methods Human placenta were deliveried from third-trimester healthy mothers. The amnion layer was mechanically peeled off of the chorion, washed several times and splitted in 1 mm x 1 mm. The amnion membrane was incubated with 0.05% trypsin containing 0.53 mM EDTA4Na and then cultured. The hAECS were obtained from the amniotic membrane by the digestion of trypsin. The stem cell markers (OCT-4^ Nanog、SSEA-4) were examined by the Immunofluorescence, Western. Result The amniotic epithelial cells express 0CT-4、Nanog^SSEA-4, which is the marker of the stem cells according to immunofluorescence staining and Westernblot results. Conclusion the hAECS express the marker of embryonic stem cell, which means it can be used as a new source of stem cells.%目的:体外分离培养人单膜上皮细胞(hAECS)并探讨其生物学特性.方法:取足月产胎儿胎盘,无菌条件下将胎盘的羊膜层与绒毛膜层分离.将洗净的羊膜组织用眼科剪剪成约1 mm×1 mm大小,加入0.05%的含有0.53 mM EDTA4Na的胰酶制备羊膜上皮细胞并进行培养.用免疫荧光、Westem blot等方法检测干细胞相关表面标记物(0CT-4、Nanog、SSEA-4)等的表达情况.结果:根据免疫荧光组织化学,Westernblot的结果显示,羊膜上皮细胞OCT-4、Nanog、ssea-4、Nestin表达阳性.结论:羊膜上皮细胞表达类似胚胎干细胞的表面标志物,可以作为干细胞的新来源.

  18. Intracerebroventricular transplantation of human amniotic epithelial cells ameliorates spatial memory deficit in the doubly transgenic mice coexpressing APPswe and PS1ΔE9-deleted genes

    Institute of Scientific and Technical Information of China (English)

    XUE Shou-ru; CHEN Chong-fang; DONG Wan-li; HUI Guo-zhen; LIU Tian-jun; GUO Li-he

    2011-01-01

    Background Human amniotic epithelial cells (HAECs),which have characteristics of both embryonic and pluripotent stem cells,are therefore a candidate in cell therapy without creating legal or ethical problems.In the present study,we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer's disease (AD) coexpressing presenilin-1 (PS1) and mutant Sweden amyloid precursor protein (APPswe)genes.Methods The offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene.The doubly transgenic (TG) mice (n=20) and wild-type (WT) mice (n=20) were randomly divided into two groups respectively:the transplantation group treated with HAECs and the control group with phosphate buffered saline.Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice.Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively.Immunofluorescence cytochemistry was used to track the survival of HAECs.Immunohistochemistry was used to determine the expression of octamer-binding protein 4 (Oct-4) and Nanog in the HAECs.High performance liquid chromatography was used to measure acetylcholine in hippocampus.The density of cholinergic neurons in basal forebrain and nerve fibers in hippocampus was measured using acetylcholinesterase staining.Results Amyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months,but not in WT mice.The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection.The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell.Compared with the control group,transplantation of HAECs can not only significantly improve the spatial memory of the TG mice,but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites.Conclusions These

  19. Reprogramming of mouse amniotic fluid cells using a PiggyBac transposon system

    Directory of Open Access Journals (Sweden)

    E. Bertin

    2015-11-01

    Full Text Available Induced pluripotent stem (iPS cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Amniotic fluid (AF cells are easy to obtain from routinely scheduled procedures for prenatal diagnosis and iPS cells have been generated from human AF. Here, we generated iPS cells from mouse AF cells, using a non-viral-based approach constituted by the PiggyBac (PB transposon system. All iPS cell lines obtained exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo.

  20. 应用羊膜上皮干细胞微环境培养人角膜内皮细胞的研究%Microenvironment of amniotic epithelium cells enhances the proliferation of human corneal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    王忠浩; 陈玮; 宋莉; 沙翔垠; 梁轩伟

    2013-01-01

    Objective To establish an effective method to enhance the proliferation of human corneal endothelial cells (HCECs). Methods The culture conditions of HCEC were optimized by utilizing the totipotent characteristics of human amniotic membrane epithelial stem cells (HAEC) to establish the optimal culture microenvironment of HAEC to promote the proliferation of HCEC. The morphology of HCEC was observed by using phase-contrast microscope and transmission electron microscope. MTT assay and Giemsa staining were performed to detect the proliferation of HCEC. The rate of apoptotic cells was investigated by using Hoechst33342 staining assay. Results Compared to the corneal endothelial cells medium (CEM), the microenvironment containing 20% HAEC-conditioned medium and HAEC-HCEC co-culture microenvironment could promote the proliferation of HCEC and could reduce the apoptosis of HCEC. The cells in HAEC-HCEC microenvironment group could be passaged 4 times without lossing their polygonal appearance. Conclusion The HAEC microenvironment could effectively enhance the proliferation of HCEC, maintain the morphology of HCEC, and inhibit the process of apoptosis of HCEC.%目的:建立一种利用羊膜上皮干细胞(human amniotic membrane epithelial cell,HAEC)微环境培养人角膜内皮细胞(human corneal endothelial cells,HCEC)的方法.方法:制备羊膜上皮干细胞微环境培养HCEC,并探讨诱导HCEC增殖的最佳培养微环境,倒置相差显微镜和透射电镜观察培养过程中细胞的形态学变化,MTT和Giemsa染色观察细胞增殖情况,Hoechst33342检测凋亡细胞比例.结果:在HCEC基本培养液(corneal endothelial cell medium,CEM)的基础上添加20% HAEC上清、HAEC-HCEC的微环境可促进HCEC的增殖,减少凋亡,细胞传代能力显著增强,HAEC-HCEC组传至4代仍保持多角形的内皮细胞形态.结论:羊膜上皮干细胞微环境培养可有效提高HCEE的增殖能力,更好地维持HCEC的形态,并能抑制其凋亡进程.

  1. Study on isolation and culture of human amniotic mesenchymal cells and its stem cells characteristics%人羊膜间充质细胞分离培养及其干细胞特性研究

    Institute of Scientific and Technical Information of China (English)

    李海建; 张广静; 江兰; 余春艳; 金岩

    2011-01-01

    Objective: To investigate the isolation, culture and stem cells characteristics of human amniotic mesenchymal cells (HAMCs)for its potential application in regenerative medicine. Methods:Term placentas fiom healthy donor mothers obtained from caesarean sections were minced under sterile conditions. After sequential trypsin and coilagenase digestion, cultivated in DMEM/F12, the morphololgy of HAMCs was observed under the microscope. Growth regularity was observed by MTT assay,the cell was identified by immunofiuorescence, and it has multi-potential ability under specific conditions. Results: Mesenchymal cells derived from human amnion membrane, SSEA-4 and OCT-4 positive by immunofluorescence, It displays a strong proliferation and mulfipotential capacity .The cells can differentiate into fat cells and osteoblasts. Conclusions: HAMCs can isolation, culture and amplification in vitro, and these cells have stem cell properties. HAMCs has good prospects applications in regenerative medicine and tissue engineering.%目的:研究人羊膜间充质细胞(Human amniotic mesenchymal cells,HAMCs)的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础.方法:无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能.结果:来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论:羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性.羊膜间充质细胞在再生医学和组织工程应用有很好的前景.

  2. 人羊水干细胞分离方法及其生物学特性研究%Isolation and Biological Characterization of Human Amniotic Fluid-derived Stem Cells

    Institute of Scientific and Technical Information of China (English)

    关婷; 谢晓砚; 刘珊玲; 陈新莲; 魏杨君; 赖怡; 谢良玉; 刘之英; 张雪梅; 刘洪倩; 张建军

    2012-01-01

    Objective To establish in vitro culture procedure for human amniotic fluid-derived CD117 positive stem cells, and to identify the characteristics of CD117 positive stem cells. Methods 86 amniotic fluid samples (10 mL of each) were obtained by second-trimester amniocentesis. Isolation of amniotic fluid-derived stem cells expressing CD117 antigen was performed via magnetic cell sorting using the CD117 MicroBead Kit. The karyotype of CD117 positive stem cells was analysed throughrepeated freezing. Adipogenic differentiation of these CD117 positive stem cells was displayed by Oil Red O staining. Osteogeneic differentiation of these CD117 positive stem cells was confirmed by Alizarin Red staining. Results The CD117 positive stem cells were successfully isolated and cultured from 61 samples, with all showing normal karyotype. Product analysis of specific staining confirmed that under specific culture mediums, these cells could be successfully induced to differentiate into adipocytes and osteocytes. Conclusion Based on this study, we estimate that isolating CD117 positive stem cells from second-trimester amniotic fluid obtained by amniocentesis has a success rate of 70. 93%. These cells maintain morphological and genetic stability in vitro. Human amniotic fluid-derived CD117 positive stem cells have the ability to differentiate in vitro into adipocytes and osteocytes under specific cuLture mediums and may be applied in cell transplantation and regenerative medicine.%目的 建立体外培养人羊水来源CD117阳性干细胞的方法,初步探讨CD117阳性干细胞的特性.方法 通过孕中期羊膜腔穿刺获得86例羊水标本.采用CD117磁珠分选表达CD117抗原的羊水干细胞.对经过反复冻存的CD117阳性干细胞进行核型分析.分别经成脂诱导和成骨诱导分化,再分别使用油红O染色、茜素红染色.结果 从61例标本中成功分离培养出CD117阳性细胞,经核型分析显示其染色体核型正常.CD117阳性细胞经

  3. Corneal haze induced by excimer laser photoablation in rabbits is reduced by preserved human amniotic membrane graft

    Science.gov (United States)

    Wang, Ming X.; Gray, Trevor; Prabhasawat, Pinnita; Ma, Xiong; Culbertson, William; Forster, Richard; Hanna, Khalil; Tseng, Scheffer C. G.

    1998-06-01

    We conducted a study to determine if preserved human amniotic membrane can reduce corneal haze induced by excimer laser photoablation. Excimer photoablation was performed bilaterally on 40 New Zealand white rabbits with a 6 mm ablation zone and 120 micrometer depth (PTK) using the VISX Star. One eye was randomly covered with a preserved human amniotic membrane and secured using four interrupted 10 - 0 nylon sutures; the other eye served as control. The amniotic membranes were removed at one week, and the corneal haze was graded with a slit-lamp biomicroscopy by three masked corneal specialists (WC, KH and RF) biweekly for the ensuing 12 weeks. Histology and in situ TUNEL staining (for fragmented DNA as an index for apoptosis) was performed at days 1, 3 and 7 and at 12 weeks. One week after excimer photoablation, the amniotic membrane-covered corneas showed more anterior stromal edema, which resolved at the second week. A consistent grading of organized reticular corneal haze was noted among the three masked observers. Such corneal haze peaked at the seventh week in both groups. The amniotic membrane-covered group showed statistically significant less corneal haze (0.50 plus or minus 0.15) than the control groups (1.25 plus or minus 0.35) (p less than 0.001). The amniotic membrane-covered corneas had less inflammatory response at days 1 and 3, showing nearly nil DNA fragmentation on keratocytes on the ablated anterior stromal and less stromal fibroblast activation. There is less altered epithelial cell morphology and less epithelial hyperplasia at 1 week in these amniotic membrane-treated eyes. We concluded from this study that amniotic membrane matrix is effective in reducing corneal haze induced by excimer photoablation in rabbits and may have clinical applications.

  4. Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

    Science.gov (United States)

    Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio

    2014-01-01

    Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

  5. An update clinical application of amniotic fluid-derived stem cells (AFSCs) in cancer cell therapy and tissue engineering.

    Science.gov (United States)

    Gholizadeh-Ghaleh Aziz, Shiva; Fathi, Ezzatollah; Rahmati-Yamchi, Mohammad; Akbarzadeh, Abolfazl; Fardyazar, Zahra; Pashaiasl, Maryam

    2017-06-01

    Recent studies have elucidated that cell-based therapies are promising for cancer treatments. The human amniotic fluid stem (AFS) cells are advantageous cells for such therapeutic schemes that can be innately changed to express therapeutic proteins. HAFSCs display a natural tropism to cancer cells in vivo. They can be useful in cancer cells targeting. Moreover, they are easily available from surplus diagnostic samples during pregnancy and less ethical and legal concern are associated with the collection and application than other putative cells are subjected. This review will designate representatives of amniotic fluid and stem cell derived from amniotic fluid. For this propose, we collect state of human AFS cells data applicable in cancer therapy by dividing this approach into two main classes (nonengineered and engineered based approaches). Our study shows the advantage of AFS cells over other putative cells types in terms differentiation ability to a wide range of cells by potential and effective use in preclinical studies for a variety of diseases. This study has shown the elasticity of human AFS cells and their favorable potential as a multipotent cell source for regenerative stem cell therapy and capable of giving rise to multiple lineages including such as osteoblasts and adipocyte.

  6. Partial or complete coverage of experimental spina bifida by simple intra-amniotic injection of concentrated amniotic mesenchymal stem cells.

    Science.gov (United States)

    Dionigi, Beatrice; Ahmed, Azra; Brazzo, Joseph; Connors, John Patrick; Zurakowski, David; Fauza, Dario O

    2015-01-01

    We sought to determine whether simple intra-amniotic delivery of concentrated amniotic mesenchymal stem cells (afMSCs) may elicit prenatal coverage of experimental spina bifida. Time-dated pregnant Sprague-Dawley dams (n=24) exposed to retinoic acid for the induction of fetal neural tube defects were divided in three groups. Group I had no further manipulations. Groups II and III received volume-matched intra-amniotic injections of either saline (Group II) or a suspension of syngeneic afMSCs labeled with green fluorescent protein (Group III) in all fetuses (n=202) on gestational day 17 (term=21-22 days). Animals were killed before term. Statistical comparisons were by ANOVA (Pspina bifida was present in 58% (96/165), with no significant differences in defect dimension across the groups (P=0.19). However, variable degrees of coverage of the defect by a rudimentary skin confirmed histologically were only present in Group III (Pspina bifida after concentrated intra-amniotic injection. Trans-amniotic stem cell therapy (TRASCET) may become a practical option in the prenatal management of spina bifida. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Human amniotic membrane as an intestinal patch for neomucosal growth in the rabbit model.

    Science.gov (United States)

    Barlas, M; Gökçora, H; Erekul, S; Dindar, H; Yücesan, S

    1992-05-01

    This experiment was carried out as a preliminary study, an attempt to grow new intestinal mucosa on human amniotic membrane in the terminal ileum in 37 rabbits. After ketamin sulfate anesthesia at laparatomy, 5-cm ileal defects were patched with human amniotic membrane (5 x 2 cm). These patched intestines were investigated on the first postoperative day and the 2nd, 5th, 10th, and 20th weeks corresponding to 4, 5, 5, 10, and 10 rabbits, respectively. Only three rabbits died in the early postoperative period. There was no evidence of intestinal obstruction or dilatation with barium meal. Microscopically, the neomucosa consisted of a thin layer of columnar epithelial cells at 2 weeks with more maturity of the villi and less irregularity and branching by 20 weeks. All patches were covered with neomucosa commencing at 2 weeks and covering the whole patch area by 20 weeks. This technique's advantages are the large size and the ease of the availability of the human amniotic membrane for neonates at risk without jeopardizing the neonates tissues. It is hoped that this method might be considered when neonatal material is scarce.

  8. A novel method to derive amniotic fluid stem cells for therapeutic purposes

    Directory of Open Access Journals (Sweden)

    Vantanasiri Chanchai

    2010-10-01

    Full Text Available Abstract Background Human amniotic fluid stem (hAFS cells have become an attractive stem cell source for medical therapy due to both their ability to propagate as stem cells and the lack of ethical debate that comes with the use of embryonic stem cells. Although techniques to derive stem cells from amniotic fluid are available, the techniques have limitations for clinical uses, including a requirement of long periods of time for stem cell production, population heterogeneity and xeno-contamination from using animal antibody-coated magnetic beads. Herein we describe a novel isolation method that fits for hAFS derivation for cell-based therapy. Methods and Results With our method, single hAFS cells generate colonies in a primary culture of amniotic fluid cells. Individual hAFS colonies are then expanded by subculturing in order to make a clonal hAFS cell line. This method allows derivation of a substantial amount of a pure stem cell population within a short period of time. Indeed, 108 cells from a clonal hAFS line can be derived in two weeks using our method, while previous techniques require two months. The resultant hAFS cells show a 2-5 times greater proliferative ability than with previous techniques and a population doubling time of 0.8 days. The hAFS cells exhibit typical hAFS cell characteristics including the ability to differentiate into adipogenic-, osteogenic- and neurogenic lineages, expression of specific stem cell markers including Oct4, SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, and maintenance of a normal karyotype over long culture periods. Conclusions We have created a novel hAFS cell derivation method that can produce a vast amount of high quality stem cells within a short period of time. Our technique makes possibility for providing autogenic fetal stem cells and allogeneic cells for future cell-based therapy.

  9. Quantitative mapping of intracellular cations in the human amniotic membrane

    Science.gov (United States)

    Moretto, Ph.; Llabador, Y.; Simonoff, M.; Razafindrabe, L.; Bara, M.; Guiet-Bara, A.

    1993-05-01

    The effect of magnesium and taurine on the permeability of cell membranes to monovalent cations has been investigated using the Bordeaux nuclear microprobe. PIXE and RBS techniques have been used to provide quantitative measurements and ion distributions in the isolated amniotic membrane. This physiological model for cellular exchanges allowed us to reveal the distribution of most elements involved in cellular pathways and the modifications under different experimental conditions of incubation in physiological fluids. The PIXE microanalysis provided an original viewpoint on these mechanisms. Following this first study, the amnion compact lamina was found to play a role which was not, up to now, taken into account in the interpretation of electrophysiological experimentations. The release of some ionic species, such as K +, from the epithelial cells, during immersion in isotonic fluids, could have been hitherto underestimated.

  10. Immunosuppressive activity of human amniotic fluid of normal and abnormal pregnancies.

    Science.gov (United States)

    Shohat, B; Faktor, J M

    1988-01-01

    Twenty specimens of amniotic fluid (AF) obtained between week 16 and 18 of gestation from normal pregnant women and six specimens from pregnant women in which trisomia of chromosome 21 was found were tested for immunosuppressive activity. Incubation of normal human donor lymphocytes with 0.2-1 mL of AF from normal pregnant women for one hour at 37 degrees C was sufficient for induction of significant inhibition of the ability of these cells to induce a local xenogeneic graft-versus-host reaction (GVHR) as well as inhibition of E and E-active rosette formation, the GVHR being the most sensitive test. On the other hand, amniotic fluid obtained from the six pregnant women in which trisomia of chromosome 21 was found showed no inhibitory activity in either the E or E-active rosette formation, nor in the local xenogeneic graft-versus-host reaction. AF from all the women tested was found to have no effect on phenotype expression of the lymphocytes, as tested by the monoclonal antibodies OKT4+ and OKT8+, nor on B-lymphocytes, as tested by surface immunoglobulins. No correlation was found between the alpha-fetoprotein levels in the sera of those women and the immunosuppressive activity. These findings indicate that genetic defects of the conceptus are not limited to the embryo but may affect the composition of immunosuppressive components present in normal amniotic fluid.

  11. Autologous nerve anastomosis versus human amniotic membrane anastomosis A rheological comparison following simulated sciatic nerve injury

    Institute of Scientific and Technical Information of China (English)

    Guangyao Liu; Qiao Zhang; Yan Jin; Zhongli Gao

    2011-01-01

    The sciatic nerve is biological viscoelastic solid, with stress relaxation and creep characteristics. In this study, a comparative analysis of the stress relaxation and creep characteristics of the sciatic nerve was conducted after simulating sciatic nerve injury and anastomosing with autologous nerve or human amniotic membrane. The results demonstrate that, at the 7 200-second time point, both stress reduction and strain increase in the human amniotic membrane anastomosis group were significantly greater than in the autologous nerve anastomosis group. Our findings indicate that human amniotic membrane anastomosis for sciatic nerve injury has excellent rheological characteristics and is conducive to regeneration of the injured nerve.

  12. Amniotic fluid derived stem cells give rise to neuron-like cells without a further differentiation potential into retina-like cells.

    Science.gov (United States)

    Hartmann, K; Raabe, O; Wenisch, S; Arnhold, S

    2013-01-01

    Amniotic fluid contains heterogeneous cell types and has become an interesting source for obtaining fetal stem cells. These stem cells have a high proliferative capacity and a good differentiation potential and may thus be suitable for regenerative medicine. As there is increasing evidence, that these stem cells are also able to be directed into the neural lineage, in our study we investigated the neuronal and glial differentiation potential of these cells, so that they may also be applied to cure degenerative diseases of the retina. Mesenchymal stem cells were isolated from routine prenatal amniocentesis at 15 to 18 weeks of pregnancy of human amniotic fluid and expanded in the cell culture. Cells were cultivated according to standard procedures for mesenchymal stem cells and were differentiated along the neural lineage using various protocols. Furthermore, it was also tried to direct them into cell types of the retina as well as into endothelial cells. Cells of more than 72 amniotic fluid samples were collected and characterized. While after induction neural-like phenotypes could actually be detected, which was confirmed using neural marker proteins such as GFAP and ßIII tubulina further differentiation into retinal like cells could not reliably be shown. These data suggest that amniotic fluid derived cells are an interesting cell source, which may also give rise to neural-like cells. However, a more specific differentiation into neuronal and glial cells could not unequivocally be shown, so that further investigations have to becarried out.

  13. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    Science.gov (United States)

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-04-30

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis.

  14. Quantitative analysis of the toxicity of human amniotic fluid to cultured rat spinal cord.

    Science.gov (United States)

    Drewek, M J; Bruner, J P; Whetsell, W O; Tulipan, N

    1997-10-01

    It has been proposed that the myelodysplastic components of a myelomeningocele are secondarily damaged as the result of exposure to amniotic fluid, the so-called 'two-hit' hypothesis. The critical time at which this secondary insult might occur has not been clearly defined. The present study addresses this issue by quantitatively assessing the toxic effects of human amniotic fluid of various gestational ages upon organotypic cultures of rat spinal cord. Using an assay for lactate dehydrogenase efflux to evaluate toxicity in such spinal cord cultures, we found that the amniotic fluid became toxic at approximately 34 weeks' gestation. This toxic effect of amniotic fluid appears to emerge rather suddenly. Accordingly, it seems reasonable to suggest that prevention of exposure of vulnerable spinal cord tissue to this toxicity by surgical closure of a myelomeningocele defect prior to the emergence of toxicity in amniotic fluid may prevent injury to vulnerable myelodysplastic spinal cord tissue.

  15. Phthalates and perfluorooctanesulfonic acid in human amniotic fluid

    DEFF Research Database (Denmark)

    Jensen, Morten Søndergaard; Nørgaard-Pedersen, Bent; Toft, Gunnar;

    2012-01-01

    Measures of prenatal environmental exposures are important, and amniotic fluid levels may directly reflect fetal exposures during hypothesized windows of vulnerability.......Measures of prenatal environmental exposures are important, and amniotic fluid levels may directly reflect fetal exposures during hypothesized windows of vulnerability....

  16. Engineering Replacement Tissues with Amniotic Stem Cells

    Science.gov (United States)

    2012-10-01

    chondrogenic conditions (2D) formed tight nodes after 14 days. From left to right, nodes stained positive for alcian blue, safranin -O, and collagen II. 3...potential (alcian blue, safranin -O, collagen II staining) and colony forming ability were established (Figure 2). We were able to expand the cell...groups appear to be negative for Safranin -O stain (Figure 16) and weakly positive for alcian blue stain (Figure 17). Upon examination at a higher

  17. Biological characteristics and dopaminergic neural-like cell differentiation potential of human amniotic membrane-derived mesenchymal stem cells%人羊膜间充质干细胞生物学特征及向多巴胺能神经元样细胞的分化

    Institute of Scientific and Technical Information of China (English)

    周文然; 李新; 王文波; 谢燕霞; 唐娜; 阎影

    2014-01-01

    干细胞所保留的多向分化潜能和有效分化成为多巴胺能神经元样细胞的特性。%BACKGROUND:Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in celltherapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are stil poorly characterized. OBJECTIVE:To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like celldifferentiation potential of the human amniotic membrane-derived mesenchymal stem cells. METHODS:Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and col agenase based enzymic digestion, and purified by percol mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural celldifferentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cellassociated bio-markers were determined by immunofluoresence staining and confocal microscope. RESULTS AND CONCLUSION:In this study, we performed a practical method to isolate and purify human

  18. Mesenchymal Stem Cells from Wharton's Jelly and Amniotic Fluid.

    Science.gov (United States)

    Joerger-Messerli, Marianne S; Marx, Caterina; Oppliger, Byron; Mueller, Martin; Surbek, Daniel V; Schoeberlein, Andreina

    2016-02-01

    The discovery of mesenchymal stem cells (MSCs) in perinatal sources, such as the amniotic fluid (AF) and the umbilical connective tissue, the so-called Wharton's jelly (WJ), has transformed them into promising stem cell grafts for the application in regenerative medicine. The advantages of AF-MSCs and WJ-MSCs over adult MSCs, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), include their minimally invasive isolation procedure, their more primitive cell character without being tumourigenic, their low immunogenicity and their potential autologous application in congenital disorders and when cryopreserved in adulthood. This chapter gives an overview of the biology of AF-MSCs and WJ-MSCs, and their regenerative potential based on the results of recent preclinical and clinical studies. In the end, open questions concerning the use of WJ-MSCs and AF-MSCs in regenerative medicine will be emphasized.

  19. Human placental lactogen levels in amniotic fluid in normal and toxemic pregnancies.

    Science.gov (United States)

    Lolis, D; Kaskarelis, D

    1978-01-01

    Amniotic fluid human placental lactogen (HPL) levels were measured by radioimmunoassay in 162 cases of women with normal pregnancy and 43 with toxemic pregnancy, in the last trimester of pregnancy. A significant differences in levels was observed.

  20. Amniotic fluid-derived stem cells as a cell source for bone tissue engineering.

    Science.gov (United States)

    Rodrigues, Márcia T; Lee, Sang Jin; Gomes, Manuela E; Reis, Rui L; Atala, Anthony; Yoo, James J

    2012-12-01

    In tissue engineering, stem cells have become an ideal cell source that can differentiate into most human cell types. Among the stem cells, bone marrow-derived stem cells (BMSCs) have been widely studied, and there is strong evidence that these cells can be differentiated into cells of the osteogenic lineage. Thus, BMSCs have become the gold standard for studies of tissue engineering in orthopedics. However, novel stem cell sources, such as amniotic fluid-derived stem cells (AFSCs) have been identified, and these have important and unique features that may lead to novel and successful applications toward the regeneration of bone tissue. This study was designed to originally compare the osteogenic potential of both BMSCs and AFSCs under distinct culture environments to determine whether the osteogenic differentiation process of both types of stem cells is related to the origin of the cells. Osteogenic differentiation was carried out in both two and three dimensions using a tissue culture plate and by means of seeding the cells onto microfibrous starch and poly(ɛ-caprolactone) scaffolds (a blend of starch and polycaprolactone), respectively. BMSCs and AFSCs were successfully differentiated into the osteogenic cell type, as cells derived from them produced a mineralized extracellular matrix. Nevertheless, the two types of cells presented different expression patterns of bone-related markers as well as different timing of differentiation, indicating that both cell origin and the culture environment have a significant impact on the differentiation into the osteogenic phenotype in AFSCs and BMSCs.

  1. Repair of an oroantral communication by a human amniotic membrane: a novel technique.

    Science.gov (United States)

    Lakshmi, Subha; Bharani, Siva; Ambardar, Kalhan

    2015-08-01

    The amniotic membrane is the innermost layer of fetal membrane and is attached to the chorion in the placenta. This membrane has been used for nearly a century in varied fields such as ophthalmology, reconstructive surgery, and burn treatment. In this case report, we used a human amniotic membrane to repair an iatrogenic oroantral communication that occurred during the extraction of the patient's right upper second molar. A splint was given after the perforation was covered with human amniotic membrane and healing was clinically evaluated at various intervals. The outcome of the study revealed that the human amniotic membrane was an efficient graft material for repairing the defect caused by an iatrogenic oroantral communication following tooth extraction.

  2. Adaptation of group A Streptococcus to human amniotic fluid.

    Directory of Open Access Journals (Sweden)

    Izabela Sitkiewicz

    Full Text Available BACKGROUND: For more than 100 years, group A Streptococcus has been identified as a cause of severe and, in many cases, fatal infections of the female urogenital tract. Due to advances in hospital hygiene and the advent of antibiotics, this type of infection has been virtually eradicated. However, within the last three decades there has been an increase in severe intra- and post-partum infections attributed to GAS. METHODOLOGY: We hypothesized that GAS alters its transcriptome to survive in human amniotic fluid (AF and cause disease. To identify genes that were up or down regulated in response to growth in AF, GAS was grown in human AF or standard laboratory media (THY and samples for expression microarray analysis were collected during mid-logarithmic, late-logarithmic, and stationary growth phases. Microarray analysis was performed using a custom Affymetrix chip and normalized hybridization values derived from three biological replicates were collected at each growth point. Ratios of AF/THY above a 2-fold change and P-value <0.05 were considered significant. PRINCIPAL FINDINGS: The majority of changes in the GAS transcriptome involved down regulation of multiple adhesins and virulence factors and activation of the stress response. We observed significant changes in genes involved in the arginine deiminase pathway and in the nucleotide de novo synthesis pathway. CONCLUSIONS/SIGNIFICANCE: Our work provides new insight into how pathogenic bacteria respond to their environment to establish infection and cause disease.

  3. Corneal Neovascularization Suppressed by TIMP2 Released from Human Amniotic Membranes

    Institute of Scientific and Technical Information of China (English)

    Xiang Ma; Jun Li

    2005-01-01

    Purpose: To investigate the effects of culture medium of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice.Methods: Culture medium of amniotic membrane was prepared by cultivating AM (with epithelium side up) in EGM basic medium for 3 days, and was collected separately to three groups, e.g. Control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were performed in Boyden chambers and by using the CyQUANT fluorescence binding assay respectively.The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP1, TIMP2) in culture medium were determined by ELISA assay.Results: CNV induced by bFGF was significantly suppressed by culture medium of amniotic membrane. When the medium was applied as an eyedrop 4 times a day for 7 days,the area of CNV was (2.48±0.76) mm2,(0.64±0.52) mm2 and (1.96±0.65) mm2 incontrol, AM and De-AM group respectively. The migration and proliferation of HUVEC were strongly inhibited by culture medium of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. The high level of TIMP2 was found in AM group, but not in De-AM group, while there was no difference in the amount of TIMP1 in medium among three groups.Conclusion: Culture medium of amniotic membrane significantly suppresses the corneal nevovascularization induced by bFGF. The mechanism of which at least in part is that high level of TIMP2 protein secreted or released into the culture medium of AM and inhibition of migration and growth of vascular endothelial cells.

  4. Therapeutic outcomes of transplantation of amniotic fluid-derived stem cells in experimental ischemic stroke.

    Science.gov (United States)

    Tajiri, Naoki; Acosta, Sandra; Portillo-Gonzales, Gabriel S; Aguirre, Daniela; Reyes, Stephanny; Lozano, Diego; Pabon, Mibel; Dela Peña, Ike; Ji, Xunming; Yasuhara, Takao; Date, Isao; Solomita, Marianna A; Antonucci, Ivana; Stuppia, Liborio; Kaneko, Yuji; Borlongan, Cesar V

    2014-01-01

    Accumulating preclinical evidence suggests the use of amnion as a source of stem cells for investigations of basic science concepts related to developmental cell biology, but also for stem cells' therapeutic applications in treating human disorders. We previously reported isolation of viable rat amniotic fluid-derived stem (AFS) cells. Subsequently, we recently reported the therapeutic benefits of intravenous transplantation of AFS cells in a rodent model of ischemic stroke. Parallel lines of investigations have provided safety and efficacy of stem cell therapy for treating stroke and other neurological disorders. This review article highlights the need for investigations of mechanisms underlying AFS cells' therapeutic benefits and discusses lab-to-clinic translational gating items in an effort to optimize the clinical application of the cell transplantation for stroke.

  5. A Mini Overview of Isolation, Characterization and Application of Amniotic Fluid Stem Cells.

    Science.gov (United States)

    Gholizadeh-Ghalehaziz, Shiva; Farahzadi, Raheleh; Fathi, Ezzatollah; Pashaiasl, Maryam

    2015-11-01

    Amniotic fluid represents rich sources of stem cells that can be used in treatments for a wide range of diseases. Amniotic fluid- stem cells have properties intermediate between embryonic and adult mesenchymal stem cells which make them particularly attractive for cellular regeneration and tissue engineering. Furthermore, scientists are interested in these cells because they come from the amniotic fluid that is routinely discarded after birth. In this review we give a brief introduction of amniotic fluid followed by a description of the cells present within this fluid and aim to summarize the all existing isolation methods, culturing, characterization and application of these cells. Finally, we elaborate on the differentiation and potential for these cells to promote regeneration of various tissue defects, including fetal tissue, the nervous system, heart, lungs, kidneys, bones, and cartilage in the form of table.

  6. Term Amniotic membrane is a high throughput source for multipotent Mesenchymal Stem Cells with the ability to differentiate into endothelial cells in vitro

    DEFF Research Database (Denmark)

    Alviano, Francesco; Fossati, Valentina; Marchionni, Cosetta

    2007-01-01

    BACKGROUND: Term Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM......, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy......, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC). RESULTS: The recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical...

  7. Therapeutic outcomes of transplantation of amniotic fluid-derived stem cells in experimental ischemic stroke

    Directory of Open Access Journals (Sweden)

    Naoki eTajiri

    2014-08-01

    Full Text Available Accumulating preclinical evidence suggests the use of amnion as a source of stem cells for investigations of basic science concepts related to developmental cell biology, but also for stem cells’ therapeutic applications in treating human disorders. We previously reported isolation of viable rat amniotic fluid-derived stem (AFS cells. Subsequently, we recently reported the therapeutic benefits of intravenous transplantation of AFS cells in a rodent model of ischemic stroke. Parallel lines of investiagtions have provided safety and efficacy of stem cell therapy for treating stroke and other neurological disorders. This review article highlights characterization of AFS cells’ phenotype and their transplant-mediated functional effects, the need for investigations of mechanisms underlying AFS cells’ therapeutic benefits and discusses lab-to-clinic translational gating items in an effort to optimize the clinical application of cell transplantation for stroke.

  8. 利用人胚胎干细胞和人羊水干细胞的神经分化模型建立体外神经毒性测试的实验研究%Establishment of an in vitro system evaluating neurotoxicity using neural differentiation of human ES cells and amniotic fluid stem cells

    Institute of Scientific and Technical Information of China (English)

    周君梅; 张胜利; 陈方

    2011-01-01

    目的 许多药物具有神经毒性,尤其是对于儿童的影响更大.目前常用的药物体内毒性实验既耗时又昂贵.人胚胎干细胞和人羊水干细胞的神经分化有望建立一种理想的用于评价药物神经毒性的体外评估系统.此项研究旨在利用红藻氨酸的神经毒性验证此系统的可行性.方法 单层贴壁状态下,人胚胎干细胞和人羊水干细胞在化学成分明确的诱导条件下向神经细胞诱导.这些细胞经神经细胞特异性的抗体染色证实其特性.诱导得到的神经细胞在不同浓度红藻氨酸的培养液中进行培养和扩增,每代细胞进行计数,绘制细胞的生长曲线.结果 人胚胎干细胞和人羊水干细胞均可被高效地诱导为神经细胞;人胚胎干细胞的神经诱导效率高于人羊水干细胞;红藻氨酸对诱导得到的神经细胞具有毒性,其浓度与毒性呈正相关.结论 人胚胎干细胞和人羊水干细胞的体外神经分化可作为用于评价药物神经毒性的体外评估系统.%Objective A lot of drugs have side effects on the central nerves system. Especially in children. In vivo neurotoxicity tests are time-consuming and expensive. The neural differentiation of human embryonic stem cells and amniotic fluid stem cells provides all ideal in vitro system that Can be applied to evaluate neurotoxicity of drugs. This study was to try to establish such a system. The kainie acid was selected to test the neurotoxicity. Methods The human embryonic stem cells and amniotic fluid stem cells were indueed to differentiate into neural cells by a chemically defined neural induction medium. The induced neural cells were propagated in the presence of basic fibroblast growth factor. Immunocytochemical staining Was applied to confirm these cells' neural identity. The induced cells were propagated under different concentration of kainic acid, then the gosh curve were made based on the cell numbers. Results Both of the human

  9. The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy.

    Science.gov (United States)

    Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu

    2014-03-01

    The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases.

  10. Amniotic Fluid Stem Cells and Their Application in Cell-Based Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Mohamadreza Baghaban Eslaminejad

    2012-01-01

    Full Text Available Advances in stem cell biotechnology hold great promise in the field of tissue engineering andregenerative medicine. Of interest are marrow mesenchymal stem cells (MSCs, embryonic stemcells (ESCs, and induced pluripotent stem cells (iPSCs. In addition, amniotic fluid stem cells (AFSCshave attracted attention as a viable choice following the search for an alternative stem cellsource. Investigators are interested in these cells because they come from the amniotic fluid that isroutinely discarded after birth. There have been multiple investigations conducted worldwide in anattempt to better understand AF-SCs in terms of their potential use in regenerative medicine. In thisreview we give a brief introduction of amniotic fluid followed by a description of the cells presentwithin this fluid. Their history related to stem cell discovery in the amniotic fluid as well as themain characteristics of AF-SCs are discussed. Finally, we elaborate on the potential for these cellsto promote regeneration of various tissue defects, including fetal tissue, the nervous system, heart,lungs, kidneys, bones, and cartilage.

  11. The role of amniotic fluid in force transfer during human birth

    Science.gov (United States)

    Baumer, Alexa; Lehn, Andrea; Leftwich, Megan

    2013-11-01

    This study seeks to understand the fundamental fluid dynamic processes involved in human birth. We begin by examining the importance of amniotic fluid. This is done using two experimental techniques that approximate the laboring human uterus to different degrees of anatomical correctness. The first, in which a latex uterus is filled with fluid and a solid fetus is extracted, investigates the importance of both amniotic fluid properties and fetal position in the force required to remove a fetus. The second experiment simplifies the geometry of birth even more. In this case, a solid cylindrical rod is pulled through a highly flexible outer tube. The force to pull the inner cylinder as a function of the gap fluid properties is measured. By carefully controlling the fluid properties of the experiment, the study will provide further insight into the roles of amniotic fluid in human birth.

  12. Hypoxic Conditioned Medium from Human Amniotic Fluid-Derived Mesenchymal Stem Cells Accelerates Skin Wound Healing through TGF-β/SMAD2 and PI3K/Akt Pathways

    Directory of Open Access Journals (Sweden)

    Eun Kyoung Jun

    2014-01-01

    Full Text Available In a previous study, we isolated human amniotic fluid (AF-derived mesenchymal stem cells (AF-MSCs and utilized normoxic conditioned medium (AF-MSC-norCM which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM, it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials. Notably, more paracrine factors, VEGF and TGF-β1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-β/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-β/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-β/SMAD2 and PI3K/AKT pathways.

  13. A new approach to the pathomechanism of amniotic fluid embolism:unknown role of amniotic cells in the induction of disseminated intravascular coagulation

    Institute of Scientific and Technical Information of China (English)

    Mieczyslaw Uszyński; Waldemar Uszyński

    2012-01-01

    There are four concepts (theories) of amniotic fluid embolism (AFE). The aim of the study was to perform their critical review and to popularize a novel integrated concept. We searched Medline (from its inception to 2011), using key words:amniotic fluid embolism, amniotic cells, tissue factor, leukotriene and microparticles. Articles most eligible for the study of etiopathomechanism of AFE were chosen by title and/or abstract contents. The analysis of the publications revealed that:(i) the integrated concept of AFE is an adequate tool to interpret the complication, being particularly useful for taking direct therapeutic decisions; (ii) disseminated intravascular coagulation (DIC) in this complication is induced not by tissue factor (TF) of amniotic origin but by spectacular procoagulant activity of apoptosis-affected amniotic cells. Descriptions of molecular processes were provided. In clonclusioin, there are two independent pathways of AFE-the DIC pathway and the leukotriene pathway. It is not the TF but the apoptosis-affected amniotic cells that are responsible for the process of DIC in AFE. 3. One of the therapeutic conclusions of the new approach to the concept of AFE indicates that attempts to use heparin in AFE are justified (at the onset of the complication).

  14. Both Freshly Prepared and Frozen-Stored Amniotic Membrane Cells Express the Complement Inhibitor CD59

    Directory of Open Access Journals (Sweden)

    Ágnes Füst

    2012-01-01

    Full Text Available Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation.

  15. Amniotic fluid elicits appetitive responses in human newborns: fatty acids and appetitive responses.

    Science.gov (United States)

    Contreras, Carlos M; Gutiérrez-García, Ana G; Mendoza-López, Remedios; Rodríguez-Landa, Juan Francisco; Bernal-Morales, Blandina; Díaz-Marte, Cynthia

    2013-04-01

    In humans, maternal cues guide newborns to the maternal breast, and transitional cues may be present in maternal-fetal fluids. The aim of the present study was to determine the consistent presence of sensorial cues in three maternal-fetal fluids--amniotic fluid, colostrum, and milk--and test the ability of these cues to produce appetitive responses in newborns. In the analytical study, gas chromatography-mass spectrometry (GC-MS) detected eight fatty acids consistently present in the amniotic fluid, colostrum, and milk from 12 healthy volunteers, but we do not find a mammalian pheromone, identified in another mammalian species (rabbits), in another 30 volunteers. In the behavioral study, we explored the ability of amniotic fluid or its fatty acids to produce appetitive responses in 19 human newborns fluid or an artificial fatty acid mixture produced a longer duration of facial reactions that suggested appetitive (sucking) movements compared with respective vehicles (i.e., propylene glycol or centrifuged amniotic fluid with a low fatty acid content verified by GC-MS). We conclude that the fatty acids contained in amniotic fluid may constitute a transitional sensorial cue that guides newborns to the maternal breast.

  16. Satellited 4q identified in amniotic fluid cells

    Energy Technology Data Exchange (ETDEWEB)

    Miller, I.; Hsieh, C.L.; Songster, G. [Stanford Univ. Medical Center, Stanford, CA (United States)] [and others

    1995-01-16

    Extra material was identified on the distal long arm of a chromosome 4 in an amniotic fluid specimen sampled at 16.6 weeks of gestational age. There was no visible loss of material from chromosome 4, and no evidence for a balanced rearrangement. The primary counseling issue in this case was advanced maternal age. Ultrasound findings were normal, and family history was unremarkable. The identical 4qs chromosome was observed in cells from a paternal peripheral blood specimen and appeared to be an unbalanced rearrangement. This extra material was NOR positive in lymphocytes from the father, but was negative in the fetal amniocytes. Father`s relatives were studied to verify the familial origin of this anomaly. In situ hybridization with both exon and intron sequences of ribosomal DNA demonstrated that ribosomal DNA is present at the terminus of the 4qs chromosome in the fetus, father, and paternal grandmother. This satellited 4q might have been derived from a translocation event that resulted in very little or no loss from the 4q and no specific phenotype. This derivative chromosome 4 has been inherited through at least 3 generations of phenotypically normal individuals. 8 refs., 3 figs.

  17. Effects of topical human amniotic fluid and human serum in a mouse model of keratoconjunctivitis sicca.

    Science.gov (United States)

    Quinto, Guilherme G; Camacho, Walter; Castro-Combs, Juan; Li, Li; Martins, Suy Anne R; Wittmann, Priscila; Campos, Mauro; Behrens, Ashley

    2012-04-01

    To compare the effects of topical human amniotic fluid (HAF), topical human serum (HS), and topical artificial tears in a mouse model of dry eye. Thirty C57BL/6 mice were divided into 3 treatment groups: HAF, HS, and preservative-free artificial tears. Dry eye was induced by an injection of botulinum toxin B (BTX-B) into the lacrimal gland. Tear production and ocular surface fluorescein staining were evaluated in each mouse at 6 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Apoptotic keratocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test assay. A significant decrease in tear production was observed 3 days after BTX-B injection in all groups. At week 1, the HAF and HS groups had improved tear production compared with the control group (P < 0.001 and P = 0.003, respectively). HAF had a significantly improved fluorescein staining score compared with the HS (P = 0.043) and control (P = 0.007) groups at week 2. Goblet cell density was significantly decreased in the control group compared with the HAF and HS groups (P < 0.001). No difference in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive keratocytes was observed among the groups. HAF was superior to HS and artificial tears for improving corneal staining within 2 weeks of therapy in this induced mouse model of keratoconjunctivitis sicca. Clinical studies are needed to ascertain the benefits of these therapies in patients with ocular surface disorders associated with dry eye.

  18. AMNIOTIC FLUID STEM CELLS: THE KNOWN, THE UNKNOWN AND POTENTIAL REGENERATIVE MEDICINE APPLICATIONS.

    Science.gov (United States)

    Loukogeorgakis, Stavros P; De Coppi, Paolo

    2016-12-23

    The amniotic fluid has been identified as an untapped source of cells with broad potential, which possess immunomodulatory properties and don't have the ethical and legal limitations of embryonic stem cells. CD117(c-Kit)+ cells selected from amniotic fluid have been shown to differentiate into cell lineages representing all three embryonic germ layers without generating tumours, making them ideal candidates for regenerative medicine applications. Moreover, their ability to engraft in injured organs and modulate immune and repair responses of host tissues, suggest that transplantation of such cells may be useful for the treatment of various degenerative and inflammatory diseases. Although significant questions remain regarding the origin, heterogeneous phenotype and expansion potential of amniotic fluid stem cells, evidence to date supports their potential role as a valuable stem cell source for the field of regenerative medicine. This article is protected by copyright. All rights reserved.

  19. 人羊水间充质干细胞分离培养方法的优化%Optimization of the isolation and cultivation method of human amniotic fluid-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    郭素红; 苑春华; 侯毅鞠; 刘会玲

    2011-01-01

    背景:目前关于人羊水间充质干细胞的体外分离培养方法不一,获得较多数量的间充质干细胞仍有困难.目的:分离80份羊水标本,比较不同的体外培养方法,优化筛选到一种合适的培养纯化条件,为人羊水间充质干细胞的基础研究和临床广泛应用奠定基础.方法:无菌条件下采集足月分娩和终止妊娠引产的羊水,分离羊水细胞,比较孕期、不同培养基、接种密度、首次换液时间对人羊水间充质干细胞原代培养过程的影响,通过细胞化学染色方法检测细胞代谢情况,观察人羊水间充质干细胞的生物学特性.结果与结论:相同培养条件下,采用AmnioMAXⅡcomplete羊水专用细胞培养基,以5×104/cm2 的密度接种,首次换液时间为6 d时,人羊水间充质干细胞原代培养成功率较高.孕期为14~20周的人羊水间充质干细胞培养成功率高于晚期妊娠(21~36周)及足月分娩(37~42周)的羊水.人羊水间充质干细胞强表达过碘酸-雪夫、酸性磷酸酶、酸性α-醋酸萘酚酯酶染色,弱表达中性粒细胞碱性磷酸酶,不表达过氧化酶、苏丹黑染色.提示,优化筛选到一种合适的人羊水间充质干细胞培养纯化条件.%BACKGROUND: There are different methods of in vitro isolating culture for human amniotic fluid-derived mesenchymal stem cells (AF-MSCs). To gain most AF-MSCs is still difficult.OBJECTIVE: We had isolated 80 cases AF, and compared different in vitro culture methods for optimization and bolted a better method to establish foundation of AF-MSCs in clinical widespread application.METHODS: Amniotic fluid was collected from full-term deliveries and artificial labors under aseptic condition. The amniotic fluid cells were isolated by centrifugation. The influences of different pregnancy ages, culture media, incubation densities and the time points of the first medium change on the growth of AF-MSCs were analyzed. The cell metabolism was

  20. Current situation of research and prospects of human amniotic fluid-derived multipotential stem cells%羊水多潜能干细胞的研究现状及前景展望

    Institute of Scientific and Technical Information of China (English)

    顾潇; 陈必良

    2011-01-01

    由于强大的自我更新能力和多向分化的潜能,多潜能干细胞被广泛应用于再生医学的研究.胚胎干细胞的研究因伦理道德问题而受到限制.羊水多潜能干细胞的发现为多潜能干细胞的研究开辟了新的领域,引起了广大学者的关注.该文就羊水多潜能干细胞的生物学性状、分化潜能、临床应用前景等予以综述.%Because of a strong serf-renewal ability and multiplex differentiation potential, multipotential stem cells are widely used in researches on regenerative medicine. The research on embryonic stem cell is restricted because of ethical issues. The discovery of amniotic fluid-derived multipotential stem cells has open up a new field for multipotential stem cell research, and attracted attention of scholars. The article reviewed biological characteristics, differentiation potentiality and future clinical application of amniotic fluid-derived multipotential stem cells.

  1. Pregnancy-specific beta-1-glycoprotein (SP1) in cultured amniotic fluid cells.

    Science.gov (United States)

    Heikinheimo, M; Wahlström, T; Aula, P; Virtanen, I; Seppälä, M

    1980-12-01

    The synthesis of pregnancy-specific beta-1-glycoprotein (SP1) was studied in amniotic fluid cell cultures using RIA, immunoperoxidase, and immunofluorescence techniques. SP1 was found by RIA in all 11 sonicates and in 21 of 26 culture media. The SP1-immunoreactive material was immunologically similar to maternal serum SP1. Immunoperoxidase and indirect immunofluorescence staining were positive in large cells identified as epithelial amniotic cells by labeling with antikeratin antibodies. Fibroblast-like cells were occasionally found in cultures, but they did not contain demonstrable amounts of SP1. The physiological significance of the findings presented remains unclear.

  2. Inhibition of glycogen synthase kinase-3 (GSK3) promotes the neural differentiation of full-term amniotic fluid-derived stem cells towards neural progenitor cells.

    Science.gov (United States)

    Gao, Liyang; Zhao, Mingyan; Ye, Wei; Huang, Jinzhi; Chu, Jiaqi; Yan, Shouquan; Wang, Chaojun; Zeng, Rong

    2016-08-01

    The amniotic fluid has a heterogeneous population of cells. Some human amniotic fluid-derived stem (hAFS) cells have been shown to harbor the potential to differentiate into neural cells. However, the neural differentiation efficiency of hAFS cells remains low. In this study, we isolated CD117-positive hAFS cells from amniotic fluid and then examined the pluripotency of these cells through the formation of embryoid bodies (EBs). Additionally, we induced the neural differentiation of these cells using neuroectodermal medium. This study revealed that the GSK3-beta inhibitor SB216763 was able to stimulate the proliferation of CD117-positive hAFS cells without influencing their undifferentiated state. Moreover, SB216763 can efficiently promote the neural differentiation of CD117-positive hAFS cells towards neural progenitor cells in the presence of DMEM/F12 and N2 supplement. These findings provide an easy and low-cost method to maintain the proliferation of hAFS cells, as well as induce an efficacious generation of neural progenitor cells from hAFS cells. Such induction of the neural commitment of hAFS cells may provide an option for the treatment of neurodegenerative diseases by hAFS cells-based therapies.

  3. Cryopreserved amniotic fluid-derived cells: a lifelong autologous fetal stem cell source for heart valve tissue engineering.

    Science.gov (United States)

    Schmidt, Dörthe; Achermann, Josef; Odermatt, Bernhard; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

    2008-07-01

    Fetal stem cells represent a promising cell source for heart valve tissue engineering. In particular, amniotic fluid-derived cells (AFDC) have been shown to lead to autologous fetal-like heart valve tissues in vitro for pediatric application. In order to expand the versatility of these cells also for adult application, cryopreserved AFDC were investigated as a potential life-long available cell source for heart valve tissue engineering. Human AFDC were isolated using CD133 magnetic beads, and then differentiated and analyzed. After expansion of CD133- as well as CD133+ cells up to passage 7, a part of the cells was cryopreserved. After four months, the cells were re-cultured and phenotyped by flow cytometry and immunohistochemistry, including expression of CD44, CD105, CD90, CD34, CD31, CD141, eNOS and vWF, and compared to their non-cryopreserved counterparts. The stem cell potential was investigated in differentiation assays. The viability of cryopreserved AFDC for heart valve tissue engineering was assessed by creating heart valve leaflets in vitro. After cryopreservation, amniotic fluid-derived CD133- and CD133+ cells retained their stem cell-like phenotype, expressing mainly CD44, CD90 and CD105. This staining pattern was comparable to that of their non-cryopreserved counterparts. Moreover, CD133- cells demonstrated differentiation potential into osteoblast-like and adipocyte-like cells. CD133+ cells showed characteristics of endothelial-like cells by eNOS, CD141 and beginning vWF expression. When used for the fabrication of heart valve leaflets, cryopreserved CD133- cells produced extracellular matrix elements comparable to their non-cryopreserved counterparts. Moreover, the resulting tissues showed a cellular layered tissue formation covered by functional endothelia. The mechanical properties were similar to those of tissues fabricated from non-cryopreserved cells. The study results suggest that the use of cell bank technology fetal amniotic fluid

  4. Amniotic fluid as a source of multipotent cells for clinical use.

    Science.gov (United States)

    Young, Bruce K; Chan, Michael K; Liu, Li; Basch, Ross S

    2016-04-01

    Amniotic fluid cells (AFC) from 2nd trimester amniocentesis have been found to be a source of multipotent stem cells which might overcome the limitations of expansion, histocompatibility, tumorigenesis, and ethical issues associated with using human embryonic cells, umbilical cord, cord blood, bone marrow, and induced pluripotent cells. Previous work by our group and others demonstrated multipotency and the ability to grow well in culture. However, all these studies were done in media containing fetal calf serum. We sought to observe the properties of AFC grown in serum-free media as that would be required for clinical transplantation in humans. Fresh samples were obtained from three patients, and each sample divided into a culture whose cells were not exposed to fetal calf serum, and the other half into a standard culture medium containing fetal calf serum. Doubling time and stem cell marker expression by flow cytometry were assessed. Differentiation to neural, osteoid, and chondrogenic lineages was induced using appropriate media and confirmed by fluorescent microscopy, histology, and immunohistochemistry. There were no statistically significant differences between cells grown serum-free and in standard media in any of these parameters. The data supports the possibility of clinical use of AFC in stem cell transplantation.

  5. PRELIMINARY STUDY ON TRANSDIFFERENTIATION OF HUMAN AMNIOTIC EPITHELIAL CELLS AND ITS INTRASLENIC TRANSPLANTATION%人羊膜上皮细胞横向分化及脾内移植的初步研究

    Institute of Scientific and Technical Information of China (English)

    罗宏武; 黄湘俊; 黄飞舟; 刘浔阳

    2011-01-01

    Objective The human amniotic epithelial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes.The hAECs may be a candidate seed cell for liver regeneration.To research the survival and migration in vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and to explore the expression ofhepatocyte-like function.Methods Thirty nude mice (aging 6-8 weeks, halfmales and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10).The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy.The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal saline was injected in group C.At 4 hours, the nude mice were sacrificed and the samples of liver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes.Results No tumor tissue was found in liver and spleen of 3 groups, and HE staining showed no tumor cells.There were a lot of roundlike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the liver of group A; no abnormal cells were found in liver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C.The GFP+ cells were detected in the spleen and liver of group A with expressing human albumin, but no GFP+ cells was found in liver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C.Conclusion AAV-GFP infected hAECs transplanted into SCID nude

  6. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential.

    Science.gov (United States)

    Pipino, Caterina; Pandolfi, Assunta

    2015-05-26

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.

  7. Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent.

    Science.gov (United States)

    Sanluis-Verdes, Anahí; Yebra-Pimentel Vilar, Maria Teresa; García-Barreiro, Juan Javier; García-Camba, Marta; Ibáñez, Jacinto Sánchez; Doménech, Nieves; Rendal-Vázquez, Maria Esther

    2015-09-01

    Human amniotic membrane (HAM) has useful properties as a dermal matrix substitute. The objective of our work was to obtain, using different enzymatic or chemical treatments to eliminate cells, a scaffold of acellular HAM for later use as a support for the development of a skin equivalent. The HAM was separated from the chorion, incubated and cryopreserved. The membrane underwent different enzymatic and chemical treatments to eliminate the cells. Fibroblasts and keratinocytes were separately obtained from skin biopsies of patients following a sequential double digestion with first collagenase and then trypsin-EDTA (T/E). A skin equivalent was then constructed by seeding keratinocytes on the epithelial side and fibroblasts on the chorionic side of the decellularizated HAM. Histological, immunohistochemical, inmunofluorescent and molecular biology studies were performed. Treatment with 1% T/E at 37 °C for 30 min totally removed epithelial and mesenchymal cells. The HAM thus treated proved to be a good matrix to support adherence of cells and allowed the achievement of an integral and intact scaffold for development of a skin equivalent, which could be useful as a skin substitute for clinical use.

  8. Trans-amniotic stem cell therapy (TRASCET) minimizes Chiari-II malformation in experimental spina bifida.

    Science.gov (United States)

    Dionigi, Beatrice; Brazzo, Joseph A; Ahmed, Azra; Feng, Christina; Wu, Yaotang; Zurakowski, David; Fauza, Dario O

    2015-06-01

    We sought to study the impact of trans-amniotic stem cell therapy (TRASCET) in the Chiari-II malformation in experimental spina bifida. Sprague-Dawley fetuses (n=62) exposed to retinoic acid were divided into three groups at term (21-22 days gestation): untreated isolated spina bifida (n=21), isolated spina bifida treated with intra-amniotic injection of concentrated, syngeneic, labeled amniotic fluid mesenchymal stem cells (afMSCs) on gestational day 17 (n=28), and normal controls (n=13). Analyses included measurements of brainstem and cerebellar placement on high resolution MRI and histology. Statistical comparisons included ANOVA. In parallel to the expected induced coverage of the spina bifida in the afMSC-treated group (Pspina bifida by TRASCET minimizes the Chiari-II malformation in the retinoic acid rodent model, further suggesting it as a practical alternative for the prenatal management of spina bifida. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Regulation of amniotic fluid volume.

    Science.gov (United States)

    Beall, M H; van den Wijngaard, J P H M; van Gemert, M J C; Ross, M G

    2007-01-01

    Water arrives in the mammalian gestation from the maternal circulation across the placenta. It then circulates between the fetal water compartments, including the fetal body compartments, the placenta and the amniotic fluid. Amniotic fluid is created by the flow of fluid from the fetal lung and bladder. A major pathway for amniotic fluid resorption is fetal swallowing; however, in many cases the amounts of fluid produced and absorbed do not balance. A second resorption pathway, the intramembranous pathway (across the amnion to the fetal circulation), has been proposed to explain the maintenance of normal amniotic fluid volume. Amniotic fluid volume is thus a function both of the amount of water transferred to the gestation across the placental membrane, and the flux of water across the amnion. Water flux across biologic membranes may be driven by osmotic or hydrostatic forces; existing data suggest that intramembranous flow in humans is driven by the osmotic difference between the amniotic fluid and the fetal serum. The driving force for placental flow is more controversial, and both forces may be in effect. The mechanism(s) responsible for regulating water flow to and from the amniotic fluid is unknown. In other parts of the body, notably the kidney, water flux is regulated by the expression of aquaporin water channels on the cell membrane. We hypothesize that aquaporins have a role in regulating water flux across both the amnion and the placenta, and present evidence in support of this theory. Current knowledge of gestational water flow is sufficient to allow prediction of fetal outcome when water flow is abnormal, as in twin-twin transfusion syndrome. Further insight into these mechanisms may allow novel treatments for amniotic fluid volume abnormalities with resultant improvement in clinical outcome.

  10. Osteogenic differentiation of amniotic fluid mesenchymalstromal cells and their bone regeneration potential

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    In orthopedics, tissue engineering approach usingstem cells is a valid line of treatment for patients withbone defects. In this context, mesenchymal stromalcells of various origins have been extensively studiedand continue to be a matter of debate. Although mesenchymalstromal cells from bone marrow are alreadyclinically applied, recent evidence suggests that one mayuse mesenchymal stromal cells from extra-embryonictissues, such as amniotic fluid, as an innovative andadvantageous resource for bone regeneration. Theuse of cells from amniotic fluid does not raise ethicalproblems and provides a sufficientnumber of cellswithout invasive procedures. Furthermore, they donot develop into teratomas when transplanted, aconsequence observed with pluripotent stem cells.In addition, their multipotent differentiation ability,low immunogenicity, and anti-inflammatory propertiesmake them ideal candidates for bone regenerativemedicine. We here present an overview of the featuresof amniotic fluid mesenchymal stromal cells and theirpotential in the osteogenic differentiation process.We have examined the papers actually availableonthis regard, with particular interest in the strategiesapplied to improve in vitro osteogenesis. Importantly, adetailed understanding of the behavior of amniotic fluidmesenchymal stromal cells and their osteogenic abilityis desirable considering a feasible application in boneregenerative medicine.

  11. Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders.

    Science.gov (United States)

    Hattori, Yuka; Mukaide, Takahiro; Jiang, Li; Kotani, Tomomi; Tsuda, Hiroyuki; Mano, Yukio; Sumigama, Seiji; Hirayama, Tasuku; Nagasawa, Hideko; Kikkawa, Fumitaka; Toyokuni, Shinya

    2015-01-01

    Amniotic fluid contains numerous biomolecules derived from fetus and mother, thus providing precious information on pregnancy. Here, we evaluated oxidative stress of human amniotic fluid and measured the concentration of catalytic Fe(II). Amniotic fluid samples were collected with consent from a total of 89 subjects in Nagoya University Hospital, under necessary medical interventions: normal pregnancy at term, normal pregnancy at the 2nd trimester, preterm delivery with maternal disorders but without fetal disorders, congenital diaphragmatic hernia, fetal growth restriction, pregnancy-induced hypertension, gestational diabetes mellitus, Down syndrome and trisomy 18. Catalytic Fe(II) and oxidative stress markers (8-hydroxy-2'-deoxyguanosine, 8-OHdG; dityrosine) were determined with RhoNox-1 and specific antibodies, respectively, using plate assays. Levels of 8-OHdG and dityrosine were higher in the 3rd trimester compared with the 2nd trimester in normal subjects, and the abnormal groups generally showed lower levels than the controls, thus suggesting that they represent fetal metabolic activities. In contrast, catalytic Fe(II) was higher in the 2nd trimester than the 3rd trimester in the normal subjects, and overall the abnormal groups showed higher levels than the controls, suggesting that high catalytic Fe(II) at late gestation reflects fetal pathologic alterations. Notably, products of H2O2 and catalytic Fe(II) remained almost constant in amniotic fluid.

  12. Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane

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    Fahmida Binte Atique

    2013-01-01

    Full Text Available Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D10 values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4×102 to 1.2×105 cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%. The D10 values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD of 11.4 KGy for a bioburden level of 1000. To compare the differences, D10 values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes.

  13. Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane

    Science.gov (United States)

    Binte Atique, Fahmida; Ahmed, Kazi Tahsin; Asaduzzaman, S. M.; Hasan, Kazi Nadim

    2013-01-01

    Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D10) values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4 × 102 to 1.2 × 105 cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%). The D10 values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD) of 11.4 KGy for a bioburden level of 1000. To compare the differences, D10 values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes. PMID:24063009

  14. 人羊膜上皮细胞移植急性肝损伤小鼠的定量效果分析☆%Quantitative analysis of transplanted effect of human amniotic epithelial cells in mice with acute liver injury

    Institute of Scientific and Technical Information of China (English)

    罗宏武; 寻权; 黄湘俊; 黄飞舟

    2013-01-01

      背景:目前已有较多关于人羊膜上皮细胞移植入动物体内的存活、迁徙及相关特性的初步研究,但其对移植效果的定量分析尚未见报道。目的:对脾内移植传代的人羊膜上皮细胞小鼠血清肝生化功能及人血白蛋白的定量分析。方法:40只裸小鼠随机分为4组,每组各10只。肝叶切除+细胞移植2周组、肝叶切除+细胞移植4周组、肝叶切除+盐水组,行半肝叶切除,肝叶切除+细胞移植组自脾下极移植密度为5×106传代的人羊膜上皮细胞约0.2 mL,分别于移植后2周和4周采血;肝叶切除+盐水组自脾下极注射生理盐水0.2 mL;单纯细胞移植组:不行肝叶切除,自脾下极移植密度为5×106传代的人羊膜上皮细胞约0.2 mL。检测其各组肝脾组织学、形态学的改变及各组血清谷丙转氨酶、谷草转氨酶、人血白蛋白的变化和人血白蛋白表达定量分析。结果与结论:人羊膜上皮细胞移植急性肝损伤小鼠4周后肝脾形态未见明显改变,组织学可检测到特异性细胞,血清谷丙转氨酶、谷草转氨酶、人血白蛋白有明显改善,血清中能检测到人血白蛋白且移植后4周较移植后2周有明显升高。因此,人羊膜上皮细胞移植入肝受损小鼠体内能存活超过4周且仍表达肝细胞样细胞的部分特性及功能,改善小鼠的肝功能,治疗小鼠急性肝损伤。%BACKGROUND:There are many preliminary studies on the survival, metaptosis, and correlation characteristics of human amniotic epithelial cel s after transplanted into the animals, but there are no reports on the quantitative analysis of the transplantation effect. OBJECTIVE:To make quantitative analysis on serum biochemical function of liver and the expression of human albumin in mice received passaged human amniotic epithelial cel s transplantation in spleen. METHODS:Forty nude mice were randomly divided into four groups (n=10

  15. CM-Dil与DAPI联合标记人羊膜上皮细胞的可行性研究%Feasibility of CM-Dil combined with DAPI double-labeling human amniotic epithelial cells

    Institute of Scientific and Technical Information of China (English)

    王黎; 周清; 杨艳; 陈剑; 徐锦堂

    2013-01-01

    目的 建立人羊膜上皮细胞(human amniotic epithelial cell,HAEC)的体外培养方法,并探讨氯甲基苯甲酰胺(CM-Dil)与4’,6-二脒基-2-苯基吲哚(DAPI)对HAEC进行联合标记示踪的可行性.方法 运用酶消化法获取HAEC,收集第2代细胞,流式细胞仪检测CD29、CD34、CD44、CD45和CD105的表达率,SP免疫化学法鉴定HAEC.CM-Dil与DAPI对HAEC进行体外标记,荧光倒置显微镜下观察1d、7d和14 d的标记情况,台盼蓝染色检测细胞活力,CCK-8法检测细胞增殖以明确联合标记对体外培养HAEC生长特性的影响.结果 HAEC贴壁培养后呈扁平多角形,CD29、CD34、CD44、CD45和CD105的阳性率分别为99.64%、2.21%、32.41%、0.84%、36.70%,细胞角蛋白Keratin阳性表达.HAEC在CM-Dil和DAPI联合标记1d后,荧光显微镜下可观察到细胞膜和细胞核分别在不同波长下呈红色和蓝色荧光,标记率为100%;14 d后,经传代培养的HAEC荧光强度与1d时相近,细胞形态无改变.台盼蓝染色显示标记细胞存活率为96.8% ~ 98.9%,CCK-8检测标记细胞的增殖力较未标记组差异无统计学意义(P>0.05).结论 CM-Dil和DAPI可有效标记HAEC,染色简单、无细胞毒性,荧光衰减较慢,可作为HAEC的标记及示踪方法.

  16. Amniotic Mesenchymal Stem Cells Can Enhance Angiogenic Capacity via MMPs In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Fei Jiang

    2015-01-01

    Full Text Available The aim of this study was to evaluate the angiogenic capacity and proteolytic mechanism of coculture using human amniotic mesenchymal stem cells (hAMSCs with human umbilical vein endothelial cells (HUVECs in vivo and in vitro by comparing to those of coculture using bone marrow mesenchymal stem cells with HUVEC. For the in vivo experiment, cells (HUVEC-monoculture, HUVEC-hAMSC coculture, and HUVEC-BMMSC coculture were seeded in fibrin gels and injected subcutaneously in nude mice. The samples were collected on days 7 and 14 and histologically analyzed by H&E and CD31 staining. CD31-positive staining percentage and vessel-like structure (VLS density were evaluated as quantitative parameters for angiogenesis. The increases of CD31-positive staining area and VLS density in both HUVEC-hAMSC group and HUVEC-BMMSC group were found between two time points, while obvious decline of those was observed in HUVEC-only group. For the in vitro experiment, we utilized the same 3D culture model to investigate the proteolytic mechanism related to capillary formation. Intensive vascular networks formed by HUVECs were associated with hAMSCs or BMMSCs and related to MMP2 and MMP9. In conclusion, hAMSCs shared similar capacity and proteolytic mechanism with BMMSCs on neovascularization.

  17. Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation

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    Fatima A

    2006-01-01

    Full Text Available Background : The technique of transplantation of cultivated limbal epithelium rather than direct limbal tissue isa novel method of "cell therapy" involved in reconstructing the ocular surface in severe limbal stem celldeficiency [LSCD], caused by chemical burns. Aim : To describe a simple feeder-cell free technique of cultivating limbal epithelium on human amniotic membrane[HAM]. Materials and Methods : The limbal tissues (2 mm were harvested from patients with LSCD. These tissueswere proliferated in vitro on HAM supplemented by human corneal epithelial cell medium and autologousserum. Cultures covering more ?50% area of 2.5x5 cm HAM were considered adequate for clinical use. Thecultured epithelium was characterized by histopathology and immunophenotyping.Results: A total of 542 cultures out of 250 limbal tissues were cultivated in the laboratory from January 2001through July 2005. The culture explants showed that clusters of cells emerging from the edge of the explantsin one-three days formed a complete monolayer within 10-14 days. In 86% of cultures (464 of 542, thegrowth was observed within one-two days. Successful explant cultures were observed in 98.5% (534 of 542cultures with 91% explant cultures showing an area of ?6.25 cm2 (6.25 - 12.5 cm2 range. The cultivatedepithelium was terminated between 10-14 days for clinical transplantation. The problems encountered wereinadequate growth (2 of 542 and contamination (2 of 542. Conclusions : We demonstrate a simple technique of generating a sheet of corneal epithelium from a limbalbiopsy. This new technique could pave the way for a novel form of cell therapy.

  18. Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

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    Caterina Morabito

    2015-05-01

    Full Text Available Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family. This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules.

  19. Applications of Amniotic Membrane and Fluid in Stem Cell Biology and Regenerative Medicine

    Directory of Open Access Journals (Sweden)

    Kerry Rennie

    2012-01-01

    Full Text Available The amniotic membrane (AM and amniotic fluid (AF have a long history of use in surgical and prenatal diagnostic applications, respectively. In addition, the discovery of cell populations in AM and AF which are widely accessible, nontumorigenic and capable of differentiating into a variety of cell types has stimulated a flurry of research aimed at characterizing the cells and evaluating their potential utility in regenerative medicine. While a major focus of research has been the use of amniotic membrane and fluid in tissue engineering and cell replacement, AM- and AF-derived cells may also have capabilities in protecting and stimulating the repair of injured tissues via paracrine actions, and acting as vectors for biodelivery of exogenous factors to treat injury and diseases. Much progress has been made since the discovery of AM and AF cells with stem cell characteristics nearly a decade ago, but there remain a number of problematic issues stemming from the inherent heterogeneity of these cells as well as inconsistencies in isolation and culturing methods which must be addressed to advance the field towards the development of cell-based therapies. Here, we provide an overview of the recent progress and future perspectives in the use of AM- and AF-derived cells for therapeutic applications.

  20. Human Amniotic Membrane-Derived Products in Sports Medicine: Basic Science, Early Results, and Potential Clinical Applications.

    Science.gov (United States)

    Riboh, Jonathan C; Saltzman, Bryan M; Yanke, Adam B; Cole, Brian J

    2016-09-01

    Amniotic membrane (AM)-derived products have been successfully used in ophthalmology, plastic surgery, and wound care, but little is known about their potential applications in orthopaedic sports medicine. To provide an updated review of the basic science and preclinical and clinical data supporting the use of AM-derived products and to review their current applications in sports medicine. Systematic review. A systematic search of the literature was conducted using the Medline, EMBASE, and Cochrane databases. The search term amniotic membrane was used alone and in conjunction with stem cell, orthopaedic, tissue engineering, scaffold, and sports medicine. The search identified 6870 articles, 80 of which, after screening of the titles and abstracts, were considered relevant to this study. Fifty-five articles described the anatomy, basic science, and nonorthopaedic applications of AM-derived products. Twenty-five articles described preclinical and clinical trials of AM-derived products for orthopaedic sports medicine. Because the level of evidence obtained from this search was not adequate for systematic review or meta-analysis, a current concepts review on the anatomy, physiology, and clinical uses of AM-derived products is presented. Amniotic membranes have many promising applications in sports medicine. They are a source of pluripotent cells, highly organized collagen, antifibrotic and anti-inflammatory cytokines, immunomodulators, and matrix proteins. These properties may make it beneficial when applied as tissue engineering scaffolds, improving tissue organization in healing, and treatment of the arthritic joint. The current body of evidence in sports medicine is heavily biased toward in vitro and animal studies, with little to no human clinical data. Nonetheless, 14 companies or distributors offer commercial AM products. The preparation and formulation of these products alter their biological and mechanical properties, and a thorough understanding of these

  1. Drug delivery human amniotic membrane scaffold combined with neural stem cells transplantation in the treatment of spinal cord injury%人羊膜缓释支架联合神经干细胞治疗脊髓损伤

    Institute of Scientific and Technical Information of China (English)

    王沛; 祁全; 王挽涛; 张铮

    2016-01-01

    of a new medical biomaterial drug delivery denuded human amniotic membrane (DHAM) scaffold combined with neural stem cells transplantation in rat spinal cord injury model.Methods The NSCs were separated,cultured,purified and identified from bilateral hippocampus of 24-hour-old SD rats (n =8).The human amniotic membrane was obtained from the healthy pregnant women and was prepared to DHAM.Two DHAM was cut into 4 by 4 cm in size.The NSCs (5 × 109 cells/L) were seeded on two DHAM.The mixture of fibrin-glue (2.5 ml) and nerve growth factor (18 μg) were aspersed on one no cell side of DHAM to construct drug delivery scaffold.The nerve growth factor (18 μg) were purely aspersed on one no cell side of another DHAM to construct no drug delivery scaffold.Thirty-six rats were made into spinal cord injury models through spinal cord complete section,and were randomly divided into three groups.In group A,the drug delivery scaffold was transplanted in rat spinal cord injury model.In group B,the no drug delivery scaffold was transplanted.In group C,rats received no treatment.After 1,2,4,and 8 weeks of surgery,the hindlimbs functional recovery was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score.Morphological changes were tested by Hematoxylin-Eosin (HE) staining,silver staining and immunohistochemistry staining after 8 weeks of surgery.Results The BBB score was higher in group A (11.63 ± 0.58) than in both other groups(P <0.05).HE staining showed different appearance in each group.In group A,most of DHAM arranged parallel.In the scaffold,there were many living cells.Some cells erupted silkiness fibers.And the silkiness fibers were growing in a directional manner.In group B,the DHAM did not keep the parallel arrangement.The DHAM arranged like waves or net.The silkiness fibers erupted by cells tended to tangle and did not have a directional manner.In group C,there were cavities in broken ends of spinal cords.The new born silkiness fibers tangled together

  2. Long-term immunomodulatory effect of amniotic stem cells in an Alzheimer's disease model.

    Science.gov (United States)

    Kim, Kyung-Sul; Kim, Hyun Sook; Park, Ji-Min; Kim, Han Wool; Park, Mi-Kyung; Lee, Hyun-Seob; Lim, Dae Seog; Lee, Tae Hee; Chopp, Michael; Moon, Jisook

    2013-10-01

    Amyloid beta (Aβ) plays a major role in Alzheimer's disease (AD), and neuroinflammatory processes mediated by Aβ plaque-induced microglial cells and astrocytes contribute to AD pathogenesis. The present study examined human placenta amniotic membrane-derived mesenchymal stem cells (AMSCs), which have potent immunomodulatory and paracrine effects in a Tg2576 (APPswe) transgenic mouse model of AD. AMSCs secreted high levels of transforming growth factor-β under in vitro inflammatory environment conditions. Six weeks after the intravenous injection of AMSCs, APPswe mice showed evidence of improved spatial learning, which significantly correlated with the observation of fewer Aβ plaques in brain. The number of ED1-positive phagocytic microglial cells associated with Aβ plaques was higher in AMSC-injected mice than in phosphate-buffered saline-injected mice, and the level of Aβ-degrading enzymes (matrix metallopeptidase-9 and insulin-degrading enzyme) was also significantly higher. Furthermore, the level of proinflammatory cytokines, interleukin-1 and tumor necrosis factor-α, was lower and that of anti-inflammatory cytokines, interleukin-10 and transforming growth factor-β, was higher in AMSC-injected mice than phosphate-buffered saline-injected mice. These effects lasted until 12 weeks after AMSC injection. Taken together, these results collectively suggest that injection of AMSCs might show significant long-lasting improvement in AD pathology and memory function via immunomodulatory and paracrine mechanisms. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation.

    Science.gov (United States)

    Weber, Benedikt; Emmert, Maximilian Y; Behr, Luc; Schoenauer, Roman; Brokopp, Chad; Drögemüller, Cord; Modregger, Peter; Stampanoni, Marco; Vats, Divya; Rudin, Markus; Bürzle, Wilfried; Farine, Marc; Mazza, Edoardo; Frauenfelder, Thomas; Zannettino, Andrew C; Zünd, Gregor; Kretschmar, Oliver; Falk, Volkmar; Hoerstrup, Simon P

    2012-06-01

    Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation

    Directory of Open Access Journals (Sweden)

    Vidane AS

    2014-08-01

    Full Text Available Atanasio S Vidane,1 Aline F Souza,1 Rafael V Sampaio,1 Fabiana F Bressan,2 Naira C Pieri,1 Daniele S Martins,2 Flavio V Meirelles,2 Maria A Miglino,1 Carlos E Ambrósio2 1Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil; 2Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, São Paulo, Brazil Abstract: Amnion-derived mesenchymal stem cells (AMSCs are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine. The cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. In culture medium, the cat AMSCs adhered to a plastic culture dish and displayed a fibroblast-like morphology. Immunophenotyping assays were positive for the mesenchymal stem cell-specific markers CD73 and CD90 but not the hematopoietic markers CD34, CD45, and CD79. Under appropriate conditions, the cat AMSCs differentiated into osteogenic, chondrogenic, and adipogenic cell lineages. One advantage of cat AMSCs was nonteratogenicity, assessed 4 weeks post injection of undifferentiated AMSCs into immunodeficient mice. These findings suggest that cat amniotic membranes may be an important and useful source of mesenchymal stem cells for clinical applications, especially for cell or tissue replacement in chronic and degenerative diseases. Keywords: amnion, cats, cell differentiation, fetal membranes, mesenchymal cells

  5. 人羊水来源干细胞治疗大鼠卵巢早衰的初步研究%Preliminary study on the effects of human amniotic fluid-derived stem cells on rats'premature ovarian failure

    Institute of Scientific and Technical Information of China (English)

    李洁; 张建芳; 白璐; 陈必良

    2012-01-01

    目的:尾静脉注射移植人羊水干细胞(AFS),评价其治疗大鼠卵巢早衰(POF)的效果.方法:采用SD大鼠,按体重随机分为四组:A组正常组、B组模型组、C组一次移植组和D组两次移植组,除A组灌胃生理盐水外,其余各组均连续14天灌胃雷公藤多苷片;C组在第14天灌胃后移植一次AFS,D组在第1天与第7天灌胃后移植两次AFS;观察大鼠阴道涂片、血清性激素水平、大鼠卵巢及子宫组织形态学变化.结果:(1)B组动情周期较A组显著延长,C组与D组均有不同程度恢复,D组恢复状况优于C组;(2)HE染色结果显示,D组大鼠卵巢均有新生卵泡;(3)性激素检测结果显示,各组之间E2值差异均有统计学意义(P均<0.01).结论:AFS对POF有修复作用,可能通过促进卵泡生成,调节雌激素分泌,从而改善了卵巢功能.%Objective; To evaluate it's the treatment effects on rats'premature ovarian failure. With transplant human amniotic fluid-derived stem cells by caudal vein injection, Methods ; SD rats were randomly divided into four groups by weight: group A ( normal group ) , group B(model group) ,group C(once transplantation group) ,group D(twice transplantation group). Except group A was perfused with physiological saline, other groups were perfused with Tripterygium Glucosides tablet for 14 days;then group C transplanted AFS once on the 14th day after gastric perfusion, group D transplanted AFS twice on the 1st day and the 7 th day after gastric perfusion. Rat's vaginal smears,serum sex hormone levels and the morphological changes of rats'ovary organizations were observed. Results; The treatment effects of AFS on Rats'premature ovarian failure: (1) The estrous cycle of group B was significantly prolonger than that of group A. Both group C and group D recovered to different degrees, and the recovery of group D was better than that of group C; (2) The results of HE coloration showed that new follicles appeared in all rats

  6. 基因转染人羊膜细胞对颅脑创伤大鼠海马的作用%The effects of human amniotic cells transfected with GDNF gene on hippocampal neuron following traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    卢奕; 惠国桢; 刘天津; 刘凤强; 吴智远; 李向东; 暨荀鹤; 郭礼和

    2010-01-01

    Objective To investigate the effect of grafting transfecting GDNF gene human amniotic cells (HACs) on hippocampal neuron following traumatic brain injury (TBI) in rats. Methods The model of hippocampal neuronal death following TBI was built up by improved Feeney's weight drop techniques. 5 μl transfecting HACs were injected into the margin of contusion 24 h after injured by microsyringe and stereotactic frame. Groups consisted of transfecting GDNF gene HACs group(TBI + GDNF), transfecting eGFP gene HACs group(TBI +eGFP), PBS group (TBI + PBS)and sham TBI group. The spatial memory performance was evaluated on 5 consecutive days by Morris water maze on 7 days after transplantation. After the probe trial, the rats were sacrificed for hippocampal neuron and brain tissue around target morphological analysis by cresyl violet staining under light microscopy. Expression of GDNF was detected by PCR in mRNA level. Results Transfecting GDNF gene HACs showed red fluorescence by immunofluorescence. The rats of TBI + GDNF group exhibited less escape latencies than those of TBI + eGFP group and TBI + PBS group, but had longer escape latencies than those of sham TBI group. Cresyl violet staining showed hippocampal neuronal loss, shrinkage and dark staining of neurons in TBI + eGFP group and TBI + PBS group. Compared with other groups, GDNF expression of TBI + GDNF group obviously increased in mRNA leveL Conclusion Grafting HACs transfected by GDNF gene plays a role in protecting against hippocampal neuronal death following TBI.%目的 观察移植转染胶质源性神经营养因子(GDNF)基因人羊膜细胞(HACs)对创伤性脑损伤(TBI)大鼠海马神经元的影响.方法 采用改进Feeney法制作TBI致海马神经元损伤模型.TBI 24 h后于挫伤灶边缘移植,移植处距硬脑膜4 mm和2 mm深浅两点移植,共5μl含5×10~5个细胞.TBI+GDNF组移植转染GDNF基因HACs;TBI+eGFP组移植转染eGFP基因HACs;TBI+PBS组注射5μl PBS;假TBI

  7. Transcriptome adaptation of group B Streptococcus to growth in human amniotic fluid.

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    Izabela Sitkiewicz

    Full Text Available BACKGROUND: Streptococcus agalactiae (group B Streptococcus is a bacterial pathogen that causes severe intrauterine infections leading to fetal morbidity and mortality. The pathogenesis of GBS infection in this environment is poorly understood, in part because we lack a detailed understanding of the adaptation of this pathogen to growth in amniotic fluid. To address this knowledge deficit, we characterized the transcriptome of GBS grown in human amniotic fluid (AF and compared it with the transcriptome in rich laboratory medium. METHODS: GBS was grown in Todd Hewitt-yeast extract medium and human AF. Bacteria were collected at mid-logarithmic, late-logarithmic and stationary growth phase. We performed global expression microarray analysis using a custom-made Affymetrix GeneChip. The normalized hybridization values derived from three biological replicates at each growth point were obtained. AF/THY transcript ratios representing greater than a 2-fold change and P-value exceeding 0.05 were considered to be statistically significant. PRINCIPAL FINDINGS: We have discovered that GBS significantly remodels its transcriptome in response to exposure to human amniotic fluid. GBS grew rapidly in human AF and did not exhibit a global stress response. The majority of changes in GBS transcripts in AF compared to THY medium were related to genes mediating metabolism of amino acids, carbohydrates, and nucleotides. The majority of the observed changes in transcripts affects genes involved in basic bacterial metabolism and is connected to AF composition and nutritional requirements of the bacterium. Importantly, the response to growth in human AF included significant changes in transcripts of multiple virulence genes such as adhesins, capsule, and hemolysin and IL-8 proteinase what might have consequences for the outcome of host-pathogen interactions. CONCLUSIONS/SIGNIFICANCE: Our work provides extensive new information about how the transcriptome of GBS responds

  8. Placental amniotic epithelial cells and their therapeutic potential in liver diseases

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    Asli eTahan

    2014-12-01

    Full Text Available As a unique source of stem cells, there is a growing interest in amniotic epithelial (AE cells. Placenta is readily available; in fact, it is often discarded following delivery. As such, it is without the ethical concerns of embryonic stem cells. Further advantages to AE include that AE cells do not demonstrate tumorigenicity upon transplantation, and are gifted with immunomodulatory and anti-inflamatory properties. Thus, AE cells have exceptional features for use as cell-based therapies for liver disease.

  9. Sternal Repair with Bone Grafts Engineered from Amniotic Mesenchymal Stem Cells

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    Steigman, Shaun A.; Ahmed, Azra; Shanti, Rabie M.; Tuan, Rocky S.; Valim, Clarissa; Fauza, Dario O.

    2013-01-01

    Background We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be employed in postnatal sternal repair. Methods Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n=6). Constructs were dynamically maintained in an osteogenic medium and equally divided into two groups with respect to time in vitro, namely 14.6 or 33.9 weeks. They were then used to repair full thickness sternal defects spanning 2–3 intercostal spaces in allogeneic kits (n=6). Grafts were submitted to multiple analyses 2 months thereafter. Results Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by micro-CT scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements pre- and post-implantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the two construct groups in any comparison. Conclusions Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction. PMID:19524727

  10. Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos

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    Fini, Jean-Baptiste; Mughal, Bilal B.; Le Mével, Sébastien; Leemans, Michelle; Lettmann, Mélodie; Spirhanzlova, Petra; Affaticati, Pierre; Jenett, Arnim; Demeneix, Barbara A.

    2017-03-01

    Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.

  11. Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos

    Science.gov (United States)

    Fini, Jean-Baptiste; Mughal, Bilal B.; Le Mével, Sébastien; Leemans, Michelle; Lettmann, Mélodie; Spirhanzlova, Petra; Affaticati, Pierre; Jenett, Arnim; Demeneix, Barbara A.

    2017-01-01

    Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development. PMID:28266608

  12. Senescence of primary amniotic cells via oxidative DNA damage.

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    Ramkumar Menon

    Full Text Available OBJECTIVE: Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB and preterm prelabor rupture of the membranes (pPROM; however, the precise mechanistic role of reactive oxygen species (ROS in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE, a predicted cause of pregnancy complications, on human amnion epithelial cells. METHODS: Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2'7'-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1, phospho-p38 (P-p38 MAPK, and p19(arf. Expression of senescence-associated β-galectosidase (SAβ-gal was used to confirm cell ageing in situ. RESULTS: ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min and significantly (p<0.01 at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19(Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19(Arf were not significantly affected by NAC. CONCLUSIONS: The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.

  13. Amniotic fluid

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    ... carefully. Removing a sample of the fluid through amniocentesis can provide information about the sex, health, and development of the fetus. Images Amniocentesis Amniotic fluid Polyhydramnios Amniotic fluid References Cunningham FG, ...

  14. Amniotic Mesenchymal Stem Cells: A New Source for Hepatocyte-Like Cells and Induction of CFTR Expression by Coculture with Cystic Fibrosis Airway Epithelial Cells

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    Valentina Paracchini

    2012-01-01

    Full Text Available Cystic fibrosis (CF is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33–50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

  15. Mesenchymal stem cells from amnion and amniotic fluid in the bovine.

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    Corradetti, B; Meucci, A; Bizzaro, D; Cremonesi, F; Lange Consiglio, A

    2013-04-01

    Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.

  16. The effects of cryopreservation on angiogenesis modulation activity of human amniotic membrane.

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    Yazdanpanah, Ghasem; Paeini-Vayghan, Ghodsieh; Asadi, Samira; Niknejad, Hassan

    2015-12-01

    Amniotic membrane (AM), as the innermost layer of placenta, has side dependent effects on the angiogenesis. Cryopreservation is a necessary process to avoid the challenging problems of fresh tissues; a procedure which makes the AM ready-to-use. Since the cryopreservation can influence the AM characteristics for experimental and clinical purposes, in this study the effects of cryopreservation were evaluated on angiogenesis modulation activity of the AM compared to fresh tissues in an animal model. The AM was implanted mesenchymal side up or epithelial side up in a rat dorsal skinfold chamber. The length and number of branches of formed capillaries were measured via intravital microscopy after 7 days. The amount of IL-8 (interleukin-8) and TIMP-2 (Tissue Inhibitor of Matrix Metalloproteinase-2) as two factors in amniotic cells which have great impacts on angiogenesis were evaluated using ELISA assay. The epithelial surface of cryopreserved AM had inhibitory effects on vessel formation. The cryopreserved amniotic mesenchymal side increased the vessel length and sprout. The result of cryopreserved AM on angiogenesis was similar to that of fresh tissues. The levels of IL-8 and TIMP-2 in cryopreserved samples were significantly less than fresh AMs which shows that angio-modulatory properties are not limited to the effects of amnion epithelial and mesenchymal stem cells and the other components such as extracellular matrix may contribute in angio-modulatory effects. These promising results show that inducing and inhibitory effects of the AM, which make it an appropriate candidate for different clinical situations, were maintained after cryopreservation.

  17. 两步法分离和培养人羊水来源胚胎间充质干细胞及其向神经元样细胞诱导分化的实验研究%Isolation of human pluripotent mesenchymal stem cells from second-trimester amniotic fluid using two kinds of culture protocol and their differentiation into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    冯建勋; 腊晓林; 马艳; 毕晓娟; 温浩

    2009-01-01

    fetal karyotyping. Flow cytometry analysis showed that they were positive for CD44, CD29, and CD105, but negative for CD45, CD34, or human leucocyte antigen-DR (HLA-DR). Importantly, a subpopulation of transcription factor Oct-4 positive cells could be detectable in the cultured MSCs from amniotic fluid. Moreover, MSCs from amniotic fluid obtained by two-stage culture protocol showed higher proliferation rate [(15.0±2.3)% vs. (10.0±1.8)%] and higher Oct-4 positive cell rate [(1.2±0.3)% vs. (0.9±0.2)%, both P<0.05]. After the induction, MSCs from amniotic fluid displayed processes that formed extensive networks. Positive cells for neurone specific enolase (NSE) constituted (54.76±3.65)%, and glial fibrillary acidic protein (GFAP) (36.28±4.27)%, respectively. Conclusion We demonstrate that human pluripotent MSCs are present in second-trimester amniotic fluid. The two-stage culture protocol could be a kind of simple one with high performance and it does not interfere with the routine fetal karyotyping. MSCs from amniotic fluid can be induced to differentiate into neuron-like cells in vitro by β-mercaptoethanol in optimal medium.

  18. Hyaluronic acid, an important factor in the wound healing properties of amniotic fluid: in vitro studies of re-epithelialisation in human skin wounds.

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    Nyman, Erika; Huss, Fredrik; Nyman, Torbjörn; Junker, Johan; Kratz, Gunnar

    2013-04-01

    Foetal wounds are unique in their ability to heal rapidly without forming scars. The amniotic fluid, rich in nutrients, growth factors, and hyaluronic acid, surrounds the foetus and is essential to foetal wound healing. The wound healing properties of foetal wounds may be the result of high concentrations of hyaluronic acid. This study aimed to verify that amniotic fluid induces re-epithelialisation in human skin wounds in vitro and to study whether this ability is dependent on hyaluronic acid. Standard deep dermal wounds were produced in vitro in human skin. The skin samples, with a central wound, were incubated in different culture media. Varying concentrations of amniotic fluid and amniotic fluid with added hyaluronidase were tested, and re-epithelialisation was assessed at 3, 7, and 12 days using light microscopy, after staining with haematoxylin and eosin. Amniotic fluid 50% resulted in a significantly higher (p < 0.05) grade of re-epithelialisation than Dulbecco's modified Eagle's medium and 10% amniotic fluid at all time points. When 50% amniotic fluid was compared with 10% foetal calf serum, no significant difference was found in grades of re-epithelialisation on days 3 and 12 and significantly higher grades of re-epithelialisation on day 7 (p < 0.05). Degradation of hyaluronic acid in the medium that contained 50% amniotic fluid gave significantly impaired re-epithelialisation (p < 0.05) on culture days 3 and 7. In conclusion, amniotic fluid promotes accelerated re-epithelialisation and hyaluronic acid is an important ingredient.

  19. Amniotic Fluid-Derived Stem Cells for Cardiovascular Tissue Engineering Applications

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    Petsche Connell, Jennifer; Camci-Unal, Gulden; Khademhosseini, Ali

    2013-01-01

    Recent research has demonstrated that a population of stem cells can be isolated from amniotic fluid removed by amniocentesis that are broadly multipotent and nontumorogenic. These amniotic fluid-derived stem cells (AFSC) could potentially provide an autologous cell source for treatment of congenital defects identified during gestation, particularly cardiovascular defects. In this review, the various methods of isolating, sorting, and culturing AFSC are compared, along with techniques for inducing differentiation into cardiac myocytes and endothelial cells. Although research has not demonstrated complete and high-yield cardiac differentiation, AFSC have been shown to effectively differentiate into endothelial cells and can effectively support cardiac tissue. Additionally, several tissue engineering and regenerative therapeutic approaches for the use of these cells in heart patches, injection after myocardial infarction, heart valves, vascularized scaffolds, and blood vessels are summarized. These applications show great promise in the treatment of congenital cardiovascular defects, and further studies of isolation, culture, and differentiation of AFSC will help to develop their use for tissue engineering, regenerative medicine, and cardiovascular therapies. PMID:23350771

  20. Microvesicles secreted from equine amniotic cells and their potential role in in vitro cell tendon repair

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    Claudia Perrini

    2015-07-01

    Full Text Available The regenerative mechanisms ascribed to mesenchymal stem cells (MSCs are classified into 3 categories: differentiating into damaged cell types, supplying nutrients, and improving survival/functions of the endogenous cells via paracrine actions. However, because of the inhospitable microenvironment of the injured tissues, a proportion of the implanted MSCs may quickly die, suggesting that other mechanisms might be present. This notion is supported by the overlapping beneficial effect (in terms of time of healing resulted  after the injection of AMCs or of amniotic mesenchymal cells - conditioned medium (AMC-CM  in equine spontaneous injured tendons and ligaments. Microvesicles (MVs released by cells are an integral component of the cell-to-cell communication network involved in tissue regeneration.In the present study, MVs secreted by AMCs were investigated with Nanosigth instrument and TEM. Then, the in vitro incorporation of MVs into equine tendon cells was studied by a dose-response curve. Lastly, the ability of MVs to counteract an in vitro inflammatory process induced by lipolysaccaride on tendon cells was studied evaluating the expression of pro-inflammatory genes like metallopeptidase (MPP 1 and 13, and prostaglandin-endoperoxide synthase 2 (COX2. Results demonstrated that AMCs secreted MVs ranging in size from 100 to 1000 nm with a prevalence of 100-200 nm large MVs. Tendon cells were able to uptake them with an inverse relationship between concentration and time. The greatest incorporation was detectable at 40x106 MVs/ml after 72h. MVs induced down-regulation of MMP1 and MMP13, suggesting that they may have contributed, along with soluble factors, to in vivo tendon regeneration.

  1. The development of a radioimmunoassay for reverse triiodothyronine sulfate in human serum and amniotic fluid

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    Wu, Sing-Yung (Veterans Administration Medical Center, Long Beach, CA (United States)); Huang, Wen-Sheng; Chen, Wei-Lian (Tri-Service General Hospital, Taipei (Taiwan, Province of China)); Polk, D.; Reviczky, A.; Williams, J. III; Chopra, I.J.; Fisher, D.A. (Univ. of California, Los Angeles (United States))

    1993-06-01

    Sulfated iodothyronines including T[sub 4]-sulfate (T[sub 4]S) and T[sub 3]-sulfate (T[sub 3]S) have been identified in human serum and amniotic fluid. Little is know, however, about the existence of sulfate conjugation of reverse T[sub 3] (rT[sub 3]S) in man. In this report, the authors employed a novel, sensitive, and specific rT[sub 3]S RIA to address this question. The rabbit antiserum to rT[sub 3]S was highly specific; T[sub 4], T[sub 3], rT[sub 3], and 3,3'-T[sub 2] showed less than 0.002% cross-reaction with the antiserum. Only T[sub 4]S and T[sub 3]S cross-reacted significantly (0.3% and 0.01%, respectively); other analogs cross-reacted less than 0.0001%. The detection threshold of the RIA was 14 pmol/L (1.0 ng/dL). The mean serum rT[sub 3]S concentration (pmol/L) was 40 in euthyroid subjects. Values were similar in hypothyroid patients (38) and pregnant women (52) but significantly (P < 0.01) elevated to 176 in hyperthyroid patient, 74 in patients with nonthyroid illnesses, and 684 in cord sera of newborns. Serum rT[sub 3]S increased significantly in hyperthyroid patients 1 day after administration of 1 g sodium ipodate orally. Reverse T[sub 3]S was detected consistently in amniotic fluid at 14 to 22 weeks of gestation and showed a marked rise 1-3 weeks after intraamniotic administration of 500-1000 [mu]g T[sub 4]. The various data suggest that : (1) rT[sub 3]S is a normal component of human serum and amniotic fluid; (2) it is derived from metabolism of T[sub 4] or rT[sub 3]; (3) circulating rT[sub 3]S increases in hyperthyroidism and in circumstances where type I 5'-monodeiodinating activity is low, e.g. nonthyroid illnesses, fetal life, and after administration of ipodate. 20 refs., 4 figs.

  2. Acute hypotension associated with intraoperative cell salvage using a leukocyte depletion filter during management of obstetric hemorrhage due to amniotic fluid embolism.

    Science.gov (United States)

    Rogers, William Kirke; Wernimont, Sarah A; Kumar, Girish C; Bennett, Eliza; Chestnut, David H

    2013-08-01

    Amniotic fluid embolism (AFE) is a rare but catastrophic obstetric complication that can lead to profound coagulopathy and hemorrhage. The role of cell salvage and recombinant human Factor VIIa (rFVIIa) administration in such cases remains unclear. We present a case of AFE and describe our experience with the use of cell salvage and rFVIIa administration during the resuscitation. Cell salvage and transfusion through a leukocyte depletion filter was attempted after the diagnosis of AFE was made, but the attempted transfusion was immediately followed by hypotension and a worsening of hemodynamics. rFVIIa, on the contrary, was used with clinical improvement in coagulopathy and without apparent adverse thrombotic effect.

  3. Amniotic fluid stem cells morph into a cardiovascular lineage: analysis of a chemically induced cardiac and vascular commitment.

    Science.gov (United States)

    Maioli, Margherita; Contini, Giovanni; Santaniello, Sara; Bandiera, Pasquale; Pigliaru, Gianfranco; Sanna, Raimonda; Rinaldi, Salvatore; Delitala, Alessandro P; Montella, Andrea; Bagella, Luigi; Ventura, Carlo

    2013-01-01

    Mouse embryonic stem cells were previously observed along with mesenchymal stem cells from different sources, after being treated with a mixed ester of hyaluronan with butyric and retinoic acids, to show a significant increase in the yield of cardiogenic and vascular differentiated elements. The aim of the present study was to determine if stem cells derived from primitive fetal cells present in human amniotic fluid (hAFSCs) and cultured in the presence of a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids show a higher yield of differentiation toward the cardiovascular phenotype as compared with untreated cells. During the differentiation process elicited by exposure to HA + BU + RA, genes controlling pluripotency and plasticity of stem cells, such as Sox2, Nanog, and Oct4, were significantly downregulated at the transcriptional level. At this point, a significant increase in expression of genes controlling the appearance of cardiogenic and vascular lineages in HA + BU + RA-treated cells was observed. The protein expression levels typical of cardiac and vascular phenotypes, evaluated by Western blotting, immunofluorescence, and flow cytometry, were higher in hAFSCs cultured in the presence of HA + BU + RA, as compared with untreated control cells. Appearance of the cardiac phenotype was further inferred by ultrastructural analysis using transmission and scanning electron microscopy. These results demonstrate that a mixture of HA + BU + RA significantly increased the yield of elements committed toward cardiac and vascular phenotypes, confirming what we have previously observed in other cellular types.

  4. Evaluation of the effects of acyclovir and/or human amniotic membrane on herpes virus culture and quantitative virus inactivity by real-time polymerase chain reaction

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    Feride Aylin Kantarci

    2014-08-01

    Full Text Available AIM: To investigate the permeability of amniotic membrane in herpes virus cell culture to acyclovir with real time polymerase chain reaction (RT-PCR.METHODS: Madin-Darby Bovine Kidney (MDBK cell culture and Bovine Herpes Virus (BHV1 type 1 were used in the study. Cell cultures were grouped into two on the basis of herpes virus inoculation. Each group was sub-grouped into three. Amniotic membrane (V-HAM, acyclovir (V-A, and amniotic membrane and acyclovir (V-HAM-A were applied to these subgroup cultures, respectively. After the application of the membrane and the drug, the cultures were evaluated at 24 and 48h for cytopathic effect positive (CPE+ with a tissue culture microscope. In the CPE (+ samples, the DNA was extracted for viral DNA analysis by RT-PCR.RESULTS: In control cultures without herpes virus CPE was not detected. Besides, amniotic membrane and acyclovir did not have cytotoxic effect on cell cultures. CPE were detected in Bovine Herpesvirus type-1 inoculated cell cultures after amniotic membrane and/or acyclovir application. DNA analysis with RT-PCR indicated that Cycle threshold (Ct values were lower in the BHV1 and membrane applied group (amniotic membrane group< acyclovir group< membrane and acyclovir group. This showed that membrane did not have antiviral effect. The membrane and acyclovir cell culture groups with high Ct values indicated that membrane was permeable and had a low barrier effect to drug,CONCLUSION: In our in-vitro study, we found that amniotic membrane, which can be used in the treatment of corneal diseases, did not have antiviral effect. Besides, we detected that amniotic membrane was permeable to acyclovir in BHV-1 inoculated MDBK cell culture. However, more studies are necessary to investigate the quantitative effects of amniotic membrane and acyclovir.

  5. Evaluation of the effects of acyclovir and/or human amniotic membrane on herpes virus culture and quantitative virus inactivity by real-time polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Feride; Aylin; Kantarci; Ali; Reza; Faraji; Aykut; Ozkul; Fikret; Akata

    2014-01-01

    ·AIM: To investigate the permeability of amniotic membrane in herpes virus cell culture to acyclovir with real time polymerase chain reaction(RT-PCR).·METHODS: Madin-Darby Bovine Kidney(MDBK) cell culture and Bovine Herpes Virus(BHV1) type 1 were used in the study. Cell cultures were grouped into two on the basis of herpes virus inoculation. Each group was sub-grouped into three. Amniotic membrane(V-HAM),acyclovir(V-A), and amniotic membrane and acyclovir(V-HAM-A) were applied to these subgroup cultures,respectively. After the application of the membrane and the drug, the cultures were evaluated at 24 and 48 h for cytopathic effect positive(CPE +) with a tissue culture microscope. In the CPE(+) samples, the DNA was extracted for viral DNA analysis by RT-PCR.·RESULTS: In control cultures without herpes virus CPE was not detected. Besides, amniotic membrane and acyclovir did not have cytotoxic effect on cell cultures.CPE were detected in Bovine Herpesvirus type-1inoculated cell cultures after amniotic membrane and/or acyclovir application. DNA analysis with RT-PCR indicated that Cycle threshold(Ct) values were lower in the BHV1 and membrane applied group(amniotic membrane group < acyclovir group < membrane and acyclovir group). This showed that membrane did not have antiviral effect. The membrane and acyclovir cell culture groups with high Ct values indicated thatmembrane was permeable and had a low barrier effect to drug.·CONCLUSION: In our in-vitro study, we found that amniotic membrane, which can be used in the treatment of corneal diseases, did not have antiviral effect. Besides,we detected that amniotic membrane was permeable to acyclovir in BHV-1 inoculated MDBK cell culture.However, more studies are necessary to investigate the quantitative effects of amniotic membrane and acyclovir.

  6. Amniotic fluid deficiency and congenital abnormalities both influence fluctuating asymmetry in developing limbs of human deceased fetuses.

    Directory of Open Access Journals (Sweden)

    Clara Mariquita Antoinette ten Broek

    Full Text Available Fluctuating asymmetry (FA, as an indirect measure of developmental instability (DI, has been intensively studied for associations with stress and fitness. Patterns, however, appear heterogeneous and the underlying causes remain largely unknown. One aspect that has received relatively little attention in the literature is the consequence of direct mechanical effects on asymmetries. The crucial prerequisite for FA to reflect DI is that environmental conditions on both sides should be identical. This condition may be violated during early human development if amniotic fluid volume is deficient, as the resulting mechanical pressures may increase asymmetries. Indeed, we showed that limb bones of deceased human fetuses exhibited increased asymmetry, when there was not sufficient amniotic fluid (and, thus, space in the uterine cavity. As amniotic fluid deficiency is known to cause substantial asymmetries and abnormal limb development, these subtle asymmetries are probably at least in part caused by the mechanical pressures. On the other hand, deficiencies in amniotic fluid volume are known to be associated with other congenital abnormalities that may disturb DI. More specifically, urogenital abnormalities can directly affect/reduce amniotic fluid volume. We disentangled the direct mechanical effects on FA from the indirect effects of urogenital abnormalities, the latter presumably representing DI. We discovered that both factors contributed significantly to the increase in FA. However, the direct mechanical effect of uterine pressure, albeit statistically significant, appeared less important than the effects of urogenital abnormalities, with an effect size only two-third as large. We, thus, conclude that correcting for the relevant direct factors allowed for a representative test of the association between DI and stress, and confirmed that fetuses form a suitable model system to increase our understanding in patterns of FA and symmetry development.

  7. 人类羊水源类ES细胞多能性干细胞的分离培养%ISOLATION AND CUTURE EMBRYONIC STEM-LIKE CELLS DERIVED HUMAN AMNIOTIC FLUID CELLS

    Institute of Scientific and Technical Information of China (English)

    华进联; 刘雨潇; 董武子

    2004-01-01

    @@ Stem cells are classified into embryonic stem (ES) cells and adult stem cells, which are the progenitors of any cell types of the body or tissues, and have the ability to self-renew and produce one or more differentiated cell types.

  8. Amniotic fluid water dynamics.

    Science.gov (United States)

    Beall, M H; van den Wijngaard, J P H M; van Gemert, M J C; Ross, M G

    2007-01-01

    Water arrives in the mammalian gestation from the maternal circulation across the placenta. It then circulates between the fetal water compartments, including the fetal body compartments, the placenta and the amniotic fluid. Amniotic fluid is created by the flow of fluid from the fetal lung and bladder. A major pathway for amniotic fluid resorption is fetal swallowing; however in many cases the amounts of fluid produced and absorbed do not balance. A second resorption pathway, the intramembranous pathway (across the amnion to the fetal circulation), has been proposed to explain the maintenance of normal amniotic fluid volume. Amniotic fluid volume is thus a function both of the amount of water transferred to the gestation across the placental membrane, and the flux of water across the amnion. Membrane water flux is a function of the water permeability of the membrane; available data suggests that the amnion is the structure limiting intramembranous water flow. In the placenta, the syncytiotrophoblast is likely to be responsible for limiting water flow across the placenta. In human tissues, placental trophoblast membrane permeability increases with gestational age, suggesting a mechanism for the increased water flow necessary in late gestation. Membrane water flow can be driven by both hydrostatic and osmotic forces. Changes in both osmotic/oncotic and hydrostatic forces in the placenta my alter maternal-fetal water flow. A normal amniotic fluid volume is critical for normal fetal growth and development. The study of amniotic fluid volume regulation may yield important insights into the mechanisms used by the fetus to maintain water homeostasis. Knowledge of these mechanisms may allow novel treatments for amniotic fluid volume abnormalities with resultant improvement in clinical outcome.

  9. Histochemical, Biochemical and Cell Biological aspects of tail regeneration in lizard, an amniote model for studies on tissue regeneration.

    Science.gov (United States)

    Alibardi, Lorenzo

    2014-01-01

    and inflammatory course, an inspiring model for understanding failure of tissue regeneration in higher vertebrates and humans. The participation of 5-Bromo-deoxyuridine (5BrdU) long retention cells, indicated as putative stem cells, for the following regeneration is analyzed and it shows that different tissue sites of the original tail contain putative stem cells that are likely activated from the wounding signal. In particular, the permanence of stem cells in the central canal of the spinal cord can explain the limited but important neurogenesis present in the caudal but also in the lumbar-thoracic spinal cord. In the latter, the limited number of glial and neurons regenerated is however sufficient to recover some limited hind limb movement after injury or spinal transection. Finally, the presence of stem cells in the spinal cord, in the regenerative blastema and skin allow to use these organs as a source of cells for studies on gene activation during cell differentiation in the new spinal cord, tail and in the epidermis. The above information form the basic knowledge for the future molecular studies on the specific gene activation capable to determine tail regeneration in lizards, and more in general genes involved in the reactivation of regeneration process in amniotes and humans.

  10. A comparative study on rabbit bone mesenchymal stem cells and human amniotic epithelial cells transplantation for rabbit limbal stem cell deficiency%兔骨髓间充质干细胞及人羊膜上皮细胞移植治疗兔角膜缘干细胞缺损的研究

    Institute of Scientific and Technical Information of China (English)

    卢建民; 吕秀丽; 马翔

    2011-01-01

    Background Limbal stem cell deficiency usually leads to blindness, and traditional therapy is limited. Recent research demonstrated that bone mesenchymal stem cells ( BMSCs ) and human amniotic epithelial cells(AECs) could differentiate into many kinds of cells including corneal epithelial cells, but the outcome and effect of these cells on corneal stem cell deficiency are still unclear. Objective This study aimed to observe and compare the effects of rabbit BMSCs and human AECs transplantation for rabbit limbal stem cell deficiency. Methods Eighteen clean New Zealand rabbits were randomly divided into the amniotic stroma(AS) group, rabbit BMSCs group and human AECs group with 6 rabbits for each group. Limbal stem cell deficiency models were established by putting a piece of filter paper that had been soaked in a NaOH solution at the corneal center. Rabbit BMSCs were isolated and purified by density gradient centrifugation combined with the attachment culture method, and human AECs were collected by a sequential trypsin digestion technique,and the third generation rabbit BMSCs and the first generation human AECs were identified with RT-PCR. After that,cells were inoculated onto the denuded AS and grafted to the corneal surface of the experimental animals. Twenty-eight days after cell transplantation, the therapeutic effects were evaluated based on the corneal neovascularization and opacity scores. Corneal histopathological examination and immunohistochemistry were performed to evaluate and compare the effectiveness among AS,rabbit BMSCs and human AECs on corneal stem cell deficiency. The procedure complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The third generation of rabbit BMSCs grew well after 12 hours, and the first generation of human AECs formed a membrane-like monolayer after 48 hours of incubation on AS. Immunohistochemistry staining showed that, 28 days after

  11. Cell sourcing for bone tissue engineering: amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells.

    Science.gov (United States)

    Peister, Alexandra; Woodruff, Maria A; Prince, Jarod J; Gray, Derwin P; Hutmacher, Dietmar W; Guldberg, Robert E

    2011-07-01

    Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem cells (AFS) and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-ε-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Fetal stem cells obtained from amniotic fluid and wharton's jelly expanded using platelet lysate for tissue engineering applications

    OpenAIRE

    Pinto, A. R.; Aleixo, I; Frias, A.M.; Fernandes, S.; Rocha, L; Reis, R. L.; Neves, N.M

    2012-01-01

    Extra-embryonic tissues, such as amniotic fluid (AF) and Wharton´s Jelly (WJ) of umbilical cord, offer many advantages over both embryonic and adult stem cell sources. These tissues are routinely discarded at parturition and the extracorporeal nature of these cell sources facilitates isolation, as well as the comparatively large volume and ease of physical manipulation theoretically increases the number of stem cells that can be isolated. Autologous approaches to use MSCs, n...

  13. Evaluation of Human Amniotic Membrane as a Wound Dressing for Split-Thickness Skin-Graft Donor Sites

    Directory of Open Access Journals (Sweden)

    Denys J. Loeffelbein

    2014-01-01

    Full Text Available Human amniotic membrane (HAM has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG donor sites in a swine model (Part A and a clinical trial (Part B. Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU foil (n=8 each. Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker, von Willebrand factor (vWF: angiogenesis, Ki-67 (cell proliferation, and laminin (basement membrane integrity. Part B: STSG donor sites in 45 adult patients (16 female/29 male were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n=15 each. Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative.

  14. Amniotic mesenchymal stem cells enhance wound healing in diabetic NOD/SCID mice through high angiogenic and engraftment capabilities.

    Directory of Open Access Journals (Sweden)

    Sung-Whan Kim

    Full Text Available Although human amniotic mesenchymal stem cells (AMMs have been recognised as a promising stem cell resource, their therapeutic potential for wound healing has not been widely investigated. In this study, we evaluated the therapeutic potential of AMMs using a diabetic mouse wound model. Quantitative real-time PCR and ELISA results revealed that the angiogenic factors, IGF-1, EGF and IL-8 were markedly upregulated in AMMs when compared with adipose-derived mesenchymal stem cells (ADMs and dermal fibroblasts. In vitro scratch wound assays also showed that AMM-derived conditioned media (CM significantly accelerated wound closure. Diabetic mice were generated using streptozotocin and wounds were created by skin excision, followed by AMM transplantation. AMM transplantation significantly promoted wound healing and increased re-epithelialization and cellularity. Notably, transplanted AMMs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, indicating a direct contribution to cutaneous closure. Taken together, these data suggest that AMMs possess considerable therapeutic potential for chronic wounds through the secretion of angiogenic factors and enhanced engraftment/differentiation capabilities.

  15. Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure.

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    Yu-Bao Zheng

    Full Text Available Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF. The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid-derived mesenchymal stem cells (AF-MSCs have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1-receptor antagonist (IL-1Ra plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra-expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine-induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6, and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.

  16. Amniotic fluid stem cell-based models to study the effects of gene mutations and toxicants on male germ cell formation

    Institute of Scientific and Technical Information of China (English)

    Claudia Gundacker; Helmut Dolznig; Mario Mikula; Margit Rosner; Oliver Brandau; Markus Hengstschl(a)ger

    2012-01-01

    Male infertility is a major public health issue predominantly caused by defects in germ cell development.In the past,studies on the genetic regulation of spermatogenesis as well as on negative environmental impacts have been hampered by the fact that human germ cell development is intractable to direct analysis in vivo.Compared with model organisms including mice,there are fundamental differences in the molecular processes of human germ cell development.Therefore,an in vitro model mimicking human sperm formation would be an extremely valuable research tool.In the recent past,both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been reported to harbour the potential to differentiate into primordial germ cells and gametes.We here discuss the pessibility to use human amniotic fluid stem (AFS) cells as a biological model.Since their discovery in 2003,AFS cells have been characterized to differentiate into cells of all three germ layers,to be genomically stable,to have a high proliferative potential and to be non-tumourigenic.In addition,AFS cells are not subject of ethical concerns.In contrast to iPS cells,AFSs cells do not need ectopic induction of pluripotency,which is often associated with only imperfectly cleared epigenetic memory of the source cells.Since AFS cells can be derived from amniocentesis with disease-causing mutations and can be transfected with high efficiency,they could be used in probing gene functions for spermatogenesis and in screening for male reproductive toxicity.

  17. 羊水预处理在羊水细胞培养瓶法中应用效果研究%Application effect research with amniotic fluid pretreatment in amniotic fluid cell culture bottle

    Institute of Scientific and Technical Information of China (English)

    向文秀

    2013-01-01

    目的 研究羊水预处理在羊水细胞培养瓶法中的应用效果.方法 采用羊水预处理培养瓶法(实验组)和未经处理羊水细胞培养瓶法(对照组)对100例18-24周的孕中期孕妇和152例25-32周的孕晚期孕妇进行羊水细胞培养.结果 100例孕18-24周羊水细胞培养实验组培养成功率高于对照组(P<0.05),实验组羊水细胞培养时间比对照组短(P<0.001),实验组有效核分裂数比对照组多(P <0.001);152例孕25-32周羊水细胞培养实验组培养成功率高于对照组(P<0.01),实验组羊水细胞培养时间比对照组短(P <0.001),实验组有效核分裂数比对照组多(P <0.001).结论 羊水预处理羊水细胞培养瓶法羊水细胞培养成功率高,细胞培养时间短,有效核分裂数多,可进行羊水产前诊断孕妇孕周范围增宽.%Objective:Research with amniotic fluid pretreatment in amniotic fluid cell culture bottle of application effect.Methods:100 samples of amniotic fluid from pregnant women during 18-24th-week gestation and 152 samples of amniotic fluid from pregnant women during 25-32th-week gestation were cultured in fluid pretreatment amniotic fluid cell culture bottle and untreated amniotic fluid cell culture bottle.Results:In 100 samples of pregnant women during 18-24th-week gestation.Culture success rate of the experimental group than the control group (P < 0.05).Amniotic fluid cell culture time of the experimental group than the control group short (P < 0.001).Effective mitotic count of the experimental group than the control group (P < 0.001).In 152 samples of pregnant women during 25-32th-week gestation,Culture success rate of the experimental group than the control group (P <0.01),Amniotic fluid cell culture time of the experimental group than the control group short (P < 0.001).Effective mitotic count of the experimental group than the control group (P < 0.001).Conclusion:Amniotic fluid pretreatment in amniotic fluid cell

  18. Analysis of perchlorate, thiocyanate, nitrate and iodide in human amniotic fluid using ion chromatography and electrospray tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Blount, Benjamin C. [Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341 (United States)]. E-mail: bblount@cdc.gov; Valentin-Blasini, Liza [Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341 (United States)

    2006-05-10

    Because of health concerns surrounding in utero exposure to perchlorate, we developed a sensitive and selective method for quantifying iodide, as well as perchlorate and other sodium-iodide symporter (NIS) inhibitors in human amniotic fluid using ion chromatography coupled with electrospray ionization tandem mass spectrometry. Iodide and NIS inhibitors were quantified using a stable isotope-labeled internal standards (Cl{sup 18}O{sub 4} {sup -}, S{sup 13}CN{sup -} and {sup 15}NO{sub 3} {sup -} with excellent assay accuracy of 100%, 98%, 99%, 95% for perchlorate, thiocyanate, nitrate and iodide, respectively, in triplicate analysis of spiked amniotic fluid sample). Excellent analytical precision (<5.2% RSD for all analytes) was found when amniotic fluid quality control pools were repetitively analyzed for iodide and NIS-inhibitors. Selective chromatography and tandem mass spectrometry reduced the need for sample cleanup, resulting in a rugged and rapid method capable of routinely analyzing 75 samples/day. Analytical response was linear across the physiologically relevant concentration range for the analytes. Analysis of a set of 48 amniotic fluid samples identified the range and median levels for perchlorate (0.057-0.71, 0.18 {mu}g/L), thiocyanate (<10-5860, 89 {mu}g/L), nitrate (650-8900, 1620 {mu}g/L) and iodide (1.7-170, 8.1 {mu}g/L). This selective, sensitive, and rapid method will help assess exposure of the developing fetus to low levels of NIS-inhibitors and their potential to inhibit thyroid function.

  19. Lectin-based analysis of fucose and sialic acid expressions on human amniotic IgA during normal pregnancy.

    Science.gov (United States)

    Orczyk-Pawiłowicz, Magdalena; Augustyniak, Daria; Hirnle, Lidia; Kątnik-Prastowska, Iwona

    2013-08-01

    The sugar moiety of IgA is known to provide a link between the innate and adaptive immune systems. Terminally located glycotopes on IgA are potential ligands engaged in the interactions which may modulate the biological activities of IgA. In the present work the expressions of Maackia amurensis (MAA), Sambucus nigra (SNA), Lens culinaris (LCA), Tetragonolobus purpureus (LTA), and Ulex europaeus (UEA) reactive glycotopes on maternal plasma and amniotic IgA were evaluated in relation to the progression of a normal human pregnancy, from the 2nd trimester, throughout the 3rd trimester, perinatal period, post-date pregnancy and delivery, by lectin-IgA-ELISA, using specific biotinylated lectins. The amniotic and maternal plasma IgA concentrations and a degree of SNA and LCA reactivity of maternal plasma IgA were almost unaltered during the normal pregnancy. The amniotic IgA from the 2nd trimester was decorated by MAA-, SNA-reactive and LCA-, LTA-, and UEA-reactive glycotopes. At the turn of the 2nd and 3rd trimesters the expression of MAA-, SNA-, LTA-, and UEA-reactive glycotopes, except for LCA-reactive, increased and remained almost at unaltered levels throughout the perinatal period and delivery. However, in the post-date pregnancy the expression of LCA-, LTA-, and UEA-reactive and SNA-reactive glycotopes were significantly higher. The unique fucosylated and sialylated glycovariants of amniotic IgA associated with the progression of the normal pregnancy may illustrate a general importance of carbohydrate-lectin receptor interactions in the control and modulation of biological events to ensuring homeostasis during pregnancy, protection and well-being of fetus.

  20. Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes.

    Science.gov (United States)

    Krause, Christopher D; Pestka, Sidney

    2015-12-01

    Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent's location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.

  1. Amniotic fluid embolism

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    Kiranpreet Kaur

    2016-01-01

    Full Text Available Amniotic fluid embolism (AFE is one of the catastrophic complications of pregnancy in which amniotic fluid, fetal cells, hair, or other debris enters into the maternal pulmonary circulation, causing cardiovascular collapse. Etiology largely remains unknown, but may occur in healthy women during labour, during cesarean section, after abnormal vaginal delivery, or during the second trimester of pregnancy. It may also occur up to 48 hours post-delivery. It can also occur during abortion, after abdominal trauma, and during amnio-infusion. The pathophysiology of AFE is not completely understood. Possible historical cause is that any breach of the barrier between maternal blood and amniotic fluid forces the entry of amniotic fluid into the systemic circulation and results in a physical obstruction of the pulmonary circulation. The presenting signs and symptoms of AFE involve many organ systems. Clinical signs and symptoms are acute dyspnea, cough, hypotension, cyanosis, fetal bradycardia, encephalopathy, acute pulmonary hypertension, coagulopathy etc. Besides basic investigations lung scan, serum tryptase levels, serum levels of C3 and C4 complements, zinc coproporphyrin, serum sialyl Tn etc are helpful in establishing the diagnosis. Treatment is mainly supportive, but exchange transfusion, extracorporeal membrane oxygenation, and uterine artery embolization have been tried from time to time. The maternal prognosis after amniotic fluid embolism is very poor though infant survival rate is around 70%.

  2. THE CONCENTRATION OF SURFACTANT PROTEIN-A IN AMNIOTIC FLUID DECREASES IN SPONTANEOUS HUMAN PARTURITION AT TERM

    Science.gov (United States)

    Chaiworapongsa, Tinnakorn; Hong, Joon-Seok; Hull, William M.; Kim, Chong Jai; Gomez, Ricardo; Mazor, Moshe; Romero, Roberto; Whitsett, Jeffrey A.

    2012-01-01

    OBJECTIVE The fetus is thought to play a central role in the onset of labor. Pulmonary surfactant protein (SP)-A, secreted by the maturing fetal lung, has been implicated in the mechanisms initiating parturition in mice. The present study was conducted to determine whether amniotic fluid concentrations of SP-A and SP-B change during human parturition. STUDY DESIGN Amniotic fluid SP-A and SP-B concentrations were measured with sensitive and specific ELISA in the following groups of pregnant women: 1) mid-trimester of pregnancy between 15th and 18th weeks of gestation (n=29); 2) term pregnancy not in labor (n=28); and 3) term pregnancy in spontaneous labor (n=26). Non-parametric statistics were used for analysis. RESULTS SP-A was detected in all amniotic fluid samples. SP-B was detected in 24.1% (7/29) of mid-trimester samples and in all samples at term. The median amniotic fluid concentration of SP-A and SP-B were significantly higher in women at term than in women in the mid-trimester (SP-A term no labor: median 5.6 μg/ml, range 2.2–15.2 μg/ml vs. mid-trimester: median 1.64 μg/ml, range 0.1–4.7 μg/ml; and SP-B term no labor: median 0.54 μg/ml, range 0.17–1.99 μg/ml vs. mid-trimester: median 0 μg/ml, range 0–0.35 μg/ml; both plabor was significantly lower than that in women at term not in labor (term in labor: median 2.7 μg/ml, range 1.2–10.1 μg/ml vs. term no labor: median 5.6 μg/ml, range 2.2–15.2 μg/ml; plabor and those not in labor (term in labor: median 0.47 μg/ml range 0.04–1.32 μg/ml vs. term no labor: median 0.54 μg/ml range 0.17–1.99 μg/ml; p=0.2). CONCLUSION The amniotic fluid concentration of surfactant protein-A decreases in spontaneous human parturition at term. PMID:18828058

  3. Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury.

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    Shih-Tao Wen

    Full Text Available High concentrations of oxygen aggravate the severity of lung injury in patients requiring mechanical ventilation. Although mesenchymal stem cells have been shown to effectively attenuate various injured tissues, there is limited information regarding a role for amniotic fluid stem cells (AFSCs in treating acute lung injury. We hypothesized that intravenous delivery of AFSCs would attenuate lung injury in an experimental model of hyperoxia-induced lung injury. AFSCs were isolated from EGFP transgenic mice. The in vitro differentiation, surface markers, and migration of the AFSCs were assessed by specific staining, flow cytometry, and a co-culture system, respectively. The in vivo therapeutic potential of AFSCs was evaluated in a model of acute hyperoxia-induced lung injury in mice. The administration of AFSCs significantly reduced the hyperoxia-induced pulmonary inflammation, as reflected by significant reductions in lung wet/dry ratio, neutrophil counts, and the level of apoptosis, as well as reducing the levels of inflammatory cytokine (IL-1β, IL-6, and TNF-α and early-stage fibrosis in lung tissues. Moreover, EGFP-expressing AFSCs were detected and engrafted into a peripheral lung epithelial cell lineage by fluorescence microscopy and DAPI stain. Intravenous administration of AFSCs may offer a new therapeutic strategy for acute lung injury (ALI, for which efficient treatments are currently unavailable.

  4. Positive effects of bFGF modified rat amniotic epithelial cells transplantation on transected rat optic nerve.

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    Jia-Xin Xie

    Full Text Available Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs with basic fibroblast growth factor (bFGF gene, preliminarily investigating its effect on transected optic nerve.A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs were observed and counted by employing biotin dextran amine (BDA and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43 within the injury site was examined with immunohistochemical staining.The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group.AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.

  5. Allogeneic amniotic membrane-derived mesenchymal stromal cell transplantation in a porcine model of chronic myocardial ischemia

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    Kimura M

    2012-01-01

    Full Text Available Introduction. Amniotic membrane contains a multipotential stem cell population and is expected to possess the machinery to regulate immunological reactions. We investigated the safety and efficacy of allogeneic amniotic membrane-derived mesenchymal stromal cell (AMSC transplantation in a porcine model of chronic myocardial ischemia as a preclinical trial. Methods. Porcine AMSCs were isolated from amniotic membranes obtained by cesarean section just before delivery and were cultured to increase their numbers before transplantation. Chronic myocardial ischemia was induced by implantation of an ameroid constrictor around the left circumflex coronary artery. Four weeks after ischemia induction, nine swine were assigned to undergo either allogeneic AMSC transplantation or normal saline injection. Functional analysis was performed by echocardiography, and histological examinations were carried out by immunohistochemistry 4 weeks after AMSC transplantation. Results. Echocardiography demonstrated that left ventricular ejection fraction was significantly improved and left ventricular dilatation was well attenuated 4 weeks after AMSC transplantation. Histological assessment showed a significant reduction in percentage of fibrosis in the AMSC transplantation group. Injected allogeneic green fluorescent protein (GFP-expressing AMSCs were identified in the immunocompetent host heart without the use of any immunosuppressants 4 weeks after transplantation. Immunohistochemistry revealed that GFP colocalized with cardiac troponin T and cardiac troponin I. Conclusions. We have demonstrated that allogeneic AMSC transplantation produced histological and functional improvement in the impaired myocardium in a porcine model of chronic myocardial ischemia. The transplanted allogeneic AMSCs survived without the use of any immunosuppressants and gained cardiac phenotype through either their transdifferentiation or cell fusion.

  6. Expression and effect of Notch1 during differentiation of human amniotic epithelial cells into neuron-like cells induced by salvia miltiorrhiza bunge in vitro%复方丹参注射液体外诱导人羊膜上皮细胞分化为神经元样细胞及Notch1表达的变化及意义

    Institute of Scientific and Technical Information of China (English)

    陈旭东; 王晓兰; 华新宇

    2012-01-01

    Objective:To explore the expression and effect of Notchl during differentiation of human amniotic epithelial cells (hAECs) into neurons induced by salvia miltiorrhiza bunge (SMB) in vitro. Methods:hAECs were divided into a SMB pre-treatment group and a control group, and SMB group had 5 subgroups. The morphological changes of hAECs were oberved under an optical microscope and the expressions of NSE, MAP-2 and Notchl were identified with the immunocytochemical method. RT-PCR was further used to detect the expression of multiple genes Oct4, Notchl and NSE. The cell viability was measured with methyl thiazolyl tetrazolium (MTT) methods. Results: Under the convert microscope, it was observed that the shape of hAECs started to change, and there were several axon or dendrite-like processes out from the cell body induced by SMB after 24 h. The NSE and MAP-2 positive straining was the strongest in the group of cells in 1. 5 μl/ml at 48 h, while the Notchl positive straining was less than that in the control group. The cell survival rate of every subgroup was decreased compared with the control group. RT-PCR showed that the expression of Oct4 and Notchl was less than that of the control group, wlile NSE was higher than that of the control group. Conclusion: SMB can induce hAECs into neuron-like cells, and the expression of Notchl decreased in the progress (especially at 1. 5μl/ml-48 h). Its possible mechanism may rely on the inhibition of Notch signal pathway.g%目的:探讨复方丹参注射液体外诱导入羊膜上皮细胞(hAECs)向神经细胞分化中Notch1的表达变化的意义.方法:将hAECs分为丹参诱导组和对照组.丹参注射液组设立5个浓度亚组,诱导hAECs分化为神经细胞,观察不同浓度丹参诱导组间细胞形态变化,应用免疫细胞化学显色鉴定神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP-2)、Notch1.RT-PCR进一步鉴定细胞多能基因Oct4、Notch1、神经元标记物NSE,四甲基偶氮唑盐比

  7. Differential Expression of Extracellular Matrix and Adhesion Molecules in Fetal-Origin Amniotic Epithelial Cells of Preeclamptic Pregnancy.

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    Myung-Sun Kim

    Full Text Available Preeclampsia is a common disease that can occur during human pregnancy and is a leading cause of both maternal and neonatal morbidity and mortality. Inadequate trophoblast invasion and deficient remodeling of uterine spiral arteries are associated with preeclampsia (PE. The development of this syndrome is thought to be related to multiple factors. Recently, we isolated patient-specific human amniotic epithelial cells (AECs from the placentas of 3 women with normal pregnancy and 3 with preeclamptic pregnancy. Since the characteristics of human AECs in PE are different from those in normal pregnancy, we sought to confirm the genes differentially expressed between preeclamptic pregnancy and normal pregnancy. Therefore, we performed transcriptome analysis to investigate the candidate genes associated with the possible pathophysiology of preeclampsia. Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID and Kyoto Encyclopedia of Genes and Genomes (KEGG online resource. In this study, we selected a total of 12 pathways and focused on extracellular matrix-related and biological adhesion molecules. Using RT-PCR array and real-time PCR, we confirmed that COL16A1, ITGB2, and LAMA3 were significantly up-regulated, but ITGA1, ITGA3, ITGA6, MMP1, MMP3, MMP10 and MMP11 were significantly down-regulated in preeclamptic fetal origin cells. Taken together, we suggest that the genes and pathways identified here may be responsible for the occurrence and development of PE, and controlling their expression may play a role in communication with fetal-maternal placenta to keep normal pregnancy.

  8. Differential Expression of Extracellular Matrix and Adhesion Molecules in Fetal-Origin Amniotic Epithelial Cells of Preeclamptic Pregnancy.

    Science.gov (United States)

    Kim, Myung-Sun; Yu, Ji Hea; Lee, Min-Young; Kim, Ah Leum; Jo, Mi Hyun; Kim, MinGi; Cho, Sung-Rae; Kim, Young-Han

    2016-01-01

    Preeclampsia is a common disease that can occur during human pregnancy and is a leading cause of both maternal and neonatal morbidity and mortality. Inadequate trophoblast invasion and deficient remodeling of uterine spiral arteries are associated with preeclampsia (PE). The development of this syndrome is thought to be related to multiple factors. Recently, we isolated patient-specific human amniotic epithelial cells (AECs) from the placentas of 3 women with normal pregnancy and 3 with preeclamptic pregnancy. Since the characteristics of human AECs in PE are different from those in normal pregnancy, we sought to confirm the genes differentially expressed between preeclamptic pregnancy and normal pregnancy. Therefore, we performed transcriptome analysis to investigate the candidate genes associated with the possible pathophysiology of preeclampsia. Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) online resource. In this study, we selected a total of 12 pathways and focused on extracellular matrix-related and biological adhesion molecules. Using RT-PCR array and real-time PCR, we confirmed that COL16A1, ITGB2, and LAMA3 were significantly up-regulated, but ITGA1, ITGA3, ITGA6, MMP1, MMP3, MMP10 and MMP11 were significantly down-regulated in preeclamptic fetal origin cells. Taken together, we suggest that the genes and pathways identified here may be responsible for the occurrence and development of PE, and controlling their expression may play a role in communication with fetal-maternal placenta to keep normal pregnancy.

  9. Amniotic fluid stem cells with low γ-interferon response showed behavioral improvement in Parkinsonism rat model.

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    Yu-Jen Chang

    Full Text Available Amniotic fluid stem cells (AFSCs are multipotent stem cells that may be used in transplantation medicine. In this study, AFSCs established from amniocentesis were characterized on the basis of surface marker expression and differentiation potential. To further investigate the properties of AFSCs for translational applications, we examined the cell surface expression of human leukocyte antigens (HLA of these cells and estimated the therapeutic effect of AFSCs in parkinsonian rats. The expression profiles of HLA-II and transcription factors were compared between AFSCs and bone marrow-derived mesenchymal stem cells (BMMSCs following treatment with γ-IFN. We found that stimulation of AFSCs with γ-IFN prompted only a slight increase in the expression of HLA-Ia and HLA-E, and the rare HLA-II expression could also be observed in most AFSCs samples. Consequently, the expression of CIITA and RFX5 was weakly induced by γ-IFN stimulation of AFSCs compared to that of BMMSCs. In the transplantation test, Sprague Dawley rats with 6-hydroxydopamine lesioning of the substantia nigra were used as a parkinsonian-animal model. Following the negative γ-IFN response AFSCs injection, apomorphine-induced rotation was reduced by 75% in AFSCs engrafted parkinsonian rats but was increased by 53% in the control group after 12-weeks post-transplantation. The implanted AFSCs were viable, and were able to migrate into the brain's circuitry and express specific proteins of dopamine neurons, such as tyrosine hydroxylase and dopamine transporter. In conclusion, the relative insensitivity AFSCs to γ-IFN implies that AFSCs might have immune-tolerance in γ-IFN inflammatory conditions. Furthermore, the effective improvement of AFSCs transplantation for apomorphine-induced rotation paves the way for the clinical application in parkinsonian therapy.

  10. Peculiarity of Porcine Amniotic Membrane and Its Derived Cells: A Contribution to the Study of Cell Therapy from a Large Animal Model.

    Science.gov (United States)

    Lange-Consiglio, Anna; Corradetti, Bruna; Bertani, Sabrina; Notarstefano, Valentina; Perrini, Claudia; Marini, Maria Giovanna; Arrighi, Silvana; Bosi, Giampaolo; Belloli, Angelo; Pravettoni, Davide; Locatelli, Valentina; Cremonesi, Fausto; Bizzaro, Davide

    2015-12-01

    The aim of this work was to provide, for the first time, a protocol for isolation and characterization of stem cells from porcine amniotic membrane in view of their potential uses in regenerative medicine. From three samples of allanto-amnion recovered at delivery, the amniotic membrane was stripped from overlying allantois and digested with trypsin and collagenase to isolate epithelial (amniotic epithelial cells [AECs]) and mesenchymal cells, respectively. Proliferation, differentiation, and characterization studies by molecular biology and flow cytometry were performed. Histological examination revealed very few mesenchymal cells in the stromal layer, and a cellular yield of AECs of 10 × 10(6)/gram of digested tissue was achieved. AECs readily attached to plastic culture dishes displaying typical cuboidal morphology and, although their proliferative capacity decreased to the fifth passage, AECs showed a mean doubling time of 24.77 ± 6 h and a mean frequency of one fibroblast colony-forming unit (CFU-F) for every 116.75 plated cells. AECs expressed mesenchymal stem cell (MSC) mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct 4), whereas they were negative for CD34 and MHCII. Mesodermic, ectodermic, and endodermic differentiation was confirmed by staining and expression of specific markers. We conclude that porcine amniotic membrane can provide an attractive source of stem cells that may be a useful tool for biomedical research.

  11. Photo-cross-linking of amniotic membranes for limbal epithelial cell cultivation

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Jui-Yang, E-mail: jylai@mail.cgu.edu.tw

    2014-12-01

    In the present study, we developed photo-cross-linked amniotic membrane (AM) as a limbal stem cell niche. After ultraviolet (UV) irradiation for varying time periods, the biological tissues were studied by determinations of cross-linking structure, degradability, and nutrient permeation ability. Our results showed that the number of cross-links per unit mass of AM significantly increased with increasing illumination time from 5 to 50 min. However, the cross-link formation was inhibited by longer irradiation time (i.e., 150 min), probably due to the scission of tissue collagen chains through irradiation. The biological stability and matrix permeability of photo-cross-linked AM materials strongly depended on their cross-linking densities affected by the UV irradiation. In vitro biocompatibility studies including cell viability and pro-inflammatory gene expression analyses demonstrated that, irrespective of the irradiation time employed, the physically cross-linked biological tissues exhibited negligible cytotoxicity and similar interleukin-6 (IL-6) mRNA levels. The data clearly indicate that these AM matrices do not cause potential harm to the corneal epithelial cells. After the growth of limbal epithelial cells (LECs) on AM substrates, Western blot analyses were conducted to examine the expression of ABCG2. It was found that the ability of UV-irradiated AM to maintain the undifferentiated precursor cell phenotype was significantly enhanced with increasing extent of photo-cross-linking. In summary, the UV irradiation time may have a profound influence on the fabrication of photo-cross-linked AM matrices for LEC cultivation. - Highlights: • We report the development of photo-cross-linked AM as a limbal stem cell niche. • Cross-linked structure of tissue materials was controlled by UV irradiation time. • Biostability and matrix permeability of AM depended on cross-linking density. • All the studied photo-cross-linked AM showed good in vitro biocompatibility.

  12. Culture of mouse amniotic fluid-derived cells on irradiated STO feeders results in the generation of primitive endoderm cell lines capable of self-renewal in vitro.

    Science.gov (United States)

    Babic, Aleksandar M; Jang, Sunyoung; Nicolov, Eugenia; Voicu, Horatiu; Luckey, Chance J

    2013-01-01

    The cells present in amniotic fluid (AF) are currently used for prenatal diagnosis of fetal anomalies but are also a potential source of cells for cell therapy. To better characterize putative progenitor cell populations present in AF, we used culture conditions that support self-renewal to determine if these promoted the generation of stable cell lines from AF-derived cells (AFC). Cells isolated from E11.5 mouse were cultured on irradiated STO fibroblast feeder layers in human embryonic germ cell derivation conditions. The cultures grew multicellular epithelial colonies that could be repropagated from single cells. Reverse transcription semiquantitative polymerase chain reaction of established cell lines revealed that they belonged to the extraembryonic endoderm (ExEn) expressing high levels of Gata6, Gata4, Sox17, Foxa2 and Sox7 mRNA. Hierarchical clustering based on the whole transcriptome expression profile of the AFC lines (AFCL) shows significant correlation between transcription profiles of AFCL and blastocyst-derived XEN, an ExEn cell line. In vitro differentiation of AFCL results in the generation of cells expressing albumin and α-fetoprotein (AFP), while intramuscular injection of AFCL into immunodeficient mice produced AFP-positive tumors with primitive endodermal appearance. Hence, E11.5 mouse AF contains cells that efficiently produce XEN lines. These AF-derived XEN lines do not spontaneously differentiate into embryonic-type cells but are phenotypically stable and have the capacity for extensive expansion. The lack of requirement for reprogramming factors to turn AF-derived progenitor cells into stable cell lines capable of massive expansion together with the known ability of ExEn to contribute to embryonic tissue suggests that this cell type may be a candidate for banking for cell therapies. © 2013 S. Karger AG, Basel

  13. Células-tronco do líquido amniótico Amniotic fluid stem cells

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    Sergio P. Bydlowski

    2009-05-01

    Full Text Available Desde o primeiro isolamento e cultivo de células-tronco embrionárias humanas, há mais de 10 anos, seu uso na pesquisa e terapia foi inibida por considerações éticas complexas e pelo risco de transformação maligna destas células indiferenciadas após transplante no paciente. As células-tronco adultas são eticamente aceitas e o risco de transformação maligna é muito baixo. Entretanto, seu potencial de diferenciação e sua capacidade proliferativa são limitados. Cerca de 6 anos atrás, a descoberta de célulastronco no líquido amniótico que expressavam Oct-4, um marcador específico de pluripotencialidade, com alta capacidade de proliferação e diferenciação, iniciou um novo campo promissor na área das células-tronco. Estas células têm potencial de se diferenciar em células dos três folhetos germinativos. Não formam tumores in vivo e não levantam os questionamentos éticos associados com as células-tronco embrionárias humanas. Futuras investigações revelarão se as células-tronco do líquido amniótico realmente irão representar um tipo intermediário com vantagens em relação tanto às células-tronco embrionárias quanto às adultas. Este artigo faz uma revisão acerca destes tópicos e das características biológicas das células-tronco do líquido amniótico.Since the first successful isolation and cultivation of human embryonic stem cells about 10 years ago, their use for research and therapy has been constrained by complex ethical considerations as well as by the risk of development of malignancies of undifferentiated embryonic stem cells after transplantation into the patient. Adult stem cells are ethically acceptable and the risk of tumor development is low. However, their differentiation potential and proliferative capacity are limited. About 6 years ago, the discovery of Oct-4 expressing amniotic fluid stem cells, a specific marker of pluripotency, with a high proliferative capacity, and multilineage

  14. 聚乳酸/羟基磷灰石膜与人羊膜基质细胞联合构建骨组织工程细胞/支架复合体%Combination application of human amniotic mesenchymal cells and poly (L-lactic acid)/hydroxyapatite membranes for construction of cells/scaffold complexes in bone tissue engineering

    Institute of Scientific and Technical Information of China (English)

    钟金晟; 梅芳; 齐伟宏; 杨小平

    2012-01-01

    背景 前期研究通过静电纺丝技术获得的聚乳酸/羟基磷灰石膜有利于细胞的贴附和生长.目的 分析电纺聚乳酸/羟基磷灰石膜和人羊膜基质细胞构建骨组织工程细胞/支架复合体的可行性.方法 利用MTT 法检测聚乳酸和聚乳酸/羟基磷灰石膜浸提液对人羊膜基质细胞增殖的影响;将第3 代人羊膜基质细胞培养于含聚乳酸和聚乳酸/羟基磷灰石膜的成骨诱导培养液中,进行组织学检查及免疫荧光细胞化学染色检测.结果 与结论 聚乳酸和聚乳酸/羟基磷灰石膜浸提液对人羊膜基质细胞均无明显细胞毒性.与两种膜材料复合培养后,人羊膜基质细胞细胞增殖明显,可观察到钙化结节的形成,钙化结节处细胞Ⅰ型胶原和碱性磷酸酶表达阳性,且聚乳酸/羟基磷灰石膜组细胞钙化结节数量及成熟程度优于聚乳酸组.说明电纺聚乳酸/羟基磷灰石膜与人羊膜基质细胞可以共同构建成细胞/支架复合体,具有应用于骨组织工程的潜力.%BACKGROUND: Previous study has shown that electrospun poly (L-lactic acid)/hydroxyapatite (PLLA/HA) membranes aresuitable for cellular adhesion and proliferation.OBJECTIVE: To investigate the feasibility of the human amniotic mesenchymal cells (hAMCs) and PLLA/HA membranesconstructing the complex of seed cells/scaffold in bone tissue engineering.METHODS: Effect of leaching liquor from the PLLA and PLLA/HA membranes on the proliferation of hAMCs was detected by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay method. The third passage of hAMCs were cultured in theosteogenic induce media containing PLLA and PLLA/HA membranes, and then were detected by histological andimmunofluorescence cytochemical staining.RESULTS AND CONCLUSION: The leaching liquor of the PLLA and PLLA/HA had no cytotoxicity on the hAMCs. After seededonto two kinds of the membranes, hAMCs proliferated actively and calcified nodules were

  15. Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model.

    Science.gov (United States)

    Piccoli, Martina; Franzin, Chiara; Bertin, Enrica; Urbani, Luca; Blaauw, Bert; Repele, Andrea; Taschin, Elisa; Cenedese, Angelo; Zanon, Giovanni Franco; André-Schmutz, Isabelle; Rosato, Antonio; Melki, Judith; Cavazzana-Calvo, Marina; Pozzobon, Michela; De Coppi, Paolo

    2012-08-01

    Mutations in the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophy, a fatal neuromuscular disorder. Mice carrying a homozygous deletion of Smn exon 7 directed to skeletal muscle (HSA-Cre, Smn(F7/F7) mice) present clinical features of human muscular dystrophies for which new therapeutic approaches are highly warranted. Herein we demonstrate that tail vein transplantation of mouse amniotic fluid stem (AFS) cells enhances the muscle strength and improves the survival rate of the affected animals. Second, after cardiotoxin injury of the Tibialis Anterior, only AFS-transplanted mice efficiently regenerate. Most importantly, secondary transplants of satellite cells (SCs) derived from treated mice show that AFS cells integrate into the muscle stem cell compartment and have long-term muscle regeneration capacity indistinguishable from that of wild-type-derived SC. This is the first study demonstrating the functional and stable integration of AFS cells into the skeletal muscle, highlighting their value as cell source for the treatment of muscular dystrophies. Copyright © 2012 AlphaMed Press.

  16. Construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells in vitro%人羊膜间充质干细胞体外构建组织工程角膜上皮层的实验研究

    Institute of Scientific and Technical Information of China (English)

    肖盼; 陈剑

    2015-01-01

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

  17. Fetal antigen 2: an amniotic protein identified as the aminopropeptide of the alpha 1 chain of human procollagen type I

    DEFF Research Database (Denmark)

    Teisner, B; Rasmussen, H B; Højrup, P

    1992-01-01

    -PAGE analysis gave an M(r) = 27 kDa under reducing and non-reducing conditions for both forms, whereas the exact M(r) determined by mass spectrometry was 14,343 +/- 3 Da. FA2 was N-terminally blocked and after tryptic digestion the amino acid composition and sequences of the peptides showed identity...... with the aminopropeptide of the alpha 1 chain of human procollagen type I as determined by nucleotide sequences. After oxidative procedures normally employed for radio-iodination (iodogen and chloramine-T), FA2 lost its immunoreactivity. An antigen which cross-reacted with polyclonal rabbit anti-human FA2 was demonstrated...... in fetal calf serum. Gel filtration with analysis of fractions by inhibition ELISA showed that the bovine homologue was present in the same molecular forms as those in human amniotic fluid, and immunohistochemical analysis with anti-human FA2 showed that its distribution in bovine skin was identical...

  18. Proteome analysis of human amnion and amniotic fluid by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Park, Soo-Jin; Yoon, Won-Gap; Song, Jin-Su; Jung, Hyun Sook; Kim, Chong Jai; Oh, Soo Young; Yoon, Bo Hyun; Jung, Guhung; Kim, Hie-Joon; Nirasawa, Takashi

    2006-01-01

    Proteome analysis by 2-DE and PMF by MALDI-TOF MS was performed on human amnion and amniotic fluid at term. Ninety-two soluble and nineteen membrane proteins were identified from amnion. Thirty-five proteins were identified from amniotic fluid. Calgranulin A and B were found in all patients infected with Ureaplasma urealyticum, but not in any of the patients without infection, indicating that they are potential markers of intrauterine infection. Identity of calgranulin A and B was confirmed by MALDI-TOF/TOF MS. This study represents the first extensive analysis of the human amnion and amniotic fluid proteome at term and demonstrates that 2-DE and MALDI-TOF MS is a useful tool for identifying clinically significant biomarkers of problematic pregnancies.

  19. Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor.

    Science.gov (United States)

    Maddipati, Krishna Rao; Romero, Roberto; Chaiworapongsa, Tinnakorn; Zhou, Sen-Lin; Xu, Zhonghui; Tarca, Adi L; Kusanovic, Juan Pedro; Munoz, Hernan; Honn, Kenneth V

    2014-11-01

    Lipid mediators play an important role in reproductive biology, especially, in parturition. Enhanced biosynthesis of eicosanoids, such as prostaglandin E2 (PGE2) and PGF2α, precedes the onset of labor as a result of increased expression of inducible cyclooxygenase 2 (COX-2) in placental tissues. Metabolism of arachidonic acid results in bioactive lipid mediators beyond prostaglandins that could significantly influence myometrial activity. Therefore, an unbiased lipidomic approach was used to profile the arachidonic acid metabolome of amniotic fluid. In this study, liquid chromatography-mass spectrometry was used for the first time to quantitate these metabolites in human amniotic fluid by comparing patients at midtrimester, at term but not in labor, and at term and in spontaneous labor. In addition to exposing novel aspects of COX pathway metabolism, this lipidomic study revealed a dramatic increase in epoxygenase- and lipoxygenase-pathway-derived lipid mediators in spontaneous labor with remarkable product selectivity. Despite their recognition as anti-inflammatory lipid mediators and regulators of ion channels, little is known about the epoxygenase pathway in labor. Epoxygenase pathway metabolites are established regulators of vascular homeostasis in cardiovascular and renal physiology. Their presence as the dominant lipid mediators in spontaneous labor at term portends a yet undiscovered physiological function in parturition. © FASEB.

  20. Metabolomics of Human Amniotic Fluid and Maternal Plasma during Normal Pregnancy.

    Science.gov (United States)

    Orczyk-Pawilowicz, Magdalena; Jawien, Ewa; Deja, Stanislaw; Hirnle, Lidia; Zabek, Adam; Mlynarz, Piotr

    2016-01-01

    Metabolic profiles of amniotic fluid and maternal blood are sources of valuable information about fetus development and can be potentially useful in diagnosis of pregnancy disorders. In this study, we applied 1H NMR-based metabolic profiling to track metabolic changes occurring in amniotic fluid (AF) and plasma (PL) of healthy mothers over the course of pregnancy. AF and PL samples were collected in the 2nd (T2) and 3rd (T3) trimester, prolonged pregnancy (PP) until time of delivery (TD). A multivariate data analysis of both biofluids reviled a metabolic switch-like transition between 2nd and 3rd trimester, which was followed by metabolic stabilization throughout the rest of pregnancy probably reflecting the stabilization of fetal maturation and development. The differences were further tested using univariate statistics at α = 0.001. In plasma the progression from T2 to T3 was related to increasing levels of glycerol, choline and ketone bodies (3-hydroxybutyrate and acetoacetate) while pyruvate concentration was significantly decreased. In amniotic fluid, T2 to T3 transition was associated with decreasing levels of glucose, carnitine, amino acids (valine, leucine, isoleucine, alanine, methionine, tyrosine, and phenylalanine) and increasing levels of creatinine, succinate, pyruvate, choline, N,N-dimethylglycine and urocanate. Lactate to pyruvate ratio was decreased in AF and conversely increased in PL. The results of our study, show that metabolomics profiling can be used to better understand physiological changes of the complex interdependencies of the mother, the placenta and the fetus during pregnancy. In the future, these results might be a useful reference point for analysis of complicated pregnancies.

  1. Synthetic bone substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation.

    Directory of Open Access Journals (Sweden)

    Barbara Barboni

    Full Text Available BACKGROUND: Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. AIM: In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC, loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT technique, was evaluated in an animal study. MATERIAL AND METHODS: Two blocks of synthetic bone substitute (∼0.14 cm(3, alone or engineered with 1×10(6 ovine AEC (oAEC, were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.. Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT, morphological, morphometric and biochemical analyses. RESULTS AND CONCLUSIONS: The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation, data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their

  2. Amniotic fluid stem cells inhibit the progression of bleomycin-induced pulmonary fibrosis via CCL2 modulation in bronchoalveolar lavage.

    Directory of Open Access Journals (Sweden)

    Orquidea Garcia

    Full Text Available The potential for amniotic fluid stem cell (AFSC treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF, is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0 or chronic (day 14 intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL, but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.

  3. A Study on the Preservation of Fresh Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    Liying Xu; Shiyou Zhou; Jiaqi Chen; Longshan Chen; Mei Zhang

    2001-01-01

    Objective: To establish the standard preservation methods of fresh amniotic membranefor clinical use.Methods: Human placentas were collected aseptically from selective caesarean sectionsin normal women in time. Amniotic or placental membrane were peeled and preserved inN.S, P.B. SorDMEM at4°C or cultured in DMEM at 37°C, 5% CO2. Trypan-bluestaining, light and electronic microscopy were observed every six hours after preservation.Results: Seventy percent of amniotic epithelial cells survived after preservation in N. Sfor 6 hours, PBS 12 hours, DMEM 24 hours and 1 week in tissue culture. The amountof living epithelial cells maintained in placental membrane preservation was less thanthat in amniotic membrane preservation at the same time (t-test, P < 0. 01) . Nocollagen degeneration was found during preservation.Conclusion: Preservative solution and time will affect the maintenance time of freshamniotic membrane greatly. Fresh amniotic membrane should be preserved within 6hours in N.S, 12 hours in P.B.S, 24 hours in DMEM at 4 °C and 1 week in tissteculture for clinical use.

  4. Treatment effects of human amniotic mesenchymal stem cells in a rat model of parkinson's disease with different transplantation approaches:a comparative study%人羊膜间充质干细胞移植治疗帕金森病模型大鼠的疗效评价

    Institute of Scientific and Technical Information of China (English)

    陈勇; 方宁; 陈代雄; 赵春华

    2015-01-01

    目的 观察不同途径移植人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)对帕金森病(Parkinson's disease,PD)模型大鼠的生物学效应及其在体内的分化.方法 采用胰蛋白酶-胶原酶消化法分离hAMSCs,流式细胞术分析表型.40只雌性Wistar大鼠随机分为假手术组、模型组、hAMSCs静脉移植组和原位移植组.采用单侧前脑内侧束(MFB)注射6-羟基多巴胺建立PD大鼠模型.通过舌下静脉或于MFB原位移植3 ×105个hAMSCs.腹腔注射阿朴吗啡诱导旋转观察大鼠的行为变化,免疫荧光染色法检测人细胞核抗原及神经元微管结合蛋白(microtubule-associated protein 2,MAP-2)的表达,免疫组化染色法检测酪氨酸羟化酶(tyrosinehydroxylase,TH)的表达.结果 与模型组比较,hAMSCs静脉移植组和原位移植组大鼠旋转次数均明显减少(均P<0.05),前者行为学改善可持续至移植后6 w,后者则至8 w;免疫荧光染色显示,hAMSCs在原位移植区可存活至少12 w,并表达MAP-2;免疫组化染色显示静脉和原位移植hAMSCs均可上调PD模型大鼠黑质TH表达,但后者强于前者.结论 hAMSCs能改善PD模型大鼠的运动行为,原位移植优于静脉移植,其机制可能与上调黑质TH表达有关.hAMSCs可在原位移植部位分化为多巴胺能神经元样细胞.

  5. Human Amniotic Membrane Dressing: an Excellent Method for Outpatient Management of Burn Wounds

    Directory of Open Access Journals (Sweden)

    Ali Akbar Mohammadi

    2009-03-01

    Full Text Available Background: Burns are among the most common traumas indeveloping countries, which consume large amounts of medicalresources. It is important to find an appropriate materialfor dressing of burn wounds that improves healing and is readilyavailable, easily applicable, and economical.Methods: In a single-blind randomized controlled clinicaltrial from March to October 2006, 211 patients with less than20% burn were enrolled into two groups. The first group contained104 patients with average burn of 11.90± 3.80% of totalbody surface area (TBSA for whom amnion dressing wasused. The second group composed of 107 patients with averageburn of 12.30± 4.14% of TBSA treated with routine silversulfadiazine dressing.Results: Amniotic membrane usage was accompanied by accelerationin wound healing, less need for skin graft, and lesspain. The mean healing time in superficial parts of burnwounds in the amnion group was significantly shorter than thecontrol group (9.50±2.13 v 14.30±2.60 days; P value < 0.01.The extent of the wound with granulation tissue which neededskin graft was less in the amnion group (2.10 ± 2.21% v 4.20±1.44%; P value < 0.01.Conclusion: Widespread use of amniotic membrane dressingis recommended for limited burn wound management.

  6. A comparison between placental and amniotic mesenchymal stem cells for transamniotic stem cell therapy (TRASCET) in experimental spina bifida.

    Science.gov (United States)

    Feng, Christina; D Graham, Christopher; Connors, John Patrick; Brazzo, Joseph; Zurakowski, David; Fauza, Dario O

    2016-06-01

    We compared placental-derived and amniotic fluid-derived mesenchymal stem cells (pMSCs and afMSCs, respectively) in transamniotic stem cell therapy (TRASCET) for experimental spina bifida. Pregnant dams (n=29) exposed to retinoic acid for the induction of fetal spina bifida were divided into four groups. Three groups received volume-matched intraamniotic injections of either saline (n=38 fetuses) or a suspension of 2×10(6) cells/mL of syngeneic, labeled afMSCs (n=73) or pMSCs (n=115) on gestational day 17 (term=21-22days). Untreated fetuses served as controls. Animals were killed before term. Statistical comparisons were by Fisher's exact test (pspina bifida (n=100), there were higher percentages of defect coverage (either partial or complete) in both afMSC and pMSC groups compared with saline and untreated groups (pspina bifida after concentrated intraamniotic injection in the rodent model. This broadens the options for timing and cell source for TRASCET as a potential alternative in the prenatal management of spina bifida. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. In vitro fabrication of autologous living tissue-engineered vascular grafts based on prenatally harvested ovine amniotic fluid-derived stem cells.

    Science.gov (United States)

    Weber, Benedikt; Kehl, Debora; Bleul, Ulrich; Behr, Luc; Sammut, Sébastien; Frese, Laura; Ksiazek, Agnieszka; Achermann, Josef; Stranzinger, Gerald; Robert, Jérôme; Sanders, Bart; Sidler, Michele; Brokopp, Chad E; Proulx, Steven T; Frauenfelder, Thomas; Schoenauer, Roman; Emmert, Maximilian Y; Falk, Volkmar; Hoerstrup, Simon P

    2016-01-01

    Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications.

  8. Combination of melatonin and Wnt-4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury.

    Science.gov (United States)

    Gao, Yuhua; Bai, Chunyu; Zheng, Dong; Li, Changli; Zhang, Wenxiu; Li, Mei; Guan, Weijun; Ma, Yuehui

    2016-04-01

    Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promoting differentiation of neural cells remain unknown. Wnt signaling mediates major developmental processes during embryogenesis and regulates maintenance, self-renewal, and differentiation of adult mammalian stem cells. However, the role of the noncanonical Wnt pathway during neurogenesis remains poorly understood. In this study, the amniotic epithelial cells ( AECs) were isolated from bovine amnion and incubated with various melatonin concentrations (0.01, 0.1, 1, 10, or 100 μm) and 5 × 10(-5) m all-trans retinoic acid (RA) for screening optimum culture medium of neural differentiation, compared with each groups, 1 μm melatonin and 5 × 10(-5) m RA were selected to induce neural differentiation of AECs, and then siMT1, siMT2, oWnt-4, and siWnt-4 were expressed in AECs to research role of these genes in neural differentiation. Efficiency of neural differentiation was evaluated after expressed above genes using flow cytometry. Cell function of neural cells was demonstrated in vivo using spinal cord injury model after cell transplantation, and damage repair of spinal cord was assessed using cell tracking and Basso, Beattie, Bresnahan Locomotor Rating Scale scores. Results demonstrated that melatonin stimulated melatonin receptor 1, which subsequently increased bovine amniotic epithelial cell vitality and promoted differentiation into neural cells. This took place through cooperation with Wnt-4. Additionally, following cotreatment with melatonin and Wnt-4, neurogenesis gene expression was significantly altered. Furthermore, single inhibition of melatonin receptor 1 or Wnt-4 expression decreased expression of neurogenesis-related genes, and bovine amniotic epithelial cell-derived neural cells were successfully colonized into injured spinal cord, which suggested participation in tissue repair.

  9. The influence of amniotic membrane extracts on cell growth depends on the part of membrane and childbirth mode selected: a proof-of-concept study.

    Science.gov (United States)

    Litwiniuk, M; Radowicka, M; Krejner, A; Grzela, T

    2017-08-02

    The amniotic membrane (AM) is a rich source of biologically active factors, important for wound healing and is widely used in various clinical applications, including tissue engineering, reconstructive surgery and wound management. The aim of the present proof-of-concept study was to assess the influence of amniotic membrane extracts on in vitro proliferation of main cells involved in tissue regeneration. The assessment was done in regards to the content of selected biologically active factors in amniotic membrane extracts. The quantitative analysis of EGF, TGF-β and TIMP-1 in tested samples was assayed by enzyme-linked immunosorbent assay (ELISA) tests. The influence of amniotic membrane extracts on proliferation of keratinocytes (HaCaT), fibroblasts (Wi-38) and endothelial cell lines (HECa-10) was assessed using a colorimetric tetrazolium salt reduction assay. In all of the amnion samples high amounts of EGF, TGF-β and TIMP-1 were detected. However, the content of these factors varied between placental and cervical portions of the same membrane. Moreover, various concentrations of biologically active factors between physiological at-term delivery and caesarean section-derived membranes were also observed. All of the assessed amnion extracts stimulated proliferation of HaCaT and Wi-38 cells, although samples prepared from caesarean section-derived cervical portion of amniotic membrane stimulated more proliferation of keratinocytes than of fibroblasts. In contrast to HaCaT and Wi-38 cells, proliferation of HECa-10 cell line was inhibited by all tested extracts. The results of our proof-of-concept study confirm that biological dressings prepared from amniotic membrane, especially its placental portion, since they stimulated both fibroblasts and keratinocytes, may provide relevant support for wound healing. On the other hand, dressings prepared from caesarean section-derived cervical portion of amniotic membrane, since they stimulate mainly epidermal cells, may be

  10. Amniotic fluid (image)

    Science.gov (United States)

    Amniotic fluid surrounds the growing fetus in the womb and protects the fetus from injury and temperature changes. ... of fetal movement and permits musculoskeletal development. The amniotic fluid can be withdrawn in a procedure called amniocentsis ...

  11. Amniotic constriction bands

    Science.gov (United States)

    ... of function of an arm or a leg. Congenital bands affecting the hand often cause the most problems. Alternative Names Pseudo-ainhum; Streeter dysplasia; Amniotic band sequence; Amniotic constriction bands; Constriction band ...

  12. 葛根芩连汤联合人羊膜间充质干细胞移植治疗糖尿病早期视网膜病变%Gegen Qin Lian Tang combined with human amniotic mesenchymal stem cell transplantation for early diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    刘亮; 王金梁

    2016-01-01

    背景:研究发现人羊膜间充质干细胞可以向胰岛素分泌细胞分化,还具有免疫调节作用。临床证实葛根芩连汤有明确降低血糖的作用。  目的:探讨葛根芩连汤联合人羊膜间充质干细胞移植对糖尿病大鼠早期视网膜病变形态学、相关血清学指标及血管内皮生长因子基因表达的影响。  方法:以腹腔注射链脲佐菌素方式制作糖尿病模型,将造模成功的42只Wistar大鼠随机分为模型组、人羊膜间充质干细胞移植组和联合组(人羊膜间充质干细胞移植+葛根芩连汤),移植后4周测定糖尿病大鼠血糖、血清胰岛素水平;移植后4周苏木精-伊红染色镜下观察糖尿病大鼠视网膜的病理形态;免疫组织化学方法检测视网膜组织CD45的表达水平;RT-PCR法检测视网膜血管内皮生长因子mRNA的表达。结果与结论:①与模型组比较,人羊膜间充质干细胞移植组和联合组大鼠血糖降低,血清胰岛素浓度增加,联合组改善更为明显,组间比较差异有显著性意义(P<0.05);②模型组大鼠有明显视网膜水肿、层次紊乱,细胞排列不规则;与模型组比较,人羊膜间充质干细胞移植组大鼠视网膜病变相对轻微,联合组视网膜病变明显好转;③模型组视网膜CD45的表达明显增加,人羊膜间充质干细胞移植组CD45的表达量明显小于模型组(P<0.05),但大于联合组(P<0.05);④联合组和人羊膜间充质干细胞移植组血管内皮生长因子mRNA表达低于模型组,联合组降低幅度更大,差异有显著性意义(P<0.05);⑤结果表明,葛根芩连汤联合人羊膜间充质干细胞移植可能通过抑制 CD45和血管内皮生长因子在视网膜组织表达,从而改善糖尿病视网膜病损严重程度。%BACKGROUND:Human amniotic mesenchymal stem cel s have the potential to differentiate into insulin-secreting cel s and have

  13. Levels of CD105(+) cells increase and cell proliferation decreases during S-phase arrest of amniotic fluid cells in long-term culture.

    Science.gov (United States)

    Wang, Ding; Chen, Rui; Zhong, Xuan; Fan, Yong; Lai, Weiqiang; Sun, Xiaofang

    2014-11-01

    The present study aimed to improve the characterization of amniotic fluid cells (AFCs) in order to optimize their use in chromosomal prenatal diagnosis and as seed or stem cells for tissue engineering. The AFCs used in the current study were obtained from three females in their second trimester of pregnancy. The cells were cultured independently and characterized by cell morphology, cell markers, cell cycle distribution and chromosome Giemsa banding in an early- and late-passage. The AFCs remained homogeneous in culture and expressed mesenchymal markers, but not endothelial markers along the culture process. In addition, compared with the early-passage cells, the late-passage cells exhibit an increase in CD105 expression, a decrease in cell division and a delay in the cell cycle, and a number of cells underwent cell cycle arrest. However, the cells retained a normal karyotype. Therefore, the current study characterized AFCs in a clinical culture and confirmed that AFCs are mesenchymal precursors. The results obtained may be useful for the application of AFCs in prenatal diagnosis.

  14. Evaluation of correlation between nucleated red blood cell count in term newborns and meconium-stained amniotic fluid

    Directory of Open Access Journals (Sweden)

    Esmaeilian L

    2001-09-01

    Full Text Available Considering the incidence of meconium-stained amniotic fluid (MSAF in newborns and its complications, and also based on indirect reports pointing out relationship between nucleated red blood cell (nRBC, as a marker of chronic hypoxia, and MSAF in term newborns, and in order to determine this relationship more accurately, this study was done on women with uncomplicated singleton term pregnancy that admitted for delivery at Shariati hospital in year 2000. After excluding confounding factors on nRBC, case group who includes infants with MSAF, and control group who have clear amniotic fluid were determined. Maternal age and parity, gestational age, birth weight, apgar score, neonatal out come, and FHR pattern as well as newborn hemoglobin values were evaluated. Venous cord blood was analyzed for nRBC counts per 100 WBC. Case and control groups were divided according to nRBC counts with cutoff point of 10 and then compared statistically. From the samples, 117 cases and 67 controls were evaluated. Maternal and neonatal factors were similar in two situations. Number of nRBC was 3.75±4.8 in control group and 12.04±11.7 in case group (P<0.01 and it was abnormal (>10 in 9 percent of newborns with clear fluid and 32 percent of MSAF (P<0.01. Cord blood nRBC count increased when meconium is passed intrauterine. This suggests that cases with MSAF may be at risk of chronic hypoxia. Cohort research is recommended to study the affect of delivery mode on the neonatal outcome in cases with MSAF and to evaluate the cause of fetal hypoxia in uncomplicated pregnancy with MSAF.

  15. Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro.

    Science.gov (United States)

    Zhang, Ruyu; Lu, Ying; Li, Ju; Wang, Jia; Liu, Caixia; Gao, Fang; Sun, Dong

    2016-02-01

    Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

  16. Application of Human Amniotic Membrane in Canine Penile Tunica Albuginea Defect: First Step toward an Innovating New Method for Treatment of Peyronie?s Disease

    Directory of Open Access Journals (Sweden)

    M. Salehipour

    2014-06-01

    Full Text Available Purposes To evaluate the efficacy of human amniotic membrane (AM grafting in the canine penile tunica albuginea defect; we developed an animal model as the first step toward an innovating new method for the treatment of Peyronie’s disease, penile cancers, and congenital deformities of the penis. Material and Methods From August to September 2011, ten healthy male dogs were selected. A rhomboid incision about 3x2cm over the tunica albuginea and its overlying squamous epithelium was made and then excised. The amniotic membrane was folded twice on itself and grafted on the defect. After 8 weeks, artificial erection was made for 5 dogs and for the other 5 dogs after 12 weeks. After artificial erection, partial penectomy was done and histopathological evaluation was performed on the grafts. Results Artificial erection performed successfully in all of the dogs. No infection or any other complication was seen. Histopathological examination showed complete re-epithelialization with squamous epithelium and collagen fiber deposition. Also, no dysplasia was seen. Conclusions The amniotic membrane can be used as a suitable substitution for tunica albuginea. It is safe, inexpensive, biodegradable, and available and may be used for the treatment of Peyronie’s disease, penile cancers, congenital penile deformities, and penile reconstructive surgery.

  17. A prospective study of 20 foot and ankle wounds treated with cryopreserved amniotic membrane and fluid allograft.

    Science.gov (United States)

    Werber, Bruce; Martin, Erin

    2013-01-01

    We reviewed the background information and previous clinical studies that considered the use of allogeneic amniotic tissue and fluid (granulized amniotic membrane and amniotic fluid) in the treatment of chronic diabetic foot wounds. This innovation represents a relatively new approach to wound management by delivering a unique allograft of live human cells in a nonimmunogenic structural tissue matrix. Developed to fill soft tissue defects and bone voids and to convey antimicrobial and anti-inflammatory capabilities, granulized amniotic membrane and amniotic fluid does not require fetal death, because its procurement is performed with maternal consent during birth. In the present investigation, 20 chronic wounds (20 patients) that had been treated with standard wound therapy for a mean of 36.6 ± 31.58 weeks and with a mean baseline area of 10.15 ± 19.54 cm(2) were followed up during a 12-week observation period or until they healed. A total of 18 of the wounds (90%) healed during the 12-week observation period, and none of the wounds progressed to amputation. From our experience with the patients in the present case series, we believe that granulized amniotic membrane and amniotic fluid represents a useful option for the treatment of chronic diabetic foot wounds.

  18. 人羊膜负载猪骨髓间充质干细胞体外生长的形态特点%Morphological study on the growth of human amniotic membrane loaded with porcine bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    闫国和; 艾国平; 汪代杰; 邹仲敏; 冉新泽; 王军平; 李蓉; 粟永萍; 程天民

    2007-01-01

    充质干细胞在人羊膜的基质面生长4 d切面,骨髓间充质干细胞在人羊膜上大多呈双层生长,细胞呈纺锤状,胞核两端胞浆突起较长,互相重叠,染色质以常染色质为主,核仁明显,可见丰富细胞器,如粗面内质网,线粒体.结论:人羊膜对骨髓间充质干细胞有明显的促增殖作用,骨髓间充质干细胞可以人羊膜为载体在体外进行培养,人羊膜是骨髓间充质干细胞的良好载体.%BACKGROUND: Human amniotic membrane (HAM) contains various ingredents such as collagen, glycoprotein,proteoglycan, integrin and laminated body, and so on, and expresses many kinds of growth factors and mRNA-associated proteins. And these ingredents can supply abundant nutriments for cellular proliferation and differentiation, and benefit cells to grow and propagate. Whether or not HAM can load porcine bone marrow-derived mesenchymal stem cells (BMSCs) to well grow on it deserves to be further investigated.OBJECTIVE: To set up a method of tissue engineering of human amniotic membrane loading porcine BMSCs and observe the morphological characteristics of growth and proliferation of BMSCs seeded on HAM.DESIGN: Randomized controlled observation.SETTING: State Key Laboratory of Trauma, Burn and Combined Injury, General Institute of Combined Injuries, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA.MATERIALS: This experiment was carried out in the State Key Laboratory of Trauma, Burn and Combined Injury,General Institute of Combined Injuries, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA between January and November 2003. Three Guizhou minipigs of either gender, aged 2 to 3 months, weighing from 6 to 8 kg, were provided by the Experimental Animal Center, Third Military Medical University of Chinese PLA. Main reagent:ISCOVE'S modified DULBECCO'S medium (IMDM) culture medium (Hyclone, USA); high-quality fetal bovine serum PAA (Germany

  19. MACROPHAGE MIGRATION INHIBITORY FACTOR IN PATIENTS WITH PRETERM PARTURITION AND MICROBIAL INVASION OF THE AMNIOTIC CAVITY

    Science.gov (United States)

    Chaiworapongsa, Tinnakorn; Romero, Roberto; Espinoza, Jimmy; Kim, Yeon Mee; Edwin, Samuel; Bujold, Emmanuel; Gomez, Ricardo; Kuivaniemi, Helena

    2006-01-01

    OBJECTIVE Macrophage migration inhibitory factor (MIF) has emerged as an important mediator of septic shock. The administration of MIF increases lethality during endotoxemia, whereas neutralization of this cytokine prevents endotoxic shock and death associated with bacterial infection. The objective of this study was to determine whether there is a change in the amniotic fluid concentration of MIF in intra-amniotic infection and human parturition. STUDY DESIGN A cross-sectional study was conducted in women in the following categories: 1) midtrimester (n=84); 2) preterm labor and intact membranes who delivered at term (n=33), who delivered preterm (n=53), and preterm labor with intra-amniotic infection (n=23); 3) preterm premature rupture of membranes (PROM) with (n=25) and without intra-amniotic infection (n=26); and 4) term with intact membranes, in labor (n=52), and not in labor (n=31). MIF concentrations in amniotic fluid were determined using a sensitive and specific immunoassay. MIF concentrations in maternal plasma were also determined in patients with preterm labor and intact membranes. Immunohistochemistry was conducted in chorioamniotic membranes obtained from a different set of patients presenting with preterm labor with (n=18) and without (n=20) histologic chorioamnionitis. Quantitative RT-PCR was used to measure MIF mRNA expression in chorioamniotic membranes of patients with preterm labor with (n=13) and without (n=13) histologic chorioamnionitis. Parametric and non-parametric, receiver-operating characteristic (ROC) curve, survival analysis, and Cox regression model were used for analysis. RESULTS Immunoreactive MIF was detectable in 96% (313/327) of amniotic fluid samples. The concentration of amniotic fluid MIF at term was higher than that in the midtrimester (p=0.004). Intra-amniotic infection in women with preterm labor and preterm PROM was associated with a significant increase in median amniotic fluid MIF concentration (p0.05). There was no

  20. Accuracy Assessment of Interphase Fluorescence In-Situ Hybridization on Uncultured Amniotic Fluid Cells

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    Hamideh Karimi

    2007-01-01

    Full Text Available Background: Parental anxiety while waiting for the results of amniocentesis has been investigatedby many authors. It seems that the implementation of faster techniques such as fluorescence in-situhybridization (FISH will have some benefits in reducing this anxiety. Besides the patients' attitudesto choosing this method, gynecologists who are the persons responsible for treatment, must feelcomfortable about prescribing FISH techniques.Materials and Methods: This study, using a simple methodology, was undertaken to evaluate theresults of FISH tests on the amniotic fluid from 40 pregnant women undergoing cesarean surgery.Two sets of probes including X/Y cocktail and 13, 21 and 18 were applied on different slides.Results: The results of FISH tests were compared with the reports of the pediatrician about thehealth condition of the newborn. Complete conformity between the two sets of findings, haveconvinced our gynecologists of the benefit of prescribing this method to reduce the anxiety ofpatients at risk of having abnormal offspring due to chromosomal anuploidies.Conclusion: As has been documented by many authors, conventional chromosome analysis hasgreat advantages over fluorescence in situ hybridization of interphase amniocytes, but reducing theanxiety of parents is a good reason for employing the FISH technique.

  1. Accuracy Assessment of Interphase Fluorescence In-Situ Hybridization on Uncultured Amniotic Fluid Cells

    Directory of Open Access Journals (Sweden)

    Hamid Gourabi

    2008-01-01

    Full Text Available Background: Parental anxiety while waiting for the results of amniocentesis has been investigatedby many authors. It seems that the implementation of faster techniques such as fluorescence in-situhybridization (FISH will have some benefits in reducing this anxiety. Besides the patients' attitudesto choosing this method, gynecologists who are the persons responsible for treatment, must feelcomfortable about prescribing FISH techniques.Materials and Methods: This study, using a simple methodology, was undertaken to evaluate theresults of FISH tests on the amniotic fluid from 40 pregnant women undergoing cesarean surgery.Two sets of probes including X/Y cocktail and 13, 21 and 18 were applied on different slides.Results: The results of FISH tests were compared with the reports of the pediatrician about thehealth condition of the newborn. Complete conformity between the two sets of findings, haveconvinced our gynecologists of the benefit of prescribing this method to reduce the anxiety ofpatients at risk of having abnormal offspring due to chromosomal anuploidies.Conclusion: As has been documented by many authors, conventional chromosome analysis hasgreat advantages over fluorescence in situ hybridization of interphase amniocytes, but reducing theanxiety of parents is a good reason for employing the FISH technique.

  2. Amniotic fluid embolism

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    Rudra A

    2009-01-01

    Full Text Available The disastrous entry of amniotic fluid into the maternal circulation leads to dramatic sequelae of clinical events, characteristically referred to as Amniotic fluid embolism (AFE. The underlying mechanism for AFE is still poorly understood. Unfortunately, this situation has very grave maternal and fetal consequences. AFE can occur during labor, caesarean section, dilatation and evacuation or in the immediate postpartum period. The pathophysiology is believed to be immune mediated which affects the respiratory, cardiovascular, neurological and hematological systems. Undetected and untreated it culminates into fulminant pulmonary edema, intractable convulsions, disseminated intravascular coagulation (DIC, malignant arrhythmias and cardiac arrest. Definite diagnosis can be confirmed by identification of lanugo, fetal hair and fetal squamous cells (squames in blood aspirated from the right ventricle. Usually the diagnosis is made clinically and by exclusion of other causes. The cornerstone of management is a multidisciplinary approach with supportive treatment of failing organs systems. Despite improved modalities for diagnosing AFE, and better intensive care support facilities, the mortality is still high.

  3. Acceleration of Regeneration of Large-Gap Peripheral Nerve Injuries Using Accellular Nerve Allografts Plus Amniotic Fluid Derived Stem Cells (AFS)

    Science.gov (United States)

    2015-09-01

    2 AD______________ AWARD NUMBER: W81XWH-13-1-0310 TITLE: Acceleration of Regeneration of Large-Gap Peripheral Nerve Injuries Using Acellular... Nerve Allografts plus amniotic Fluid Derived Stem Cells (AFS). PRINCIPAL INVESTIGATOR: Li, Zhongyu CONTRACTING ORGANIZATION: Wake Forest...DATE September 2015 2. REPORT TYPE Annual Report 3. DATES COVERED 1 Sep 2014 - 31 Aug 2015 4. TITLE AND SUBTITLE Acceleration of Regeneration of Large

  4. Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients.

    Science.gov (United States)

    Gholipourmalekabadi, M; Bandehpour, M; Mozafari, M; Hashemi, A; Ghanbarian, H; Sameni, M; Salimi, M; Gholami, M; Samadikuchaksaraei, A

    2015-11-01

    Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections.

  5. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    Science.gov (United States)

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  6. Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy

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    Jūratė Savickienė

    2016-01-01

    Full Text Available Human amniotic-fluid-derived mesenchymal stem cells (AF-MSCs are interesting for their multilineage differentiation potential and wide range of therapeutic applications due to the ease of culture expansion. However, MSCs undergo replicative senescence. So far, the molecular mechanisms that underlie fetal diseases and cell senescence are still poorly understood. Here, we analyzed senescence-associated morphologic, molecular, and epigenetic characteristics during propagation of MSCs derived from AF of normal and fetus-affected pregnancy. AF-MSCs cultures from both cell sources displayed quite similar morphology and expression of specific cell surface (CD44, CD90, and CD105 and stemness (Oct4, Nanog, Sox2, and Rex1 markers but had interindividual variability in proliferation capability and time to reach senescence. Within passages 4 and 8, senescent cultures exhibited typical morphological features, senescence-associated β-galactosidase activity, increased levels of p16, and decreased levels of miR-17 and miR-21 but showed differential expression of p21, p53, and ATM dependently on the onset of cell senescence. These differences correlated with changes in the level of chromatin modifiers (DNMT1 and HDAC1 and polycomb group proteins (EZH2, SUZ12, and BMI1 paralleling with changes in the expression of repressive histone marks (H3K9me3 and H3K27me3 and stemness markers (Oct4, Nanog, Sox2, and Rex1. Therefore epigenetic factors are important for AF-MSCs senescence process that may be related with individuality of donor or a fetus malignancy status.

  7. Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy

    Science.gov (United States)

    Savickienė, Jūratė; Baronaitė, Sandra; Zentelytė, Aistė; Treigytė, Gražina

    2016-01-01

    Human amniotic-fluid-derived mesenchymal stem cells (AF-MSCs) are interesting for their multilineage differentiation potential and wide range of therapeutic applications due to the ease of culture expansion. However, MSCs undergo replicative senescence. So far, the molecular mechanisms that underlie fetal diseases and cell senescence are still poorly understood. Here, we analyzed senescence-associated morphologic, molecular, and epigenetic characteristics during propagation of MSCs derived from AF of normal and fetus-affected pregnancy. AF-MSCs cultures from both cell sources displayed quite similar morphology and expression of specific cell surface (CD44, CD90, and CD105) and stemness (Oct4, Nanog, Sox2, and Rex1) markers but had interindividual variability in proliferation capability and time to reach senescence. Within passages 4 and 8, senescent cultures exhibited typical morphological features, senescence-associated β-galactosidase activity, increased levels of p16, and decreased levels of miR-17 and miR-21 but showed differential expression of p21, p53, and ATM dependently on the onset of cell senescence. These differences correlated with changes in the level of chromatin modifiers (DNMT1 and HDAC1) and polycomb group proteins (EZH2, SUZ12, and BMI1) paralleling with changes in the expression of repressive histone marks (H3K9me3 and H3K27me3) and stemness markers (Oct4, Nanog, Sox2, and Rex1). Therefore epigenetic factors are important for AF-MSCs senescence process that may be related with individuality of donor or a fetus malignancy status. PMID:27803714

  8. 羊水来源干细胞的研究进展%Research Progresses of Amniotic Fluid-derived Stem Cells

    Institute of Scientific and Technical Information of China (English)

    王艳

    2011-01-01

    干细胞具有自我更新和多向分化的潜能,是目前医学及组织工程学研究的热点.近来发现羊水中含有干细胞,具有向内、中、外三个胚层分化的能力,且通过了功能测试.由于羊水来源广泛、创伤小、无伦理道德方面的限制且无致瘤性,将为干细胞开辟新的研究领域,为组织工程提供新的种子细胞来源,为细胞介导的基因治疗提供新的载体.目前对其来源及生物学特征的研究还不是很清楚.现对羊水中细胞的组成、羊水来源干细胞分离培养、生物学特征及鉴定、分化潜能等问题进行综述.%Stem cells possess the potentials of self-renewal and multipotent differentiation, which has become a hotspot of medical and tissue engineering research. Recently, amniotic fluids are found to contain stem cells capable of differentiating to endoderm, mesoderm,and ectoderm as justified by functional tests. As amniotic fluids have a wide source, minimally invasive harvest,less ethnical restriction, and free of tumorigenesis,they are used to create a new research field to provide new seed cells for tissue engineering and new vectors for cell-based gene therapy. The source and biological properties of amniotic fluid-derived stem cells remain unclear until now. This article reviews the components of amniotic cells,isolation and culture of amniotic fluid-derived stem cells, biological properties, characterization, and differential potentials.

  9. Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid.

    Science.gov (United States)

    Collado, Maria Carmen; Rautava, Samuli; Aakko, Juhani; Isolauri, Erika; Salminen, Seppo

    2016-03-22

    Interaction with intestinal microbes in infancy has a profound impact on health and disease in later life through programming of immune and metabolic pathways. We collected maternal faeces, placenta, amniotic fluid, colostrum, meconium and infant faeces samples from 15 mother-infant pairs in an effort to rigorously investigate prenatal and neonatal microbial transfer and gut colonisation. To ensure sterile sampling, only deliveries at full term by elective caesarean section were studied. Microbiota composition and activity assessment by conventional bacterial culture, 16S rRNA gene pyrosequencing, quantitative PCR, and denaturing gradient gel electrophoresis revealed that the placenta and amniotic fluid harbour a distinct microbiota characterised by low richness, low diversity and the predominance of Proteobacteria. Shared features between the microbiota detected in the placenta and amniotic fluid and in infant meconium suggest microbial transfer at the foeto-maternal interface. At the age of 3-4 days, the infant gut microbiota composition begins to resemble that detected in colostrum. Based on these data, we propose that the stepwise microbial gut colonisation process may be initiated already prenatally by a distinct microbiota in the placenta and amniotic fluid. The link between the mother and the offspring is continued after birth by microbes present in breast milk.

  10. M1 and M2 macrophage recruitment during tendon regeneration induced by amniotic epithelial cell allotransplantation in ovine.

    Science.gov (United States)

    Mauro, Annunziata; Russo, Valentina; Di Marcantonio, Lisa; Berardinelli, Paolo; Martelli, Alessandra; Muttini, Aurelio; Mattioli, Mauro; Barboni, Barbara

    2016-04-01

    Recently, we have demonstrated that ovine amniotic epithelial cells (oAECs) allotransplanted into experimentally induced tendon lesions are able to stimulate tissue regeneration also by reducing leukocyte infiltration. Amongst leukocytes, macrophages (Mφ) M1 and M2 phenotype cells are known to mediate inflammatory and repairing processes, respectively. In this research it was investigated if, during tendon regeneration induced by AECs allotransplantation, M1Mφ and M2Mφ phenotype cells are recruited and differently distributed within the lesion site. Ovine AECs treated and untreated (Ctr) tendons were explanted at 7, 14, and 28 days and tissue microarchitecture was analyzed together with the distribution and quantification of leukocytes (CD45 positive), Mφ (CD68 pan positive), and M1Mφ (CD86, and IL12b) and M2Mφ (CD206, YM1 and IL10) phenotype related markers. In oAEC transplanted tendons CD45 and CD68 positive cells were always reduced in the lesion site. At day 14, oAEC treated tendons began to recover their microarchitecture, contextually a reduction of M1Mφ markers, mainly distributed close to oAECs, and an increase of M2Mφ markers was evidenced. CD206 positive cells were distributed near the regenerating areas. At day 28 oAECs treated tendons acquired a healthy-like structure with a reduction of M2Mφ. Differently, Ctr tendons maintained a disorganized morphology throughout the experimental time and constantly showed high values of M1Mφ markers. These findings indicate that M2Mφ recruitment could be correlated to tendon regeneration induced by oAECs allotransplantation. Moreover, these results demonstrate oAECs immunomodulatory role also in vivo and support novel insights into their allogeneic use underlying the resolution of tendon fibrosis.

  11. Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Wu Dan; Chi Hongbin; Shao Minjie; Wu Yao; Jin Hongyan; Wu Baiyan; Qiao Jie

    2014-01-01

    Backgroud Amniotic fluid (AF) supernatant contains cell-free fetal DNA (cffDNA) fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction (QF-PCR) to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat (STR) fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93% (26/28; confidence interval,CI:77%-98%) and the specificity was 100% (26/26; CI:88%-100%).The determination rate of the origin of the extra chromosome was 69%.The sensitivity and the specificity of the assay in the euploid were 100% (27/27).Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

  12. Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix.

    Science.gov (United States)

    Filioli Uranio, M; Valentini, L; Lange-Consiglio, A; Caira, M; Guaricci, A C; L'Abbate, A; Catacchio, C R; Ventura, M; Cremonesi, F; Dell'Aquila, M E

    2011-05-01

    The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

  13. Human amniotic membrane, best healing accelerator, and the choice of bone induction for vestibuloplasty technique (an animal study

    Directory of Open Access Journals (Sweden)

    Ahad Khoshzaban

    2010-12-01

    Full Text Available Mohammad H Samandari1, Shahriar Adibi2, Ahad Khoshzaban3, Sara Aghazadeh5, Parviz Dihimi4, Siamak S Torbaghan6, Saeed H Keshel5, Zohreh Shahabi71Department of Oral and Maxillofacial Surgery, Dentistry Faculty, 2Dental Research of Torabinejad Research Centre, 3Iranian Tissue Bank Research and Preparation Centre, Imam Khomeini Hospital Complex, 4Department of Oral and Maxillofacial Pathology, Dentistry Faculty, Isfahan University of Medical Sciences, Isfahan, Iran; 5Stem Cells Preparation Unit, Eye Research Center, Farabi Hospital, 6Department of Pathology, Imam Khomeini Medical Centre, 7BMT Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, IranObjective: To investigate the effects of amniotic membrane (AM in bone induction and wound healing after vestibuloplasty surgery on animal samples while receptacle proteins such as growth factors were considered as accelerators for wound healing and bone induction after these operations.Material and methods: Ten adult dogs (5 females, 5 males; race, Iranian mixed; weight, 44 pounds were included, which underwent surgery for transplantation on mandible and maxillary. AM was used for promoting bone induction and healing.Results: The tissue samples were obtained after 2, 8, and 12 weeks for histology survey. No significant differences were observed between male and female or left and right jaws. AM decreased fibrinoleukocytic exudates and inflammation in the experimental group, had significant effects on bone formation, considerably improves wound healing, and gives rise to bone induction (P < 0.0001.Conclusions: Our study findings indicate that the AM is a suitable cover for different injuries and acellular AM has the potential for rapid improvement and bone induction. The AM contains collagen, laminin, and fibronectin, which provide an appropriate substrate for bone induction. This substrate promoted bone induction and might contribute to induction of the progenitor cells and/or stem

  14. Amniotic Fluid Derived Stem Cells with a Renal Progenitor Phenotype Inhibit Interstitial Fibrosis in Renal Ischemia and Reperfusion Injury in Rats.

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    Marina Gabriela Monteiro Carvalho Mori da Cunha

    Full Text Available Mesenchymal stem cells derived from human amniotic fluid (hAFSCs are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R model and prevent late stage fibrosis.A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey's test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p <0.05.hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48 h. The treated group had less fibrosis and proteinuria at 2 months after injury.hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.

  15. Challenges in validating the sterilisation dose for processed human amniotic membranes

    Science.gov (United States)

    Yusof, Norimah; Hassan, Asnah; Firdaus Abd Rahman, M. N.; Hamid, Suzina A.

    2007-11-01

    Most of the tissue banks in the Asia Pacific region have been using ionising radiation at 25 kGy to sterilise human tissues for save clinical usage. Under tissue banking quality system, any dose employed for sterilisation has to be validated and the validation exercise has to be a part of quality document. Tissue grafts, unlike medical items, are not produced in large number per each processing batch and tissues relatively have a different microbial population. A Code of Practice established by the International Atomic Energy Agency (IAEA) in 2004 offers several validation methods using smaller number of samples compared to ISO 11137 (1995), which is meant for medical products. The methods emphasise on bioburden determination, followed by sterility test on samples after they were exposed to verification dose for attaining of sterility assurance level (SAL) of 10 -1. This paper describes our experience in using the IAEA Code of Practice in conducting the validation exercise for substantiating 25 kGy as sterilisation dose for both air-dried amnion and those preserved in 99% glycerol.

  16. Challenges in validating the sterilisation dose for processed human amniotic membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yusof, Norimah [Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor (Malaysia)], E-mail: norimah@mint.gov.my; Hassan, Asnah [Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor (Malaysia); Firdaus Abd Rahman, M.N.; Hamid, Suzina A. [National Tissue Bank, Hospital Universiti Sains Malaysia, Kubang Kerian, 16130 Kelantan (Malaysia)

    2007-11-15

    Most of the tissue banks in the Asia Pacific region have been using ionising radiation at 25 kGy to sterilise human tissues for save clinical usage. Under tissue banking quality system, any dose employed for sterilisation has to be validated and the validation exercise has to be a part of quality document. Tissue grafts, unlike medical items, are not produced in large number per each processing batch and tissues relatively have a different microbial population. A Code of Practice established by the International Atomic Energy Agency (IAEA) in 2004 offers several validation methods using smaller number of samples compared to ISO 11137 (1995), which is meant for medical products. The methods emphasise on bioburden determination, followed by sterility test on samples after they were exposed to verification dose for attaining of sterility assurance level (SAL) of 10{sup -1}. This paper describes our experience in using the IAEA Code of Practice in conducting the validation exercise for substantiating 25 kGy as sterilisation dose for both air-dried amnion and those preserved in 99% glycerol.

  17. Partial trisomy 8 mosaicism not detected by cultured amniotic-fluid cells

    Directory of Open Access Journals (Sweden)

    Meng-Che Tsai

    2014-12-01

    Conclusion: Conventional karyotyping through amniocentesis has limitations particularly in detecting rare trisomy mosaicism if trisomic cells show growth disadvantage. Array-CGH using uncultured cells may be of help in providing more information on genetic dosage variations in such cases.

  18. Intravenous grafts of amniotic fluid-derived stem cells induce endogenous cell proliferation and attenuate behavioral deficits in ischemic stroke rats.

    Directory of Open Access Journals (Sweden)

    Naoki Tajiri

    Full Text Available We recently reported isolation of viable rat amniotic fluid-derived stem (AFS cells [1]. Here, we tested the therapeutic benefits of AFS cells in a rodent model of ischemic stroke. Adult male Sprague-Dawley rats received a 60-minute middle cerebral artery occlusion (MCAo. Thirty-five days later, animals exhibiting significant motor deficits received intravenous transplants of rat AFS cells or vehicle. At days 60-63 post-MCAo, significant recovery of motor and cognitive function was seen in stroke animals transplanted with AFS cells compared to vehicle-infused stroke animals. Infarct volume, as revealed by hematoxylin and eosin (H&E staining, was significantly reduced, coupled with significant increments in the cell proliferation marker, Ki67, and the neuronal marker, MAP2, in the dentate gyrus (DG [2] and the subventricular zone (SVZ of AFS cell-transplanted stroke animals compared to vehicle-infused stroke animals. A significantly higher number of double-labeled Ki67/MAP2-positive cells and a similar trend towards increased Ki67/MAP2 double-labeling were observed in the DG and SVZ of AFS cell-transplanted stroke animals, respectively, compared to vehicle-infused stroke animals. This study reports the therapeutic potential of AFS cell transplantation in stroke animals, possibly via enhancement of endogenous repair mechanisms.

  19. Usos de la membrana amniótica humana en oftalmología Some uses of human amniotic membrane in Ophthalmology

    Directory of Open Access Journals (Sweden)

    Jaime Alemañy González

    2006-06-01

    Full Text Available Se realizó una revisión bibliográfica sobre el uso de la membrana amniótica en oftalmología con los propósitos de conocer sus mecanismos de acción y aplicaciones, así como los resultados publicados sobre el transplante de membrana amniótica humana en la superficie ocular. Se estudiaron textos y artículos publicados sobre este tipo de tratamiento en las enfermedades y traumas de córnea, conjuntiva, esclera y párpados. Se concluye que es una alternativa terapéutica valida que puede ser de gran utilidad en nuestro medioA literature review was made on the use of amniotic membrane in ophthalmology, with the objective of finding out its mechanisms of action and applications as well as the results of human amniotic membrane transplantation for ocular surface reconstruction. A number of texts and articles published on this type of treatment for cornea, conjunctiva, sclera and eyelids diseases and traumas were studied. It was concluded that this is a valid therapeutical alternative that may be very beneficial in our context

  20. Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203

    OpenAIRE

    Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Shin, Tae-Hoon; Seo, Yoojin; Kim, Hyung-Sik; Kang, Kyung-Sun

    2016-01-01

    Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem c...

  1. Application Value of Amniotic Cell Culture in the Prenatal Diagnosis%羊水细胞培养在产前诊断中的应用价值

    Institute of Scientific and Technical Information of China (English)

    张赫

    2016-01-01

    目的:探讨羊水细胞培养在产前诊断中的应用价值。方法整群选取2010年5月—2015年10月在医院进行产前诊断的216例高危孕妇,给予羊膜穿刺和羊水细胞培养后分析染色体核型。结果所有孕妇穿刺成功率为99.1%,经过羊水细胞培养后,211例为正常核型,占97.7%,5例为异常核型,占2.3%。结论对孕妇进行羊水细胞培养在产前诊断中意义重大,可以预防先天性缺陷患儿的发生,提高新生儿质量,值得应用。%Objective To discuss the application value of amniotic cell culture in the prenatal diagnosis. Methods 216 cas-es of high risk pregnant women with prenatal diagnosis treated in our hospital from May 2010 to October 2015 were select-ed, and the chromosome karyotypes were analyzed after giving amniocentesis and amniotic cell culture. Results The success rate of amniocentesis of all pregnant women was 99.1%, 211 cases were normal karyotypes, accounting for 97.7%, 5 cases were abnormal karyotypes, accounting for 2.3%. Conclusion Amniotic cell culture in the prenatal diagnosis is of great sig-nificance, which can prevent the occurrence of children with congenital defects and improve the quality of newborns, and it is worth application.

  2. Induced pluripotent stem (iPS) cells from human fetal stem cells.

    Science.gov (United States)

    Guillot, Pascale V

    2016-02-01

    Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications.

  3. Hydroxyapatite ocular prosthesis implants wrapped by human amniotic membrane%羊膜包裹的羟基磷灰石义眼座植入

    Institute of Scientific and Technical Information of China (English)

    刘文斌; 孙思勤; 温耀春; 万川

    2001-01-01

    Objective To observe the clinical effects of human amniotic membrane as an alternative substrate for wrapping hydroxyapatite ocular prosthesis implants.Methods After enucleation, hydroxyapatite orbital implants wrapped by human amniotic membrane were implanted with four rectus sutured to the implants .Among them, 4 cases were given primary implantation and 3 cases secondary implantation.Results After 8 to 39 week follow-up, it was found that in 6 cases, the eyelids were plump and prosthesis moved well, but one implant was removed because of failure of scleral patch graft for implant exposure.Conclusion The clinical results were satisfactory for ocular prosthesis implantation of hydroxyapatite sphere wrapped by human amniotic membrane, which may be considered as an alternative substrate for ocular prosthesis implants wrapper.%目的观察羊膜包裹的羟基磷灰石义眼座植入的临床效果。方法 7例眼球摘除术后,以库存的人羊膜缝合包裹选定的合适直径的义眼座,后段覆盖羊膜多处开窗后将义眼座植入肌椎内,再通过义眼座前1/3处穿出 4条缝线将 4条直肌缝合固定在义眼座上,然后分层缝合筋膜、结膜。结果追踪观察 8~39 wk,除 1例因局部放疗后眼结膜、筋膜萎缩,义眼座暴露,库存异体巩膜修补失败后,将义眼座取出外,其余 6例外观饱满,义眼活动良好。结论羊膜包裹的羟基磷灰石义眼座效果良好。羊膜可成为新的义眼座包裹物。

  4. Therapeutic potential of amniotic-fluid-derived stem cells on liver fibrosis model in mice

    Directory of Open Access Journals (Sweden)

    Shao-Yu Peng

    2014-06-01

    Conclusion: The possible repair mechanism from our data revealed that EGFP-mAFSCs may fuse with the recipient liver cells. Overall, EGFP-mAFSCs can ameliorate liver fibrosis in mice, thus providing insight into the future development of regenerative medicine.

  5. Release of arachidonic acid metabolites from bloodby cultivation of human amniotic fluid with oneself blood%羊水与自体血培养后刺激花生四烯酸代谢物的释放

    Institute of Scientific and Technical Information of China (English)

    杨鉴; 余艳红; 周凤琴; 钟梅

    2000-01-01

    AIM: To investigate the effect of human amniotic fluid on the release of thromboxane A2 (TXA2), prostaglandin I2 (PGI2) and Leukotriene C4 (LTC4) from blood cells. METHODS: 1 mL human amniotic fluid and 10 mL oneself blood collected from 38 - 41 weeks with cesarean section were cultured at 37℃ for 30 min, and then centrifuged. The supernatants were taken and stored at - 70℃. TXB2 and 6 - Keto - PGF1α of the supernrants were determined by radioimmunoassay and LTC4 by enzyme immunoassay. RESULTS: It was found that the levels of TXB2 and LTC4 in blood were elevated from (63.5 + 52.0) ng/L and (40.1 + 39.2) ng/L to (189.1 + 102.0) ng/L and (293.5 + 206.1) ng/L respectively ( P 0.05 ). CONCLUSION: Amniotic fluid might stimulate the release of TXA2 and LTC4 from blood, it might affect the balance of TXA2 and PGI2 in blood, which might play an important role in the pathogenesis of amniotic fluid embolism.%目的:探讨人羊水在体外刺激自体血细胞释放前列环素(PGI2)、血栓素A2(TXA2)和白三烯C4(LTC4)等花生四烯酸代谢物的作用。方法:取产妇羊水与自体血进行培养,用放射免疫分析法检测血中血栓素B2(TXB2)和6-酮前列腺素F1α(6-Keto-PGF1α)的含量,用酶联免疫法检测LTC4。结果:羊水能刺激血细胞释放TXA2和LTC4,胎粪污染的羊水作用更为明显。TXB2的含量由加羊水培养前的(63.5±52.0)ne/L增加到培养后的(189.1±l02.0)ng/L(P0.05)。结论:羊水能刺激血细胞释放花生四烯酸类生物活性物质,使其正常的平衡状态被破坏,可能与羊水栓塞的发生机理有关。

  6. A Dermal Equivalent Engineered with TGF-β3 Expressing Bone Marrow Stromal Cells and Amniotic Membrane: Cosmetic Healing of Full-Thickness Skin Wounds in Rats.

    Science.gov (United States)

    Samadikuchaksaraei, Ali; Mehdipour, Ahmad; Habibi Roudkenar, Mehryar; Verdi, Javad; Joghataei, Mohammad Taghi; As'adi, Kamran; Amiri, Fatemeh; Dehghan Harati, Mozhgan; Gholipourmalekabadi, Mazaher; Karkuki Osguei, Nushin

    2016-12-01

    Transforming growth factor beta-3 (TGF-β3) has been shown to decrease scar formation after scheduled topical applications to the cutaneous wounds. This study aimed to continuously deliver TGF-β3, during the early phase of wound healing, by engineering a dermal equivalent (DE) using TGF-β3 expressing bone marrow stromal cells (BM-SCs) and human dehydrated amniotic membrane (hDAM). To engineer a DE, rat BM-SCs were seeded on the hDAM and TGF-β3 was transiently transfected into the BM-SCs using a plasmid vector. Pieces of the dermal equivalent were transplanted onto the full-thickness excisional skin wounds in rats. The process of wound healing was assessed by image analysis, Manchester Scar Scale (MSS), and histopathological studies 7, 14, 21, and 85 days after the excision. The results confirmed accurate construction of recombinant pcDNA3.1-TGF-β3 expression system and showed that the transfected BM-SCs seeded on hDAM expressed TGF-β3 mRNA and protein from day 3 through day 7 after transfection. After implantation of the DE, contraction of the wounds was measured from day 7 through 21 and analyzed by linear regression, which revealed that the rate of wound contraction in all experimental groups was similar. Histologic evaluation demonstrated that transfected BM-SCs decreased retention and recruitment of the cells during the early stage of wound healing, decreased the formation of vascular structures and led to formation of uniformly parallel collagen bundles. MSS scores showed that TGF-β3 secreting cells significantly improved the cosmetic appearance of the healed skin and decreased the scar formation. From these results, it could be concluded that transient secretion of TGF-β3, during the early phase of healing, by BM-SCs seeded on hDAM can improve the cosmetic appearance of the scar in cutaneous wounds without negatively affecting the process of wound repair. Copyright © 2016 International Center for Artificial Organs and Transplantation and Wiley

  7. 人羊膜在牙种植术中的临床应用研究%Clinical Application of the Human Amniotic Membrane in Dental Implantation

    Institute of Scientific and Technical Information of China (English)

    汪文丰; 翟幼文; 王鑫; 黄啸林

    2014-01-01

    目的:观察人羊膜联合生物骨粉(Bio-Oss)引导骨再生术应用于牙种植的临床疗效。方法:将32例骨量不足的患者按随机数字表法分为A、B两组各16例。两组植入种植体同时植入Bio-Oss与自体骨的混合物,A组表面覆盖人羊膜,B组表面覆盖金属钛膜。术后1、3、6、12个月通过临床检查及X线检查评价临床疗效。结果:两组植骨区新骨形成均良好,牙槽嵴外形丰满,种植体无松动脱落,均完成了冠修复。长期随访示A组骨吸收量更小,与B组比较差异有统计学意义(P<0.01)。结论:人羊膜及金属钛膜联合引导骨再生术应用于种植区骨量不足的患者均可取得较好的疗效,但人羊膜组效果更佳。%Objective:To observe the clinical curative effect of amniotic membrane combined with Bio-Oss guided bone regeneration in dental implant.Method:32 cases of bone defects were randomly divided into the group A, B for 16 cases. While in the implantation of Bio-Oss and autogenous bone,the group A covered with human amniotic membrane,the group B with titanium membrane. The clinical efficacy after 1,3,6 and 12 months’ treatment were evaluated by clinical examination and X-ray examination.Result:The two groups had a good bone formation in bone graft area,alveolar ridge shape plump,no implant got loosen or missed,and finally the crown were all completed. The long term follow-up showed there was less bone absorption in the group A,and compared with the group B,the difference was statistically significant(P<0.01).Conclusion:Human amniotic membrane and titanium membrane combined with guided bone regeneration technique applied in the planting area of bone deficiency patients can affect well,and human amniotic membrane get a more significant effect than titanium membrane.

  8. Combined HLA matched limbal stem cells allograft with amniotic membrane transplantation as a prophylactic surgical procedure to prevent corneal graft rejection after penetrating keratoplasty: case report

    Directory of Open Access Journals (Sweden)

    Paolo Capozzi

    2014-09-01

    Full Text Available Purpose. To determine if the use of combined HLA matched limbal stem cells allograft with amniotic membrane transplantation (AMT is a safe and effective prophylactic surgical procedure to prevent corneal graft after penetrating keratoplasty (PK. Methods. We report the case of a 17 years old patient with a history of congenital glaucoma, trabeculectomy and multiple corneal graft rejections, presenting total limbal cell deficiency. To reduce the possibility of graft rejection in the left eye after a new PK, a two step procedure was performed. At first the patient underwent a combined HLA matched limbal stem cells allograft (LAT and AMT and then, 10 months later, a new PK. Results. During 12 months of follow-up, the corneal graft remained stable and smooth, with no sign of graft rejection. Conclusions. In our patient, the prophylactic use of LAT from HLA-matched donors and AMT before PK, may result in a better prognosis of corneal graft survival.

  9. Effect of the Mode of Application of Cryopreserved Human Amniotic Membrane on Adhesion Formation after Abdomino-Pelvic Surgery in a Mouse Model.

    Science.gov (United States)

    Nassif, Joseph; Abbasi, Sehrish A; Kechli, Mohamad Karim; Boutary, Suzan S; Ghulmiyyah, Labib; Khalifeh, Ibrahim; Abou Ghaddara, Hussein; Nassar, Anwar H

    2016-01-01

    Adhesions after abdomino-pelvic surgery are a cause of morbidity and reoperations. The use of human amniotic membrane (HAM) for adhesion prevention has given controversial results. The mode of administration of the amniotic membrane has not been well studied. This study assessed the efficacy of two modes of application of cryopreserved HAM, patch or fragmented in Lactated Ringer (LR) solution, for the prevention of pelvic adhesion formation postabdomino-pelvic surgery in a mice model. After a midline laparotomy incision, a small cautery lesion was done on each side of the abdominal wall peritoneum in mice. In Group A (control; n = 42), the abdomen was closed directly, Group B (n = 42) received 2.5 ml of LR prior to closure. In Groups C (n = 42) and D (n = 42), a 2 cm × 2 cm patch of HAM and another one fragmented and dispersed in 2.5 ml of LR were applied prior to closure, respectively. Two weeks later, a laparotomy was performed, and gross and pathological evaluation of adhesions, fibrosis, angiogenesis, and inflammation were conducted. Group D exhibited a significantly lower rate of gross adhesion formation. Fibrosis was significantly lowest in Group C as compared to the control. Group B had the lowest vascular formation in the adhesions. The use of HAM fragmented in LR solution is associated with a significantly lower incidence of postoperative adhesions in mice when compared to LR alone, HAM patch, or control. The mechanism of action of this reduction needs to be elucidated by future studies.

  10. [Amniotic membrane in conjunctivoplasty].

    Science.gov (United States)

    Samoila, O; Lacramioara, Totu; Mihu, D

    2012-01-01

    The basic principles of conjunctival reconstruction are similar in all pathologies that inflict conjunctival tissue loss. Large conjunctival defects are difficult to treat, with little conjunctival reserve that can be used to close the defect. The study had the objective to find alternatives to conjunctival autograft. Frozen amniotic membrane was investigated. From 27 cases, 22 cases were reconstructed using amniotic membrane alone, but in 5 cases conjunctival autograft was also needed. Bulbar conjunctiva was reconstructed in 23 cases, of which 19 with pterygium, 3 with conjunctival tumors, one operatory plague. Fornix reconstruction was carried out in 4 cases, one tumor and 3 symblepharons (2 after conjunctival burns and one pemfigus). The rate of pterygium recurrence was 7,14%. Amniotic membrane can heal large defects of the conjunctiva, either bulbar or forniceal.

  11. Identification of fetal ABO blood groups by analyzing amniotic fluid cells via PCR-SSP%PCR-SSP技术对羊水细胞ABO血型的基因鉴定

    Institute of Scientific and Technical Information of China (English)

    马欣; 陈江

    2013-01-01

    目的 通过PCR-SSP基因技术检测胎儿羊水细胞ABO血型基因型,产前诊断胎儿ABO血型.方法 选取了6名孕16 W以上的孕妇,抽取羊水细胞并进行分离,提取羊水细胞DNA,运用PCR-SSP技术分析其ABO血型基因型,并通过出生后的脐带血的血型鉴定进行确认.结果 6例羊水标本均通过PCR-SSP方法检测出了ABO血型的基因型;该6名胎儿的脐带血的ABO血型与羊水细胞的血型一致.结论 PCR-SSP技术可以准确地检测胎儿羊水细胞的ABO血型.%Objective To identify fetal ABO blood groups by analyzing amniotic fluid cells via PCR-SSP. Methods Six pregnant women with gestational age between 16 weeks and 25 weeks were selected. DNA was taken in amniotic fluid cells from these pregnant women and then ABO genotypes of amniotic fluid cells were detected by PCR-SSP. And the ABO genotypes were conformed by detecting the cord blood . Results The ABO genotypes were identified from the 6 samples of amniotic fluid cells, and the results were in accordance with cord blood. Conclusion Fetal ABO blood group can be accurately detected out by amniotic fluid cells.

  12. Mercury concentrations in human placenta, umbilical cord, cord blood and amniotic fluid and their relations with body parameters of newborns.

    Science.gov (United States)

    Kozikowska, Iwona; Binkowski, Łukasz J; Szczepańska, Katarzyna; Sławska, Helena; Miszczuk, Katarzyna; Śliwińska, Magdalena; Łaciak, Tomasz; Stawarz, Robert

    2013-11-01

    Studies were conducted on samples taken from giving birth women (n = 40) living in Poland, representing three age groups: 19-25, 26-30 and 31-38 years old. Mercury concentrations were measured with CV-AAS in placenta, umbilical cord, cord blood and amniotic fluid. The placentas weight did not exceed the 750 g value and was heavier than 310 g. Mean values of Hg concentrations in blood, placenta and umbilical cord were similar (c.a. 9 μg/g). High levels of mercury were noted in cord blood which in 75% of all observations exceeded (up to 17 μg/L) the safe dose set by US EPA (5.8 μg/L). No statistically significant differences in medium level of Hg in all the studied tissues among age groups of women were observed. Positive correlations between Hg concentrations in placenta and umbilical cord and cord blood were revealed as well as some negative ones between mercury concentrations and pregnancy parameters. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Amniotic Fluid Embolism

    Science.gov (United States)

    ... embolisms are rare, which makes it difficult to identify risk factors. It's estimated that there are between 1 ... Kramer MS, et al. Amniotic fluid embolism: Incidence, risk factors, and impact on perinatal outcome. BJOG: An International Journal of Obstetrics and Gynaecology. 2012;119:874. Baskett ...

  14. 拔牙创使用人羊膜的临床研究%Clinical Application of the Human Amniotic Membrane in Tooth Extraction Wound

    Institute of Scientific and Technical Information of China (English)

    翟幼文; 汪文丰; 黄啸林; 梁向新; 王鑫

    2015-01-01

    目的:观察人羊膜植入临床拔牙创后对牙槽骨愈合的影响.方法:选用来本院需拔除双侧下颌第二前磨牙正畸治疗的患者20例,右侧拔牙创植入人羊膜作为试验组,左侧拔牙创作为空白对照.术后1、2、3个月拍口腔全景片,观察新骨形成情况及牙槽嵴吸收评价拔牙创愈合情况.结果:术后1周牙龈愈合及术后1、2、3个月拔牙创新骨形成量试验组优于对照组,术后1、2、3个月牙槽嵴吸收高度试验组均明显低于对照组(P0.01).结论:临床拔牙创植入人羊膜能促进拔牙创的早期愈合,保存牙槽嵴高度.%Objective:To observe the effect of human amniotic membrane planted in tooth extraction wound in the knitting of alveolar bone.Method:20 patients who need to extract the two lower second premolars because of orthodontic treatment in our department were chosen. The right side were considered as the experimental group with the human amniotic membrane implanted in tooth extraction wound, and the left as blank control group. Then the condition of knitting after tooth extraction and the absorption of alveolar bone were observed by taking oral panoramic pieces respectively in the first,second and third months,to evaluate the knitting of the tooth extraction wound.Result:The healing of the gingiva in the experimental group was better than in the control group one week after the operation.The same happened in the new bone formation in the tooth extraction wound.The height of alveolar ridge absorbed in experimental group were significantly lower than control group in the 1st, 2nd, 3rd month(P0.01).Conclusion:Human amnion can promote the healing of tooth extraction wound in early days and keep the height of alveolar ridge.

  15. A new method for immediate derivatization of hydroxyl groups by fluoroalkyl chloroformates and its application for the determination of sterols and tocopherols in human serum and amniotic fluid by gas chromatography-mass spectrometry.

    Science.gov (United States)

    Řimnáčová, L; Hušek, P; Šimek, P

    2014-04-25

    A new method has been described for efficient derivatization of secondary alicyclic hydroxyl groups in steroids by alkyl chloroformates (RCFs). Cholesterol, an essential human sterol and a steroid precursor in eukaryotic cells, was used as a model for treatment with various RCFs in an aqueous and non-aqueous environment. While the cholesterol hydroxyl group did not react completely with any of the tested RCFs reagents in the former case, trifluoroethyl chloroformate (TFECF) or heptafluorobutyl chloroformate (HFBCF) fully converts cholesterol and related metabolites into the corresponding mixed carbonates under anhydrous conditions in seconds. The acylation reaction was combined with liquid-liquid microextraction (LLME) between isooctane and acetonitrile phase. The sample preparation requires just a stepwise addition of 50μl isooctane with 5μl of a pyridine catalyst, 100μl acetonitrile and 100μl isooctane with dissolved 5μl of the fluoroalkyl chloroformate reagent to a dried sample. The protocol developed in this study was successfully tested for GC-MS analysis of 12 important model steroids and four main tocopherols. Each analyte provided a single peak with excellent GC separation properties, well defined EI spectra containing diagnostic fragment ions suitable for their identification and quantitation. The new method was further validated for the determination of six diagnostic non-cholesterol sterols and four main tocopherols in human serum and in amniotic fluid. Satisfactory data were obtained in terms of calibration, quantitation limits (for sterols and tocopherols, 0.05 and 0.15μg/ml, respectively), within-run precision (0.9-19.5%) and between-run precision (0.2-19.0%), accuracy (82-115%) and recovery (90-110%). The validated method was successfully applied to GC-MS analysis of the analytes in woman sera and amniotic fluids and the results are well-comparable with those reported by other authors. The presented work demonstrates for the first time

  16. In Vitro Differentiation Potential of Human Placenta Derived Cells into Skin Cells

    Directory of Open Access Journals (Sweden)

    Ruhma Mahmood

    2015-01-01

    Full Text Available Skin autografting is the most viable and aesthetic technique for treatment of extensive burns; however, this practice has potential limitations. Harvesting cells from neonatal sources (such as placental tissue is a simple, inexpensive, and noninvasive procedure. In the current study authors sought to evaluate in vitro potential of human placenta derived stem cells to develop into skin-like cells. After extensive washing, amniotic membrane and umbilical cord tissue were separated to harvest amniotic epithelial cells (AECs and umbilical cord mesenchymal stem cells (UC-MSCs, respectively. Both types of cells were characterized for the expression of embryonic lineage markers and their growth characteristics were determined. AECs and UC-MSCs were induced to differentiate into keratinocytes-like and dermal fibroblasts-like cells, respectively. After induction, morphological changes were detected by microscopy. The differentiation potential was further assessed using immunostaining and RT-PCR analyses. AECs were positive for cytokeratins and E-Cadherin while UC-MSCs were positive for fibroblast specific makers. AECs differentiated into keratinocytes-like cells showed positive expression of keratinocyte specific cytokeratins, involucrin, and loricrin. UC-MSCs differentiated into dermal fibroblast-like cells indicated expression of collagen type 3, desmin, FGF-7, fibroblast activation protein alpha, procollagen-1, and vimentin. In conclusion, placenta is a potential source of cells to develop into skin-like cells.

  17. 利用抗氧化剂促进羊水细胞培养效果的研究%The study of improving the culture results of amniotic fluid cells by adding antioxidants

    Institute of Scientific and Technical Information of China (English)

    丘力功; 蔡小杰; 黄志诚

    2012-01-01

    目的 通过添加抗氧化剂促进羊水细胞的培养效果.方法 分离传代的成纤维型羊水细胞,以生长指数为检测指标,在H配方的基础上利用部分析因设计法和最陡爬坡法对促进羊水细胞生长的抗氧化剂进行筛选和优化.优化后的培养基通过原代羊水细胞培养进行效果验证.结果 添加的维生素E、L-抗坏血酸、α硫辛酸和还原型谷胱甘肽等抗氧化剂皆能促进羊水细胞的生长,且具有协同效应.与H培养基相比,经最陡爬坡法优化的培养基培养的羊水细胞在克隆总数、生长指数等指标都提高3倍以上,分裂相细胞总数增长4倍以上.结论 改良后的羊水细胞培养基培养效果要显著优于H配方培养基,在产前诊断应用中具有推广价值.%Objective To improve the culture results of amniotic fluid by adding antioxidants. Methods Using Fractional factorial designs combined with the steepest ascent path approach, the concentration of antioxidants added in H medium was optimized to increase the growth speed of the passage fibroblast cells from amniotic fluid . And the performance of the optimized culture medium was tested with the growth index, the numbers of clones and metaphase of primary amniotic fluid cells culture. Results Vitamin E, L-ascorbic acid,α-lipoic acid and reduced CSH can all promote the growth of the amniotic fluid cells with synergic effect. Total number of clones,growth index,and total number of metaphase cells from amniotic fluid cells cultured in medium optimized by the steepest ascent path approach are all 3 times higher than those from H medium. Conclusion The optimization method of adding antioxidants in H medium can improve significantly the results of amniotic fluid cells culture and the optimized culture medium is applicable to the prenatal diagnosis.

  18. To research application of the reforming of amniotic cells culture on the methods in prenatal diagnosis%改良羊水细胞培养方法在产前诊断中的应用研究

    Institute of Scientific and Technical Information of China (English)

    向文秀

    2011-01-01

    目的 研究改良羊水细胞培养法在产前诊断中的应用.方法 采用改良多种处理方式的经典羊水细胞培养法和原位培养法对280例孕16-32周的孕妇进行羊水产前诊断.结果 成功率99.7%,培养收获时间5-7天,最短收获5天,最长7天.有效的细胞染色体核型>60个/例,发现异常核型15例,染色体异常栓出率5.4%.结论 两种改良羊水细胞培养技术,培养成功率高,细胞培养时间短,可供分析染色体核型多,培养适用范围宽,可满足临床产前诊断的需要.%objective: To research application of the reforming of amniotic fluid cells culture in different methods in prenatal diagnosis. Methods: 280 samples of amniotic fluid from pregnant women during 16 -32th -week gestation were cultured in the reforming of diverse ways to deal with of amniotic cell culture in classical method of amniotic fluid cell culture and method of situ cultivation. Results: The successful rate was 99. 7%. Culture Collection time: 5 -7days, the shortest time: 5 days, The most time: 7days. Effective karyotypes in each case were more than the 60. Discovered 15 cases of abnormal karyotype. 5.4% rate of chromosomal abnormalities detected. Conclusion: The reforming of two amniotic fluid cell culture techniques were superior than the classical method of amniotic fluid cell culture in training success rate, cell culture time, providing the number of analysis of karyotype and training scope. They can meet the needs of clinical prenatal diagnosis.

  19. Chromosome karyotype analysis of amniotic fluid cells of 1 466 pregnant women in Yangzhou%扬州地区1466例孕妇羊水细胞染色体核型分析

    Institute of Scientific and Technical Information of China (English)

    陈剑; 徐贵江

    2014-01-01

    Objective To explore the clinic value of chromosome karyotype analysis of amniotic fluid cells in prenatal diagnosis . Methods 1 466 cases of pregnant women who had the prenatal diagnosis indexes were selected ,and their amniotic fluid specimens were collected through amniocentesis guiding by type‐B ultrasonic around the 16th to 24th week .Amniotic fluid cells were gained after a successful cell culture .G banding was used for the karyotype analysis of amniotic fluid cells .Results The one‐time success rate of cultivation for amniotic fluid cells was 99 .8% .In 1 466 cases of pregnant women ,there were 16 cases of abnormal karyotype polymorphism (including 12 cases of trisomy 21 ,1 case of trisomy 18 ,and 3 cases of Chromosome abnormalities) and 3 cases of chromosomal polymorphism .Conclusion The chromosome karyotype analysis of amniotic fluid cell is still an irreplaceable test in prenatal diagnosis .%目的:探讨孕妇羊水细胞染色体核型分析在产前诊断中的临床应用价值。方法选择孕16~24周、具有产前诊断指征的孕妇1466例,在B超引导下行羊膜腔穿刺抽取羊水。经过羊水细胞培养增殖成功后收获细胞,G显带检查分析羊水细胞的染色体核型。结果羊水细胞一次性培养成功率为99.8%。1466例孕妇中,检出16例异常核型(其中12例21三体,1例18三体,3例染色体异常)和3例染色体多态性。结论羊水细胞染色体核型检查仍然是产前诊断中不可替代的手段。

  20. The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane.

    Science.gov (United States)

    Russo, Alessandra; Bonci, Paola; Bonci, Paolo

    2012-06-01

    The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.

  1. Elemental analysis of human amniotic fluid and placenta by total-reflection X-ray fluorescence and energy-dispersive X-ray fluorescence: child weight and maternal age dependence

    Science.gov (United States)

    Carvalho, M. L.; Custódio, P. J.; Reus, U.; Prange, A.

    2001-11-01

    This work is an attempt to evaluate the possible influence of the mother's age in trace element concentrations in human amniotic fluid and placenta and whether these concentrations are correlated to the weight of the newborn infants. Total-reflection X-ray fluorescence (TXRF) was used to analyze 16 amniotic fluid samples, and the placenta samples were analyzed by energy dispersive X-ray fluorescence (EDXRF). The whole samples were collected during delivery from healthy mothers and healthy infants and full-term pregnancies. According to the age of the mother, three different groups were considered: 20-25, 25-30 and 30-40 years old. Only two mothers were aged more than 35 years. The weight of the infants ranged from 2.56 to 4.05 kg and three groups were also considered: 2.5-3, 3-3.5 and 3.5-4 kg. The organic matrix of the amniotic fluid samples was removed by treatment with HNO 3 followed by oxygen plasma ashing. Yttrium was used as the internal standard for TXRF analysis. Placenta samples were lyophilized and analyzed by EDXRF without any chemical treatment. Very low levels of Ni and Sr were found in the amniotic fluid samples, and were independent of the age of the mother and weight of the child. Cr, Mn, Se and Pb were at the level of the detection limit. Zn, considered one of the key elements in neonatal health, was not significantly different in the samples analyzed; however, it was weakly related to birth weigh. The concentrations obtained ranged from 0.11 to 0.92 mg/l and 30 to 65 μg/g in amniotic fluid and placenta, respectively. The only two elements which seemed to be significantly correlated with mother's age and newborn weight were Ca and Fe for both types of sample: Ca levels were increased in heavier children and older mothers; however, Fe increased with increasing maternal age, but decreased for heavier babies. The same conclusions were obtained for placenta and amniotic fluid samples. Cu is closely associated with Fe in its function in the organism

  2. Amniotic fluid embolism

    OpenAIRE

    Thongrong, Cattleya; Kasemsiri, Pornthep; Hofmann, James P; Bergese, Sergio D.; Thomas J Papadimos; Gracias, Vicente H.; Adolph, Michael D.; Stawicki, Stanislaw P A

    2013-01-01

    Amniotic fluid embolism (AFE) is an unpredictable and as-of-yet unpreventable complication of maternity. With its low incidence it is unlikely that any given practitioner will be confronted with a case of AFE. However, this rare occurrence carries a high probability of serious sequelae including cardiac arrest, ARDS, coagulopathy with massive hemorrhage, encephalopathy, seizures, and both maternal and infant mortality. In this review the current state of medical knowledge about AFE is outline...

  3. EXPERIMENTAL STUDY ON HUMAN AMNIOTIC MEMBRANE FOR REPAIRING TENDON SHEATH DEFECT%人羊膜修复腱鞘缺损的实验研究

    Institute of Scientific and Technical Information of China (English)

    高鸣; 赵红芳; 田德虎; 于昆仑; 白江博; 董瑞一; 张国辉

    2013-01-01

    目的 探讨人羊膜修复鸡足趾腱鞘缺损后防止肌腱粘连的可行性和有效性.方法 取行剖腹产术产妇自愿捐赠的胎盘,制备大小为1.5 cm×1.0 cm的羊膜片.3~6月龄健康雄性来亨鸡40只,体重(1.86±0.04)kg,取双足第3趾制备肌腱、腱鞘损伤模型.“8”字缝合修复肌腱后,右足采用羊膜片修复缺损腱鞘(A组),左足缺损腱鞘不作处理(B组).术后1、2、4、6周各取10只实验动物行大体及组织学观察,并按照Tang等肌腱粘连大体观察分级标准进行分级,生物力学试验测定肌腱滑移度及总屈趾角度.结果 术后实验动物均存活至实验完成,切口均愈合良好.随术后时间延长,大体及组织学观察显示两组均有假鞘(新生腱鞘)形成,但A组假鞘较B组成熟、光滑.术后1、6周A组肌腱粘连分级均明显优于B组,差异有统计学意义(P< 0.05).生物力学试验测定示,术后1、2周两组肌腱滑移度比较,差异均无统计学意义(P> 0.05),4、6周时A组肌腱滑移度均较B组长(P<0.05).术后1、2、4、6周A组总屈趾角度均小于B组,差异有统计学意义(P<0.05).结论 采用人羊膜修复鸡腱鞘缺损能有效预防肌腱粘连,利于肌腱滑动功能的恢复.%Objective To investigate the feasibility and effect of human amniotic membrane in prevention of tendon adhension after tendon sheat defect repair. Methods The amniotic membrane in size of 1.5 cm × 1.0 cm was harvested from human placenta which was voluntary donated from maternal after cesarean. Forty healthy male Leghorn chicken (aged 3-6 months) were selected, weighing (1.86 ± 0.04) kg. The model of flexor digitorum profundus tendon and tendon sheath defects was established at the third toe. After repair of the flexor digitorum profundus tendon, the human amniotic membrane was used to repair the tendon sheath defect in the right foot (group A), but tendon sheath defect was not repaired in the left foot (group B) . At 1, 2, 4

  4. 羊水干细胞的研究现状及应用前景%Current status in research on amniotic fluid-derived stem cells and its application persnective

    Institute of Scientific and Technical Information of China (English)

    白璐; 陈必良

    2011-01-01

    Stem cells which emerged at the beginning of human life, have been widely used in medicine and many other fields. Compared with embryonic stem cells (ESC) and adult stem cells (ASC), amniotic fluid-derived stem cells (AFS) not only can be easily obtained but also are safe to the pregnant women and her fetus. Meanwhile, the isolated and amplified AFS have some surface markers as same as other stem cells and can differentiate into many cells. They can be used in fetus herseff or autologous transplantation, as well as other individuals which are compatible in immunity. At present, researches on AFS have attracted more and more attention. In this article, the related contents in researches on ASC were reviewed.%干细胞已被广泛用于医学及多个领域.与胚胎干细胞和成体干细胞相比,羊水取材方便,对孕妇及胎儿安全微创,分离扩增的羊水干细胞与其他干细胞一样存在一些表面标志物,且具有分化潜能.羊水干细胞不仅用于胎儿或自体移植,也可用于免疫相容的其他个体.目前对于羊水干细胞的研究已不断引起人们的关注,该文对羊水干细胞的相关内容作以综述.

  5. Amniotic fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis.

    Science.gov (United States)

    Jain, Sunil K; Baggerman, Eric W; Mohankumar, Krishnan; Namachivayam, Kopperuncholan; Jagadeeswaran, Ramasamy; Reyes, Victor E; Maheshwari, Akhil

    2014-03-01

    Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.

  6. Amniotic epithelial stem cell biocompatibility for electrospun poly(lactide-co-glycolide), poly(ε-caprolactone), poly(lactic acid) scaffolds.

    Science.gov (United States)

    Russo, Valentina; Tammaro, Loredana; Di Marcantonio, Lisa; Sorrentino, Andrea; Ancora, Massimo; Valbonetti, Luca; Turriani, Maura; Martelli, Alessandra; Cammà, Cesare; Barboni, Barbara

    2016-12-01

    Three biodegradable thermoplastic polymers, poly(ε-caprolactone) (PCL), poly(l-lactide-co-d,l-lactide) (PLA) and poly(l-lactide-co-glycolide) (PLGA), have been used to produce nonwovens scaffolds with uniform micrometer fibres. Scaffolds' physical and morphological characterization was performed by X-ray diffraction, Scanning Electron Microscopy and Contact-Angle test. Morphological investigations revealed that all produced fibres were randomly orientated with interconnected pores ranging between 5 and 12μm in diameter. An average fibre diameter of 1.5, 0.75 and 1.2μm was found for PCL, PLA and PLGA, respectively. Moreover, experiments were designed to verify whether the fabricated electrospun substrates were biocompatible for ovine amniotic epithelial stem cells (oAECs) under in vitro conditions. Cell adhesion, survival, spatial organization on fibres, proliferation index, and DNA quantification after 48h culture, showed an enhanced adhesion and proliferation, especially for PLGA scaffolds. The favourable interaction between oAECs and the fibrous scaffolds was attributed to the greatly improved porosity and pore size distribution of the electrospun scaffolds. In addition, AECs can be considered ideal for tissue engineering especially when using biocompatible and opportunely produced scaffolds. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Favaron, P O; Carvalho, R C; Borghesi, J; Anunciação, A R A; Miglino, M A

    2015-12-01

    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.

  8. Amniotic fluid stem cells rescue both in vitro and in vivo growth, innervation, and motility in nitrofen-exposed hypoplastic rat lungs through paracrine effects.

    Science.gov (United States)

    Pederiva, F; Ghionzoli, M; Pierro, A; De Coppi, P; Tovar, J A

    2013-01-01

    Lung hypoplasia can be prevented in vitro by retinoic acid (RA). Recent evidence suggests that amniotic fluid stem (AFS) cells may integrate injured lungs and influence their recovery. We tested the hypothesis that AFS cells might improve lung growth and motility by paracrine mechanisms. Pregnant rats received either nitrofen or vehicle on E9.5. In vitro E13 embryonic lungs were cultured in the presence of culture medium alone or with RA, basophils, or AFS cells. In vivo green fluorescent protein-expressing (GFP(+)) rat AFS cells were transplanted in nitrofen-exposed rats on E10.5. E13 lung explants were cultured before analysis. The surface, the number of terminal buds, and the frequency of bronchial contractions were assessed. Protein gene product 9.5 (PGP 9.5) and α-actin protein levels were measured. The lung explants transplanted with AFS cells were stained for α-actin, PGP 9.5, and TTF-1. The levels of FGF-10, VEGFα, and TGF-β1 secreted by the AFS cells in the culture medium were measured. Comparison between groups was made by ANOVA. In vitro, the surface, the number of terminal buds, and the bronchial peristalsis were increased in nitrofen+AFS cell explants in comparison with nitrofen-exposed lungs. While nitrofen+RA lungs were similar to nitrofen+AFS ones, basophils did not normalize these measurements. PGP 9.5 protein was decreased in nitrofen lungs, but after adding AFS cells, the value was similar to controls. No differences were found in the expression of α-actin. In vivo, the surface, number of terminal buds, and peristalsis were similar to control after injection of AFS cells in nitrofen-exposed rats. Colocalization with TTF-1-positive cells was found. The levels of FGF-10 and VEGFα were increased in nitrofen+AFS cell explants, while the levels of TGF-β1 were similar to controls. Lung growth, bronchial motility, and innervation were decreased in nitrofen explants and rescued by AFS cells both in vitro and in vivo, similarly to that observed

  9. The transplantation of Akt-overexpressing amniotic fluid-derived mesenchymal stem cells protects the heart against ischemia-reperfusion injury in rabbits.

    Science.gov (United States)

    Wang, Yan; Li, Yigang; Song, Lei; Li, Yanyan; Jiang, Shan; Zhang, Song

    2016-07-01

    Amniotic fluid-derived mesenchymal stem cells (AFMSCs) are an attractive cell source for applications in regenerative medicine, due to characteristics such as proliferative capacity and multipotency. In addition, Akt, a serine‑threonine kinase, maintains stem cells by promoting viability and proliferation. Whether the transplantation of Akt-overexpressing AFMSCs protects the heart against ischemia‑reperfusion (I/R) injury has yet to be elucidated. Accordingly, the Akt gene was overexpressed in AFMSCs using lentiviral transduction, and Akt‑AFMSCs were transplanted into the ischemic myocardium of rabbits prior to reperfusion. Any protective effects resulting from this procedure were subsequently sought after three weeks later. A histological examination revealed that there was a decrease in intramyocardial inflammation and ultrastructural damage, and an increase in capillary density and in the levels of GATA binding protein 4, connexin 43 and cardiac troponin T in the Akt‑AFMSC group compared with the control group. A significant decrease in cardiomyocyte apoptosis, accompanying an increase in phosphorylated Akt and B‑cell lymphoma 2 (Bcl-2) and a decrease in caspase‑3, was also observed. Furthermore, the left ventricular function was markedly augmented in the Akt‑AFMSC group compared with the control group. These observations suggested that the protective effect of AFMSCs may be due to the delivery of secreted cytokines, promotion of neoangiogenesis, prevention of cardiomyocyte apoptosis, transdifferentiation into cardiomyocytes and promotion of the viability of AFMSCs, which are assisted by Akt gene modification. Taken together, the results of the present study have indicated that transplantation of Akt-AFMSCs is able to alleviate myocardial I/R injury and improve cardiac function.

  10. Isolation and characterization of amniotic fluid-derived stem cells in Turner’s syndrome%Turner综合征羊水干细胞的分离及特征*★◆

    Institute of Scientific and Technical Information of China (English)

    龚余; 骆玉梅; 田霖; 刘海波; 陈欣洁; 孙筱放; 陈耀勇

    2013-01-01

    light microscope. The karyotype was analyzed. Specific cel surface antigens and cel cycle of the clonal amniotic fluid-derived mesenchymal stem cel s at passage 4 were characterized by flow cytometry. Osteogenic differentiation of amniotic fluid-derived mesenchymal stem cel s was confirmed by alkaline phosphatase staining and alizarin red staining. RESULTS AND CONCLUSION: The cultured human amniotic fluid-derived mesenchymal stem cel s proliferated rapidly after passage. Karyotype mapping showed abnormal female chromosome type with 45, X/46, XX observed. The amniotic fluid-derived mesenchymal stem cel s had an immunophenotype similar to that of common mesenchymal stem cel s and were positive for CD29, CD44, CD90 and CD105, but negative for CD34 and CD45. The cel cycle measurement showed that amniotic fluid-derived mesenchymal stem cel s cultured in vitro could maintain strong proliferation ability. Alkaline phosphatase staining and alizarin red staining results confirmed that amniotic fluid-derived mesenchymal stem cel s could be successful y induced to differentiate into osteocytes under specific culture media. These results demonstrated that amniotic fluid-derived mesenchymal stem cel s in 45, X/46, XX (Turner’s syndrome) were successful y isolated and the cel s had a great potential of proliferation and showed the characteristics of mesenchymal stem cel s.

  11. 荷斯坦牛羊水来源干细胞分离培养及其表面标志检测%Isolation of amniotic fluid-derived stem cells from Holstein cow and test of the cell markers

    Institute of Scientific and Technical Information of China (English)

    贺小英; 张涌; 郑月茂

    2011-01-01

    利用羊水来源干细胞培养技术体系,从胎龄285 d荷斯坦奶牛胎儿羊水中分离培养干细胞;采用RT-PCR技术检测干细胞表面标志或相关基因.成功分离培养出奶牛羊水采源干细胞,干细胞中CD-90、Nanog、Oct4、TERT、Sox2、Desmin和HES1表达阳性.从奶牛胎儿羊水中分离干细胞具有可行性和有效性,为进行转基因研究提供靶细胞奠定了基础.%The aim of this study was to get amniotic fluid-derived stem (AFS) cells from cow which may be used as target cells for transgenic research in future. AFS cells were isolated from amniotic fluid of a 285-day-old Holstein cow fetus. Expanded AFS cells were harvested for analysis of markers using reverse transcription-polymerase chain reaction (RT-PCR). The results showed that AFS cells were abtained from amniotic fluid. AFS cells expressed CD-90, Nanog,Oct4,TERT, Sox2,Desmin and HES1. We have demonstrated that stem cells isolated from amniotic fluid of cow may be used as target cells for transgenic research in future.

  12. Optimizing amniotic membrane tissue banking protocols for ophthalmic use.

    Science.gov (United States)

    Hettiarachchi, D; Dissanayake, V H W; Goonasekera, H W W

    2016-09-01

    Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco's modified eagle's medium (DMEM) and glycerol at -80 °C and at -196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at -196 and -80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10(6); 1.59, after 6 weeks in DMSO:FBS at -196 °C 3.0 × 10(6); 2.38 and at -80 °C 2.1 × 10(6); 1.60, in DMEM:Glycerol at -196 °C 3.6 × 10(6); 2.33 at -80 °C 23 × 10(6); 1.66 and at 4 °C 3.3 × 10(6); 2.14. Histology analysis of the fresh AM showed an intact epithelial

  13. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  14. Effects of ionizing radiation on glycerolated amniotic membranes as a substract for cultured human epithelium; Efeitos da radiacao ionizante em membranas amnioticas gliceroladas empregadas como substrato ao cultivo de epitelio humano

    Energy Technology Data Exchange (ETDEWEB)

    Paggiaro, Andre Oliveira

    2011-07-01

    The amniotic membrane (AM) is a biomaterial with biological properties that are beneficial to tissue repair. It has been used as a temporary coverage to threat burns and chronic wounds. Recently, it has been served as a substrate for keratinocytes culture to construct a living skin equivalent. However, MA is a biological material, and its transplantation could cause infectious disease for receptors. So, it must be preserved and sterilized before clinical use. The aim of this study was to evaluate the radiation effects on glycerol-preserved MA, considering its compatibility to support human keratinocytes culture. Four MA were stored in high concentrations of glycerol (> 85%) and half of them were radio sterilized with a dose of 25 kGy. Then, we established two groups: nonirradiated MA (MA-ni) and irradiated MA (MA-i). Both groups was deepithelialized by a standardized protocol and was investigated morphologically, immunohistochemical and ultrastructural. Subsequently human keratinocytes were cultivated immersed and in air-liquid interface on denuded surface of MA-i and MA-ni. The results were compared at 14 and 21 days of culture by light and electron microscopy. After epithelial denudation, analyses demonstrated the continuity of the basement membrane in MA-ni group, whereas in the irradiated group, there was no indication of the basement membrane’s presence on the surface of MA. The cell cultures showed that in the non-irradiated group, there was growth of a multi-layered and differentiated epithelium, with a stratum corneum’s formation in air-liquid interface. In the irradiated group, the epithelium had only two or three layer, little cell differentiation, with the same results immersed or air-liquid interface system. Glycerol-preserved MA was biocompatible with the growth of a cultivated epithelium, showing its potential as a skin substitute. Irradiation at 25 kGy cause structural damage to the tissue, making changes in basement membrane, that facilitates

  15. Amniotic Tissues for the Treatment of Chronic Plantar Fasciosis and Achilles Tendinosis

    Directory of Open Access Journals (Sweden)

    Bruce Werber

    2015-01-01

    Full Text Available Introduction. Allogeneic amniotic tissue and fluid may be used to treat chronic plantar fasciosis and Achilles tendinosis. This innovative approach involves delivering a unique allograft of live human cells in a nonimmunogenic structural tissue matrix to treat chronic tendon injury. These tissues convey very positive regenerative attributes; procurement is performed with maternal consent during elective caesarian birth. Materials and Methods. In the present investigation all patients were unresponsive to multiple standard therapies for a minimum of 6 months and were treated with one implantation of PalinGen SportFLOW around the plantar fascia and/or around the Achilles paratenon. The patients were given a standard protocol for postimplant active rehabilitation. Results. The analogue pretreatment pain score (VAS of 8. By the fourth week after treatment, all patients had significantly reduced self-reported pain. Twelve weeks following the procedure the average pain level had reduced to only 2. No adverse reactions were reported in any of the patients. Conclusion. All patients in this study experienced heel or Achilles pain, unresponsive to standard therapy protocols. After treatment all patients noted significant pain reduction, indicating that granulized amniotic membrane and amniotic fluid can be successfully used to treat both chronic plantar fasciosis and Achilles tendinosis.

  16. Membrana amniótica na reconstrução da superfície ocular após exérese de carcinoma de células escamosas da conjuntiva Amniotic membrane for ocular surface reconstruction after conjunctival squamous cell carcinoma resection

    Directory of Open Access Journals (Sweden)

    Paulo Roberto de Carvalho-Rêgo

    2008-02-01

    Full Text Available OBJETIVO: Investigar a utilização da membrana amniótica humana na reconstrução da superfície ocular após exérese de carcinoma de células escamosas da conjuntiva. MÉTODOS: A membrana amniótica foi captada a partir de parto cesárea, conservada em meio de preservação de córnea e glicerol 1:1 e armazenada a -80º C. Os critérios de inclusão foram: pacientes portadores de lesão proliferativa conjuntival com características de carcinoma de células escamosas (lesão esbranquiçada plana ou elevada com aspecto em "carne de peixe", envolvendo conjuntiva, limbo e córnea. Oito olhos de 8 pacientes portadores de "carcinoma de células escamosas" da conjuntiva foram submetidos a cirurgia de exérese do tumor conjuntival, associado a transplante de membrana amniótica. Destes, 3 foram submetidos a epiteliectomia total da córnea e a transplante de limbo do olho contralateral. RESULTADOS: O tempo médio de seguimento foi de 17,8 meses (variação entre 10 e 35 meses. Cinco pacientes (71,4% obtiveram sucesso com o tratamento cirúrgico, com adequada reconstrução da área conjuntival retirada. Dois pacientes (28,6% obtiveram sucesso parcial, com presença de alterações cicatriciais leves (simbléfaro pequeno ou pequena fibrose. Um paciente foi excluído porque apresentou recidiva agressiva do tumor, com invasão intraocular, necessitando ser submetido a exenteração. CONCLUSÃO: O uso de membrana amniótica humana pode constituir uma importante opção para a reconstrução da superfície ocular após remoção de carcinoma de células escamosas.PURPOSE: This study was designed to evaluate the use of human amniotic membrane for ocular surface reconstruction after conjunctival squamous cell carcinoma resection. METHODS: Amniotic membrane was obtained at the time of cesarean section and was preserved at -80ºC in glycerol and cornea culture media at a ratio of 1:1. The inclusion criteria were patients presenting proliferating lesions

  17. Perfluorooctane Sulfonate Concentrations in Amniotic Fluid, Biomarkers of Fetal Leydig Cell Function, and Cryptorchidism and Hypospadias in Danish Boys (1980-1996)

    DEFF Research Database (Denmark)

    Toft, Gunnar; Jönsson, Bo A G; Bonde, Jens Peter

    2016-01-01

    associations with fetal hormone levels may have long-term implications for reproductive health. CITATION: Toft G, Jönsson BA, Bonde JP, Nørgaard-Pedersen B, Hougaard DM, Cohen A, Lindh CH, Ivell R, Anand-Ivell R, Lindhard MS. 2016. Perfluorooctane sulfonate concentrations in amniotic fluid, biomarkers of fetal...

  18. Ex vivo expansion of hematopoietic stem- and progenitor cells from cord blood in coculture with mesenchymal stroma cells from amnion, chorion, Wharton's jelly, amniotic fluid, cord blood, and bone marrow.

    Science.gov (United States)

    Klein, Caroline; Strobel, Julian; Zingsem, Jürgen; Richter, Richard H; Goecke, Tamme W; Beckmann, Matthias W; Eckstein, Reinhold; Weisbach, Volker

    2013-12-01

    In most cases, the amount of hematopoietic stem and progenitor cells (HSPCs) in a single cord blood (CB) unit is not sufficient for allogenic transplantation of adults. Therefore, two CB units are usually required. The ex vivo expansion of HSPCs from CB in coculture with mesenchymal stroma cells (MSCs) might be an alternative. It was investigated, whether bone marrow-derived MSCs, which have to be obtained in an invasive procedure, introduce a further donor and increases the risk of transmissible infectious diseases for the patient can be replaced by MSCs from amnion, chorion, Wharton's jelly, amniotic fluid, and CB, which can be isolated from placental tissue which is readily available when CB is sampled. In a two-step ex vivo coculture mononuclear cells from cryopreserved CB were cultured with different MSC-feederlayers in a medium supplemented with cytokines (stem cell factor, thrombopoietin [TPO], and granulocyte colony-stimulating factor). Expansion rates were analyzed as well, by long-term culture-initiating cell (LTC-IC) and colony-forming unit (CFU) assays, as by measuring CD34(+)- and CD45(+)-cells. Due to the comparably low number of 5×10(2) to 1×10(4) CD34(+)-cells per cm(2) MSC-monolayer, we observed comparably high expansion rates from 80 to 391,000 for CFU, 70 to 313,000 for CD34(+)-, and 200 to 352,000 for CD45(+)-cells. Expansion of LTC-IC was partly observed. Compared to the literature, we found a better expansion rate of CD34(+)-cells with MSCs from all different sources. This is probably due to the comparably low number of 5×10(2) to 1×10 CD34(+)-cells per cm(2) MSC-monolayer we used. Comparably, high expansion rates were observed from 80 to 391,000 for CFUs, 70 to 313,000 for CD34(+)-, and 200 to 352,000 for CD45(+)-cells. However, the expansion of CD34(+)-cells was significantly more effective with MSCs from bone marrow compared to MSCs from amnion, chorion, and Wharton's jelly. The comparison of MSCs from bone marrow with MSCs from CB and

  19. Characterization of Side Cell Populations Obtained from Human Amnion Mesenchymal Cells

    Institute of Scientific and Technical Information of China (English)

    LI Ning; PIAO Zhengfu; Mamoru Kobayashi; Koji Sasaki; DING Shu-qin; Aiko Kikuchi; Isao Kamo; Norio Sakuragawa

    2009-01-01

    Human amnion mesenchymal cells (AMCs) contain multipotent cells. To enrich such multipotent stem cells, we applied to AMCs the new method for the isolation of side population (SP) cells used for the enrichment of multipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs (AMC-SP cells). AMC-SP cells were found in 0.2% of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses uggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.

  20. Amniotic cells culture and its application in the prenatal diagnosis%孕中期羊水细胞培养及其在产前诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    武其文; 范含萍; 江峰; 浦春

    2012-01-01

    Objective :To explore an amniotic cell culture technology with higher harvests and verify its validity in prenatal diagnosis. Methods : The amniotic fluid samples were obtained in 238 pregnant women with gesta-tional weeks of 16 to 30 and indications for prenatal diagnosis by aseptic amniocentesis and cultured for determination of the chromosome karyo-type. In real performance, moderate improvement was done to the sample collection,cell inoculation and harvesting as well as aspects involved in the procedures. Results: Amniotic cells were finally cultured in 231 of 238 cases( 97. 1% )whose abnormalities were identified in 8( 3. 5% )in which 6 were abnormal number of chromosomes, 1 mosaic and another 1 , chromosomal balance translocation. Conclusion-. The modified amniotic cell culturing technique for kargotyping is effective in prenatal diagnosis of fetal chromosomal disease for its safety and reliability.%目的:探索一种具有较高培养成功率的羊水细胞培养技术及其在产前诊断中的应用.方法:238例孕16~30周且有产前诊断指征的孕妇,在无菌条件下行羊膜腔穿刺术采集羊水进行细胞培养和染色体核型分析.实验过程对标本的采集、细胞接种收获和制片等环节进行了改进.结果:238例孕妇羊水成功培养231例,成功率97.1%,发现染色体异常核型8例,异常率占3.5%;其中染色体数目异常6例,嵌合体1例,平衡易位1例.结论:改进后的羊水细胞培养染色体核型分析技术是孕中期产前诊断胎儿染色体病的一种安全、有效、可靠的方法.

  1. Application of chromosomal karyotype analysis of amniotic cells for pregnant women with advanced maternal age%羊水细胞染色体核型分析在高龄孕妇产前诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    许多

    2012-01-01

    Objective; To conduct prenatal diagnosis among pregnant women with advanced maternal age through chromosomal karyotype analysis of amniotic cells. Methods; A total of 910 pregnant women with advanced maternal age during 16-27 gestational weeks were selected, then amniotic cells were obtained by amniocentesis and cultured, chromosomal karyotype analysis was conducted after preparing chromosomes. Results; Among 910 pregnant women, amniotic cells culture succeeded in 891 , the successful rate was 97. 91%. Postoperative abortion occurred in 5 women, the abortion rate was 0.55%. A total of 43 pregnant women were found with abnormal karyotypes, including 24 pregnant women with autosomal chromosomal numerical abnormality, 6 pregnant women with sex chromosomal numerical abnormality, 2 pregnant women with chimera, and 11 pregnant women with chromosomal structural abnormality. Conclusion; Chromosomal karyotype analysis of amniotic cells is safe, effective, and essential prenatal diagnosis of pregnant women with advanced maternal age.%目的:利用羊水细胞染色体核型分析对高龄孕妇进行产前诊断.方法:对910例孕16 ~ 27周高龄孕妇进行羊膜腔穿刺抽取羊水细胞培养,制备染色体并分析染色体核型.结果:910例高龄孕妇,羊水培养成功891例,成功率为97.91%.5例术后流产,流产率为0.55%.检出43例异常核型,其中常染色体数目异常24例,性染色体数目异常6例,嵌合体2例,结构异常11例.结论:羊水细胞染色体核型分析高龄孕妇产前诊断是安全、有效且必要的.

  2. Significance of amniotic fluid cells culture in the prenatal diagnosis for chromosomal diseases%羊水细胞培养用于染色体病产前诊断分析

    Institute of Scientific and Technical Information of China (English)

    刘慈; 刘义辉; 刘学军; 王振海; 张晓艳; 辛虹

    2014-01-01

    Objective To investigate the feasibility and necessity of amniotic fluid cells culture in the prenatal diagnosis for chromosomal diseases in order to prevent the birth of fetus with chromosomal diseases .Methods The 293 specimens of amniotic cell obtained by amniocentesis from 293 pregnant women with prenatal diagnosis indications were cultured and analyzed .Results Among the 293 specimens of amniotic cell ,14 cases of chromosome abnormality were found , with the chromosome abnormality rate being 4.78%,including 9 cases of chromosome number abnormality and 5 cases of chromosomal structure abnormality .The chromosome abnormality rates were different in different groups with different prenatal diagnosis indications ,in which the chromosome abnormality rates were the highest in the group with one party of the couple with chromosome abnormality .Conclusion The amniotic cell culture and chromosome karyotype analysis are the effective means in prenatal diagnosis for chromosomal diseases in the pregnant women with prenatal diagnosis indications at mid trimester of pregnancy, moreover, the combined detection of serological screening , chromosome examination and ultrasonic inspection plays an important role in the prevention of child birth defects .%目的:探讨羊水细胞培养对孕中期孕妇进行产前诊断的可行性及必要性,防止染色体病患儿的出生。方法对293例有产前诊断指征的孕妇进行羊膜腔穿刺,采取羊水细胞培养,制备中期染色体,分析胎儿核型,进行产前诊断。结果发现染色体异常14例,异常率4.78%,其中数目异常9例,结构异常5例,不同产前诊断指征分组中的异常率不同,以夫妇一方为染色体异常组异常率最高。结论妊娠中期对有产前诊断指征的孕妇进行羊水细胞培养染色体核型分析是产前诊断的重要手段,而血清学筛查,染色体检查和超声检查三者相互结合对于预防出生缺陷意义重大。

  3. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  4. Application of FISH in prenatal diagnosis of chromosome number abnormality in amniotic fluid cells%FISH在产前羊水细胞染色体数目异常诊断中的应用观察

    Institute of Scientific and Technical Information of China (English)

    张艳丽; 李华锋; 高刚

    2011-01-01

    Objective To observe effect of fluorescence in situ hybridization(FISH) on prenatal diagnosis of abnormal number of chromosomes in amniotic fluid cells. Methods The amniotic fluid of 1 121 cases of pregnant women with down syndrome screening in high-risk or age higher than 35 years old, were got by amniocentesis, and udenvent rapid prenatal diagnosis by FISH. Then the G banding karyotypes from standard cytogenetic analysis after cultured amniotic fluid cells were compared to the FISH results. Results 16 cases were found abnormal result, including 7 cases of trisomy 21 , 4 cases of trisomy 21, and other 5 cases with abnormal. It was consistent with G banding karyotypes results. Conclusion Prenatal diagnosis of chromosome humber sbnormality by FISH is satisfactory.%目的 观察应用荧光原位杂交( FISH)技术产前诊断羊水细胞染色体数目异常的效果.方法 唐氏综合征筛查高危或高龄(≥35岁)孕妇1 121例,经腹部穿刺抽取羊水,应用FISH技术进行羊水细胞染色体数目检测,并将其结果与羊水细胞常规G显带核型分析结果作比较.结果 均获得诊断结果,发现16例异常胎儿,其中7例为21三体,4例为18三体,5例为其他异常.FISH检测与核型分析结果一致.结论 用FISH产前诊断羊水细胞染色体数目异常效果满意.

  5. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  6. Incidence, diagnosis and pathophysiology of amniotic fluid embolism.

    Science.gov (United States)

    Ito, F; Akasaka, J; Koike, N; Uekuri, C; Shigemitsu, A; Kobayashi, H

    2014-10-01

    Amniotic fluid embolism (AFE) is a rare clinical entity, sometimes fatal. A review was conducted to describe the frequency, diagnosis and pathophysiology of AFE. The reported incidences ranged from 1.9 cases per 100,000 maternities (UK) to 6.1 per 100,000 maternities (Australia), which can vary considerably, depending on the period, region of study and the definition. Although the development of amniotic fluid-specific markers would have an impact on early diagnosis, definition of AFE based on these markers is not widely accepted. To date, immunological mechanisms, amniotic fluid-dependent anaphylactic reaction and complement activation, have been proposed as potential pathogenetic and pathophysiological mechanisms. Immune cell activation induced through complement activation may be associated with the mechanism that immediately initiates maternal death, only in susceptible individuals. This review will focus on advances in the field of AFE biology and discuss the prevalence, diagnosis and pathophysiology of AFE.

  7. Amniotic membrane allografts: development and clinical utility in ophthalmology

    Directory of Open Access Journals (Sweden)

    Rizzuti A

    2014-12-01

    Full Text Available Allison Rizzuti,1,2 Adam Goldenberg,1 Douglas R Lazzaro1,2 1SUNY Downstate Medical Center, 2Kings County Hospital Center, Brooklyn, NY, USA Abstract: Amniotic membrane, the innermost layer of the placenta, is a tissue that promotes epithelialization, while decreasing inflammation, neovascularization, and scarring. It is used in the surgical management of a wide variety of ophthalmic conditions where it functions as a graft or patch in ocular surface reconstruction. The development of new preservation techniques, as well as a sutureless amniotic membrane, has allowed for easier, in-office placement, without the disadvantages of an operating room procedure. The purpose of this review is to describe the historical development of amniotic membrane in ophthalmology and to describe its current clinical applications, particularly focusing on recent advances. Keywords: ocular surface, cornea, stem cells, prokera, allograft, patch, transplantation

  8. 以人羊膜为支架的表皮黑素细胞培养与生物活性鉴定%Culture of human epidermal melanocytes with human amniotic membrane as a scaffold and their biological ability identification

    Institute of Scientific and Technical Information of China (English)

    佘小光; 刘小明; 雷铁池

    2011-01-01

    目的 从吸引疱疱顶微小皮片建立人表皮黑素细胞(MC)原代培养,以人羊膜为支架培养MC,观察羊膜对MC黏附、增殖和树突发育的影响.方法 用负压吸疱法分离疱顶微小皮片,多巴染色后对取自同一个体下腹部和前臂屈侧皮片的MC进行计数.皮片经胰酶消化后自基底面轻刮表皮细胞建立原代MC培养,将培养的第2~4代MC接种至新鲜或冻存羊膜上共孵育不同时间后,倒置显微镜下观察MC形态和树突发育.行石蜡包埋组织切片,HE染色后观察MC对羊膜的黏附.用MTT比色法测定接种在羊膜上的MC存活率.结果 前臂屈侧吸引疱疱顶皮片多巴染色阳性细胞个数为(1543.1±113.3)个/mm2,腹部皮片为(857.4±101.7)个/mm2.在4周内建立MC原代培养至少需要从2个疱顶(面积约25.1 mm2)的前臂皮片获取的表皮细胞.倒置显微镜下观察,与单纯接种细胞培养皿的MC比较,接种至新鲜或冻存羊膜上继续培养4、8、12 d的MC形态多为两极,树突细长延伸.HE染色示MC能黏附并均匀分布在羊膜的基底膜层表面.MTT法测定结果示,羊膜对MC的分裂增殖有抑制,但冻存羊膜与新鲜羊膜对MC增殖抑制差异无统计学意义(P>0.05).结论 人前臂屈侧皮肤富含活性MC,是获取吸引疱疱顶微小皮片建立MC原代培养的较好部位.人羊膜支持MC黏附生长和树突发育,有望成为一种能荷载体外培养扩增MC的人源生物支架.%Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were

  9. The evolution of amniote gastrulation: the blastopore-primitive streak transition.

    Science.gov (United States)

    Stower, Matthew J; Bertocchini, Federica

    2017-03-01

    In the animal kingdom, gastrulation, the process by which the primary germ layers are formed involves a dramatic transformation in the topology of the cells that give rise to all of the tissues of the adult. Initially formed as a mono-layer, this tissue, the epiblast, becomes subdivided through the internalization of cells, thereby forming a two (bi-laminar) or three (tri-laminar) layered embryo. This morphogenetic process coordinates the development of the fundamental body plan and the three-body axes (antero-posterior, dorso-ventral, and left-right) and begins a fundamental segregation of cells toward divergent developmental fates. In humans and other mammals, as well as in avians, gastrulating cells internalize along a structure, called the primitive streak, which builds from the periphery toward the center of the embryo. How these morphogenetic movements are orchestrated and evolved has been a question for developmental biologists for many years. Is the primitive streak a feature shared by the whole amniote clade? Insights from reptiles suggest that the primitive streak arose independently in mammals and avians, while the reptilian internalization site is a structure half-way between an amphibian blastopore and a primitive streak. The molecular machinery driving primitive streak formation has been partially dissected using mainly the avian embryo, revealing a paramount role of the planar cell polarity (PCP) pathway in streak formation. How did the employment of this machinery evolve? The reptilian branch of the amniote clade might provide us with useful tools to investigate the evolution of the amniote internalization site up to the formation of the primitive streak. WIREs Dev Biol 2017, 6:e262. doi: 10.1002/wdev.262 For further resources related to this article, please visit the WIREs website.

  10. Early mesozoic coexistence of amniotes and hepadnaviridae.

    Directory of Open Access Journals (Sweden)

    Alexander Suh

    2014-12-01

    Full Text Available Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic "fossil" is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote-HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.

  11. 紫外光-核黄素诱导羊膜胶原交联的实验研究%Cross-linking of human amniotic membrane by ultraviolet A-riboflavin

    Institute of Scientific and Technical Information of China (English)

    赵红红; 孙旭光; 梁庆丰; 邓世靖; 王智群; 李然

    2012-01-01

    Background Studies confirmed that ultraviolet A (UVA)- riboflavin photodynamic therapy can control keratoconus progresses by altering the physicochemical property of cornea.The collagen components of amniotic membrane transplantation is similar to that of cornea and amniotic membrane transplantation has been widely used to ocular surface reconstruction.However,the study on UVA riboflavin-induced-collagen crosslinking for amniotic tissue is less now. Objective This study was to investigate the role of UVA-riboflavin on frozen-preserved human amniotic membrane. Methods Human amnions were obtained in informed consent and prepared into 2 mm×15 mm pieces and were then divided into 4 groups using lottery method and 6 pieces for each group.The first 3 groups were treated with the photosensitizer riboflavin and UVA-irradiation ( wavelength:370 nm ; irradiation energy:1,2 or 3 mW/cm2,distance:10 mm) for 30 minutes,and the untreated fourth group was as control group.Biomechanical stress-strain test was performed using a microcomputer-controlled biomaterial tester and the stress(mN) was recorded when the strains were set to 5%,10% and 15%.The 7 mm diameter of human amniotic membrane pieces were trephined and divided into 4 groups(5 pieces for each group) with the treated method as mentioned above,and then the buttons were exposed to 0.1% collagenase Ⅰ solution.The transparency was scored and the complete dissolving time was record.In histological evaluation,three groups (3 pieces for each group) of human amniotic membranes were treated using UVAriboflavin(3 mW/cm2),0.1% riboflavin,normal saline for 30 minutes respectively and examined under the transmission electron microscopy.This study was performed under the permission of the Ethic Commission of Beijing Tongren Hospital. Results When the strain was 5%,10%,15%,the stress of control group and 1,2,3 mW/cm2UVA group were statistically signifcantly different ( F =3.411,P =0.037; F =9.927,P =0.001;F=11.118,P=0

  12. 1016例孕中期孕妇羊水细胞的培养及染色体核型分析%Amniotic cell culture and karyotype analysis of 1 016 pregnant women in second trimester

    Institute of Scientific and Technical Information of China (English)

    黎永鉴; 闫丽琼; 庞义坚

    2014-01-01

    Objective To investigate the application value of amniotic cell culture and karyotype analysis in prenatal diagnosis . Methods 1 016 pregnant women in second trimester were subject to amniocentesis under the guidance of B-type ultrasonic inspec-tion ,and the cell culture and karyotype analysis were performed on the amniotic fluid which had been drawn out .Results Among 1 016 pregnant women ,1 011(99 .5% ) succeeded in the first operation of amniocentesis and cell culture .The detection rate of fetal chromosomal abnormal karyotype was 10 .3% (105/1 016) ,in which 8 .3% (85/1 016) of structural abnormality and 2 .0%(20/1 016) of quantity abnormality .856 cases were received follow-up .Conclusion Amniotic fluid cell culture and karyotype analy-sis is a safe and reliable method for prenatal diagnosis .%目的:探讨羊水细胞培养和染色体核型分析在产前诊断中的的应用价值。方法对1016例孕中期孕妇,在B型超声波检查的监护下行羊膜腔穿刺术,对抽出的羊水进行细胞培养及染色体核型分析。结果 1016例孕妇中,一次性羊水穿刺、细胞培养成功1011例(99.5%)。胎儿染色体异常核型检出率为10.3%(105/1016),其中,结构异常8.3%(85/1016);数目异常2.0%(20/1016)。856例接受随访。结论羊水细胞培养及染色体核型分析是安全、可靠的产前诊断方法。

  13. 羊水间充质干细胞在肿瘤治疗中的研究进展%The research progress of amniotic fluid-derived mesenchymal stem cells in cancer therapy

    Institute of Scientific and Technical Information of China (English)

    王德君(综述); 张广美(审校)

    2014-01-01

    羊水间充质干细胞是多能的非造血祖细胞,具有自我修复及多向分化潜能,如成骨细胞、成软骨细胞及成脂肪细胞。尽管其具有高度增殖能力,但是其在体内并没有成瘤的报道,甚至多次传代后其核型也正常。 AF-MSCs对肿瘤的趋向性及其作为治疗多种肿瘤的细胞载体已经被很多研究证实。本文总结近期国内外的相关文献,就羊水间充质干细胞在肿瘤治疗中的研究进展进行综述。%Amniotic fluid-derived mesenchymal stem cells(AF-MSCs)are multipotential,nonhemato-poietic progenitor cells that have an inherent ability to self -renew and to differentiate into many cell types ,inclu-ding osteoblasts,chondrocytes and adipocytes.Despite their high proliferation rate,no in vivo tumorigenicity has been reported and their karyotype was normal even after extensive expansion in culture .Several studies have dem-onstrated the inherent tumortropic properties of MSCs and their successful use as cellular vehicles of therapeutic agents for the treatment of several types of cancer .This article reviews recent articles on studying the role of amni-otic fluid-derived mesenchymal stem cells in cancer therapy .

  14. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  15. Meta-analysis of limbal stem cell transplantation and amniotic membrane transplantation for pterygium%角膜缘干细胞移植与羊膜移植治疗翼状胬肉的Meta分析

    Institute of Scientific and Technical Information of China (English)

    于晓明; 吴欣怡

    2013-01-01

    Objective To evaluate the clinical effect of limbal stem cell transplantation and amniotic membrane transplantation in the treatment of pterygium by evidence based medicine. Methods Clinical data of effect in limbal stem cell transplantation and amniotic membrane transplantation were collected from computer literature searching, which was carried out in PubMed, EMBASE, Wanfang and VIP database etc, also performed searching based on www. baidu. com and www. google. com. Meta analysis that recommended by Cochrane Collaboration was done for the data obtained that 3 126 eyes that met with the eleven criteria of enrollment were included in this study. Publication bias was assessed by using a funnel plot. Results The results of meta-analysis showed that 35 RCTs evaluated the pterygium recurrent rate after grafting including 3 126 eyes, the incidence rate including 1 108 eyes, and the mean time of corneal epithelium wound healing including 900 eyes. In comparison with the amniotic membrane transplantation group, the recurrent rates of pterygium (P < 0.000 01) and the total complication rate (P = 0.01) were lower in the limbal stem cell transplantation group. Moreover , the mean time of corneal epithelium wound healing was shorter in the limbal stem cell transplantation group. Conclusion Corneal limbal stem cell autograft is an effective way to reduce the recurrent rate of pterygium and the incidence of symblepharon and to accelerate the healing of corneal wound comparing with amniotic membrane transplantation.%目的 以循证医学证据评价角膜缘干细胞移植和羊膜移植治疗翼状胬肉的临床疗效.方法 通过计算机检索Pubmed数据库、EMBASE、万方数据库、中国期刊全文数据库和维普数据库,并结合文献追溯的方法,收集关于角膜缘干细胞移植和羊膜移植治疗翼状胬肉临床疗效的随机前瞻性对照试验(RCT),并按Cochrane协作网推荐的方法对符合纳入标准的35个研究,共3 126只眼进

  16. AMNIOTIC MEMBRANE TRANSPLANTATION FOR KERATITIS.

    Directory of Open Access Journals (Sweden)

    Snezhana Murgova

    2015-06-01

    Full Text Available Keratitis without proper management tends to perforate the cornea, resulting in severe adverse consequences. In recent studies, amniotic membrane is reported to have anti-inflammatory effect and promote wound healing of corneal ulcer. Purpose: To report on the efficacy of permanent amniotic membrane transplantation (AMT in the treatment of keratitis. Case report: A 58-year-old man with severe keratitis in both eyes caused by long term administration of topical anesthetic (alcaine for electric ophthalmia. Single layer of amniotic membrane (AM was placed on the defect and secured to the limbus with interrupted 10-0 nylon sutures. A bandage contact lens was applied on the AM. Postoperative medication included topical antibiotic, artificial tears and mydriatic. Three months later corticosteroid was included. There was an immediate decrease of patient’s pain after surgery. Complete epithelialization was noted after 1 month. Conclusion: AMT is an alternative adjunctive method of treatment of keratitis; it promotes epithelialization process, decreased inflammation, corneal haze and neovascularization.

  17. Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

    Science.gov (United States)

    Iglesias, Diana M; El-Kares, Reyhan; Taranta, Anna; Bellomo, Francesco; Emma, Francesco; Besouw, Martine; Levtchenko, Elena; Toelen, Jaan; van den Heuvel, Lambertus; Chu, Leelee; Zhao, Jing; Young, Yoon Kow; Eliopoulos, Nicoletta; Goodyer, Paul

    2012-01-01

    Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

  18. Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

    Directory of Open Access Journals (Sweden)

    Diana M Iglesias

    Full Text Available Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

  19. Stem Cell Microvesicles Transfer Cystinosin to Human Cystinotic Cells and Reduce Cystine Accumulation In Vitro

    Science.gov (United States)

    Taranta, Anna; Bellomo, Francesco; Emma, Francesco; Besouw, Martine; Levtchenko, Elena; Toelen, Jaan; van den Heuvel, Lambertus; Chu, LeeLee; Zhao, Jing; Young, Yoon Kow; Eliopoulos, Nicoletta; Goodyer, Paul

    2012-01-01

    Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules. PMID:22912749

  20. Visfatin/Pre-B Cell Colony-Enhancing Factor in Amniotic Fluid in Normal Pregnancy, Spontaneous Labor at Term, Preterm Labor and Prelabor Rupture of Membranes: an Association with Subclinical Intrauterine Infection in Preterm Parturition

    Science.gov (United States)

    Mazaki-Tovi, Shali; Romero, Roberto; Kusanovic, Juan Pedro; Erez, Offer; Gotsch, Francesca; Mittal, Pooja; Than, Nandor Gabor; Nhan-Chang, Chia-lang; Hamill, Neil; Vaisbuch, Edi; Chaiworapongsa, Tinnakorn; Edwin, Samuel S.; Nien, Jyh Kae; Gomez, Ricardo; Espinoza, Jimmy; Kendal-Wright, Claire; Hassan, Sonia S.; Bryant-Greenwood, Gillian

    2008-01-01

    Objective Visfatin, a novel adipokine originally discovered as a pre-B-cell colony enhancing factor, is expressed by amniotic epithelium, cytotrophoblast, and decidua and is over-expressed when fetal membranes are exposed to mechanical stress and/or pro-inflammatory stimuli. Visfatin expression by fetal membranes is dramatically up-regulated after normal spontaneous labor. The aims of this study were to determine if visfatin is detectable in amniotic fluid (AF) and whether its concentration changes with gestational age, spontaneous labor, preterm prelabor rupture of membranes (preterm PROM) and in the presence of microbial invasion of the amniotic cavity (MIAC). Methods In this cross-sectional study, visfatin concentration in AF was determined in patients in the following groups: 1) mid-trimester (n=75); 2) term not in labor (n=27); 3) term in spontaneous labor (n=51); 4) patients with preterm labor with intact membranes (PTL) without MIAC who delivered at term (n=35); 5) patients with PTL without MIAC who delivered preterm (n=52); 6) patients with PTL with MIAC (n=25); 7) women with preterm PROM without MIAC (n=26); and 8) women with preterm PROM with MIAC (n=26). Non-parametric statistics were used for analysis. Results 1) The median AF concentration of visfatin was significantly higher in patients at term than in midtrimester; 2) Among women with PTL who delivered preterm, the median visfatin concentration was significantly higher in patients with MIAC than those without MIAC; 3) Similarly, patients with PTL and MIAC had a higher median AF visfatin concentration than those with PTL who delivered at term; 4) Among women with preterm PROM, the median AF visfatin concentration was significantly higher in patients with MIAC than those without MIAC. Conclusions 1) Visfatin is a physiologic constituent of AF; 2) The concentration of AF visfatin increases with advancing gestational age; 3) AF visfatin concentration is elevated in patients with MIAC, regardless of the

  1. Spectral luminescence analysis of amniotic fluid

    Science.gov (United States)

    Slobozhanina, Ekaterina I.; Kozlova, Nataly M.; Kasko, Leonid P.; Mamontova, Marina V.; Chernitsky, Eugene A.

    1997-12-01

    It is shown that the amniotic fluid has intensive ultra-violet luminescence caused by proteins. Along with it amniotic fluid radiated in the field of 380 - 650 nm with maxima at 430 - 450 nm and 520 - 560 nm. The first peak of luminescence ((lambda) exc equals 350 nm; (lambda) em equals 430 - 440 nm) is caused (most probably) by the presence in amniotic fluid of some hormones, NADH2 and NADPH2. A more long-wave component ((lambda) exc equals 460 nm; (lambda) em equals 520 - 560 nm) is most likely connected with the presence in amniotic fluid pigments (bilirubin connected with protein and other). It is shown that intensity and maximum of ultra-violet luminescence spectra of amniotic fluid in normality and at pathology are identical. However both emission spectra and excitation spectra of long-wave ((lambda) greater than 450 nm) luminescence of amniotic fluid from pregnant women with such prenatal abnormal developments of a fetus as anencephaly and spina bifida are too long-wave region in comparison with the norm. Results of research testify that spectral luminescent analysis of amniotic fluid can be used for screening of malformations of the neural tube. It is very difficult for a practical obstetrician to reveal pregnant women with a high risk of congenital malformations of the fetus. Apart from ultrasonic examination, cytogenetic examination of amniotic fluid and defumination of concentrations of alpha-fetoprotein and acetylcholin-esterases in the amniotic fluid and blood plasma are the most widely used diagnostic approaches. However, biochemical and cytogenetic diagnostic methods are time-consuming. In the present work spectral luminescence properties of the amniotic fluid are investigated to determine spectral parameters that can be used to reveal pregnant women with a high risk of congenital malformations of their offsprings.

  2. 21 CFR 884.1550 - Amniotic fluid sampler (amniocentesis tray).

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Amniotic fluid sampler (amniocentesis tray). 884... Diagnostic Devices § 884.1550 Amniotic fluid sampler (amniocentesis tray). (a) Identification. The amniotic fluid sampler (amniocentesis tray) is a collection of devices used to aspirate amniotic fluid from...

  3. Proposed diagnostic criteria for the case definition of amniotic fluid embolism in research studies.

    Science.gov (United States)

    Clark, Steven L; Romero, Roberto; Dildy, Gary A; Callaghan, William M; Smiley, Richard M; Bracey, Arthur W; Hankins, Gary D; D'Alton, Mary E; Foley, Mike; Pacheco, Luis D; Vadhera, Rakesh B; Herlihy, J Patrick; Berkowitz, Richard L; Belfort, Michael A

    2016-10-01

    Amniotic fluid embolism is a leading cause of maternal mortality in developed countries. Our understanding of risk factors, diagnosis, treatment, and prognosis is hampered by a lack of uniform clinical case definition; neither histologic nor laboratory findings have been identified unique to this condition. Amniotic fluid embolism is often overdiagnosed in critically ill peripartum women, particularly when an element of coagulopathy is involved. Previously proposed case definitions for amniotic fluid embolism are nonspecific, and when viewed through the eyes of individuals with experience in critical care obstetrics, would include women with a number of medical conditions much more common than amniotic fluid embolism. We convened a working group under the auspices of a committee of the Society for Maternal-Fetal Medicine and the Amniotic Fluid Embolism Foundation whose task was to develop uniform diagnostic criteria for the research reporting of amniotic fluid embolism. These criteria rely on the presence of the classic triad of hemodynamic and respiratory compromise accompanied by strictly defined disseminated intravascular coagulopathy. It is anticipated that limiting research reports involving amniotic fluid embolism to women who meet these criteria will enhance the validity of published data and assist in the identification of risk factors, effective treatments, and possibly useful biomarkers for this condition. A registry has been established in conjunction with the Perinatal Research Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development to collect both clinical information and laboratory specimens of women with suspected amniotic fluid embolism in the hopes of identifying unique biomarkers of this condition.

  4. Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model

    National Research Council Canada - National Science Library

    Piccoli, Martina; Franzin, Chiara; Bertin, Enrica; Urbani, Luca; Blaauw, Bert; Repele, Andrea; Taschin, Elisa; Cenedese, Angelo; Zanon, Giovanni Franco; André-Schmutz, Isabelle; Rosato, Antonio; Melki, Judith; Cavazzana-Calvo, Marina; Pozzobon, Michela; De Coppi, Paolo

    2012-01-01

    .... Mice carrying a homozygous deletion of Smn exon 7 directed to skeletal muscle (HSA-Cre, Smn(F7/F7) mice) present clinical features of human muscular dystrophies for which new therapeutic approaches are highly warranted...

  5. Effect of Amniotic Membrane Combined with Ciprofloxacin in Curing the Primary Stages of Pseudomonal Keratitis

    Directory of Open Access Journals (Sweden)

    Mohammad Kazem Sharifi Yazdi

    2012-03-01

    Full Text Available Background: Keratitis caused by Pseudomonas aeruginosa is often resulted in severe corneal ulcers and perforation, which leads to losses of vision. Human amniotic membrane (HAM forms the inner wall of the membranous sac which surrounds and protects the embryo during gestation. The purpose of this study was to evaluate the effectiveness of the amniotic membrane's healing in rabbits with pseudomonas keratitis.Methods: In total 14 rabbits divided in 2 groups of: 1 as Control and 2 as experimental amniotic membrane combined with ciprofloxacin. A 0.05 ml suspension of Pseudomonas aeruginosa ATCC 27853 was injected into rabbit’s corneal stroma, with no interference in control group. In the second group, the amniotic membrane in pieces of 1.5 × 1.5 cm transplanted to the entire corneal surface by eight interrupted 10.0 nylon sutures. In the first day ciprofloxacin drop was injected to the second group every 30 minutes and through second to seventh days every 2 hours. The results of perforation in cornea and the amount of infiltration were registered.Results: The results showed that amniotic membrane transplantation (AMT + ciprofloxacin group had 0% perforation and the control group 85.6%. Average infiltrations were 5 mm in AMT + ciprofloxacin groups and 23.75 mm in control.Conclusion: The use of amniotic membrane with ciprofloxacin was effective in prevention of cornea perforation and controlling the process of pseudomonal keratitis remission. The improvement of inflammation rapidly happened in ciprofloxacin + AMT group.

  6. Genome Wide Expression of Amniotic Fluid Stem Cells in Early and Late Passage during in Vitro Culture%羊水干细胞体外培养早期和后期基因表达谱分析

    Institute of Scientific and Technical Information of China (English)

    焦扬; 刘建军; 冯国华; 于明; 赵洪波

    2013-01-01

    Objective This study was purposed to investigate the changes of biological properties and expression patterns of the amniotic fluid stem cells (AFSCs) during in vitro culture.Methods The gene chip data of amniotic fluid stem cells were obtained from GEO database and statistically analyzed using R and Bioconductor to identify the differentially expressed genes,then do the Gene Ontology analysis and KEGG pathway analysis.Results We did not find differentially expressed genes by linear model using limma package,but 495 differentially expressed probes were identified by RankProd,including 217 up-regulated and 278 down-regulated probes.Further analysis with Gene Ontology functional categories showed that the up-regulated genes were concentrated in those related to collagen fibril organization,extracellular matrix organization,extracellular structure organization,skeletal system development,cell adhesion and biological adhesion and the down-regulated probes in late passage were associated with nuclear division,mitosis,and cell cycle.The up-regulated pathway was ECM-receptor interaction and focal adhesion.Cell cycle,cytokine-cytokine receptor interaction,p53 signaling pathway and oocyte meiosis were down-regulated.Conclusion AFSCs maintain their genome-wide expression profile during in-vitro culture.%目的 探讨羊水干细胞(amniotic fluid stem cells,AFSCs)体外培养早期和后期全基因组表达规律.方法 在公共基因芯片数据库GEO中找到羊水干细胞相关的基因芯片数据,使用R和Bioconductor软件包对其进行统计学分析,筛选差异表达基因,并进行GO分析和KEGG通路分析.结果 通过线性模型没有找到差异表达基因,而通过秩积法找到217个上调探针,278个下调探针.对这些差异表达基因进行功能富集,上调的生物过程是胶原纤维组织相关过程、细胞外基质组织、胞外结构组织、骨骼系统发育、细胞粘附等.下调过程最显著的是核分裂过程,有丝分

  7. Tentative research on the human amniotic fluid proteomics%人羊水特异蛋白质组学的探讨研究

    Institute of Scientific and Technical Information of China (English)

    朱斌; 陈芳; 侯常; 黎丽红; 黄素华

    2014-01-01

    目的:寻找人羊水中特异性表达的蛋白质。方法羊膜腔穿刺获取3份正常孕妇的羊水,同时采集其外周肘静脉血,分离血清。提取羊水及血清中的蛋白质,用双向凝胶电泳观察人羊水和血清中蛋白质表达情况,并选取羊水中表达量较血清中高2倍以上的蛋白斑点行基质辅助激光解吸/离子化飞行时间质谱分析。结果在pH 4~7、相对分子质量10~55 kDa区域里,羊水中约(613±29)个蛋白点,血清中约(785±64)个蛋白点。在羊水中表达但血清中不表达的蛋白点约50个,羊水中表达量较血清高2倍以上的蛋白点约50个。结论羊水中存在特异性表达蛋白质,其在妊娠相关生理、病理学中可能起重要作用。%Objective To explore the specific expression of proteomics in amniotic fluid (AF ).Methods Three samples were collected from normal AF by amniocentesis and peripheral venous blood sam-ples were collected simultaneously from which the serum was isolated. Proteins were extracted from both sam-ples and analyzed by two-dimensional gel electrophoresis (2-DE). The protein blots expressed in normal AF twice as high as that in serum were identified by matrix-assisted laser desorption ionizafion time-of-flight mass spectrometry.Results At pH 4-7 and within a molecular mass of 10-55 kDa,(613 ±29)protein blots were i-dentified in normal AF and (785 ±64)in serum. Approximately 50 protein blots were expressed in normal AF rather than serum. Roughly 50 blots were expressed in normal AF twice as high as that in serum. Conclusion Specific protein expression was observed in normal AF,which probably plays a vital role in pregnancy-related physiology and pathology.

  8. Proteomic profiling of the amniotic fluid to detect inflammation, infection, and neonatal sepsis.

    Directory of Open Access Journals (Sweden)

    Catalin S Buhimschi

    2007-01-01

    Full Text Available BACKGROUND: Proteomic analysis of amniotic fluid shows the presence of biomarkers characteristic of intrauterine inflammation. We sought to validate prospectively the clinical utility of one such proteomic profile, the Mass Restricted (MR score. METHODS AND FINDINGS: We enrolled 169 consecutive women with singleton pregnancies admitted with preterm labor or preterm premature rupture of membranes. All women had a clinically indicated amniocentesis to rule out intra-amniotic infection. A proteomic fingerprint (MR score was generated from fresh samples of amniotic fluid using surface-enhanced laser desorption ionization (SELDI mass spectrometry. Presence or absence of the biomarkers of the MR score was interpreted in relationship to the amniocentesis-to-delivery interval, placental inflammation, and early-onset neonatal sepsis for all neonates admitted to the Newborn Special Care Unit (n = 104. Women with "severe" amniotic fluid inflammation (MR score of 3 or 4 had shorter amniocentesis-to-delivery intervals than women with "no" (MR score of 0 inflammation or even "minimal" (MR score of 1 or 2 inflammation (median [range] MR 3-4: 0.4 d [0.0-49.6 d] versus MR 1-2: 3.8 d [0.0-151.2 d] versus MR 0: 17.0 d [0.1-94.3 d], p 100 cells/mm3, whereas the combination of Gram stain and MR score was best for rapid prediction of intra-amniotic infection (positive amniotic fluid culture. CONCLUSIONS: High MR scores are associated with preterm delivery, histological chorioamnionitis, and early-onset neonatal sepsis. In this study, proteomic analysis of amniotic fluid was shown to be the most accurate test for diagnosis of intra-amniotic inflammation, whereas addition of the MR score to the Gram stain provides the best combination of tests to rapidly predict infection.

  9. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  10. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    Science.gov (United States)

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems.

  11. Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices.

    Science.gov (United States)

    Stafiej, Piotr; Küng, Florian; Thieme, Daniel; Czugala, Marta; Kruse, Friedrich E; Schubert, Dirk W; Fuchsluger, Thomas A

    2017-02-01

    In this work, polycaprolactone (PCL) was used as a basic polymer for electrospinning of random and aligned nanofiber matrices. Our aim was to develop a biocompatible substrate for ophthalmological application to improve wound closure in defects of the cornea as replacement for human amniotic membrane. We investigated whether blending the hydrophobic PCL with poly (glycerol sebacate) (PGS) or chitosan (CHI) improves the biocompatibility of the matrices for cell expansion. Human corneal epithelial cells (HCEp) and human corneal keratocytes (HCK) were used for in vitro biocompatibility studies. After optimization of the electrospinning parameters for all blends, scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and water contact angle were used to characterize the different matrices. Fluorescence staining of the F-actin cytoskeleton of the cells was performed to analyze the adherence of the cells to the different matrices. Metabolic activity of the cells was measured by cell counting kit-8 (CCK-8) for 20days to compare the biocompatibility of the materials. Our results show the feasibility of producing uniform nanofiber matrices with and without orientation for the used blends. All materials support adherence and proliferation of human corneal cell lines with oriented growth on aligned matrices. Although hydrophobicity of the materials was lowered by blending PCL, no increase in biocompatibility or proliferation, as was expected, could be measured. All tested matrices supported the expansion of human corneal cells, confirming their potential as substrates for biomedical applications.

  12. Application of middle and late pregnancy amniotic cell culture united to FISH in prenatal diagnosis%中晚孕期羊水细胞培养联合FISH在产前诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    张建芳; 徐慧; 燕凤; 陈必良

    2011-01-01

    Objective: To investigate the application value of improved amniotic cell culture united to FISH (fluorescente in situ hybridization) in prenatal diagnosis of middle and late pregnancy. Methods; 71 gravida possessing prenatal diagnosis indication ac-cepted improved amniotic cell culture and FISH to analyze fetal karyotype. Gestational weeks were among 25 to 39. Results:Achieve-ment ratio of cell culture and FISH were both up to 100%. 8 abnormal karyotype were found, including 3 trisomy 18,1 trisomy 13, 1 Trisomy X, 1 heterosome chimera, one each in chromosomal 4 and 9 pericentric inversion. Coincidence rate of karyotype and FISH was 100% , except pericentric inversion because of which exceeding detection limit of FISH. Conclusion: Amniotic cell culture and karyo-type analysis acciciated FISH was feasible in comparatively large gestational weeks. It can diagnose chromosomal disorder quickly and exactly, and extend the time window of amniocentesis, cut down the risk of venipuncture.%目的 探讨改良的羊水细胞培养技术联合荧光原位杂交( FISH)对中晚孕期孕妇进行产前诊断的应用价值.方法 改进培养方法、建立收获标准、改良染色体制备过程,对71例孕25 - 39 w具有产前诊断指征的孕妇进行羊水细胞培养并联合FISH进行核型分析.结果 羊水细胞培养和FISH分析成功率均为100%.发现异常核型8例,异常比例为11.3%.其中18三体3例,13三体1例,47,XXX 1例,4号和9号染色体臂间倒位各1例,46,XY/45,X0嵌合体1例;除4号和9号染色体臂间倒位不在FISH检测范围外,其余6个染色体异常FISH与核型均同时检出,两者符合率100%.结论较大孕周同样可以进行羊水细胞培养和染色体分析,联合FISH可快捷明确地诊断染色体病,使羊膜腔穿刺进行产前诊断的时间窗延长,降低脐静脉穿刺率和风险.

  13. Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion

    OpenAIRE

    Massee, Michelle; Chinn, Kathryn; Lim, Jeremy J; Godwin, Lisa; Young, Conan S.; Koob, Thomas J.

    2016-01-01

    Objective: Human amniotic membranes have been shown to be effective for healing diabetic foot ulcers clinically and to regulate stem cell activity in vitro and in vivo; however, diabetic stem cells may be impaired as a sequela of the disease. In this study, dehydrated human amnion/chorion membrane (dHACM) allografts (EpiFix®; MiMedx Group) were evaluated for their ability to regulate diabetic stem cells in vitro. Approach: Human adipose-derived stem cells (ADSCs) from normal, type I diabetic,...

  14. Amniotic fluid propionylcarnitine in methylmalonic aciduria.

    Science.gov (United States)

    Penn, D; Schmidt-Sommerfeld, E; Jakobs, C; Bieber, L L

    1987-01-01

    Amniotic fluid samples from pregnancies complicated by foetal methylmalonic aciduria and from metabolically normal pregnancies were obtained at 16-18 weeks of gestation and analysed for total, free and acylcarnitine and individual carnitine esters. The amniotic fluid concentrations of total acylcarnitine and propionylcarnitine were higher in pregnancies with higher in pregnancies with methylmalonic aciduria than in normal pregnancies. The predominant carnitine ester was propionylcarnitine in the methylmalonic aciduria group and acetylcarnitine in the normal group. These findings suggest that in methylmalonic aciduria, abnormalities of carnitine metabolism already occur early in gestation. The amount of propionylcarnitine in amniotic fluid may be useful as an additional indicator of foetal methylmalonic aciduria.

  15. Karyotype analysis of second-trimester amniotic fluid cells in 572 high-risk pregnant women%高危孕妇572例妊娠中期羊水细胞染色体核型分析

    Institute of Scientific and Technical Information of China (English)

    朱蕊; 曾爱群; 杜晶春

    2016-01-01

    目的:探讨产前诊断指征与羊水细胞染色体核型异常的关系及羊膜腔穿刺术的安全性,为产前遗传咨询提供实验依据。方法:对2012年1月至2015年8月期间我院572例具备产前诊断指征的高危孕妇行羊膜腔穿刺术及羊水细胞染色体核型检查。结果:羊水细胞培养一次性成功率为99.83%;在572例培养成功的羊水细胞中,异常核型20例,异常率为3.50%,其中染色体数目异常17例,占85%,染色体结构异常3例,占15%。以高龄为产前诊断指征的299例孕妇中,异常核型7例,异常率为2.34%;在273例非高龄孕妇中,异常核型13例,异常率为4.76%。结论:(1)唐氏或无创DNA筛查高危、高龄、超声异常及不良孕产史者有必要进一步行产前诊断。(2)羊水细胞核型分析是一种相对安全性高,准确性高的诊断方法,其对降低新生儿出生缺陷,提高人口素质,减轻家庭和社会负担具有重要意义。%Objective To explore the relationship between prenatal diagnosis indications and fetal chromosomal aberrations , and the security of amniocentesis. Methods The amniotic fluid cells were sampled by amniocentesis and cultured in 572 high-risk pregnant women from January 2012 to August 2015. The chromosomal karyotypes were examined by G-banding. Results The success rate of the first amniotic fluid cells culture reached 99.83%. In all the 572 valid samples , there were 20 cases of chromosomal aberrations and the abnormal rate was 3.50%, including 17 of numeric aberrations and 3 of structural aberrations. There were 7 cases of chromosomal aberrations in all the 299 elderly parturient in high-risk indications and the abnormal rate was 2.34%, and there were 13 cases of chromosomal aberrations in all the 273 non-elderly parturient and the abnormal rate was 4.76%. Conclusions (1)It is necessary to further diagnose in pregnant women with high-risk factors

  16. Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds.

    Science.gov (United States)

    Hortensius, Rebecca A; Ebens, Jill H; Harley, Brendan A C

    2016-06-01

    Adult tendon wound repair is characterized by the formation of disorganized collagen matrix which leads to decreases in mechanical properties and scar formation. Studies have linked this scar formation to the inflammatory phase of wound healing. Instructive biomaterials designed for tendon regeneration are often designed to provide both structural and cellular support. In order to facilitate regeneration, success may be found by tempering the body's inflammatory response. This work combines collagen-glycosaminoglycan scaffolds, previously developed for tissue regeneration, with matrix materials (hyaluronic acid and amniotic membrane) that have been shown to promote healing and decreased scar formation in skin studies. The results presented show that scaffolds containing amniotic membrane matrix have significantly increased mechanical properties and that tendon cells within these scaffolds have increased metabolic activity even when the media is supplemented with the pro-inflammatory cytokine interleukin-1 beta. Collagen scaffolds containing hyaluronic acid or amniotic membrane also temper the expression of genes associated with the inflammatory response in normal tendon healing (TNF-α, COLI, MMP-3). These results suggest that alterations to scaffold composition, to include matrix known to decrease scar formation in vivo, can modify the inflammatory response in tenocytes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1332-1342, 2016.

  17. Human Amnion Membrane: Potential Applications in Oral and Periodontal Field.

    Science.gov (United States)

    Mohan, Ranjana; Bajaj, Aashima; Gundappa, Mohan

    2017-01-01

    Human amniotic membrane (HAM) is derived from the fetal membranes which consist of the inner amniotic membrane made of single layer of amnion cells fixed to collagen-rich mesenchyme attached to chorion. HAM has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Amniotic membrane has biological properties which are important for the experimental and clinical applications in managing patients of various medical specialties. Abundant, natural and wonderful biomembrane not only protects the foetus but also has various clinical applications in the field of dermatology, ophthalmology, ENT surgery, orthopedics and dental surgery. As it is discarded post-partum it may be useful for regenerative medicine and cell therapy to treat damaged or diseased tissues.

  18. Human Amnion Membrane: Potential Applications in Oral and Periodontal Field

    Science.gov (United States)

    Mohan, Ranjana; Bajaj, Aashima; Gundappa, Mohan

    2017-01-01

    Human amniotic membrane (HAM) is derived from the fetal membranes which consist of the inner amniotic membrane made of single layer of amnion cells fixed to collagen-rich mesenchyme attached to chorion. HAM has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Amniotic membrane has biological properties which are important for the experimental and clinical applications in managing patients of various medical specialties. Abundant, natural and wonderful biomembrane not only protects the foetus but also has various clinical applications in the field of dermatology, ophthalmology, ENT surgery, orthopedics and dental surgery. As it is discarded post-partum it may be useful for regenerative medicine and cell therapy to treat damaged or diseased tissues. PMID:28316944

  19. Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203.

    Science.gov (United States)

    Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Shin, Tae-Hoon; Seo, Yoojin; Kim, Hyung-Sik; Kang, Kyung-Sun

    2016-07-05

    Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) treated with a retina differentiation cocktail induced gene expressions of retina development-relevant genes. Furthermore, microRNA-203 (miR-203) is abundantly expressed in human AESCs and human UCB-MSCs. This miR-203 is predicted to target multiple retina development-relevant genes, particularly DKK1, CRX, RORβ, NEUROD1, NRL and THRB. The inhibition of miR-203 induced a retina differentiation of AESCs and UCB-MSCs. Moreover, successive treatments of anti-miR-203 led to the expression of both mature photoreceptor (PR) markers, rhodopsin and opsin. In addition, we determined that CRX, NRL and DKK1 are direct targets of miR-203 using a luciferase assay. Thus, the work presented here suggests that somatic stem cells can potentially differentiate into neural retina cell types when treated with anti-miR-203. They may prove to be a source of both PR subtypes for future allogeneic stem cell-based therapies of non-regenerative retina diseases.

  20. Modulation of intestinal inflammation by minimal enteral nutrition with amniotic fluid in preterm pigs

    DEFF Research Database (Denmark)

    Østergaard, Mette V; Bering, Stine Brandt; Jensen, Michael L

    2014-01-01

    Background: Necrotizing enterocolitis (NEC) is a severe inflammatory disorder, associated with the difficult transition from parenteral to enteral feeding after preterm birth. We hypothesized that minimal enteral nutrition (MEN) with amniotic fluid (AF), prior to enteral formula feeding, would...... improve resistance to NEC in preterm pigs. Methods: Experiment 1: IEC-6 cells were incubated with porcine (pAF) and human AF (hAF) to test AF-stimulated enterocyte proliferation and migration in vitro. Experiment 2: Cesarean-delivered, preterm pigs were fed parenteral nutrition and MEN with pAF, h...... fed AF as MEN, but NEC incidences were similar (NEC-pAF) or increased (NEC-hAF) compared with controls. Conclusions: Intake of pAF or hAF improved body growth and modulated intestinal inflammatory cytokines during a period of parenteral nutrition, but did not protect against later formula-induced NEC...

  1. An analysis on karyotype of amniotic fluid cells from 1262 fetus%1262例孕中期羊水细胞染色体检查结果分析

    Institute of Scientific and Technical Information of China (English)

    李小燕; 卢守莲; 王珏; 黄欢; 孙丽洲

    2013-01-01

    Objective To understand the relationship between the abnormal karyotype and amniocentesis indication for providing a basis for genetic counseling.Methods The amniotic fluid samples were taken from 1262 outpatients with the indications of prenatal diagnosis.After the cells in amniotic fluid were cultured,the karyotyping was performed with G-banding stain and grouped according to different indications.The incidence of abnormal karyotype was analyzed and the parents of fetal structural abnormalities chromosomes were checked to search for the origin of abnormal karyotype.Results A total of 65 patients with abnormal karyotype was detected in 1262 fetuses karyotype with an abnormal rate of 5.3 %,of which aneuploidy rate was 44.6 % (29/65),chromosome structural abnormality rate was 41.5 % (27/65),and marker chromosome rate was 10.8 % (7/65).Conclusion Abnormal karyotype composition is different according to different maternal amniocentesis indications,indicating the importance of mastering the indications for amniocentesis.%目的 了解异常核型与产前诊断指征的关系,为临床遗传咨询提供依据.方法 抽取1262例有产前诊断指征的孕妇孕中期羊水,细胞培养后G显带染色,分析胎儿染色体核型,并根据不同产前诊断指征进行分组.分析不同指征的发病率,并对染色体结构异常胎儿的父母染色体进行检查,以确定结构异常来源.结果 1262例羊水细胞染色体检查者,共检出65例异常核型,异常核型发生率为5.3%.其中,以非整倍体最多见,共检出29例(44.6%);其次为染色体结构异常,共检出27例(41.5%);检出染色体多态性改变7例(10.8%).结论 不同产前诊断指征染色体异常的检出比例有所不同,说明严格把握产前诊断指征的重要性.

  2. Human Amnion-Derived Mesenchymal Stem Cells Promote Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells by Influencing the ERK1/2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yuli Wang

    2016-01-01

    Full Text Available Human amnion-derived mesenchymal stem cells (HAMSCs are considered to be an important resource in the field of tissue engineering because of their anti-inflammatory properties and fewer ethical issues associated with their use compared with other sources of stem cells. HAMSCs can be obtained from human amniotic membranes, a readily available and abundant tissue. However, the potential of HAMSCs as seed cells for treating bone deficiency is unknown. In this study, HAMSCs were used to promote proliferation and osteoblastic differentiation in human bone marrow mesenchymal stem cells (HBMSCs in a Transwell coculture system. Proliferation levels were investigated by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU. Osteoblastic differentiation and mineralization were evaluated in chromogenic alkaline phosphatase (ALP activity substrate assays, Alizarin red S staining, and RT-PCR analysis of early HBMSCs osteogenic marker expression. We demonstrated that HAMSCs stimulated increased alkaline phosphatase (ALP activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Moreover, the effect of HAMSCs was significantly inhibited by U0126, a highly selective inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2 signaling. We demonstrate that HAMSCs promote osteogenic differentiation in HBMSCs by influencing the ERK1/2 signaling pathway. These observations confirm the potential of HAMSCs as a seed cell for the treatment of bone deficiency.

  3. Diffusion inside living human cells

    DEFF Research Database (Denmark)

    Leijnse, N.; Jeon, J. -H.; Loft, Steffen

    2012-01-01

    Naturally occurring lipid granules diffuse in the cytoplasm and can be used as tracers to map out the viscoelastic landscape inside living cells. Using optical trapping and single particle tracking we found that lipid granules exhibit anomalous diffusion inside human umbilical vein endothelial...... cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center...... of the cell or within the nucleus. Also, granules in cells which are stressed by intense laser illumination or which have attached to a surface for a long period of time move in a more restricted fashion than those within healthy cells. For granules diffusing in healthy cells, in regions away from the cell...

  4. Human Wharton's Jelly Mesenchymal Stem Cells plasticity augments scar-free skin wound healing with hair growth.

    Science.gov (United States)

    Sabapathy, Vikram; Sundaram, Balasubramanian; V M, Sreelakshmi; Mankuzhy, Pratheesh; Kumar, Sanjay

    2014-01-01

    Human mesenchymal stem cells (MSCs) are a promising candidate for cell-based transplantation and regenerative medicine therapies. Thus in the present study Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) have been derived from extra embryonic umbilical cord matrix following removal of both arteries and vein. Also, to overcome the clinical limitations posed by fetal bovine serum (FBS) supplementation because of xenogeneic origin of FBS, usual FBS cell culture supplement has been replaced with human platelet lysate (HPL). Apart from general characteristic features of bone marrow-derived MSCs, wharton jelly-derived MSCs have the ability to maintain phenotypic attributes, cell growth kinetics, cell cycle pattern, in vitro multilineage differentiation plasticity, apoptotic pattern, normal karyotype-like intrinsic mesenchymal stem cell properties in long-term in vitro cultures. Moreover, the WJ-MSCs exhibited the in vitro multilineage differentiation capacity by giving rise to differentiated cells of not only mesodermal lineage but also to the cells of ectodermal and endodermal lineage. Also, WJ-MSC did not present any aberrant cell state upon in vivo transplantation in SCID mice and in vitro soft agar assays. The immunomodulatory potential assessed by gene expression levels of immunomodulatory factors upon exposure to inflammatory cytokines in the fetal WJ-MSCs was relatively higher compared to adult bone marrow-derived MSCs. WJ-MSCs seeded on decellularized amniotic membrane scaffold transplantation on the skin injury of SCID mice model demonstrates that combination of WJ-MSCs and decellularized amniotic membrane scaffold exhibited significantly better wound-healing capabilities, having reduced scar formation with hair growth and improved biomechanical properties of regenerated skin compared to WJ-MSCs alone. Further, our experimental data indicate that indocyanin green (ICG) at optimal concentration can be resourcefully used for labeling of stem cells and in vivo

  5. Human Wharton's Jelly Mesenchymal Stem Cells plasticity augments scar-free skin wound healing with hair growth.

    Directory of Open Access Journals (Sweden)

    Vikram Sabapathy

    Full Text Available Human mesenchymal stem cells (MSCs are a promising candidate for cell-based transplantation and regenerative medicine therapies. Thus in the present study Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs have been derived from extra embryonic umbilical cord matrix following removal of both arteries and vein. Also, to overcome the clinical limitations posed by fetal bovine serum (FBS supplementation because of xenogeneic origin of FBS, usual FBS cell culture supplement has been replaced with human platelet lysate (HPL. Apart from general characteristic features of bone marrow-derived MSCs, wharton jelly-derived MSCs have the ability to maintain phenotypic attributes, cell growth kinetics, cell cycle pattern, in vitro multilineage differentiation plasticity, apoptotic pattern, normal karyotype-like intrinsic mesenchymal stem cell properties in long-term in vitro cultures. Moreover, the WJ-MSCs exhibited the in vitro multilineage differentiation capacity by giving rise to differentiated cells of not only mesodermal lineage but also to the cells of ectodermal and endodermal lineage. Also, WJ-MSC did not present any aberrant cell state upon in vivo transplantation in SCID mice and in vitro soft agar assays. The immunomodulatory potential assessed by gene expression levels of immunomodulatory factors upon exposure to inflammatory cytokines in the fetal WJ-MSCs was relatively higher compared to adult bone marrow-derived MSCs. WJ-MSCs seeded on decellularized amniotic membrane scaffold transplantation on the skin injury of SCID mice model demonstrates that combination of WJ-MSCs and decellularized amniotic membrane scaffold exhibited significantly better wound-healing capabilities, having reduced scar formation with hair growth and improved biomechanical properties of regenerated skin compared to WJ-MSCs alone. Further, our experimental data indicate that indocyanin green (ICG at optimal concentration can be resourcefully used for labeling of stem cells

  6. Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus.

    Science.gov (United States)

    Marques, Patricia X; O'Donovan, James; Souda, Puneet; Gutierrez, Jorge; Williams, Erin J; Worrall, Sheila; McElroy, Maire; Proctor, Aisling; Brady, Colm; Sammin, Donal; Basset, Hugh; Whitelegge, Julian P; Markey, Bryan K; Nally, Jarlath E

    2011-03-15

    Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.

  7. Expression of specific proteins of neural cells in rat's cultured amniotic epithelial cells%神经组织细胞特异性蛋白在大鼠羊膜上皮细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    娄小倩; 孟晓婷; 王大伟; 陈东

    2006-01-01

    BACKGROUND: It has been suggested that amniotic epithelial cells (AECs) express almost all of the markers of neural cell and secret a lot of neurotrophic factors and neurotransmitters. If AECs could substitute neural cells, its neurotrophic effect will bring promising prospect in treating neuron injuries and degenerative neural disease.OBJECTIVE: To detect specific proteins of neural cells in rat's cultured AECs.DESIGN: Repeated measurement design.SETTING: Second Clinical Medical College , Jilin University; Department of Histology & Embryology, School of Basic Medical Science, Jilin University.MATERIALS: This experiment was conducted at the Department of Histology & Embryology, School of Basic Medical Science, Jilin University from October 2004 to October 2005. The rat amniotic epithelial tissue was mechanically peeled from an embryonic 12 to 14days Wistar rats. Mouse anti Nestin was purchased from Chemicon Co.,and anti-ChAT rabbit anti-NSE and anti-NT-3 antibodies from Wuhan Boshide Company. Mouse anti-Musashi antibody was donated by Pro.Okano.METHODS: AECs were dissociated and purified from the amnion of pregnancy 12-14 day rats. AECs were treated with trypsin for 5 minutes,then cultured in DMEM/F12 medium at a humidified atmosphere of 0.05 volume fraction of CO2 in air at 37 ℃. Cells were inoculated at a concentration of 5×109 cells/L in culture flask. After 3 days, cells were inoculated onto poly-lysine-treated 35 mm culture Petri dish at a density of 1 × 108 cells/L for immunocytochemically staining. The cells were fixed with 40 g/L paraformaldehyde for 20 minutes. Immunocytochemical staining method was used to detect the expression of microtubule-associated protein-2 (MAP-2),neuron specific enolase(NSE), glial fibrillary acidic protein (GFAP) and choline acetyl transferase(ChAT).MAIN OUTCOME MEASURES: ① Morphological observation of rat'AECs at different culture time. ② Expression of specific protein of neural cells in rat' cultured AECs.RESULTS:

  8. Soluble leukocyte-Associated Ig-like Receptor-1 in amniotic fluid is of fetal origin and positively associates with lung compliance.

    Science.gov (United States)

    Houben, Michiel L; Olde Nordkamp, Marloes J M; Nikkels, Peter G J; van der Ent, Cornelis K; Meyaard, Linde; Bont, Louis

    2013-01-01

    The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1) is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355) collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39). The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001). Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung compliance

  9. Soluble leukocyte-Associated Ig-like Receptor-1 in amniotic fluid is of fetal origin and positively associates with lung compliance.

    Directory of Open Access Journals (Sweden)

    Michiel L Houben

    Full Text Available The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1 is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355 collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39. The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001. Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung

  10. Clinical chorioamnionitis at term VIII: a rapid MMP-8 test for the identification of intra-amniotic inflammation.

    Science.gov (United States)

    Chaiyasit, Noppadol; Romero, Roberto; Chaemsaithong, Piya; Docheva, Nikolina; Bhatti, Gaurav; Kusanovic, Juan Pedro; Dong, Zhong; Yeo, Lami; Pacora, Percy; Hassan, Sonia S; Erez, Offer

    2017-07-26

    MMP-8 results were determined by Yoon's MMP-8 Check®. Intra-amniotic inflammation was defined as an elevated amniotic fluid IL-6 concentration ≥2.6 ng/mL, and intra-amniotic infection was diagnosed by the presence of microorganisms in the amniotic fluid accompanied by intra-amniotic inflammation. The diagnostic indices of Yoon's MMP-8 Check® for the identification of intra-amniotic inflammation were calculated. In order to objectively compare Yoon's MMP-8 Check® with the ELISA IL-6 test for the identification of intra-amniotic inflammation, we used an amniotic fluid white blood cell (WBC) count ≥50 cells/mm3 to define intra-amniotic inflammation. (1) A positive rapid MMP-8 test had a sensitivity of 82.4% (28/34), specificity of 90% (9/10), positive predictive value of 96.6% (28/29), negative predictive value of 60% (9/15), positive likelihood ratio 8.2 (95% CI 1.3-53.2), and negative likelihood ratio 0.2 (95% CI 0.1-0.4) for the identification of intra-amniotic inflammation (prevalence 77.3%); (2) a positive rapid MMP-8 test had a sensitivity of 91.7% (22/24), specificity of 65% (13/20), positive predictive value of 75.9% (22/29), negative predictive value of 86.7% (13/15), positive likelihood ratio of 2.6 (95% CI 1.4-4.8), and negative likelihood ratio of 0.1 (95% CI 0.03-0.5) for the identification of intra-amniotic infection; (3) the rapid MMP-8 test had a significantly higher specificity than the ELISA IL-6 test in the identification of intra-amniotic inflammation as determined by an amniotic fluid WBC count ≥50 cells/mm3. The sensitivity and accuracy of the rapid MMP-8 test were comparable to those of the ELISA IL-6 test; and (4) importantly, the rapid MMP-8 test had 100% sensitivity and 100% negative predictive value in the identification of neonates affected with fetal inflammatory response syndrome (FIRS). The rapid diagnosis of intra-amniotic inflammation is possible by analysis of amniotic fluid using a point-of-care test for MMP-8. Patients with a

  11. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and......Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self...... of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from...

  12. Changes of Nerve Growth Factor in Amniotic Fluid and Correlation with Ventriculomegaly

    Institute of Scientific and Technical Information of China (English)

    Xiao-yan Xia; Xing-hua Huang; Yi-xin Xia; Wei-hua Zhang

    2011-01-01

    Objective To detect the change of nerve growth iactor (NGF) level in human amniotic fluid during gestation, and to explore the relationship between this change and fetal ventriculomegaly (VM). Methods The studied subjects (collected from 2004 to 2007) were divided into four groups, including the second-trimester pregnancy group (n=113), third-trimester pregnancy group (n= 110), fetal cerebral VM group (n= 12), and health), control group (n= 12) which matched with the VM group in gestational weeks. The amniotic fluid specimens were obtained during amniocentesis or cesarean section. The NGF levels in amniotic fluid were detected with enzyme-linked immunosorbent assay.Results A significantly negative correlation was found between gestational age and the NGF level in amniotic fluid (r=-0.6149, P<0.0001). The NGF level in patients with fetal VM was significantly lower than that in healthy controls (33.95+29.24 pg/mL vs. 64.73+ 16.21 pg/mL, P=0.024). Conclusion NGF levels in amniotic fluid may be a sensitive marker for fetal VM.

  13. Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Wang, Yuli; Yin, Ying; Jiang, Fei; Chen, Ning

    2015-02-01

    Human amnion mesenchymal stem cells (HAMSCs) can be obtained from human amniotic membrane, a highly abundant and readily available tissue. HAMSC sources present fewer ethical issues, have low immunogenicity, anti-inflammatory properties, considerable advantageous characteristics, and are considered an attractive potential treatment material in the field of regenerative medicine. We used a co-culture system to determine whether HAMSCs could promote osteogenesis in human bone marrow mesenchymal stem cells (HBMSCs). We isolated HAMSCs from discarded amnion samples and collected them using pancreatin/collagenase digestion. We cultured HAMSCs and HBMSCSs in basal medium. Activity of alkaline phosphatase (ALP), an early osteogenesis marker, was increased in the co-culture system compared to the control single cultures, which we also confirmed by ALP staining. We used immunofluorescence testing to investigate the effects of co-culturing with HAMSCs on HBMSC proliferation, which revealed that the co-culturing enhanced EdU expression in HBMSCs. Western blotting and quantitative real-time PCR indicated that co-culturing promoted osteogenesis in HBMSCs. Furthermore, Alizarin red S staining revealed that extracellular matrix calcium levels in mineralized nodule formation produced by the co-cultures were higher than that in the controls. Using the same co-culture system, we further observed the effects of HAMSCs on osteogenic differentiation in primary osteoblasts by Western blotting, which better addressed the mechanism for HAMSCs in bone regeneration. The results showed HAMSCs are osteogenic and not only play a role in promoting HBMSC proliferation and osteogenic differentiation but also in osteoblasts, laying the foundation for new regenerative medicine methods.

  14. Amniotic fluid and development of the immature intestine

    DEFF Research Database (Denmark)

    Østergaard, Mette Viberg

    ), and slow advancement of enteral feeds. Human breast milk is the gold standard of enteral nutrition (EN) of the newborn and protects against NEC in premature infants, but is often unavailable or limited after preterm birth. Innovative nutrition strategies and novel sources of the first EN are therefore...... in demand. Amniotic Fluid (AF) is the natural source of fetal EN throughout gestation in ammals. Fetal AF swallowing stimulates somatic and gastrointestinal growth during fetal development, and modulates the development of the intestinal mucosa. In addition, AF protects the fetus against infections...

  15. Intraoral application of hyperdry amniotic membrane to surgically exposed bone surface.

    Science.gov (United States)

    Tsuno, Hiroaki; Arai, Naoya; Sakai, Chieko; Okabe, Motonori; Koike, Chika; Yoshida, Toshiko; Nikaido, Toshio; Noguchi, Makoto

    2014-02-01

    Hyperdry amniotic membrane, a novel preservable material derived from the human amnion, has been introduced clinically in ophthalmology and other fields. This membrane is available as a wound dressing material for surgical wounds of the tongue and buccal mucosa but has not been used on wounds of the alveolar mucosa. This paper reports 2 cases in which intraoral alveolar wounds with bone exposure were successfully treated with the use of hyperdry amniotic membrane: a 74-year-old woman with gingival leukoplakia of the edentulous mandible, and a 43-year-old man who underwent vestibuloplasty of the reconstructed mandible. The results indicate that the hyperdry amniotic membrane is a useful dressing material, not only for soft tissue wounds, but also for exposed bone in the oral cavity. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Clinical significance of human alpha-fetoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Yachnin, S.

    1978-01-01

    Deviations from the normal of alpha-fetoprotein (AFP) concentrations in fetal serum, amniotic fluid, maternal serum and adult human serum can be explained by understanding the normal physiology and the pathophysiology of AFP synthesis and metabolism. AFP is the prototype of oncofetal markers. Emphasis is given to the usefulness of elevated serum AFP levels in the diagnosis and management of primary hepatomas and tumors of germ cell origin. The ability to detect neural tube defects early in gestation by monitoring maternal serum and amniotic fluid AFP concentrations is discussed.

  17. Rat amniotic epithelial cells are induced to differentiate into neural-like stem cells in vitro%大鼠羊膜上皮细胞体外诱导可分化为类神经干细胞*★

    Institute of Scientific and Technical Information of China (English)

    郭兵; 许家军

    2013-01-01

      背景:以往研究发现修复神经损伤的细胞来源主要有许旺细胞、嗅鞘细胞、神经干细胞、激活的巨噬细胞等,但这些细胞存在着来源困难、有成瘤性、异体排斥等缺点。而羊膜上皮细胞不存在以上缺陷,且具有多向分化潜能,经诱导后可向心肌样细胞、神经干细胞、肝细胞、成骨和软骨细胞等分化。目的:体外培养大鼠羊膜上皮细胞,并诱导其向类神经干细胞方向分化。方法:取妊娠晚期大鼠,采用胰酶消化法获得羊膜上皮细胞,用无血清的神经干细胞条件培养基对细胞进行诱导分化,并用免疫细胞化学法和RT-PCR对诱导前后的细胞相关标志物进行鉴定。结果与结论:形态学观察结果显示,条件培养基诱导后的羊膜上皮细胞胞体回缩,胞核部分折光性增强,出现类似于树突及轴突样结构,这些突起可交织成网状,细胞贴壁牢靠。免疫细胞化学检测结果显示,与诱导前相比,条件培养基诱导后的羊膜上皮细胞中巢蛋白、胶质纤维酸性蛋白荧光强度较强,而特异性胚胎抗原4、波形蛋白荧光强度较弱。RT-PCR检测显示,与诱导前相比,条件培养基诱导后的羊膜上皮细胞的巢蛋白、胶质纤维酸性蛋白、微管相关蛋白 mRNA 的表达增强,而平滑肌22α、Sox-2及Nanog mRNA的表达减弱。说明体外诱导的大鼠羊膜上皮细胞可成功分化为类神经干细胞。%BACKGROUND:Previous studies have found that Schwann cel s, olfactory ensheathing cel s, neural stem cel s and activated macrophages may repair nerve injuries, but these cel s have some deficiencies, such as source restrictions, tumorigenicity, and al ograft rejection. Amniotic epithelial cel s have no above-mentioned limits, and possess multiple-differentiation potential. After the induction, amniotic epithelial cel s may differentiate into cardiomyocyte-like cel s, neural stem

  18. Induction of apoptosis, stimulation of cell-cycle arrest and inhibition of angiogenesis make human amnion-derived cells promising sources for cell therapy of cancer.

    Science.gov (United States)

    Niknejad, Hassan; Yazdanpanah, Ghasem; Ahmadiani, Abolhassan

    2016-03-01

    Amniotic membrane (AM), the nearest layer of fetal membranes to the fetus, contains two types of cells with unique characteristics that make them excellent candidates for clinical applications. Amniotic epithelial and mesenchymal cells have low immunogenicity, anti-inflammation, anti-fibrosis and anti-bacterial properties and no ethical issues. Although amniotic cells have stem cell properties and express transcription factors specific for pluripotent stem cells, they are not tumorigenic after transplantation. In the last decade, a new line of research has been initiated with a focus on the anti-proliferative effects of amniotic epithelial and mesenchymal cells on tumor growth. Amnion-derived epithelial and mesenchymal cells inhibit tumor growth and invasion through three pathways: the induction of apoptosis, the stimulation of cell-cycle arrest and the inhibition of angiogenesis. In this review, the various aspects of the anti-cancer properties of amnion-derived cells and the underlying mechanisms are discussed with emphasis on the translation of the cell therapy of cancer from experimental into clinical practice.

  19. Human embryonic stem cells handbook

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2013-03-01

    Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

  20. Effects of amniotic fluid stem cell transplantation on immune tolerance and oxidative stress in kidney transplantation Effects of amniotic fluid stem cell transplantation on immune tolerance and oxidative stress in kidney transplantation%羊水干细胞诱导肾移植免疫耐受及对氧化应激的影响

    Institute of Scientific and Technical Information of China (English)

    邓春阳; 冯建勋; 张海英; 陈庭芳; 李静

    2015-01-01

    BACKGROUND:Stem cels can induce immune tolerance, prolong graft survival time and reduce rejection in organ transplantation, which have become a hot research. OBJECTIVE:To induce immune tolerance to alogenic kidney transplantation with amniotic fluid stem cels in recipient rats and to explore the mechanism underlying immune tolerance. METHODS: Amniotic fluid stem cels were isolated from Wistar rats. Two inbred male rat strains, Wistar rats and Sprague-Dawley rats, were selected as donors and recipients of kidney transplantation. The rat models of renal orthotopic transplantation were divided into the folowing four groups: a sham-operated group (n=10, Sprague-Dawley rats); an isograft group (n=10, Sprague-Dawley to Sprague-Dawley rats); a control group (n=10, Wistar to Sprague-Dawley rats, treated with 1 mL saline); and an experimental group (n=10, Wistar to Sprague-Dawley rats, treated with 1 mL of 3×106/L amniotic fluid stem cels). Serum levels of creatinine, urea nitrogen, interleukin-2, interferon-γ, parameters of oxidative stress were detected at 5 days after operation. Flow cytometry was employed to determine the percentage of CD4+ and CD8+ lymphocytes in the peripheral blood. Kidney transplants were observed pathologicaly. RESULTS AND CONCLUSION:Compared with the control group, the levels of creatinine, urea nitrogen, interleukin-2, interferon-γ, parameters of oxidative stress and proteinuria were lower in the experimental group (P < 0.05). Percentages of CD4+, CD8+ and CD4+/CD8+ ratio were also significantly lower in the experimental group than the control group. However, the rate of cretinemia clearance in the experimental group was significantly higher than that in the control group (P < 0.05). Furthermore, the degree of kidney injury in the experimental group was significantly lower than that in the control group. Our findings demonstrate that the amniotic fluid stem cel transplantation can induce immune tolerance, extenuate oxidative stress

  1. Investigation on Chromosomal Karyotypes of Amniotic Fluid Cells for 2 414 Cases%2414例胎儿羊水细胞的体外培养及染色体核型分析

    Institute of Scientific and Technical Information of China (English)

    卢丽华; 邹德学; 胡飞雪

    2013-01-01

    Objective:To investigate the application of karyotypes of amniotic fluid cells in prenatal diagnosis.Methods:A total of 2 414 pregnant women received amniocenteiss via B ultranound in sterile condition.A volume of 20 ml amniotic fluid was extracted from each case.Then the amniotic fluid cells were in vitro cultrued and their karyotypes were analysed by G band.Results:There were 124 cases with chromosomal abnormalities and it was 5.1% over all 2 414 cases.There were 46 cases with chromosomal numberical abnormality and 38 cases with trisomy syndrome,accounting for 82.6% of chromosomal numberical abnormality.There were 56 cases with chromosomal structural abnormality and 47 cases with balanced chromosome rearrangements including balanced translocation,Rob's translocation and inversion,accounting for 83.9% of chromosomal structural abnormality.There were 22 cases with mosaicism and they had normal chromosome of umbilical cord blood follow-up.The constituent ratios of Down's high-risk group and advanced matemal age group were 48.3% and 36.6%,respectively,which were significantly higher than those of other groups (P<0.05).The abnormal ratios in fetus abnormal ultra-sonographic group,nuchal translucency (NT) enhancement group and parental balanced chromosome rearrangements group were 25.5%,19.0% and 17.2%,respectively,which were significantly different from other groups (P<0.05).Conclusion:The improvement of amniocytes cultured in vitro was more practicability,and karyotypes of amnioc fluid cells were more important for prenatal diagnosis.%目的:探讨羊水细胞染色体分析在产前诊断中的应用价值.方法:无菌条件下,经B超介导,对2 414名孕16~25周的孕妇行羊膜腔穿刺术,每例抽取羊水20 ml,经体外培养后进行G显带,显微镜下做核型分析,并了解异常核型的相应高危因素分布情况.结果:共收集到2 414例羊水标本,2 407例培养成功,成功率为99.7%.核型异常124例,异常率为5

  2. Amniotic membrane seeded with mesenchymal adipose-derived stem cell for coverage of wound in third degree burn: An experimental study

    Directory of Open Access Journals (Sweden)

    Mohammad Javad Fatemi

    2014-09-01

    Conclusion: Acellular amnion seeded with adipose-derived stem cell can result in faster wound healing and better histopathology characteristic. The amnion as a scaffold and the fat derived stem cells as healing accelerator are recommended for coverage of the 3rd degree burn wounds after excision and it may reduce the need for skin graft.

  3. 中山地区羊水细胞培养核型分析联合荧光原位杂交技术在产前诊断中的应用%The combined application of karyotype analysis of chromosome in amniotic fluid cells and fish in prenatal diagnosis in Zhongshan District

    Institute of Scientific and Technical Information of China (English)

    苏年华; 季明芳; 陈慧

    2016-01-01

    Objective:To explore the applications value of karyotype analysis of chromosome in amniotic fluid cells and fluorescence in situ hybridization (FISH) in prenatal diagnosis.Methods:369 samples of amniotic fluid from women during 16-22th-week gestation and with the indications of prenatal diagnosis were cultured and performed both karyotype analysis of chromosome and FISH using the 13/18/21/X/Y chromosome probe.Results:The success rate of karyotype analysis of chromosome in amniotic fluid cells culture was 98.37% (363 in 369 cases).336 cases of normal variation of chromosome and 27 cases of abnormal variation of chromosome (numerical abnormalities chromosome in 18 cases,and structural abnormalities chromosome in 9 cases) were detected.The success rate of FISH detection of amniotic fluid cells culture was 100.00%,which included 351 cases of normal variation of chromosome and 18 cases of numerical abnormal variation of chromosome.However,there were no cases of structural abnormalities chromosome detected in FISH,due to the limited detecting probe.Conclusion:The karyotype analysis and FISH detection showed good consistency in detection of abnormal chromosome numbers (aneuploidy) in amniotic fluid cells culture.Furthermore,the former was more comprehensive with higher detection rate,and the latter was more convenient with shorter operating time and less operating steps.To combined apply karyotype analysis and FISH in amniotic fluid cells can be more effective for fetal chromosome abnormalities in prenatal diagnosis.%目的 探索羊水细胞培养核型分析联合荧光原位杂交技术(FISH)在产前诊断中的应用价值;方法 采集16~22孕周、有产前诊断指征孕妇的羊水标本369例,进行羊水细胞培养核型分析并同时采用13/18/21/X/Y染色体探针对未培养羊水进行FISH检测;结果 369例羊水细胞培养核型分析成功363例,异常27例(数目异常18例,结构异常9例);未培养羊水细胞FISH检测

  4. Inhibitor of intramembranous absorption in ovine amniotic fluid.

    Science.gov (United States)

    Brace, Robert A; Cheung, Cecilia Y; Anderson, Debra F

    2014-02-01

    Intramembranous absorption increases during intra-amniotic infusion of physiological saline solutions. The increase may be due partly to the concomitant elevation in fetal urine production as fetal urine contains a stimulator of intramembranous absorption. In this study, we hypothesized that the increase in intramembranous absorption during intra-amniotic infusion is due, in part, to dilution of a nonrenal inhibitor of intramembranous absorption that is present in amniotic fluid. In late-gestation fetal sheep, amniotic fluid volume and the four primary amniotic inflows and outflows were determined over 2-day intervals under three conditions: 1) control conditions when fetal urine entered the amniotic sac, 2) during intra-amniotic infusion of 2 l/day of lactated Ringer solution when urine entered the amniotic sac, and 3) during the same intra-amniotic infusion when fetal urine was continuously replaced with lactated Ringer solution. Amniotic fluid volume, fetal urine production, swallowed volume, and intramembranous absorption rate increased during the infusions independent of fetal urine entry into the amniotic sac or its replacement. Lung liquid secretion rate was unchanged during infusion. Because fetal membrane stretch has been shown not to be involved and because urine replacement did not alter the response, we conclude that the increase in intramembranous absorption that occurs during intra-amniotic infusions is due primarily to dilution of a nonrenal inhibitor of intramembranous absorption that is normally present in amniotic fluid. This result combined with our previous study suggests that a nonrenal inhibitor(s) together with a renal stimulator(s) interact to regulate intramembranous absorption rate and, hence, amniotic fluid volume.

  5. Diversity of microbes in amniotic fluid.

    Science.gov (United States)

    DiGiulio, Daniel B

    2012-02-01

    Recent polymerase chain reaction (PCR)-based studies estimate the prevalence of microbial invasion of the amniotic cavity (MIAC) to be ≥30-50% higher than that detected by cultivation-based methods. Some species that have been long implicated in causing MIAC remain among the common invaders (e.g. Ureaplasma spp., Mycoplasma spp., Fusobacterium spp. Streptococcus spp., Bacteroides spp. and Prevotella spp.). Yet we now know from studies based on PCR of the 16S ribosomal DNA that cultivation-resistant anaerobes belonging to the family Fusobacteriaceae (particularly Sneathia sanguinegens, and Leptotrichia spp.) are also commonly found in amniotic fluid. Other diverse microbes detected by PCR of amniotic fluid include as-yet uncultivated and uncharacterized species. The presence of some microbial taxa is associated with specific host factors (e.g. Candida spp. and an indwelling intrauterine device). It appears that MIAC is polymicrobial in 24-67% of cases, but the potential role of pathogen synergy is poorly understood. A causal relationship between diverse microbes, as detected by PCR, and preterm birth is supported by types of association (e.g. space, time and dose) proposed as alternatives to Koch's postulates for inferring causality from molecular findings. The microbial census of the amniotic cavity remains unfinished. A more complete understanding may inform future research directions leading to improved strategies for preventing, diagnosing and treating MIAC.

  6. Corneal Differentiation Following HSC70 and HSP72 Expression in Air-Exposed Limbal Stem Cells Cultured on Denuded Amniotic Membrane

    Directory of Open Access Journals (Sweden)

    Parvaneh Mohammadi

    2010-01-01

    Full Text Available Objective: The aim of this study is to create an ex vivo model to examine the expressionof two heat shock protein 70 (HSP70 family members, heat shock protein 72 (HSP72and heat shock constitute protein 70 (HSC70, at the mRNA and protein levels in differentiatingcorneal cells from air exposed limbal stem cells.Materials and Methods: Limbal biopsies were cultured as explants on a cellular amnioticmembrane for 14 days. The cells were then exposed to air for 16 extra additional days.The proposed expression of limbal stem cell markers (p63, ABCG2, corneal markers(K3/12, connexin 43, as well as HSP72 and HSC70 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR at the mRNA level, and by immunocytochemistryand flowcytometry at the protein level both pre and post air exposure. Fresh limbal andcorneal tissues were used as control group.Results: Air exposure decreased expression of p63 and increased expression of K3/K12 indicating an increase in the number of corneal cells. Our data showed that HSP72and HSC70 were expressed at the mRNA level before and after air exposure while theirexpression significantly increased post air exposure at the protein level.Conclusion: We assume HSC70 expression may be related to early and terminal stagesof differentiation in cultured limbal stem cells. In addition, limbal stem cells were protectedduring normal development against oxidative stress thru increased HSP72 expression.These findings may have broader implications in development of therapeutic strategiesfor treating wound healing disorders by induction of HSPs.

  7. Peptidomic Analysis of Amniotic Fluid for Identification of Putative Bioactive Peptides in Ventricular Septal Defect

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    Xing Li

    2016-05-01

    Full Text Available Background: Ventricular septal defect (VSD is one of the most common congenital heart diseases and to date the role of peptides in human amniotic fluid in the pathogenesis of VSD have been rarely investigated. Methods: To gain insight into the mechanisms of protein and peptides in cardiovascular development, we constructed a comparative peptidomic profiling of human amniotic fluid between normal and VSD fetuses using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by nano liquid chromatography tandem mass spectrometry. Results: We identified and quantified 692 non-redundant peptides, 183 of which were differentially expressed in the amniotic fluid of healthy and VSD fetuses; 69 peptides were up regulated and 114 peptides were down regulated. These peptides were imported into the Ingenuity Pathway Analysis (IPA and identified putative roles in cardiovascular system morphogenesis and cardiogenesis. Conclusion: We concluded that 35 peptides located within the functional domains of their precursor proteins could be candidate bioactive peptides for VSD. The identified peptide changes in amniotic fluid of VSD fetuses may advance our current understanding of congenital heart disease and these peptides may be involved in the etiology of VSD.

  8. Human fetal mesenchymal stem cells.

    Science.gov (United States)

    O'Donoghue, Keelin; Chan, Jerry

    2006-09-01

    Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The

  9. Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

    Science.gov (United States)

    Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

    2014-09-01

    Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

  10. Acceleration of Regeneration of Large-Gap Peripheral Nerve Injuries Using Acellular Nerve Allografts Plus Amniotic Fluid Derived Stem Cells (AFS)

    Science.gov (United States)

    2014-09-01

    nerves are incorporated into other rat strains without immunologic disturbances according to the vendor (AxoGen). 15. SUBJECT TERMS 16. SECURITY...mm). The outcomes of these surgeries will be compared to those obtained with autograft nerve repairs that currently have the best outcomes for large...ANA construct with AFS cells (Group 2), or with an autograft (nerve segment is cut out, reversed, and sewn back in place)(Group 3). All surgeries

  11. Differentiated human stem cells resemble fetal, not adult, β cells.

    Science.gov (United States)

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  12. A fetal and an intra-amniotic inflammatory response is more severe in preterm labor than in preterm PROM in the context of funisitis: unexpected observation in human gestations.

    Directory of Open Access Journals (Sweden)

    Chan-Wook Park

    Full Text Available OBJECTIVE: Although intra-amniotic(IA infection is present in both preterm labor and intact membranes(PTL and preterm premature rupture of membranes(preterm-PROM, it is more common in preterm-PROM than in PTL. Microorganisms and their products in the amniotic-cavity can elicit an inflammatory-response in fetus as well as in amniotic-cavity in the progression of acute histologic chorioamnionitis(acute-HCA. A fundamental question is whether a fetal and an IA inflammatory-response is more severe in preterm-PROM than in PTL, in the same-context of acute-HCA with or without fetal-involvement. The purpose of current-study was to answer this-question. STUDYDESIGN: Study population consisted of 213 singleton preterm-gestations(.05 for each;3 however, acute-HCA with funisitis was associated with a significantly higher median AF MMP-8 and UCP CRP concentration and higher rate of cervical dilatation≥3 cm or 4 cm in PTL than in preterm-PROM(AF MMP-8, 675 ng/mlvs.417 ng/ml; UCP CRP, 969 ng/mlvs.397 ng/ml;each for p<.05, despite less common IA infection in PTL than in preterm-PROM(29%vs.57%;p<.05. CONCLUSIONS: A fetal and an IA inflammatory-response is more severe in PTL than in preterm-PROM in the context of funisitis, despite less common IA infection. This unexpected observation may indicate the fundamental difference in the pathogenesis between PTL and preterm-PROM.

  13. The origin of fetal sterols in second-trimester amniotic fluid : endogenous synthesis or maternal-fetal transport?

    NARCIS (Netherlands)

    Baardman, Maria E.; Erwich, Jan Jaap H. M.; Berger, Rolf M. F.; Hofstra, Robert M. W.; Kerstjens-Frederikse, Wilhelmina S.; Luetjohann, Dieter; Plosch, Torsten; Lutjohann, D.

    2012-01-01

    OBJECTIVE: Cholesterol is crucial for fetal development. To gain more insight into the origin of the fetal cholesterol pool in early human pregnancy, we determined cholesterol and its precursors in the amniotic fluid of uncomplicated, singleton human pregnancies. STUDY DESIGN: Total sterols were cha

  14. Association between fetal weight and amniotic fluid index in women of Central India

    Directory of Open Access Journals (Sweden)

    Nitin Wadnere

    2014-01-01

    Full Text Available Background : The placenta is important for fetal growth and well-being. Defective placentation and impaired placental circulation may result in anomalies in fetal growth. Placental volume in the second trimester appears to be closely related to the neonatal weight. The association of body weight with urine output has been observed in human neonates. Our goal is to assess the association of the amniotic fluid index (AFI with the estimated fetal weight (EFW. Materials and Methods : Thirteen hundred and ninety-three pregnant women were prospectively studied by means of an ultrasound over a 12-month period. The fetal weight (FW was estimated using a combination of fetal parameters - bi-parietal diameter, fetal trunk cross-sectional area, and femur length. AFI was assessed using the four quadrant method. The level of statistical significance was set at P ≤ 0.05. Result s: There was no statistically significant association between AFI and EFW (P > 0.05; r = 0.413. We also did not find a significant association between AFI and EFW for all subdivisions of gestation age, except in the 24 - 28 weeks and 29 - 32 weeks′ groups. Conclusion : The FW calculations and amniotic index show a variation in values in late pregnancy. There does not appear to be a linear association between the ultrasound estimate of FW and the amniotic index. The implication of this is that the fetal size need not be taken into cognizance when alterations in amniotic fluid values are noted.

  15. Amniotic membrane extract ameliorates benzalkonium chloride-induced dry eye in a murine model.

    Science.gov (United States)

    Xiao, Xinye; Luo, Pingping; Zhao, Hui; Chen, Jingyao; He, Hui; Xu, Yuxue; Lin, Zhirong; Zhou, Yueping; Xu, Jianjiang; Liu, Zuguo

    2013-10-01

    Human amniotic membrane (AM) is avascular but contains various beneficial bioactive factors, its extract (AE) is also effective in treating many ocular surface disorders. In this study, we for the first time evaluated the therapeutic effects of AE on dry eye induced by benzalkonium chloride in a BALB/c mouse model. Topical application of AE (1.5 and 3 μg/eye/day) resulted in significantly longer tear break-up time on Day 3 and 6, lower fluorescein staining scores on Day 3, and lower inflammatory index on Day 6. AE reduced corneal epithelial K10 expression, inflammatory infiltration, and levels of TNF-α, IL-1β and IL-6 in BAC treated mice than that in the control mice. Moreover, decreased TUNEL positive cells in cornea and increased goblet cells in conjunctiva were also observed in AE treated corneas. Finally, AE induced more Ki-67 positive cells in corneal epithelium of dry eye mouse. Taken together, our data provide further support for BAC induced dry eye model as a valuable for dry eye study and suggest a great potential for AE as a therapeutic agent in the clinical treatment of dry eye.

  16. Stem cell therapy ameliorates bladder dysfunction in an animal model of Parkinson disease.

    Science.gov (United States)

    Soler, Roberto; Füllhase, Claudius; Hanson, Ariel; Campeau, Lysanne; Santos, Cesar; Andersson, Karl-Erik

    2012-04-01

    Different cell based therapies have been tested, focusing on motor function. We evaluated the effect of human amniotic fluid stem cells and bone marrow derived mesenchymal stem cells (ALLCELLS, Emeryville, California) on bladder dysfunction in a rat model of Parkinson disease. A nigrostriatal lesion was induced by 6-hydroxydopamine in 96 athymic nude female rats divided into 3 treatment groups. After 2 weeks the groups were injected with human amniotic fluid stem cells, bone marrow derived mesenchymal stem cells and vehicle for sham treatment, respectively. At 3, 7, 14 and 28 days the bladder function of 8 rats per group was analyzed by conscious cystometry. Brains were extracted for immunostaining. The nigrostriatal lesion caused bladder dysfunction, which was consistent in sham treated animals throughout the study. Several cystometric parameters improved 14 days after human amniotic fluid stem cell or bone marrow derived mesenchymal stem cell injection, concomitant with the presence of human stem cells in the brain. At 14 days only a few cells were observed in a more caudal and lateral position. At 28 days the functional improvement subsided and human stem cells were no longer seen. Human stem cell injection improved the survival of dopaminergic neurons until 14 days. Human stem cells expressed superoxide dismutase-2 and seemed to modulate the expression of interleukin-6 and glial cell-derived neurotrophic factor by host cells. Cell therapy with human amniotic fluid stem cells and bone marrow derived mesenchymal stem cells temporarily ameliorated bladder dysfunction in a Parkinson disease model. In contrast to integration, cells may act on the injured environment via cell signaling. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  17. Analysis on Karyotype of Amniotic Fluid Cells from 3 800 Fetus and Related Genetic Counseling%3800例羊水细胞染色体核型分析及相关遗传咨询

    Institute of Scientific and Technical Information of China (English)

    孙立娟; 李岩; 张秀玲; 史云芳; 李晓洲; 张颖

    2011-01-01

    目的:探讨染色体异常核型与产前诊断指征的关系及羊膜腔穿刺术的安全性,为产前遗传咨询提供客观的实验依据.方法:3 800例具备产前诊断指征的妊娠妇女,在知情选择的情况下行羊膜腔穿刺术及染色体核型检测.分析相关数据,追踪羊膜腔穿刺术的结局.结果:羊水细胞一次培养成功率为99.26%(3772/3 800),两次培养成功率为99.97%(3 795/3 796).在3 795例羊水细胞培养成功的染色体核型中,检出异常核型120例,异常率为3.16%,其中染色体数目异常率1.61%(61/3 795),结构异常率O.58%(22/3 795),多态性变异异常率0.97%(37/3 795).产前诊断指征中,按羊膜腔穿刺例数.位于前3位的分别是唐氏综合征筛查高危人群组(以下简称唐筛高危组,3 54l 例)、不良妊娠分娩史组(95例)和单纯高龄组(≥35岁,83例).检出染色体异常核型例数前3位的分别是唐筛高危组(103例)、夫妻单方染色体异常组(8例)和单纯高龄组(4例).染色体核型异常率前3位的分别是夫妻单方染色体异常组(38.10%,8/21,仅1例有临床意义)、超声提示胎儿异常组(9.38%.3/32)和单纯高龄组(4.82%.4/83).唐筛高危组中,高龄和低龄妊娠妇女染色体核型异常率差异有统计学意义(x2=4.342,P0.05).胎儿丢失率0.237%(9/3 800).胎死宫内率0.053%(2/3 800).结论:①唐筛高危、高龄、超声提示胎儿异常及夫妻单方染色体异常者均有必要进行产前诊断.②羊膜腔穿刺术相对安全.③根据相关实验数据对高危妊娠妇女进行个体化遗传咨询是必要的.%Objective: In order to constitute a basis for genetic counseling, we studied the relationship between fetal chromosomal aberrations and prenatal diagnosis indications, and analyzed the security of amniocentesis. Methods:Fetal chromosomal karyotypes were examined in 3 800 pregnant women with amniotic cell culture in accordance with the indications for prenatal diagnosis. We studied the

  18. The potency of human testicular stem cells

    NARCIS (Netherlands)

    Chikhovskaya, J.V.

    2013-01-01

    In this thesis, we evaluate the stem cell state of cells present in primary human testicular cell cultures as well as their origin and relation to germ or somatic lineages within testicular tissue. We conclude that human testis-derived embryonic stem cell-like (htES-like) colonies arising in primary

  19. Prognostic potential of amniotic fluid analysis at birth on canine neonatal outcomes.

    Science.gov (United States)

    Groppetti, D; Martino, P A; Ravasio, G; Bronzo, V; Pecile, A

    2015-12-01

    Glucose, lactate and cortisol concentrations in amniotic fluid were measured at birth in 95 pups and related to neonatal viability based on Apgar scoring and to neonatal mortality. Neither amniotic parameters nor neonatal mortality were associated with the Apgar score. Stillborn pups showed high lactate (P amniotic concentrations (P amniotic fluid differences were observed between normal and malformed pups. Amniotic glucose (P amniotic fluid collected at birth could be a valuable predictor of neonatal outcomes in dogs.

  20. To study on the methods of serial subcultivation of amniotic fluid in penatal diagnosis%转瓶培养在羊水细胞产前诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    李锋; 郑红云; 童永清

    2011-01-01

    目的 建立一种成功率高,收获分裂相多的羊水细胞培养方法.方法 总共357例羊水标本用经典培养法和转瓶培养法培养.结果 经典培养成功率为94.3%(337/357),转瓶培养二次收获后成功率为97.8% (349/357),成功率提高,且所获分裂相数目多,形态好.结论 转瓶培养方法是一种简便易行的羊水细胞培养方法.%Objective; To improve the successful rate of culture of human amniotic fluid cells. Methods; The classic cultivation method and serial subcultivation method was performed in 357 cases of amniotic fluid cultivation. Results: The successful rate of classic cultivation method and serial subcultivation method was 94. 3% (337/357) and 97. 8% (349/357) respectively. The serial sub-cultivation has high successful rate. More and high - quality M - phase can be observed by serial subcultivation method. Conclusion: The serial subcultivation is convenient method in amniotic fluid culture. It has the advantages of good chromosome morphous and high successful rate.

  1. Prenatal Amniotic Fluid Cell Chromosome Examination in the Diagnosis of Fetal Abnormalities in the Elderly%羊膜腔穿刺羊水细胞染色体检查用于高龄孕妇胎儿畸形预见性诊断分析

    Institute of Scientific and Technical Information of China (English)

    王花花

    2016-01-01

    目的:探讨高龄孕妇进行孕中期羊膜腔穿刺羊水细胞培养染色体核型分析,提高对胎儿畸形的预见性诊断分析结果。方法随机选取该院2013年1月―2015年12月收治的714例高龄孕妇给予羊膜腔穿刺前的检查及超声诊断,行羊膜腔穿刺抽取羊水的孕妇,进行羊水细胞培养,制备染色体标本,分析高龄孕妇羊水胎儿细胞的染色体核型和胎儿染色体异常情况。结果该研究选取的714例高龄孕妇中染色体异常的分类有21三体综合征、18三体综合征、性染色体异常和其它的染色体异常。有23例高龄孕妇的染色体出现异常,检出率为3.22%;714例高龄孕妇中35~39岁占450例,发现胎儿染色体异常例数9例,检出率为2..00%为最低;44~46岁高龄孕妇占31例,发现胎儿染色体异常例数为3例,检出率为9.68%为最高。结论在产前对高龄孕妇进行羊膜腔穿刺羊水细胞染色体培养可以有效的检测胎儿的染色体异常情况,有效的对高龄孕妇分娩畸形胎儿进行预见性的诊断,明显降低新生儿的缺陷率。%Objective Discussion older pregnant women were second-trimester amniocentesis amniotic fluid cells of fetal malformation predictive diagnostic analysis of cultured karyotype analysis. Methods The study selected from our hospital in January 2013 to December 2015 in 714 cases of advanced maternal age to give pre amniocentesis examination and ultra-sound diagnosis, underwent amniocentesis and amniotic fluid of pregnant women, the amniotic cell culture prepared chromo-some specimen, analysis of women of advanced maternal age and fetal amniotic fluid cells stained color karyotype and fetal chromosomal abnormalities. Results The classification of chromosomal abnormalities in 714 cases of elderly women is 21, 18,, sex chromosome abnormalities and other chromosomal abnormalities. Have abnormal chromosomes in 23 cases of ad-vanced maternal age, the

  2. Stem cell differentiation and human liver disease

    Institute of Scientific and Technical Information of China (English)

    Wen-Li Zhou; Claire N Medine; Liang Zhu; David C Hay

    2012-01-01

    Human stem cells are scalable cell populations capable of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells.Such an approach has the potential to improve our understanding of human biology and treating disease.In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases.In recent years,efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own.In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology.

  3. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    Full Text Available BACKGROUND: The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. METHODOLOGY/PRINCIPAL FINDINGS: Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. CONCLUSION/SIGNIFICANCE: The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of

  4. Cytogenetic studies in human cells exposed in vitro to GSM-900 MHz radiofrequency radiation using R-banded karyotyping.

    Science.gov (United States)

    Bourthoumieu, S; Joubert, V; Marin, B; Collin, A; Leveque, P; Terro, F; Yardin, C

    2010-12-01

    It is important to determine the possible effects of exposure to radiofrequency (RF) radiation on the genetic material of cells since damage to the DNA of somatic cells may be linked to cancer development or cell death and damage to germ cells may lead to genetic damage in next and subsequent generations. The objective of this study was to investigate whether exposure to radiofrequency radiation similar to that emitted by mobile phones of second-generation standard Global System for Mobile Communication (GSM) induces genotoxic effects in cultured human cells. The cytogenetic effects of GSM-900 MHz (GSM-900) RF radiation were investigated using R-banded karyotyping after in vitro exposure of human cells (amniotic cells) for 24 h. The average specific absorption rate (SAR) was 0.25 W/kg. The exposures were carried out in wire-patch cells (WPCs) under strictly controlled conditions of temperature. The genotoxic effect was assessed immediately or 24 h after exposure using four different samples. One hundred metaphase cells were analyzed per assay. Positive controls were provided by using bleomycin. We found no direct cytogenetic effects of GSM-900 either 0 h or 24 h after exposure. To the best of our knowledge, our work is the first to study genotoxicity using complete R-banded karyotyping, which allows visualizing all the chromosomal rearrangements, either numerical or structural.

  5. Interactions among pulmonary surfactant, vernix caseosa, and intestinal enterocytes: intra-amniotic administration of fluorescently liposomes to pregnant rabbits.

    Science.gov (United States)

    Nishijima, Koji; Shukunami, Ken-ichi; Yoshinari, Hideo; Takahashi, Jin; Maeda, Hideyuki; Takagi, Hitoshi; Kotsuji, Fumikazu

    2012-08-01

    Although vernix caseosa is known to be a natural biofilm at birth, human pulmonary surfactant commences to remove the vernix from fetal skin into the amniotic fluid at gestational week 34, i.e., well before delivery. To explain this paradox, we first produced two types of fluorescently labeled liposomes displaying morphology similar to that of pulmonary surfactant and vernix caseosa complexes. We then continuously administered these liposomes into the amniotic fluid space of pregnant rabbits. In addition, we produced pulmonary surfactant and vernix caseosa complexes and administered them into the amniotic fluid space of pregnant rabbits. The intra-amniotic infused fluorescently labeled liposomes were absorbed into the fetal intestinal epithelium. However, the liposomes were not transported to the livers of fetal rabbits. We also revealed that continuous administration of micelles derived from pulmonary surfactants and vernix caseosa protected the small intestine of the rabbit fetus from damage due to surgical intervention. Our results indicate that pulmonary surfactant and vernix caseosa complexes in swallowed amniotic fluid might locally influence fetal intestinal enterocytes. Although the present studies are primarily observational and further studies are needed, our findings elucidate the physiological interactions among pulmonary, dermal-epidermal, and gastrointestinal developmental processes.

  6. Generation of Human Induced Pluripotent Stem Cells from Extraembryonic Tissues of Fetuses Affected by Monogenic Diseases.

    Science.gov (United States)

    Spitalieri, Paola; Talarico, Rosa V; Botta, Annalisa; Murdocca, Michela; D'Apice, Maria Rosaria; Orlandi, Augusto; Giardina, Emiliano; Santoro, Massimo; Brancati, Francesco; Novelli, Giuseppe; Sangiuolo, Federica

    2015-08-01

    The generation of human induced pluripotent stem cells (hiPSCs) derived from an autologous extraembryonic fetal source is an innovative personalized regenerative technology that can transform own-self cells into embryonic stem-like ones. These cells are regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug discovery. Thus, hiPSCs enable researchers to undertake studies for treating diseases or for future applications of in utero therapy. We used a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding OCT4, SOX2, KLF4, and cMYC genes and containing loxP sites, excisible by Cre recombinase, to reprogram patient-specific fetal cells derived from prenatal diagnosis for several genetic disorders, such as myotonic dystrophy type 1 (DM1), β-thalassemia (β-Thal), lymphedema-distichiasis syndrome (LDS), spinal muscular atrophy (SMA), cystic fibrosis (CF), as well as from wild-type (WT) fetal cells. Because cell types tested to create hiPSCs influence both the reprogramming process efficiency and the kinetics, we used chorionic villus (CV) and amniotic fluid (AF) cells, demonstrating how they represent an ideal cell resource for a more efficient generation of hiPSCs. The successful reprogramming of both CV and AF cells into hiPSCs was confirmed by specific morphological, molecular, and immunocytochemical markers and also by their teratogenic potential when inoculated in vivo. We further demonstrated the stability of reprogrammed cells over 10 and more passages and their capability to differentiate into the three embryonic germ layers, as well as into neural cells. These data suggest that hiPSCs-CV/AF can be considered a valid cellular model to accomplish pathogenesis studies and therapeutic applications.

  7. Quantitative analysis of nerve growth factor in the amniotic fluid during chick embryonic development.

    Science.gov (United States)

    Mashayekhi, Farhad; Dianati, Elham; Moghadam, Lotfali Masomi

    2011-04-01

    Nerve growth factor (NGF) and most neurotrophic factors support the proliferation and survival of particular types of neurons. Besidesthe pivotal role of NGF in the development of neuronal cells, it also has important functions on non-neuronal cells. The amnion surrounds the embryo, providing an aqueous environment for the embryo. A wide range of proteins has been identified in human amniotic fluid (AF). In this study, total protein concentration (TPC) and NGF level in AF samples from chick embryos were measured using a Bio-Rad protein assay, enzyme linked immunosorbent assay (ELISA) and Western blot. TPC increased from days E10 to day E18. There was a rapid increase in AF TPC on day E15 when compared to day E16. No significant changes in NGF levels have been seen from day E10 to day E14. There was a rapid increase in NGF content on days E15 and E16, and thereafter the levels decreased from day E16 to day E18. Since, NGF is important in brain development and changes in AF NGF levels have been seen in some CNS malformations, changes in the TPC and NGF levels in AF during chick embryonic development may be correlated with cerebral cortical development. It is also concluded that NGF is a constant component of the AF during chick embryogenesis.

  8. Endocannabinoids and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Giovanna Zolese

    2010-10-01

    Full Text Available N-acylethanolamides (NAEs are naturally occurring signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. Usually they are present in a very small amounts in many mammalian tissues and cells, including human reproductive tracts and fluids. Recently, the presence of N-arachidonoylethanolamide (anandamide, AEA, the most characterised member of endocannabinoids, and its congeners palmitoylethanolamide (PEA and oleylethanolamide (OEA in seminal plasma, oviductal fluid, and follicular fluids was demonstrated. AEA has been shown to bind not only type-1 (CB1 and type-2 (CB2 cannabinoid receptors, but also type-1 vanilloid receptor (TRPV1, while PEA and OEA are inactive with respect to classical cannabinoid CB1 and CB2 but activate TRPV1 or peroxisome proliferator activate receptors (PPARs. This review concerns the most recent experimental data on PEA and OEA, endocannabinoid-like molecules which appear to exert their action exclusively on sperm cells with altered features, such as membrane characteristics and kinematic parameters. Their beneficial effects on these cells could suggest a possible pharmacological use of PEA and OEA on patients affected by some forms of idiopathic infertility.

  9. A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta.

    Science.gov (United States)

    Whidbey, Christopher; Harrell, Maria Isabel; Burnside, Kellie; Ngo, Lisa; Becraft, Alexis K; Iyer, Lakshminarayan M; Aravind, L; Hitti, Jane; Waldorf, Kristina M Adams; Rajagopal, Lakshmi

    2013-06-03

    Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury.

  10. Transplante de membrana amniótica canina criopreservada para cicatrização de córnea com deficiência de células límbicas em coelhos Transplantation of cryopreserved canine amniotic membrane for cicatrisation in cornea with limbal stem cells deficiency in rabbits

    Directory of Open Access Journals (Sweden)

    D.N. Cremonini

    2007-12-01

    Full Text Available Avaliaram-se as alterações relacionadas à deficiência das células límbicas precursoras do epitélio corneano de coelhos e o efeito da membrana amniótica sobre sua cicatrização. A lesão, induzida com n-heptanol associado à peritomia conjuntival em 360°, foi recoberta com membrana amniótica canina, suturada à episclera perilímbica, criopreservada em meio para congelação de embrião ou em meio próprio, ambos com glicerol a 50% e mantida a -80°C. O grupo-controle não foi tratado com a membrana. As avaliações histológicas foram realizadas ao sétimo, 15º e 30º dias. Todos desenvolveram deficiência de células germinativas do limbo, denominada conjuntivalização, com presença de neovascularização, inflamação e defeitos epiteliais recorrentes, caracterizada na histopatologia pela presença de neovasos, edema, leucócitos e células caliciformes. O transplante de membrana amniótica não foi eficiente para o tratamento desta deficiência, entretanto auxiliou o processo de cicatrização da córnea.Changes related to limbal stem cells deficiency in corneal epithelium in rabbits, as well as the results of amniotic membrane transplant on the cicatrisation were evaluated. The ulcer was induced with n-heptanol associated to 360° conjunctival peritomy; the corneal surface was covered with canine amniotic membrane, sutured to perilimbal episclera, cryopreserved in embryo solution or own medium, both with 50% glycerol and stored at -80°C. The control group was not treated with membrane. Histological evaluations were performed at seven, 15, and 30 days. All of them developed limbal stem cells deficiency, named conjunctivalization, with neovascularization, inflammation and recurrent epithelial defects, observed in histopathology by the occurrence of neovascularization, edema, leukocytes and goblet cells. Thus amniotic membrane transplantation was not efficient in the treatment of limbal stem cells deficiency, however it helped in

  11. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  12. Prenatal diagnosis of methylmalonic aciduria by measuring methylmalonic acid in dried amniotic fluid on filter paper using gas chromatography-mass spectrometry.

    Science.gov (United States)

    Inoue, Yoshito; Ohse, Morimasa

    2011-06-01

    Methylmalonic aciduria is a common inherited metabolic disorder. Methylmalonic acid (MMA), a key indicator of methylmalonic aciduria, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the MMA in amniotic fluid can be measured by stable-isotope dilution gas chromatography-mass spectrometry. Here, we quantified the MMA in cell-free amniotic fluid samples that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. Our results indicated that the filter paper method was reproducible and accurate enough to be applied to clinical analysis. We also used the filter paper method to screen at-risk fetuses and obtained a clear diagnosis in each case. We conclude that our method enables the prenatal diagnosis of methylmalonic aciduria using practical procedures and a simplified method for transporting the samples.

  13. Satellite cells in human skeletal muscle plasticity.

    Science.gov (United States)

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  14. Detection of Bacteroides fragilis endotoxin in amniotic fluid by counterimmunoelectrophoresis

    NARCIS (Netherlands)

    I. Beckmann (Ilse); K. de Graaff (K.); F. Meisel-Mikolajczyk; H.C.S. Wallenburg (Henk)

    1994-01-01

    textabstractThe ability of counter immunoelectrophoresis (CIE) to detectBacteroides fragilis endotoxin in amniotic fluid in small concentrations was evaluated. A method was developed which, in combination with ultrafiltration, permits detection ofB. fragilis endotoxin in amniotic fluid in a concentr

  15. Does reduction of amniotic fluid affect fetal movements?

    NARCIS (Netherlands)

    Sival, D A; Visser, G H; Prechtl, H F

    1990-01-01

    The effect of the amount of amniotic fluid on the form of fetal general movements was studied longitudinally in 19 pregnancies complicated by premature rupture of the amniotic membranes (PROM). Before birth, general movements were studied weekly by means of 1-h ultrasound observations, performed und

  16. IMMUNORESPONSES OF HUMANIZED SCID MICE TO HUMAN LUNG CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    陈力真; 王树蕙; 张云; 王世真

    1996-01-01

    HuPBL-SCID mice were used to explore how they would response to human ttmoor cells of 801/MLC.Living 801/MLC cells appeared to be fetal to the the mice due to the production of human TNF. The huP-BL-SCID rniee did not generate any noticeable amotmt of specific human immunoglobttlin either by single immunization with living 801/MLC cells or by repeated immunization with irradiated 801/MLC cells. Our preliminary experiments with huPBL-SCID mice showed that such chimeras would he a very useful models for tumor immunological researches.

  17. Search for naive human pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Simone Aparecida Siqueira Fonseca; Roberta Montero Costas; Lygia Veiga Pereira

    2015-01-01

    Normal mouse pluripotent stem cells were originallyderived from the inner cell mass (ICM) of blastocystsand shown to be the in vitro equivalent of those preimplantationembryonic cells, and thus were calledembryonic stem cells (ESCs). More than a decade later,pluripotent cells were isolated from the ICM of humanblastocysts. Despite being called human ESCs, thesecells differ significantly from mouse ESCs, includingdifferent morphology and mechanisms of control ofpluripotency, suggesting distinct embryonic originsof ESCs from the two species. Subsequently, mousepluripotent stem cells were established from the ICMderivedepiblast of post-implantation embryos. Thesemouse epiblast stem cells (EpiSCs) are morphologicaland epigenetically more similar to human ESCs. Thisraised the question of whether cells from the humanICM are in a more advanced differentiation stage thantheir murine counterpart, or whether the availableculture conditions were not adequate to maintain thosehuman cells in their in vivo state, leading to a transitioninto EpiSC-like cells in vitro . More recently, novel cultureconditions allowed the conversion of human ESCs intomouse ESC-like cells called naive (or ground state)human ESCs, and the derivation of naive human ESCsfrom blastocysts. Here we will review the characteristicsof each type of pluripotent stem cells, how (andwhether) these relate to different stages of embryonicdevelopment