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Sample records for human embryonic palatal

  1. Prescreening for environmental teratogens using cultured mesenchymal cells from the human embryonic palate

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, R.M.; Grove, R.I.; Willis, W.D.

    1982-01-01

    Mesenchymal cells from the prefusion human embryonic palate have been established in culture and can be grown in either a serum-free hormone-supplemented medium or a serum-containing medium. The growth of these cells is quite rapid in culture and inhibited in a dose-dependent manner by most teratogens thus far tested, such as dexamethasone. These cells are highly sensitive to a variety of DNA synthetic and mitotic inhibitors. The responses of these cells are complementary to the ovarian tumor cell attachment assay of Braun et al (1, and in this volume). When used in conjunction with the tumor cells, the overall reliability is greater than 90% with only one false-negative, allopurinol.

  2. Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

    Science.gov (United States)

    Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

  3. Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

    Science.gov (United States)

    Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

  4. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection.

    OpenAIRE

    Yoneda, T.; Urade, M; Sakuda, M.; Miyazaki, T

    1986-01-01

    We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis...

  5. File list: Oth.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.AllAg.Embryonic_palates.bed ...

  6. File list: Oth.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.AllAg.Embryonic_palates.bed ...

  7. File list: Pol.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.50.AllAg.Embryonic_palates mm9 RNA polymerase Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.50.AllAg.Embryonic_palates.bed ...

  8. File list: Pol.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.AllAg.Embryonic_palates mm9 RNA polymerase Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.AllAg.Embryonic_palates.bed ...

  9. File list: Oth.Emb.10.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.Embryonic_palates.bed ...

  10. File list: Pol.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.AllAg.Embryonic_palates mm9 RNA polymerase Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.05.AllAg.Embryonic_palates.bed ...

  11. File list: Oth.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.Embryonic_palates.bed ...

  12. File list: ALL.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_palates.bed ...

  13. File list: NoD.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.05.AllAg.Embryonic_palates mm9 No description Embryo Embryonic palates ERX6...50310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.05.AllAg.Embryonic_palates.bed ...

  14. File list: NoD.Emb.10.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.10.AllAg.Embryonic_palates mm9 No description Embryo Embryonic palates ERX6...50310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.10.AllAg.Embryonic_palates.bed ...

  15. File list: InP.Emb.10.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Embryonic_palates.bed ...

  16. File list: ALL.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_palates.bed ...

  17. File list: InP.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.20.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.20.AllAg.Embryonic_palates.bed ...

  18. File list: InP.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.05.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.05.AllAg.Embryonic_palates.bed ...

  19. File list: NoD.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.50.AllAg.Embryonic_palates mm9 No description Embryo Embryonic palates ERX6...50310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.50.AllAg.Embryonic_palates.bed ...

  20. File list: ALL.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_palates.bed ...

  1. File list: InP.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_palates.bed ...

  2. Rat embryonic palatal shelves respond to TCDD in organ culture

    Energy Technology Data Exchange (ETDEWEB)

    Abbott, B.D.; Birnbaum, L.S. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (USA))

    1990-05-01

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in (3H)TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves.

  3. Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

    DEFF Research Database (Denmark)

    Jakobsen, L.P.; Borup, R.; Vestergaard, J.;

    2009-01-01

    . Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP...... and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed...... and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend...

  4. Dual embryonic origin of maxillary lateral incisors: clinical implications in patients with cleft lip and palate

    Directory of Open Access Journals (Sweden)

    Daniela Gamba Garib

    2015-10-01

    Full Text Available Introduction:Cleft lip and palate are craniofacial anomalies highly prevalent in the overall population. In oral clefts involving the alveolar ridge, variations of number, shape, size and position are observed in maxillary lateral incisors. The objective of this manuscript is to elucidate the embryonic origin of maxillary lateral incisors in order to understand the etiology of these variations.Contextualization: The hypothesis that orofacial clefts would split maxillary lateral incisor buds has been previously reported. However, recent studies showed that maxillary lateral incisors have dual embryonic origin, being partially formed by both the medial nasal process and the maxillary process. In other words, the mesial half of the lateral incisor seems to come from the medial nasal process while the distal half of the lateral incisor originates from the maxillary process. In cleft patients, these processes do not fuse, which results in different numerical and positional patterns for lateral incisors relating to the alveolar cleft. In addition to these considerations, this study proposes a nomenclature for maxillary lateral incisors in patients with cleft lip and palate, based on embryology and lateral incisors position in relation to the alveolar cleft.Conclusion:Embryological knowledge on the dual origin of maxillary lateral incisors and the use of a proper nomenclature for their numerical and positional variations renders appropriate communication among professionals and treatment planning easier, in addition to standardizing research analysis.

  5. Embryonic death and the creation of human embryonic stem cells

    OpenAIRE

    Landry, Donald W.; Zucker, Howard A.

    2004-01-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ ...

  6. Embryonic death and the creation of human embryonic stem cells.

    Science.gov (United States)

    Landry, Donald W; Zucker, Howard A

    2004-11-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

  7. A preliminary study on the teratogenesis of dexamethasone and the preventive effect of vitamin B12 on murine embryonic palatal shelf fusion in vitro

    Institute of Scientific and Technical Information of China (English)

    Sheng-jun LU; Wei HE; Bing SHI; Tian MENG; Xiao-yu LI; Yu-rong LIU

    2008-01-01

    Excessive dexamethasone (Dex) administrated into pregnant mice during critical periods of palatal development can produce a high incidence of cleft palate. Its mechanisms remain unknown. Vitamin B12 has been shown to antagonize the tera-togenic effects of Dex, which, however, remains controversial. In this study, we investigated the effects of Dex and vitamin B,2 on murine embryonic palatal shelf fusion using organ culture of murine embryonic shelves. The explanted palatal shelves on embryonic day 14 (E14) were cultured for 24,48,72 or 96 h in different concentrations of Dex and/or vitamin B12. The palatal shelves were examined histologically for the morphological alterations on the medial edge epithelium (MEE) and fusion rates among different groups. It was found that the palatal shelves were not fused at 72 h or less of culture in Dex group, while they were completely fused in the control and vitamin B12-treated groups at 72 and 96 h, respectively. The MEE still existed and proliferated. In Dex+vitamin B12 group the palatal shelves were fused at each time point in a similar rate to controls. These results may suggest that Dex causes teratogenesis of murine embryonic palatal shelves and vitamin B12 prevents the teratogenic effect of Dex on palatogenesis on murine embryos in vitro.

  8. 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....

  9. Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

    DEFF Research Database (Denmark)

    Jakobsen, Linda P; Borup, Rehannah; Vestergaard, Janni

    2009-01-01

    Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip...... and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed....... Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP...

  10. Divergent palate morphology in turtles and birds correlates with differences in proliferation and BMP2 expression during embryonic development.

    Science.gov (United States)

    Abramyan, John; Leung, Kelvin Jia-Mien; Richman, Joy Marion

    2014-02-01

    During embryonic development, amniotes typically form outgrowths from the medial sides of the maxillary prominences called palatal shelves or palatine processes. In mammals the shelves fuse in the midline and form a bony hard palate that completely separates the nasal and oral cavities. In birds and lizards, palatine processes develop but remain unfused, leaving a natural cleft. Adult turtles do not possess palatine processes and unlike other amniotes, the internal nares open into the oral cavity. Here we investigate craniofacial ontogeny in the turtle, Emydura subglobosa to determine whether vestigial palatine processes develop and subsequently regress, or whether development fails entirely. We found that the primary palate in turtles develops similarly to other amniotes, but secondary palate ontogeny diverges. Using histology, cellular dynamics and in situ hybridization we found no evidence of palatine process development at any time during ontogeny of the face in the turtle. Furthermore, detailed comparisons with chicken embryos (the model organism most closely related to turtles from a molecular phylogeny perspective), we identified differences in proliferation and gene expression patterns that correlate with the differences in palate morphology. We propose that, in turtles, palatine process outgrowth is never initiated due to a lack of mesenchymal bone morphogenetic protein 2 (BMP2) expression in the maxillary mesenchyme, which in turn fails to induce the relatively higher cellular proliferation required for medial tissue outgrowth. It is likely that these differences between turtles and birds arose after the divergence of the lineage leading to modern turtles.

  11. TCDD AND EGF AFFECT MAPK PATHWAY ACTIVATION IN MURINE EMBRYONIC PALATE

    Science.gov (United States)

    Palatal fusion occurs on GD 14-15 in the mouse, accompanied by a decrease in EGF receptor (EGFR) at the medial edge of the palatal shelves. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and maintains EGF and EGF receptor (EGFR) expression levels in the medial ed...

  12. Cortical network from human embryonic stem cells

    OpenAIRE

    2010-01-01

    Abstract The connection of embryonic stem cell technology and developmental biology provides valuable tools to decipher the mechanisms underlying human brain development and diseases, especially among neuronal populations, that are not readily available in primary cultures. It is obviously the case of neurons forming the human cerebral cortex. In the images that are presented, the neurons were generated in vitro from human embryonic stem cells via forebrain-like progenitors. Maintained in cul...

  13. Effects of phenytoin and Echinacea purpurea extract on proliferation and apoptosis of mouse embryonic palatal mesenchymal cells.

    Science.gov (United States)

    Hu, Xiao; Chen, Zhenguo; Mao, Xiaoyan; Tang, Shijie

    2011-05-01

    Cleft palate is one of the most common birth defects. Several environment factors are involved in the disorder, such as smoking, vitamin deficiency and teratogens. We investigated the teratogenic agent phenytoin and extract of the immunostimulant Echinacea purpurea in the etiology of cleft palate associated with the proliferation and apoptosis of mouse embryonic palatal mesenchymal (MEPM) cells. We measured the effects of phenytoin, E. purpurea extract, and the mixture of phenytoin and E. purpurea extract on the cell viability of MEPM cells by CCK-8 assay and on the proliferation and apoptosis of MEPM cells by BrdU labeling assay, flow cytometry, and TUNEL assay. Exposure to phenytoin for 24 h inhibited cell proliferation and increased cell apoptosis of MEPM cells, and E. purpurea extract had the reverse effect. Importantly, treatment with the mixture of phenytoin and E. purpurea extract increased the proliferation and decreased the apoptosis of MEPM cells as compared with treatment with phenytoin alone. The teratogenic effect of phenytoin on cleft palate is associated with the proliferation and apoptosis of MEPM cells, and E. purpurea extract may have a protective effect. Copyright © 2011 Wiley-Liss, Inc.

  14. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosom

  15. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  16. Custom-designed mouthpiece for HDR brachytherapy of embryonal rhabdomyosarcoma of the soft palate.

    Science.gov (United States)

    Ekwelundu, Emmanuel; Krasin, Matthew J; Farr, Jonathan B

    2014-10-01

    This paper describes the design and fabrication of the mouthpiece used for high-dose-rate (HDR) brachytherapy of a cancerous lesion in the soft palate of a pediatric patient. A custom mouth guard made with Thermo-forming material (Clear - Mouthguard) similar to those used by athletes, with a bite section, alveolar sulcus, hard and soft palate sections was made. Markers were placed around the lesion using a color transfer applicator and the impression transferred to the mouthpiece. Ten catheters arranged in a plane were placed on the inferior side (concave part) of the mouthpiece, and held in place by stitching each to the mouthpiece. Two pieces of lead (Pb) sheets with total thickness of 5.7 mm were placed beneath the catheters. Wax was used to create additional distance between the tongue and the catheters, and the entire assembly was covered with wax.

  17. Physiopathology of human embryonic implantation: clinical incidences.

    Directory of Open Access Journals (Sweden)

    Pauline Demailly

    2010-01-01

    Full Text Available Embryo implantation consists of a series of events promoting the invasion of the endometrium and then the uterine arterial system by the extra-embryonic trophoblast. In order for this semi-heterologous implantation to succeed, the endometrium has to first undergo a number of structural and biochemical changes (decidualization. The decidua's various constituents subsequently play a role in the embryonic implantation. The third step is the transformation of the uterine vascular system and the growth of the placenta, which will provide the foetoplacental unit with nutrients. Several physiopathological aspects will be discussed: 1 the implantation window, regulated by maternal and embryonic hormonal secretions and thus influenced by any defects in the latter: dysharmonic luteal phase, 21-hydroxylase block, abnormal integrin expression, 2 the successive trophoblast invasions of uterine vessels which, when defective, lead to early embryo loss or late-onset vascular pathologies, as preeclampsia, 3 the pregnancy's immunological equilibrium, with a spontaneously tolerated semi-allogeneic implant, 4 the impact of pro-coagulant factors (thrombophilia on the pregnancy's progression, 5 the environment of the uterus, ranging from hydrosalpinx to uterine contractions. In summary, the least anatomical or physiological perturbation can interfere with human embryonic implantation - a very particular phenomenon and a true biological paradox.

  18. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  19. Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

    DEFF Research Database (Denmark)

    Wright, A; Andrews, N; Bardsley, K

    2011-01-01

    The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...

  20. Human embryonic stem cells and patent protection

    Directory of Open Access Journals (Sweden)

    Radovanović Sanja M.

    2015-01-01

    Full Text Available Given the importance of biotechnological research in modern diagnostics and therapeutics, on the one hand, and stimulative function of a patent, on the other hand, this work deals with the question of the possibility of pa-tent protection of human embryonic stem cells. Taking into account that this is a biotechnological invention, the key question that this paper highlights is the interpretation of the provisions of their patentability. Namely, thanks to the advanced methods of isolation, purification and preparation for implementation, modern patent systems do not exclude a priori living organisms from patent protection. Therefore, the analysis of representative administrative decisions or court rulings sought to define the criteria that would be applied in order to give patent protection to a certain biotechnological invention (stem cells while others do not.

  1. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  2. Human embryonic stem cells for neuronal repair.

    Science.gov (United States)

    Ben-Hur, Tamir

    2006-02-01

    Human embryonic stem cells may serve as a potentially endeless source of transplantable cells to treat various neurologic disorders. Accumulating data have shown the therapeutic value of various neural precursor cell types in experimental models of neurologic diseases. Tailoring cell therapy for specific disorders requires the generation of cells that are committed to specific neural lineages. To this end, protocols were recently developed for the derivation of dopaminergic neurons, spinal motor neurons and oligodendrocytes from hESC. These protocols recapitulate normal development in culture conditions. However, a novel concept emerging from these studies is that the beneficial effect of transplanted stem cells is not only via cell replacement in damaged host tissue, but also by trophic and protective effects, as well as by an immunomodulatory effect that down-regulates detrimental brain inflammation.

  3. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  4. Differential expression of Bmp2, Bmp4 and Bmp3 in embryonic development of mouse anterior and posterior palate

    Institute of Scientific and Technical Information of China (English)

    NIE Xu-guang

    2005-01-01

    Background The palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage. Methods To detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18. Results The expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively. Conclusions Bmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate.

  5. Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

    Science.gov (United States)

    Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

  6. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  7. Genetic Manipulation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.

  8. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  9. Spontaneous cyclic embryonic movements in humans and guinea pigs

    NARCIS (Netherlands)

    Felt, Renee H. M.; Mulder, Eduard J. H.; Luchinger, Annemarie B.; van Kan, Colette M.; Taverne, Marcel A. M.; de Vries, J. I. P.

    2012-01-01

    Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cycl

  10. Human embryonic stem cell research: ethical and legal issues.

    Science.gov (United States)

    Robertson, J A

    2001-01-01

    The use of human embryonic stem cells to replace damaged cells and tissues promises future hope for the treatment of many diseases. However, many countries now face complex ethical and legal questions as a result of the research needed to develop these cell-replacement therapies. The challenge that must be met is how to permit research on human embryonic tissue to occur while maintaining respect for human life generally.

  11. Biological impact of human embryonic stem cells.

    Science.gov (United States)

    Martín, Miguel; Menéndez, Pablo

    2012-01-01

    Research on human embryonic stem cells (hESCs) and induced pluripotent (iPS) stem cells is currently a field of great potential in biomedicine. These cells represent a highly valuable tool for developmental biology studies, disease models, and drug screening and toxicity. The ultimate goal of hESCs and iPS cell research is the treatment of diseases or disorders for which there is currently no treatment or existing therapies are only partially effective. Despite the disproportionate short-term hopes generated, which are putting too much pressure on scientists, the international scientific community is making rapid progress in understanding hESCs and iPS cells. Nonetheless, great efforts have to be made to provide an answer to still quite basic questions concerning their biology. Moreover, translation to clinical applications in cell replacement therapy requires prior solution to ethical barriers. The recent development of iPS cells has provided a strong alternative to overcome ethical issues concerning hESCs. However, an in-depth characterization of their genetic and epigenetic features, as well as their differentiation potential still remains to be undertaken. This chapter will describe, precisely, what the critical issues are, where scientific and ethical barriers stand, and how we are to overcome them. Only then, we shall finally discover whether hESCs and iPS cells will allow building reproducible disease models, and whether they really are a safe tool, with great potential for regenerative medicine.

  12. Therapeutic potentials of human embryonic stem cells in Parkinson's disease

    National Research Council Canada - National Science Library

    Newman, Mary B; Bakay, Roy A E

    2008-01-01

    .... The isolation, differentiation, and long-term cultivation of human embryonic stem cells and the therapeutic research discovery made in relation to the beneficial properties of neurotrophic and neural...

  13. Derivation of multipotent mesenchymal precursors from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available BACKGROUND: Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. METHODS AND FINDINGS: Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. CONCLUSION: Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.

  14. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

    Full Text Available The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational

  15. Nucleosome Organization in Human Embryonic Stem Cells.

    Science.gov (United States)

    Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H

    2015-01-01

    The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome

  16. 3D evaluation of palatal rugae for human identification using digital study models

    Directory of Open Access Journals (Sweden)

    Emilia D Taneva

    2015-01-01

    Full Text Available Background: While there is literature suggesting that the palatal rugae could be used for human identification, most of these studies use two-dimensional (2D approach. Aim: The aims of this study were to evaluate palatal ruga patterns using three-dimensional (3D digital models; compare the most clinically relevant digital model conversion techniques for identification of the palatal rugae; develop a protocol for overlay registration; determine changes in palatal ruga individual patterns through time; and investigate the efficiency and accuracy of 3D matching processes between different individuals′ patterns. Material and Methods: Five cross sections in the anteroposterior dimension and four cross sections in the transverse dimension were computed which generated 18 2D variables. In addition, 13 3D variables were defined: The posterior point of incisive papilla (IP, and the most medial and lateral end points of the palatal rugae (R1MR, R1ML, R1LR, R1LL, R2MR, R2ML, R2LR, R2LL, R3MR, R3ML, R3LR, and R3LL. The deviation magnitude for each variable was statistically analyzed in this study. Five different data sets with the same 31 landmarks were evaluated in this study. Results: The results demonstrated that 2D images and linear measurements in the anteroposterior and transverse dimensions were not sufficient for comparing different digital model conversion techniques using the palatal rugae. 3D digital models proved to be a highly effective tool in evaluating different palatal ruga patterns. The 3D landmarks showed no statistically significant mean differences over time or as a result of orthodontic treatment. No statistically significant mean differences were found between different digital model conversion techniques, that is, between OrthoCAD™ and Ortho Insight 3D™, and between Ortho Insight 3D™ and the iTero; scans, when using 12 3D palatal rugae landmarks for comparison. Conclusion: Although 12 palatal 3D landmarks could be used for

  17. 3D evaluation of palatal rugae for human identification using digital study models

    Science.gov (United States)

    Taneva, Emilia D.; Johnson, Andrew; Viana, Grace; Evans, Carla A.

    2015-01-01

    Background: While there is literature suggesting that the palatal rugae could be used for human identification, most of these studies use two-dimensional (2D) approach. Aim: The aims of this study were to evaluate palatal ruga patterns using three-dimensional (3D) digital models; compare the most clinically relevant digital model conversion techniques for identification of the palatal rugae; develop a protocol for overlay registration; determine changes in palatal ruga individual patterns through time; and investigate the efficiency and accuracy of 3D matching processes between different individuals’ patterns. Material and Methods: Five cross sections in the anteroposterior dimension and four cross sections in the transverse dimension were computed which generated 18 2D variables. In addition, 13 3D variables were defined: The posterior point of incisive papilla (IP), and the most medial and lateral end points of the palatal rugae (R1MR, R1ML, R1LR, R1LL, R2MR, R2ML, R2LR, R2LL, R3MR, R3ML, R3LR, and R3LL). The deviation magnitude for each variable was statistically analyzed in this study. Five different data sets with the same 31 landmarks were evaluated in this study. Results: The results demonstrated that 2D images and linear measurements in the anteroposterior and transverse dimensions were not sufficient for comparing different digital model conversion techniques using the palatal rugae. 3D digital models proved to be a highly effective tool in evaluating different palatal ruga patterns. The 3D landmarks showed no statistically significant mean differences over time or as a result of orthodontic treatment. No statistically significant mean differences were found between different digital model conversion techniques, that is, between OrthoCAD™ and Ortho Insight 3D™, and between Ortho Insight 3D™ and the iTero® scans, when using 12 3D palatal rugae landmarks for comparison. Conclusion: Although 12 palatal 3D landmarks could be used for human

  18. 3D evaluation of palatal rugae for human identification using digital study models.

    Science.gov (United States)

    Taneva, Emilia D; Johnson, Andrew; Viana, Grace; Evans, Carla A

    2015-01-01

    While there is literature suggesting that the palatal rugae could be used for human identification, most of these studies use two-dimensional (2D) approach. The aims of this study were to evaluate palatal ruga patterns using three-dimensional (3D) digital models; compare the most clinically relevant digital model conversion techniques for identification of the palatal rugae; develop a protocol for overlay registration; determine changes in palatal ruga individual patterns through time; and investigate the efficiency and accuracy of 3D matching processes between different individuals' patterns. Five cross sections in the anteroposterior dimension and four cross sections in the transverse dimension were computed which generated 18 2D variables. In addition, 13 3D variables were defined: The posterior point of incisive papilla (IP), and the most medial and lateral end points of the palatal rugae (R1MR, R1ML, R1LR, R1LL, R2MR, R2ML, R2LR, R2LL, R3MR, R3ML, R3LR, and R3LL). The deviation magnitude for each variable was statistically analyzed in this study. Five different data sets with the same 31 landmarks were evaluated in this study. The results demonstrated that 2D images and linear measurements in the anteroposterior and transverse dimensions were not sufficient for comparing different digital model conversion techniques using the palatal rugae. 3D digital models proved to be a highly effective tool in evaluating different palatal ruga patterns. The 3D landmarks showed no statistically significant mean differences over time or as a result of orthodontic treatment. No statistically significant mean differences were found between different digital model conversion techniques, that is, between OrthoCAD™ and Ortho Insight 3D™, and between Ortho Insight 3D™ and the iTero(®) scans, when using 12 3D palatal rugae landmarks for comparison. Although 12 palatal 3D landmarks could be used for human identification, certain landmarks were especially important in the

  19. In vitro screening assay for teratogens using growth inhibition of human embryonic cells.

    Science.gov (United States)

    Pratt, R M; Willis, W D

    1985-01-01

    We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo. Images PMID:3862095

  20. In vitro screening assay for teratogens using growth inhibition of human embryonic cells

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, R.M.; Willis, W.D.

    1985-09-01

    The authors have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. The authors suggest that this assay, along with the complementary tumor cell-attachment assay of Braun may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

  1. Tbx1 regulates oral epithelial adhesion and palatal development

    Science.gov (United States)

    Funato, Noriko; Nakamura, Masataka; Richardson, James A.; Srivastava, Deepak; Yanagisawa, Hiromi

    2012-01-01

    Cleft palate, the most frequent congenital craniofacial birth defect, is a multifactorial condition induced by the interaction of genetic and environmental factors. In addition to complete cleft palate, a large number of human cases involve soft palate cleft and submucosal cleft palate. However, the etiology of these forms of cleft palate has not been well understood. T-box transcriptional factor (Tbx) family of transcriptional factors has distinct roles in a wide range of embryonic differentiation or response pathways. Here, we show that genetic disruption of Tbx1, a major candidate gene for the human congenital disorder 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome), led to abnormal epithelial adhesion between the palate and mandible in mouse, resulting in various forms of cleft palate similar to human conditions. We found that hyperproliferative epithelium failed to undergo complete differentiation in Tbx1-null mice (Tbx1−/−). Inactivation of Tbx1 specifically in the keratinocyte lineage (Tbx1KCKO) resulted in an incomplete cleft palate confined to the anterior region of the palate. Interestingly, Tbx1 overexpression resulted in decreased cell growth and promoted cell-cycle arrest in MCF7 epithelial cells. These findings suggest that Tbx1 regulates the balance between proliferation and differentiation of keratinocytes and is essential for palatal fusion and oral mucosal differentiation. The impaired adhesion separation of the oral epithelium together with compromised palatal mesenchymal growth is an underlying cause for various forms of cleft palate phenotypes in Tbx1−/− mice. Our present study reveals new pathogenesis of incomplete and submucous cleft palate during mammalian palatogenesis. PMID:22371266

  2. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    OpenAIRE

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF...

  3. Passive movement of human soft palate during respiration: A simulation of 3D fluid/structure interaction.

    Science.gov (United States)

    Zhu, Jian Hua; Lee, Heow Pueh; Lim, Kian Meng; Lee, Shu Jin; Teo, Li San Lynette; Wang, De Yun

    2012-07-26

    This study reconstructed a three dimensional fluid/structure interaction (FSI) model to investigate the compliance of human soft palate during calm respiration. Magnetic resonance imaging scans of a healthy male subject were obtained for model reconstruction of the upper airway and the soft palate. The fluid domain consists of nasal cavity, nasopharynx and oropharynx. The airflow in upper airway was assumed as laminar and incompressible. The soft palate was assumed as linear elastic. The interface between airway and soft palate was the FSI interface. Sinusoidal variation of velocity magnitude was applied at the oropharynx corresponding to ventilation rate of 7.5L/min. Simulations of fluid model in upper airway, FSI models with palatal Young's modulus of 7539Pa and 3000Pa were carried out for two cycles of respiration. The results showed that the integrated shear forces over the FSI interface were much smaller than integrated pressure forces in all the three directions (axial, coronal and sagittal). The total integrated force in sagittal direction was much smaller than that of coronal and axial directions. The soft palate was almost static during inspiration but moved towards the posterior pharyngeal wall during expiration. In conclusion, the displacement of human soft palate during respiration was mainly driven by air pressure around the surface of the soft palate with minimal contribution of shear stress of the upper airway flow. Despite inspirational negative pressure, expiratory posterior movement of soft palate could be another factor for the induction of airway collapse.

  4. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  5. 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.

  6. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  7. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  8. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  9. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...

  10. Differentiation of neuroepithelia from human embryonic stem cells

    OpenAIRE

    2009-01-01

    We describe the method for in vitro differentiation of neuroepithelial cells from human embryonic stem cells under a chemically defined condition. The protocol is established following the fundamental principle of in vivo neuroectodermal specification. The primitive neuroepithelial cells generated by this protocol can be further induced into neuronal and glia cells with forebrain, mid/hind brain, and spinal cord identities.

  11. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  12. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during different

  13. Improved genetic manipulation of human embryonic stem cells.

    NARCIS (Netherlands)

    Braam, S.R.; Denning, C.; van den Brink, S.; Kats, P.; Hochstenbach, R.; Passier, R.; Mummery, C.L.

    2008-01-01

    Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth requ

  14. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  15. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    Science.gov (United States)

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  16. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  17. Derivation, propagation and differentiation of human embryonic stem cells.

    Science.gov (United States)

    Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

    2004-04-01

    Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

  18. Derivation of naive human embryonic stem cells.

    Science.gov (United States)

    Ware, Carol B; Nelson, Angelique M; Mecham, Brigham; Hesson, Jennifer; Zhou, Wenyu; Jonlin, Erica C; Jimenez-Caliani, Antonio J; Deng, Xinxian; Cavanaugh, Christopher; Cook, Savannah; Tesar, Paul J; Okada, Jeffrey; Margaretha, Lilyana; Sperber, Henrik; Choi, Michael; Blau, C Anthony; Treuting, Piper M; Hawkins, R David; Cirulli, Vincenzo; Ruohola-Baker, Hannele

    2014-03-25

    The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

  19. Embryonic Human Life and Dignity: The French Connection

    Directory of Open Access Journals (Sweden)

    Dimitrios Tsarapatsanis

    2015-12-01

    Full Text Available Human dignity is considered by a number of commentators as a normative concept that could potentially bridge the gap between bioethics and human rights. The purpose of this article is to question this assumption insofar as it applies to embryonic human life by way of a case study. The article will chart the way dignity has been historically used in French political and legal debates since the 1990s to attempt to afford constitutional protection to human embryos. It then proposes an interpretation of why such attempts failed, which could have wider significance for current debates.

  20. Spontaneous cyclic embryonic movements in humans and guinea pigs.

    Science.gov (United States)

    Felt, Renée H M; Mulder, Eduard J H; Lüchinger, Annemarie B; van Kan, Colette M; Taverne, Marcel A M; de Vries, Johanna I P

    2012-08-01

    Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cyclic. This study explores the distribution of SB-to-SB intervals in human and guinea pig embryos before the appearance of more complex movements such as general movements. We hypothesized that the activity in both species is cyclic. We made 15-min sonographic recordings of SBs between 5 weeks and 0 days (5wk0d) and 7wk0d conceptional age (CA) in 18 human embryos of uncomplicated IVF pregnancies (term 38 weeks) and in 20 guinea pig embryos between 3wk4d and 4wk0d CA (term 9 weeks). SB-to-SB interval durations were categorized as long (≥10 s) or short (guinea pigs 38 s (range, 10-288 s) and 5 s (range, 1-9 s), respectively. During development, the duration of long intervals decreased while the number of short intervals increased for both species. The earliest embryonic motility in the human and guinea pig is performed cyclically with distinct developmental milestones. The resemblance of their interval development offers promising possibilities to use the guinea pig as a noninvasive animal model of external influences on motor and neural development.

  1. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  2. Cancer genes hypermethylated in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Vincenzo Calvanese

    Full Text Available Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

  3. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    Science.gov (United States)

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  4. Indirect modulation of Shh signaling by Dlx5 affects the oral-nasal patterning of palate and rescues cleft palate in Msx1-null mice.

    Science.gov (United States)

    Han, Jun; Mayo, Julie; Xu, Xun; Li, Jingyuan; Bringas, Pablo; Maas, Richard L; Rubenstein, John L R; Chai, Yang

    2009-12-01

    Cleft palate represents one of the most common congenital birth defects in human. During embryonic development, palatal shelves display oronasal (O-N) and anteroposterior polarity before the onset of fusion, but how the O-N pattern is established and how it relates to the expansion and fusion of the palatal shelves are unknown. Here we address these questions and show that O-N patterning is associated with the expansion and fusion of the palatal shelves and that Dlx5 is required for the O-N patterning of palatal mesenchyme. Loss of Dlx5 results in downregulation of Fgf7 and expanded Shh expression from the oral to the nasal side of the palatal shelf. This expanded Shh signaling is sufficient to restore palatal expansion and fusion in mice with compromised palatal mesenchymal cell proliferation, such as Msx1-null mutants. Exogenous Fgf7 inhibits Shh signaling and reverses the cranial neural crest (CNC) cell proliferation rescue in the Msx1/Dlx5 double knockout palatal mesenchyme. Thus, Dlx5-regulated Fgf7 signaling inhibits the expression of Shh, which in turn controls the fate of CNC cells through tissue-tissue interaction and plays a crucial role during palatogenesis. Our study shows that modulation of Shh signaling may be useful as a potential therapeutic approach for rescuing cleft palate.

  5. Non-invasive Imaging of Human Embryonic Stem Cells

    OpenAIRE

    Hong, Hao; Yang, Yunan; Zhang, Yin; Cai, Weibo

    2010-01-01

    Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography...

  6. Human embryonic growth trajectories and associations with fetal growth and birthweight

    NARCIS (Netherlands)

    van Uitert, Evelyne M.; Exalto, Niek; Burton, Graham J.; Willemsen, Sten P.; Koning, Anton H. J.; Eilers, Paul H. C.; Laven, Joop S. E.; Steegers, Eric A. P.; Steegers-Theunissen, Regine P. M.

    2013-01-01

    How do human embryonic growth trajectories evolve in the first trimester, and is first-trimester embryonic growth associated with fetal growth and birthweight (BW)? Human embryonic growth rates increase between 9 and 10 weeks of gestation and are associated with mid-pregnancy fetal growth and BW. Fe

  7. Derivation and differentiation of haploid human embryonic stem cells.

    Science.gov (United States)

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-07

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

  8. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  9. Evaluation of palatal rugoscopy in dentulous and edentulous cases for human identification in forensic dentistry

    Directory of Open Access Journals (Sweden)

    Rogério José Scandiuzzi

    2014-03-01

    The search for identity is based on a set of characteristics, which defines the uniqueness of a person. Principles such as classificability, immutability, persistence, practicability and uniqueness must be considered when applying an identification technique. This study aimed to evaluate the use of palatal rugoscopy in dentulous and edentulous volunteers, with or without upper removable denture, for purposes of human identification. In this study 60 subjects were asked to give dental casts and photography of the upper dental arch, defined in the following groups: Group A (n = 30, edentulous patients with full upper removable dentures and Group B (n = 30, dentulous without upper removable partial denture. The rugoscopy analysis method used was Martins-dos-Santos classification, for checking the applicability and success in human identification. It was found that it is possible to use this technique and it has an application of 40% in the group A and 86.66% in the group B. In conclusion, the identification method by palatal rugoscopy is satisfactory for dentulous patients, however in cases of tooth loss and friction cases generated by prosthetic devices, the region of the palate lose its characteristics, but even then it is still possible to be applied.

  10. A quantitative method for defining high-arched palate using the Tcof1(+/-) mutant mouse as a model.

    Science.gov (United States)

    Conley, Zachary R; Hague, Molly; Kurosaka, Hiroshi; Dixon, Jill; Dixon, Michael J; Trainor, Paul A

    2016-07-15

    The palate functions as the roof of the mouth in mammals, separating the oral and nasal cavities. Its complex embryonic development and assembly poses unique susceptibilities to intrinsic and extrinsic disruptions. Such disruptions may cause failure of the developing palatal shelves to fuse along the midline resulting in a cleft. In other cases the palate may fuse at an arch, resulting in a vaulted oral cavity, termed high-arched palate. There are many models available for studying the pathogenesis of cleft palate but a relative paucity for high-arched palate. One condition exhibiting either cleft palate or high-arched palate is Treacher Collins syndrome, a congenital disorder characterized by numerous craniofacial anomalies. We quantitatively analyzed palatal perturbations in the Tcof1(+/-) mouse model of Treacher Collins syndrome, which phenocopies the condition in humans. We discovered that 46% of Tcof1(+/-) mutant embryos and new born pups exhibit either soft clefts or full clefts. In addition, 17% of Tcof1(+/-) mutants were found to exhibit high-arched palate, defined as two sigma above the corresponding wild-type population mean for height and angular based arch measurements. Furthermore, palatal shelf length and shelf width were decreased in all Tcof1(+/-) mutant embryos and pups compared to controls. Interestingly, these phenotypes were subsequently ameliorated through genetic inhibition of p53. The results of our study therefore provide a simple, reproducible and quantitative method for investigating models of high-arched palate.

  11. Epigenetic Regulation of Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Qidong eHu

    2012-11-01

    Full Text Available Recently, there has been tremendous progress in characterizing the transcriptional network regulating hESCs (MacArthur et al., 2009; Loh et al., 2011, including those signaling events mediated by Oct4, Nanog and Sox2. There is growing interest in the epigenetic machinery involved in hESC self-renewal and differentiation. In general, epigenetic regulation includeschromatin reorganization, DNA modification and histone modification, which are not directly related to alterations in DNA sequences. Various protein complexes, includingPolycomb, trithorax, NuRD, SWI/SNF andOct4, have been shown to play critical roles in epigenetic control of hESC maintenance and differentiation. Hence, we will formally review recent advances in unraveling the multifaceted role of epigenetic regulation in hESC self-renewal and induced differentiation, particularly with respect to chromatin remodeling and DNA methylation events. Unraveling the molecular mechanisms underlying the maintenance/differentiation of hESCs and reprogramming of somatic cells will greatly strengthen our capacity to generate various types of cells to treat human diseases.

  12. Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes.

    Science.gov (United States)

    Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

    2013-01-15

    Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.

  13. PDGF mediates derivation of human embryonic germ cells.

    Science.gov (United States)

    Li, Yang; Hong, Wan Xing; Lan, Baojin; Xu, Xiaoyan; Liu, Yinan; Kong, Lin; Li, Yaxuan; Zhou, Shixin; Liu, Ying; Feng, Ruopeng; Jiang, Sibo; He, Qihua; Tan, Jichun

    2013-01-01

    Human embryonic germ cells (hEGCs) are a valuable and underutilized source of pluripotent stem cells. Unlike embryonic stem cells, which have been extensively studied, little is known about the factors that regulate hEGC derivation and maintenance. This study demonstrates for the first time a central role for selective activation of PDGFR signaling in the derivation and maintenance of pluripotency in hEGCs. In the study, hEGCs were found to express PDGF receptor α at high levels compared to human embryonic stem cells (hESCs). PDGF significantly improved formation of alkaline phosphatase (AP) positive hEGC colonies. We subsequently determined that PDGF activates the phosphatidylinositol-3-kinase (PI3K) pathway as phosphorylation of AKT was up-regulated in response to PDGF. Furthermore, inhibition of PI3K signaling using small molecular inhibitor LY294002 led to significantly decreased AP positive hEGC colony formation whereas inhibition of MAPK pathway using U0126 had a negligible effect. We established a primary mechanism for PDGF mediated derivation and maintenance of hEGCs by demonstrating that OCT4 was upregulated and PTEN was suppressed in a dose dependent manner in response to PDGF.

  14. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    Science.gov (United States)

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well.

  15. Human embryonic stem cells carrying mutations for severe genetic disorders.

    Science.gov (United States)

    Frumkin, Tsvia; Malcov, Mira; Telias, Michael; Gold, Veronica; Schwartz, Tamar; Azem, Foad; Amit, Ami; Yaron, Yuval; Ben-Yosef, Dalit

    2010-04-01

    Human embryonic stem cells (HESCs) carrying specific mutations potentially provide a valuable tool for studying genetic disorders in humans. One preferable approach for obtaining these cell lines is by deriving them from affected preimplantation genetically diagnosed embryos. These unique cells are especially important for modeling human genetic disorders for which there are no adequate research models. They can be further used to gain new insights into developmentally regulated events that occur during human embryo development and that are responsible for the manifestation of genetically inherited disorders. They also have great value for the exploration of new therapeutic protocols, including gene-therapy-based treatments and disease-oriented drug screening and discovery. Here, we report the establishment of 15 different mutant human embryonic stem cell lines derived from genetically affected embryos, all donated by couples undergoing preimplantation genetic diagnosis in our in vitro fertilization unit. For further information regarding access to HESC lines from our repository, for research purposes, please email dalitb@tasmc.health.gov.il.

  16. Human embryonic epidermis contains a diverse Langerhans cell precursor pool.

    Science.gov (United States)

    Schuster, Christopher; Mildner, Michael; Mairhofer, Mario; Bauer, Wolfgang; Fiala, Christian; Prior, Marion; Eppel, Wolfgang; Kolbus, Andrea; Tschachler, Erwin; Stingl, Georg; Elbe-Bürger, Adelheid

    2014-02-01

    Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207(-) cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14(+)CD16(-) blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14(-) haematopoietic precursors possess a much higher differentiation potential than CD14(+) precursor cells.

  17. Connexin mutant embryonic stem cells and human diseases

    Institute of Scientific and Technical Information of China (English)

    Kiyomasa; Nishii; Yosaburo; Shibata; Yasushi; Kobayashi

    2014-01-01

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  18. Connexin mutant embryonic stem cells and human diseases.

    Science.gov (United States)

    Nishii, Kiyomasa; Shibata, Yosaburo; Kobayashi, Yasushi

    2014-11-26

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  19. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  20. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.;

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  1. Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

    Science.gov (United States)

    Banda, Erin; Grabel, Laura

    2016-01-01

    A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

  2. Human embryonic stem cell research debates: a confucian argument.

    Science.gov (United States)

    Tsai, D F-C

    2005-11-01

    Human embryonic stem cell research can bring about major biomedical breakthroughs and thus contribute enormously to human welfare, yet it raises serious moral problems because it involves using human embryos for experiment. The "moral status of the human embryo" remains the core of such debates. Three different positions regarding the moral status of the human embryo can be categorised: the "all" position, the "none" position, and the "gradualist" position. The author proposes that the "gradualist" position is more plausible than the other two positions. Confucius's moral principle of jen, which proposes a unique theory of "love of gradation", and the principle of yi, which advocates "due treatment for persons", are then explored. The author then argues that our moral obligations to do good to other living organisms, persons, and our families are different. Putting together the "gradualist" position on the human embryo, and Confucius's theories of "love of gradation" and "due treatment for persons", the author concludes that the early embryo has less ethical significance than the later fetus and adult human. The moral obligation we have toward persons is clearer and stronger than that which we have toward human embryos. Embryo research is justifiable if it brings enormous welfare to human persons that cannot be otherwise achieved. The "love of gradation" requires us, however, to extend love and respect towards other entities according to their different status. We should therefore be very cautious in using human embryos for research, acknowledging the gradualist nature of their moral status.

  3. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

  4. Enhancement of cardiomyocyte differentiation from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2’-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2’-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.

  5. Numerical simulation of soft palate movement and airflow in human upper airway by fluid-structure interaction method

    Science.gov (United States)

    Sun, Xiuzhen; Yu, Chi; Wang, Yuefang; Liu, Yingxi

    2007-08-01

    In this paper, the authors present airflow field characteristics of human upper airway and soft palate movement attitude during breathing. On the basis of the data taken from the spiral computerized tomography images of a healthy person and a patient with Obstructive Sleep Apnea-Hypopnea Syndrome (OSAHS), three-dimensional models of upper airway cavity and soft palate are reconstructed by the method of surface rendering. Numerical simulation is performed for airflow in the upper airway and displacement of soft palate by fluid-structure interaction analysis. The reconstructed three-dimensional models precisely preserve the original configuration of upper airways and soft palate. The results of the pressure and velocity distributions in the airflow field are quantitatively determined, and the displacement of soft palate is presented. Pressure gradients of airway are lower for the healthy person and the airflow distribution is quite uniform in the case of free breathing. However, the OSAHS patient remarkably escalates both the pressure and velocity in the upper airway, and causes higher displacement of the soft palate. The present study is useful in revealing pathogenesis and quantitative mutual relationship between configuration and function of the upper airway as well as in diagnosing diseases related to anatomical structure and function of the upper airway.

  6. Numerical simulation of soft palate movement and airflow in human upper airway by fluid-structure interaction method

    Institute of Scientific and Technical Information of China (English)

    Xiuzhen Sun; Chi Yu; Yuefang Wang; Yingxi Liu

    2007-01-01

    In this paper, the authors present airflow field characteristics of human upper airway and soft palate move-ment attitude during breathing. On the basis of the data taken from the spiral computerized tomography images of a healthy person and a patient with Obstructive Sleep Apnea-Hypopnea Syndrome (OSAHS), three-dimensional models of upper air-way cavity and soft palate are reconstructed by the method of surface rendering. Numerical simulation is performed for airflow in the upper airway and displacement of soft palate by fluid-structure interaction analysis. The reconstructed three-dimensional models precisely preserve the original config-uration of upper airways and soft palate. The results of the pressure and velocity distributions in the airflow field are quantitatively determined, and the displacement of soft palate is presented. Pressure gradients of airway are lower for the healthy person and the airflow distribution is quite uniform in the case of free breathing. However, the OSAHS patient remarkably escalates both the pressure and velocity in the upper airway, and causes higher displacement of the soft palate. The present study is useful in revealing pathogenesis and quantitative mutual relationship between configuration and function of the upper airway as well as in diagnosing diseases related to anatomical structure and function of the upper airway.

  7. Enabling research with human embryonic and fetal tissue resources

    Science.gov (United States)

    Gerrelli, Dianne; Lisgo, Steven; Copp, Andrew J.; Lindsay, Susan

    2015-01-01

    Summary Congenital anomalies are a significant burden on human health. Understanding the developmental origins of such anomalies is key to developing potential therapies. The Human Developmental Biology Resource (HDBR), based in London and Newcastle UK, was established to provide embryonic and fetal material for a variety of human studies ranging from single gene expression analysis to large scale genomic/transcriptomic studies. Increasingly HDBR material is enabling the derivation of stem cell lines and contributing towards developments in tissue engineering. Use of the HDBR and other fetal tissue resources discussed here will contribute to the long term aims of understanding the causation and pathogenesis of congenital anomalies, and developing new methods for their treatment and prevention. PMID:26395135

  8. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  9. Differences in Oral Structure and Tissue Interactions during Mouse vs. Human Palatogenesis: Implications for the Translation of Findings from Mice

    Science.gov (United States)

    Yu, Kai; Deng, Mei; Naluai-Cecchini, Theresa; Glass, Ian A.; Cox, Timothy C.

    2017-01-01

    Clefting of the secondary palate is one of the most common human birth defects and results from failure of the palatal shelves to fuse during embryonic development. Palatogenesis is traditionally considered to be a highly conserved developmental process among mammalian species. However, cleft palate phenotypes in humans are considerably more variable than those seen in mice, the most common animal model for studying palatal development and pathogenesis of cleft palate. In this investigation, we utilized macroscopic observations, histology and 3D imaging techniques to directly compare palate morphology and the oral-nasal cavity during palate closure in mouse embryos and human conceptuses. We showed that mouse and human palates display distinct morphologies attributable to the structural differences of the oral-nasal cavity. We further showed that the palatal shelves interact differently with the primary palate and nasal septum in the hard palate region and with pharyngeal walls in the soft palate region during palate closure in mice and humans. Knowledge of these morphological differences is important for improved translation of findings in mouse models of human cleft lip/palate and, as such, should ultimately enhance our understanding of human palatal morphogenesis and the pathogenesis of cleft lip/palate in humans. PMID:28360863

  10. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  11. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    Science.gov (United States)

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  12. [Development of human embryonic stem cell model for toxicity evaluation].

    Science.gov (United States)

    Yu, Guang-yan; Cao, Tong; Ouyang, Hong-wei; Peng, Shuang-qing; Deng, Xu-liang; Li, Sheng-lin; Liu, He; Zou, Xiao-hui; Fu, Xin; Peng, Hui; Wang, Xiao-ying; Zhan, Yuan

    2013-02-18

    The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.

  13. Ethical issues of the research on human embryonic stem cells.

    Science.gov (United States)

    Hotta, Yoshitaro

    2008-09-01

    This paper examines the debate on human embryonic stem cell (hES) research. In Japan, as in many Western countries, the moral status of the human embryo has been the main focus. There is a dichotomy between the advocates of research on hES cells and the advocates of the dignity of the embryos. Advocates of research on hES cells claim that the act of abandoning the embryo and the act of using it for research have the same moral status. Critics of research using embryos reject this position, claiming that the embryo has a status incommensurable with other values. This paper points out that the standpoint of the woman providing the embryos has been overlooked in this discussion.

  14. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Science.gov (United States)

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta

    2010-09-01

    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

  15. Human embryonic stem cell derivation and directed differentiation.

    Science.gov (United States)

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  16. Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

    Science.gov (United States)

    Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser

    2016-01-01

    Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

  17. A quantitative electron microscopic analysis of the keratinizing epithelium of noral human hard palate.

    Science.gov (United States)

    Meyer, M; Schroeder, H E

    1975-01-01

    The epithelium of normal human hard palate was subjected to sterologic analysis. Ten biosies were selected from a total of twenty specimens collected from 9 to 16 year old females, and processed for light- and electron microscopy. At two levels of magnification, electron micrographs were sampled from three strata (basale, spinosum, granulosum) in two locations (epithelial ridges and portions over connective tissue papillae). Stereologic point counting procedures were employed to analyse a total 1560 electron micrographs. In general, the thickness of the palate epithelium was 0.12 mm (over papillae) and 0.31 mm (in ridges), the epithelium is distinctly stratified, and homogeneously ortho-keratinized. From basal to granular layers, the composition of strata revealed decreasing densities of nuclei, mitochondria, membrane-bound organelles and aggregates of free ribosomes. Keratohyalin bodies and membrane coating granules increased, and cytoplasmic filaments with a constant diameter of about 85 A increased from 14 to 30% of cytoplasmic unit volume. The cytoplasmic ground substance occupied a stable 50% of the epithelial cytoplasm in all strata. The composition of basal layers in ridges differed from that over connective tissue papillae. The data are discussed in relation to the observations that (1) an increasing gradient of filament density is not the most characteristic feature of ortho-keratinizing oral epithelium and (2) differences in the degree of differentiation in cells of the stratum basale coincided with the comparable frequency distribution pattern of dividing cells.

  18. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

    OpenAIRE

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4...

  19. Cryopreservation of human embryonic stem cells by vitrification

    Institute of Scientific and Technical Information of China (English)

    周灿权; 麦庆云; 李涛; 庄广伦

    2004-01-01

    Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.Methods Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.Results Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P<0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.Conclusions Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

  20. Biobanking human embryonic stem cell lines: policy, ethics and efficiency.

    Science.gov (United States)

    Holm, Søren

    2015-12-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy discussions relating to their establishment or maintenance. It is argued (1) that 'ethical arguments' are often more important in the establishment phase and 'efficiency arguments' more important in the maintenance phase, and (2) that arguments relating to the interests of embryo and gamete donors are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being ignored in the policy making process.

  1. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  2. Genetic engineering of human embryonic stem cells with lentiviral vectors.

    Science.gov (United States)

    Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

    2005-08-01

    Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

  3. Raman microscopy of individual living human embryonic stem cells

    Science.gov (United States)

    Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

    2010-04-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

  4. Random mitotic activities across human embryonic stem cell colonies.

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L. (Biosciences Division)

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  5. Measurement of human embryonic stem cell in the growing cycle

    Science.gov (United States)

    Li, X.; Zhao, L.; Oh, Steve K. W.; Chong, W. K.; Ong, J. K.; Chen, Allen K.; Choo, Andre B. H.

    2008-09-01

    A measurement and imaging system has been developed for in-line continuous measurement of live, unmodified, human embryonic stem cells (hESC). The measurement will not affect cell growth, structure, sterility and suitability for clinical use. The stem cell imaging system (SCIS) can be used to support the optimization of automated stem cell growth for invitro study and for high-volume bio-manufacture. This paper present the experimental and analysis for the optimization of system parameters. A non-linear lighting is developed to obtain a clear images. The individual cluster can be traced from day one to day two. The whole system is calibrated with measurement microscope and haemacytometer. The measurement accuracy is better than 90%.

  6. The production and directed differentiation of human embryonic stem cells.

    Science.gov (United States)

    Trounson, Alan

    2006-04-01

    Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of

  7. Establishment and characterization of two new human embryonic stem cell lines, SYSU-1 and SYSU-2

    Institute of Scientific and Technical Information of China (English)

    HUANG Guo; Andy Peng Xiang; LI Wei-qiang; CHEN Rui; CHEN Zhen-guang; ZHANG Xiu-ming; MAO Fu-xiang; HUANG Shao-liang; LI Shu-nong; Bruce T Lahn

    2007-01-01

    Background Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stem cell lines are highly warranted.Methods Surplus embryos (blastocysts) from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation.Results Two human embryonic stem cell lines (SYSU-1 and SYSU-2) were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA- 4, Tra-1-60, Oct-4, Nanog and Rex-1.They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo.Conclusion Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.

  8. Enriched retinal ganglion cells derived from human embryonic stem cells

    Science.gov (United States)

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  9. Ethical issues in neurografting of human embryonic cells.

    Science.gov (United States)

    Boer, G J

    1999-09-01

    During the last decade neurotransplantation has developed into a technique with the possible potential to repair damaged or degenerating human brain. Effective neurotransplantation has so far been based on the use of fetal brain tissue derived from aborted embryos or fetuses. The ethical issues related to this new therapeutic approach therefore not only concern the possible adverse side effects for a neural graft-receiving patient, but also the relationship between the requirements for fetal tissue and the decision-making process for induced abortion. Although for decades human embryos and fetuses have been the subject of biomedical studies, and, in principle, their use has therefore not been seen as ethically objectionable, the above points made it necessary to reconsider the moral issues. The present paper points out several of these issues, both from the donor and acceptor (patient) point of view. The conclusion is that under a series of restrictions intended to prevent the use of grafts from encouraging induced abortions and to maintain high standards of respect for life and human dignity, neurotransplantation using embryonic or fetal brain tissue parts cannot be rejected on moral grounds.

  10. Human Embryonic Stem Cells Form Functional Thyroid Follicles

    Science.gov (United States)

    Latif, Rauf; Davies, Terry F.

    2015-01-01

    Objective: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Methods: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Results: Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. Conclusion: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be

  11. The N-glycome of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Olonen Anne

    2009-06-01

    Full Text Available Abstract Background Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC, the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. Results The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. Conclusion The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

  12. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

    Science.gov (United States)

    2013-02-28

    ... Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be... within 60 days of the date of this publication. Proposed Collection: Request for Human Embryonic Stem...

  13. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

    Science.gov (United States)

    2013-04-29

    ... for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded Research SUMMARY: Under the... be requested in writing. Proposed Collection: Request for Human Embryonic Stem Cell Line to be... used by applicants to request that human embryonic stem cell lines be approved for use in NIH-funded...

  14. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG DongHui; JIANG Wei; SHI Yan; DENG HongKui

    2009-01-01

    Efficiently obtaining functional pancreaUc islet cells derived from human embryonic stem (hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology. In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro. Since then, many strategies (such as overexpression of key transcription factors,delivery of key proteins for pancreatic development, co-transplantation of differentiated hES cells along with fetal pancreas, stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells. Moreover, patient-specific induced pluripotent stem (iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection. In this review, we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  15. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Efficiently obtaining functional pancreatic islet cells derived from human embryonic stem(hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology.In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro.Since then,many strategies(such as overexpression of key transcription factors,delivery of key proteins for pancreatic development,co-transplantation of differentiated hES cells along with fetal pancreas,stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells.Moreover,patient-specific induced pluripotent stem(iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection.In this review,we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  16. Cytotoxic Effect Of Verapamil On Human Embryonic Kidney Cell Line

    Directory of Open Access Journals (Sweden)

    Jamil L Ahmad

    2015-08-01

    Full Text Available Introduction The link between long term use of verapamil and cancer development has been suggested in literature many years back. However there are numerous controversies surrounding this association with several epidemiological studies in the positive negative and non-association between verapamil and cancer development. Aim To investigate in mechanistic terms the link between chronic use of a calcium channel blocker verapamil and cancer development using human embryonic kidney HEK293 cell line. Method Trypan blue dye exclusion cell counting and 3-amp615314 5-Dimethylthiazol-2-ylamp61533-2 5-diphenyl-tetrazolium bromide MTT assays were used to determine the proliferative as well as cytotoxic effects of verapamil. Results Verapamil had a growth inhibitory rather than proliferative effect on HEK293 cells and the growth inhibition was found to be significant p0.05. Conclusion The long term use of verapamil is associated with cellular growth inhibition and this possibly explained the rationale behind its use as part of combination chemotherapy for some human cancers.

  17. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

    2012-09-01

    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  18. Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

    2017-01-01

    NSSCs in ex vivo organotypic cultures of embryonic chick femurs. Isolated embryonic chick femurs (E10 and E11) were cultured for 10 days together with micro-mass cell pellets of hNSSCs, human umbilical vein endothelial cells (HUVEC) or a combination of the two cell types. Changes in femurs gross morphology...

  19. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    Science.gov (United States)

    Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

    2015-01-01

    In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

  20. Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

    OpenAIRE

    Wang, Xiaosheng

    2011-01-01

    Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class comparison analy...

  1. Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

    OpenAIRE

    Wang Xiaosheng

    2011-01-01

    Abstract Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class compari...

  2. Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Reynolds, Paul M.; Petersen, Dorthe Roenn

    2016-01-01

    Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have...... of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.000X). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of defi nitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure...

  3. Human embryonic stem cell technologies and drug discovery.

    Science.gov (United States)

    Jensen, Janne; Hyllner, Johan; Björquist, Petter

    2009-06-01

    Development of new drugs is costly and takes huge resources into consideration. The big pharmaceutical companies are currently facing increasing developmental costs and a lower success-rate of bringing new compounds to the market. Therefore, it is now of outmost importance that the drug-hunting companies minimize late attritions due to sub-optimal pharmacokinetic properties or unexpected toxicity when entering the clinical programs. To achieve this, a strong need to test new candidate drugs in assays of high human relevance in vitro as early as possible has been identified. The traditionally used cell systems are however remarkably limited in this sense, and new improved technologies are of greatest importance. The human embryonic stem cells (hESC) is one of the most powerful cell types known. They have not only the possibility to divide indefinitely; these cells can also differentiate into all mature cell types of the human body. This makes them potentially very valuable for pharmaceutical development, spanning from use as tools in early target studies, DMPK or safety assessment, as screening models to find new chemical entities modulating adult stem cell fate, or as the direct use in cell therapies. This review illustrates the use of hESC in the drug discovery process, today, as well as in a future perspective. This will specifically be exemplified with the most important cell type for pharmaceutical development-the hepatocyte. We discuss how hESC-derived hepatocyte-like cells could improve this process, and how these cells should be cultured if optimized functionality and usefulness should be achieved. J. Cell. Physiol. 219: 513-519, 2009. (c) 2009 Wiley-Liss, Inc.

  4. Molecular basis of cleft palates in mice

    Institute of Scientific and Technical Information of China (English)

    Noriko; Funato; Masataka; Nakamura; Hiromi; Yanagisawa

    2015-01-01

    Cleft palate, including complete or incomplete cleft palates, soft palate clefts, and submucosal cleft palates, is the most frequent congenital craniofacial anomaly in humans. Multifactorial conditions, including genetic and environmental factors, induce the formation of cleft palates. The process of palatogenesis is temporospatially regulated by transcription factors, growth factors, extracellular matrix proteins, and membranous molecules; a single ablation of these molecules can result in a cleft palate in vivo. Studies on knockout mice were reviewed in order to identify genetic errors that lead to cleft palates. In this review, we systematically describe these mutant mice and discuss the molecular mechanisms of palatogenesis.

  5. Human embryonic stem cell therapies for neurodegenerative diseases.

    Science.gov (United States)

    Tomaskovic-Crook, Eva; Crook, Jeremy M

    2011-06-01

    There is a renewed enthusiasm for the clinical translation of human embryonic stem (hES) cells. This is abetted by putative clinically-compliant strategies for hES cell maintenance and directed differentiation, greater understanding of and accessibility to cells through formal cell registries and centralized cell banking for distribution, the revised US government policy on funding hES cell research, and paradoxically the discovery of induced pluripotent stem (iPS) cells. Additionally, as we consider the constraints (practical and fiscal) of delivering cell therapies for global healthcare, the more efficient and economical application of allogeneic vs autologous treatments will bolster the clinical entry of hES cell derivatives. Neurodegenerative disorders such as Parkinson's disease are primary candidates for hES cell therapy, although there are significant hurdles to be overcome. The present review considers key advances and challenges to translating hES cells into novel therapies for neurodegenerative diseases, with special consideration given to Parkinson's disease and Alzheimer's disease. Importantly, despite the focus on degenerative brain disorders and hES cells, many of the issues canvassed by this review are relevant to systemic application of hES cells and other pluripotent stem cells such as iPS cells.

  6. Generative models: Human embryonic stem cells and multiple modeling relations.

    Science.gov (United States)

    Fagan, Melinda Bonnie

    2016-04-01

    Model organisms are at once scientific models and concrete living things. It is widely assumed by philosophers of science that (1) model organisms function much like other kinds of models, and (2) that insofar as their scientific role is distinctive, it is in virtue of representing a wide range of biological species and providing a basis for generalizations about those targets. This paper uses the case of human embryonic stem cells (hESC) to challenge both assumptions. I first argue that hESC can be considered model organisms, analogous to classic examples such as Escherichia coli and Drosophila melanogaster. I then discuss four contrasts between the epistemic role of hESC in practice, and the assumptions about model organisms noted above. These contrasts motivate an alternative view of model organisms as a network of systems related constructively and developmentally to one another. I conclude by relating this result to other accounts of model organisms in recent philosophy of science. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Morphogenesis of the spleen during the human embryonic period.

    Science.gov (United States)

    Endo, Aya; Ueno, Saki; Yamada, Shigehito; Uwabe, Chigako; Takakuwa, Tetsuya

    2015-05-01

    We aimed to observe morphological changes in the spleen from the emergence of the primordium to the end of the embryonic period using histological serial sections of 228 samples. Between Carnegie stages (CSs) 14 and 17, the spleen was usually recognized as a bulge in the dorsal mesogastrium (DM), and after CS 20, the spleen became apparent. Intrasplenic folds were observed later. A high-density area was first recognized in 6 of the 58 cases at CS 16 and in all cases examined after CS 18. The spleen was recognized neither as a bulge nor as a high-density area at CS 13. The mesothelium was pseudostratified until CS 16 and was replaced with high columnar cells and then with low columnar cells. The basement membrane was obvious after CS 17. The mesenchymal cells differentiated from cells in the DM, and sinus formation started at CS 20. Hematopoietic cells were detected after CS 18. The vessels were observed at CS 14 in the DM. Hilus formation was observed after CS 20. The parallel entries of the arteries and veins were observed at CS 23. The rate of increase in spleen length in relation to that of stomach length along the cranial-caudal direction was 0.51 ± 0.11, which remained constant during CSs 19 and 23, indicating that their growths were similar. These data may help to better understand the development of normal human embryos and to detect abnormal embryos in the early stages of development.

  8. [Embryonic mechanisms in development of spina bifida in humans].

    Science.gov (United States)

    Vostrikov, O V; Zotov, V A; Nikitenko, E V

    2004-01-01

    The study of 86 human embryos and fetuses beginning from 23rdday of development up to 18th week after fecundation has detected 23 cases of pathologic development which correspond to the modern definition of spina bifida (SB). It is shown that the cause of various forms of the anomaly is the disturbance or temporary delay of a movement of caudal neurulation wave forming the spinal cord. The anomaly size and type are determined by the time and duration of a pathogenic action on neurulation. Postnatal or the 1st type of SB develops in neurulation wave disturbance not longer than 4-6 hours. Anomaly consequence may be compensated surgically. The 2nd or fetal type of SB arises when neurulation delay is from 6 to 20 hours. If the delay occurs on the 22-24th day of development, embryos die by the neurulation end. If the delay takes place on the 26-28th day embryos may survive till the late fetal period. In embryonal or type III of SB embryos die by the end of the 8th week and do not enter the medical statistics. Their death is associated with delayed movement of the caudal neurulation wave for 24 hours and longer. This results in a spontaneous abortion.

  9. A novel marker for undifferentiated human embryonic stem cells.

    Science.gov (United States)

    Higashi, Kiyoshi; Yagi, Masaki; Arakawa, Tatsuhiko; Asano, Kouji; Kobayashi, Kumiko; Tachibana, Taro; Saito, Koichi

    2015-02-01

    Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs. The antigen recognized by MAb2 is expressed on the cell surface of undifferentiated hESCs; three diffused bands with molecular mass between 30 and 60 kDa in the lysates of hESCs were diminished during hESC differentiation into neural cells. The expression of MAb2 antigen was also observed on the plasma membrane of lung cancer cells, and MAb2 detected 55, 50, and 35 kDa protein bands in the cell lysates. Immunoprecipitation followed by proteomics analyses identified CD147/basigin as a MAb2 antigen. Finally, the positive expression of CD147/basigin protein in undifferentiated hESCs was confirmed. These results suggested that CD147/basigin could be another undifferentiated hESC marker.

  10. HIF induces human embryonic stem cell markers in cancer cells.

    Science.gov (United States)

    Mathieu, Julie; Zhang, Zhan; Zhou, Wenyu; Wang, Amy J; Heddleston, John M; Pinna, Claudia M A; Hubaud, Alexis; Stadler, Bradford; Choi, Michael; Bar, Merav; Tewari, Muneesh; Liu, Alvin; Vessella, Robert; Rostomily, Robert; Born, Donald; Horwitz, Marshall; Ware, Carol; Blau, C Anthony; Cleary, Michele A; Rich, Jeremy N; Ruohola-Baker, Hannele

    2011-07-01

    Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

  11. Non-invasive imaging of human embryonic stem cells.

    Science.gov (United States)

    Hong, Hao; Yang, Yunan; Zhang, Yin; Cai, Weibo

    2010-09-01

    Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography (PET), and multimodality approaches. Although the focus of this review article is primarily on hESCs, the labeling/tracking strategies described here can be readily applied to other (stem) cell types as well. Non-invasive imaging can provide convenient means to monitor hESC survival, proliferation, function, as well as overgrowth (such as teratoma formation), which could not be readily investigated previously. The requirement for hESC tracking techniques depends on the clinical scenario and each imaging technique will have its own niche in preclinical/clinical research. Continued evolvement of non-invasive imaging techniques will undoubtedly contribute to significant advances in understanding stem cell biology and mechanisms of action.

  12. Maintenance of human embryonic stem cells on gelatin

    Institute of Scientific and Technical Information of China (English)

    LI Yang; LIN ChangSheng; WANG Li; LIU Ying; MU XiaoNing; MAYue; LI LingSong

    2009-01-01

    Matrigel is routinely used as a coating material in the feeder-free culture system of human embryonic stem cells (hESCs).However,matrigel is costive and inconvenient to use.In this study,the possibility of using gelatin as an alternative coating material was investigated.The results showed that,after trypsinization,hESCs were maintained undifferentiated on gelatin.These hESCs expressed pluripotent markers,formed teratoma and maintained a normal karyotype.As measured at passage 10,the hESCs expressed a high level of Oct4 on both gelatin and Matrigel.hESCs growing on gelatin formed AP-positive colonies in similar size and number to those growing on Matrigel (P>0.05).Moreover,hESCs growing on gelatin contained a comparable percentage of SSEA-4-positive cells to those growing on Matrigel (95.1% vs.94.3%,P>0.05).H-1 hESCs were maintained undifferentiated on gelatin for 20 passages and remained the stable normal karyotype.This gelatin-based culture protocol may allow us to propagate hESCs in large scale,with less cost.

  13. Neural precursors derived from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Peng Hongmei; Chen Gui'an

    2005-01-01

    Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells, hES cells (Line PKU-1 and Line PKU-2) were cultured in suspension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs).The EBs were then cultured in N2 medium containing bFGF in poly- L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2-3 short processes of the spreading outgrowth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4-5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotypeⅢ, GABA, serotonin and synaptophysin.Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oligodendrocytes expressing O4. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS) in vitro.

  14. Translational Research: Palatal-derived Ecto-mesenchymal Stem Cells from Human Palate: A New Hope for Alveolar Bone and Cranio-Facial Bone Reconstruction.

    Science.gov (United States)

    Grimm, Wolf Dieter; Dannan, Aous; Giesenhagen, Bernd; Schau, Ingmar; Varga, Gabor; Vukovic, Mark Alexander; Sirak, Sergey Vladimirovich

    2014-05-01

    The management of facial defects has rapidly changed in the last decade. Functional and esthetic requirements have steadily increased along with the refinements of surgery. In the case of advanced atrophy or jaw defects, extensive horizontal and vertical bone augmentation is often unavoidable to enable patients to be fitted with implants. Loss of vertical alveolar bone height is the most common cause for a non primary stability of dental implants in adults. At present, there is no ideal therapeutic approach to cure loss of vertical alveolar bone height and achieve optimal pre-implantological bone regeneration before dental implant placement. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from different dental resources, namely the dental follicle, the dental pulp and the periodontal ligament. Our research group has cultured palatal-derived stem cells (paldSCs) as dentospheres and further differentiated into various cells of the neuronal and osteogenic lineage, thereby demonstrating their stem cell state. In this publication will be shown whether paldSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate alveolar bone tissue in vivo in an athymic rat model. Furthermore, using these data we have started a proof of principle clinical- and histological controlled study using stem cell-rich palatal tissues for improving the vertical alveolar bone augmentation in critical size defects. The initial results of the study demonstrate the feasibility of using stem cell-mediated tissue engineering to treat alveolar bone defects in humans.

  15. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    Science.gov (United States)

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process.

  16. Primitive cardiac cells from human embryonic stem cells.

    Science.gov (United States)

    Hudson, James; Titmarsh, Drew; Hidalgo, Alejandro; Wolvetang, Ernst; Cooper-White, Justin

    2012-06-10

    Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

  17. Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

    Science.gov (United States)

    Sleeboom-Faulkner, Margaret

    2010-01-01

    This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

  18. RETRACTION - In Vitro Derivation of Human Sperm from Embryonic Stem Cells.

    Science.gov (United States)

    Nayernia, Karim; Lee, Jae Ho; Lako, Majlinda; Armstrong, Lyle; Herbert, Mary; Li, Manyu; Engel, Wolfgang; Elliott, David; Stojkovic, Miodrag; Parrington, John; Murdoch, Alison; Strachan, Tom; Zhang, Xin

    2009-07-07

    This article, "In Vitro Derivation of Human Sperm from Embryonic Stem Cells," is being retracted from Stem Cells and Development. Further details will follow online, and in a subsequent issue of the Journal.

  19. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    Science.gov (United States)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  20. Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

    Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

  1. Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Jin YANG; Guo-qiang LIU; Rui WEI; Wen-fang HOU; Mei-juan GAO; Ming-xia ZHU; Hai-ning WANG; Gui-an CHEN; Tian-pei HONG

    2011-01-01

    Aim:Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells.The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and,if so,whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α).Methods:Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol.The cumulative percentage of beating EBs was calculated.The expression of cardiac-specific markers including cardiac troponin Ⅰ (cTnl) and α-myosin heavy chain (α-MHC) was detected using RT-PCR,real-time PCR and Western blot.The dispersed beating EBs were examined using immunofluorescent staining.Results:The percentage of beating EBs and the expression of cTnl were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium.From 6 to 18 d of differentiation,the increased expression of cTnl and α-MHC by ghrelin (1 nmol/L)was time-dependent,and in line with the alteration of the percentages of beating EBs.Furthermore,the dispersed beating EBs were double-positively immunostained with antibodies against cTnl and α-actinin.However,blockage of GHS-R1α with its specific antagonist D-[lys3]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation.Conclusion:Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells,which is not mediated via GHS-R1α.

  2. Toxicity study of reclaimed water on human embryonic kidney cells.

    Science.gov (United States)

    Ren, Xianghao; Kou, Ying-Ying; Kim, Taeeung; Chae, Kyu-Jung; Ng, How Yong

    2017-08-28

    The importance of evaluating the toxic effects associated with the use of reclaimed water has been increasing. The purpose of this research was to investigate the cytotoxicity and molecular toxicity of reclaimed water on the human embryonic kidney 293 (HEK293) cells. The culture medium was synthesized using the reclaimed water samples. Wastewater treatment plant influent (WTI) and effluent (WTE), containing micropollutants at the nanogram per liter level, decreased cell proliferation (93.4-98.9% and 91.5-96.6% of the control, respectively) and increased cell damage (103.6-117.5% and 100.7-109% of the control, respectively) at all exposure times, except for a decrease in cell damage observed after an 8-h exposure to WTE. Membrane bioreactor permeate (MBRP) increased cell proliferation (102.1-106.7% of the control) and decreased cell damage at 8 and 12 h (92.4 and 98.4% of the control, respectively), but slightly increased cell damage at 24 h and later time points (101.1-104.9% of the control). All three water samples induced cell apoptosis (120.9-123.4% of the control). They also affected the expression of cell-cycle regulatory proteins (p16(INK4a), p27(Kip1), cyclin-dependent kinases 2 and 4, cyclin D1, and cyclin E) and apoptosis-related regulatory proteins (p-JNK, Bcl-2, caspase-9, and caspase-3). In conclusion, all three water samples showed cytotoxicity and molecular toxicity in the HEK293 cells, and the results of the cell-cycle and apoptosis regulatory protein expression after WTI and WTE treatments were consistent with the results of the cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

  4. TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

    Science.gov (United States)

    Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

    2015-05-01

    The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

  5. [Expert consensus on induction of human embryonic stem cells into tenocytes].

    Science.gov (United States)

    Chen, Xiao; Zou, Xiaohui; Yu, Guangyan; Fu, Xin; Cao, Tong; Xiao, Yin; Ouyang, Hongwei

    2016-03-01

    Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.

  6. Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

    Science.gov (United States)

    d'Amaro, Rocca; Scheidegger, Rolf; Blumer, Susan; Pazera, Pawel; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf

  7. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

    DEFF Research Database (Denmark)

    Kiprilov, Enko N; Awan, Aashir; Desprat, Romain

    2008-01-01

    Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human h...

  8. Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

    Directory of Open Access Journals (Sweden)

    Wang Xiaosheng

    2011-10-01

    Full Text Available Abstract Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class comparison analysis and survival analysis algorithms to identify differentially expressed genes and their associated transcription factors, pathways and microRNAs among normal vs. tumor or good prognosis vs. poor prognosis phenotypes classes based on numerous human cancer gene expression data. We found that most of the human embryonic stem cell- associated signatures were frequently identified in the analysis, suggesting a strong linkage between human embryonic stem cells and cancer cells. Conclusions The present study revealed the close linkage between the human embryonic stem cell associated gene expression profiles and cancer-associated gene expression profiles, and therefore offered an indirect support for the cancer stem cell theory. However, many interest issues remain to be addressed further.

  9. Implications of TGFβ on transcriptome and cellular biofunctions of palatal mesenchyme

    Directory of Open Access Journals (Sweden)

    Xiujuan eZhu

    2012-04-01

    Full Text Available Development of the palate comprises sequential stages of growth, elevation and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFβ-mediated regulation of palate development. We hypothesized that the isoforms of TGFβ regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM cells were treated with TGFβ1, β2, and β3 for microarray-based gene expression studies in order to identify the roles of TGFβ in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFβ-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them were previously identified as being crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFβ1, TGFβ2, and TGFβR1. These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFβ treatment in HEPM cells. Our results suggest that both TGFβ1 and TGFβ2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by

  10. Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cell.

    Science.gov (United States)

    Walsh, James; Andrews, Peter W

    2003-01-01

    Embryonal carcinoma (EC) cells, the pluripotent stem cells of teratocarcinomas, show many similar-ities to embryonic stem (ES) cells. Since EC cells are malignant but their terminally differentiated derivatives are not, understanding the molecular mechanisms that regulate their differentiation maybe of value for diagnostic and therapeutic purposes. We have examined the expression of multiple components of two developmentally important cell-cell signalling pathways, Wnt and Notch, in the pluripotent human EC cell line, NTERA2, and the human ES cell line, H7. Both pathways have well-documented roles in controlling neurogenesis, a process that occurs largely in response to retinoicacid (RA) treatment of NTERA2 cultures and spontaneously in H7 cultures. In NTERA2, many ofthe genes tested showed altered transcriptional regulation following treatment with RA. These include members of the frizzled gene family (FZDI, FZD3, FZD4, FZD5, FZD6), encoding receptors forWnt proteins, the Frizzled Related Protein family (SFRPI, SFRP2, FRZB, SFRP4), encoding solubleWnt antagonists and also ligands and receptors of the Notch pathway (Dlkl, Jaggedl; Notchl, Notch2, Notch3). Few differences were found in the repertoire of Wnt and Notch pathway genes expressed by NTERA2 EC cells and H7 ES cells. We present a model in which interactions between and regulation of Wnt and Notch signalling are important in maintaining EC/ES stem cells and also controlling their differentiation.

  11. MicroRNAs in human embryonic and cancer stem cells.

    Science.gov (United States)

    Navarro, Alfons; Monzo, Mariano

    2010-09-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate messenger RNAs at the post-transcriptional level. They play an important role in the control of cell physiological functions, and their alterations have been related to cancer, where they can function as oncogenes or tumor suppressor genes. Recently, they have emerged as key regulators of "stemness", collaborating in the maintenance of pluripotency, control of self-renewal, and differentiation of stem cells. The miRNA pathway has been shown to be crucial in embryonic development and in embryonic stem (ES) cells, as shown by Dicer knockout analysis. Specific patterns of miRNAs have been reported to be expressed only in ES cells and in early phases of embryonic development. Moreover, many cancers present small populations of cells with stem cell characteristics, called cancer stem cells (CSCs). CSCs are responsible for relapse and treatment failure in many cancer patients, and the comparative analysis of expression patterns between ES cells and tumors can lead to the identification of a miRNA signature to define CSCs. Most of the key miRNAs identified to date in ES cells have been shown to play a role in tumor diagnosis or prognosis, and may well prove to be essential in cancer therapy in the foreseeable future.

  12. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  13. Intraflagellar transport 88 (IFT88) is crucial for craniofacial development in mice and is a candidate gene for human cleft lip and palate.

    Science.gov (United States)

    Tian, Hua; Feng, Jifan; Li, Jingyuan; Ho, Thach-Vu; Yuan, Yuan; Liu, Yang; Brindopke, Frederick; Figueiredo, Jane C; Magee, William; Sanchez-Lara, Pedro A; Chai, Yang

    2017-03-01

    Ciliopathies are pleiotropic human diseases resulting from defects of the primary cilium, and these patients often have cleft lip and palate. IFT88 is required for the assembly and function of the primary cilia, which mediate the activity of key developmental signaling pathways. Through whole exome sequencing of a family of three affected siblings with isolated cleft lip and palate, we discovered that they share a novel missense mutation in IFT88 (c.915G > C, p.E305D), suggesting this gene should be considered a candidate for isolated orofacial clefting. In order to evaluate the function of IFT88 in regulating craniofacial development, we generated Wnt1-Cre;Ift88fl/fl mice to eliminate Ift88 specifically in cranial neural crest (CNC) cells. Wnt1-Cre;Ift88fl/flpups died at birth due to severe craniofacial defects including bilateral cleft lip and palate and tongue agenesis, following the loss of the primary cilia in the CNC-derived palatal mesenchyme. Loss of Ift88 also resulted in a decrease in neural crest cell proliferation during early stages of palatogenesis as well as a downregulation of the Shh signaling pathway in the palatal mesenchyme. Importantly, Osr2KI-Cre;Ift88fl/flmice, in which Ift88 is lost specifically in the palatal mesenchyme, exhibit isolated cleft palate. Taken together, our results demonstrate that IFT88 has a highly conserved function within the primary cilia of the CNC-derived mesenchyme in the lip and palate region in mice and is a strong candidate as an orofacial clefting gene in humans. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Characterization of human PGD blastocysts with unbalanced chromosomal translocations and human embryonic stem cell line derivation?

    Science.gov (United States)

    Frydman, N; Féraud, O; Bas, C; Amit, M; Frydman, R; Bennaceur-Griscelli, A; Tachdjian, G

    2009-01-01

    Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

  15. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells.

    Science.gov (United States)

    Yang, Chao; Ji, Lei; Yue, Wen; Shi, Shuang-Shuang; Wang, Ruo-Yong; Li, Yan-Hua; Xie, Xiao-Yan; Xi, Jia-Fei; He, Li-Juan; Nan, Xue; Pei, Xue-Tao

    2012-02-01

    Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

  16. Maturation of spinal motor neurons derived from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tomonori Takazawa

    Full Text Available Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

  17. Human Embryonic Stem Cell derived Cardiomyocytes self-arrange with areas of different subtypes during differentiation

    DEFF Research Database (Denmark)

    Vestergaard, Maj Linea; Grubb, Søren J; Rasmussen, Karen Koefod

    2017-01-01

    The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESC) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of CMs derived from hESCs (h...

  18. Human embryonic stem cells as a model for cardiac gene discovery : from chip to chap

    NARCIS (Netherlands)

    Beqqali, A.

    2008-01-01

    Here we described the use of human embryonic stem cells (hESCs) as a model to obtain insights into commitment to the mesoderm and endoderm lineages and the early steps in human cardiac cell differentiation by means of whole-genome temporal expression profiling. Furthermore, we used it as an approach

  19. PGC-1α and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function

    NARCIS (Netherlands)

    M.J. Birket (Matthew); S. Casini (Simona); G. Kosmidis (Georgios); D.J. Elliott (David); A.A. Gerencser (Akos); A. Baartscheer (Antonius); C. Schumacher (Cees); P.G. Mastroberardino (Pier); A.G. Elefanty (Andrew); E.G. Stanley (Ed); C.L. Mummery (Christine)

    2013-01-01

    textabstractDiminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, wh

  20. Human embryonic stem cells : advancing biology and cardiogenesis towards functional applications l

    NARCIS (Netherlands)

    Braam, Stefan Robbert

    2010-01-01

    Human embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on comprehensive knowledge of their biology and control of their signaling mechanisms and fate choices. To begin to a

  1. Apoptotic gene expression in the neural tube during early human embryonic development

    Institute of Scientific and Technical Information of China (English)

    Guifang Chen; Tiandong Li; Peipei Ding; Ping Yang; Xiao Zhang

    2011-01-01

    Neural tube development comprises neural induction,neural epithelial cell proliferation,and apoptosis,as well as migration of nerve cells.Too much or too little apoptosis leads to abnormal nervous system development.The present study analyzed expression and distribution of apoptotic-related factors,including Fas,FasL,and caspase-3,during human embryonic neural tube development.Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondriai-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development.Subsequently,Fas and FasL expression increased during embryonic development.The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways.Therefore,neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.

  2. Differential resistance of human embryonic stem cells and somatic cell types to hydrogen peroxide-induced genotoxicity may be dependent on innate basal intracellular ROS levels.

    Science.gov (United States)

    Vinoth, Kumar Jayaseelan; Manikandan, Jayapal; Sethu, Swaminathan; Balakrishnan, Lakshmidevi; Heng, Alexis; Lu, Kai; Poonepalli, Anuradha; Hande, Manoor Prakash; Cao, Tong

    2015-01-01

    Previously, we demonstrated that undifferentiated human embryonic stem cells (hESC) displayed higher resistance to oxidative and genotoxic stress compared to somatic cells, but did not further probe the underlying mechanisms. Using H₂O₂-induced genotoxicity as a model, this study investigated whether higher resistance of hESC to oxidative and genotoxic stress could be due to lower innate basal intracellular levels of reactive oxygen species (ROS), as compared to their differentiated fibroblastic progenies (H1F) and two other somatic cell types - human embryonic palatal mesenchymal (HEPM) cells and peripheral blood lymphocytes (PBL). Comet assay demonstrated that undifferentiated hESC consistently sustained lower levels of DNA damage upon acute exposure to H₂O₂ for 30 min, compared to somatic cells. DCFDA and HE staining with flow cytometry showed that undifferentiated hESC had lower innate basal intracellular levels of reactive oxygen species compared to somatic cells, which could lead to their higher resistance to genotoxic stress upon acute exposure to H₂O₂.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice.

    Science.gov (United States)

    Zhang, Jian; Yang, Rui; Liu, Ziyi; Hou, Congzhe; Zong, Wen; Zhang, Aizhen; Sun, Xiaoyang; Gao, Jiangang

    2015-11-01

    In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.

  6. Scale-up of human embryonic stem cell culture using a hollow fibre bioreactor.

    Science.gov (United States)

    Roberts, Iwan; Baila, Stefano; Rice, R Brent; Janssens, Michiel Etienne; Nguyen, Kim; Moens, Nathalie; Ruban, Ludmila; Hernandez, Diana; Coffey, Pete; Mason, Chris

    2012-12-01

    The commercialisation of human embryonic stem cell derived cell therapies for large patient populations is reliant on both minimising expensive and variable manual-handling methods whilst realising economies of scale. The Quantum Cell Expansion System, a hollow fibre bioreactor (Terumo BCT), was used in a pilot study to expand 60 million human embryonic stem cells to 708 million cells. Further improvements can be expected with optimisation of media flow rates throughout the run to better control the cellular microenvironment. High levels of pluripotency marker expression were maintained on the bioreactor, with 97.7 % of cells expressing SSEA-4 when harvested.

  7. Vitamin B-complex initiates growth and development of human embryonic brain cells in vitro.

    Science.gov (United States)

    Danielyan, K E; Abramyan, R A; Galoyan, A A; Kevorkian, G A

    2011-09-01

    We studied a combined effect of subcomponents of vitamin B complex on the growth, development, and death of human embryonic brain-derived cells (E90) cultured using a modified method of Matson. Cell death was detected by trypan blue staining. According to our results, vitamin B-complex in low-doses promote the development, maturation, and enlargement of human embryonic brain cells, on the one hand, and increases the percent of cell death, which attests to accelerated maturation and metabolism, on the other.

  8. Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

    Directory of Open Access Journals (Sweden)

    Yi-Lin Wu

    2014-08-01

    Full Text Available Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP, stage-specific embryonic antigen-3 (SSEA-3, SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

  9. Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

    Science.gov (United States)

    Furukawa, Jun-Ichi; Okada, Kazue; Shinohara, Yasuro

    2016-10-01

    Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

  10. Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.

    Science.gov (United States)

    Lai, Dongmei; Wang, Yongwei; Sun, Jian; Chen, Yifei; Li, Ting; Wu, Yi; Guo, Lihe; Wei, Chunsheng

    2015-05-07

    Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.

  11. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    Science.gov (United States)

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.

  12. BDE-209 inhibits pluripotent genes expression and induces apoptosis in human embryonic stem cells.

    Science.gov (United States)

    Du, Lili; Sun, Wen; Zhang, Huili; Chen, Dunjin

    2016-05-01

    Decabromodiphenyl ether (BDE-209) has been detected in human serum, semen, placenta, cord blood and milk worldwide. However, little is known regarding the potential effects on the early human embryonic development of BDE-209. In this study, human embryonic stem cell lines FY-hES-10 and FY-hES-26 were used to evaluate the potential effects and explore the toxification mechanisms using low-level BDE-209 exposure. Our data showed that BDE-209 exposure (1, 10 and 100 nM) reduced the expression of pluripotent genes such as OCT4, SOX2 and NANOG and induced human embryonic stem cells (hESCs) apoptosis. The downregulation of BIRC5/BCL2 and upregulation of BAX were related to apoptosis of hESCs induced by BDE-209 exposure. A mechanism study showed that OCT4 down-regulation accompanied by OCT4 promoter hypermethylation and increasing miR-145/miR-335 levels, OCT4 inhibitors. Moreover, BDE-209 could increase the generation of intracellular reactive oxygen species (ROS) and decrease SOD2 expression. The ROS increase and OCT4 downregulation after BDE-209 exposure could be reversed partly by antioxidant N-acetylcysteine supplement. These findings showed that BDE-209 exposure could decrease pluripotent genes expression via epigenetic regulation and induce apoptosis through ROS generation in human embryonic stem cells in vitro.

  13. Generation of human female reproductive tract epithelium from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Louie Ye

    Full Text Available BACKGROUND: Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD, the primordial female reproductive tract (FRT. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM is recombined with green fluorescent protein (GFP-tagged human embryonic stem cells (hESCs; GFP-hESC (ENVY. We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1(+ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA. CONCLUSIONS/SIGNIFICANCE: These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.

  14. Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

    2011-01-01

    the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...

  15. Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells.

    Science.gov (United States)

    Daniels, Brian R; Hale, Christopher M; Khatau, Shyam B; Kusuma, Sravanti; Dobrowsky, Terrence M; Gerecht, Sharon; Wirtz, Denis

    2010-12-01

    Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell "softness" is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.

  16. miR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Heiko Fuchs

    Full Text Available Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG, epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3 of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8 axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3 of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

  17. Growth trajectories of the human embryonic head and periconceptional maternal conditions

    NARCIS (Netherlands)

    Koning, I V; Baken, L; Groenenberg, I A L; Husen, S C; Dudink, J; Willemsen, S P; Gijtenbeek, M; Koning, A H J; Reiss, I K M; Steegers, E A P; Steegers-Theunissen, R P M

    2016-01-01

    STUDY QUESTION: Can growth trajectories of the human embryonic head be created using 3D ultrasound (3D-US) and virtual reality (VR) technology, and be associated with second trimester fetal head size and periconceptional maternal conditions? SUMMARY ANSWER: Serial first trimester head circumference

  18. Periconception Maternal Folate Status and Human Embryonic Cerebellum Growth Trajectories : The Rotterdam Predict Study

    NARCIS (Netherlands)

    Koning, Irene V; Groenenberg, Irene A L; Gotink, Anniek W; Willemsen, Sten P; Gijtenbeek, Manon; Dudink, Jeroen; Go, Attie T J I; Reiss, Irwin K M; Steegers, Eric A P; Steegers-Theunissen, Régine P M

    2015-01-01

    We aimed to investigate whether periconceptional maternal folate status affects human embryonic cerebellar size and growth trajectories. In a prospective periconceptional cohort participants filled out questionnaires and received weekly transvaginal 3D-ultrasounds between 7+0 and 12+6 weeks

  19. Periconception maternal folate status and human embryonic cerebellum growth trajectories: The Rotterdam predict study

    NARCIS (Netherlands)

    I.V. Koning (Irene); I.A.L. Groenenberg (Irene); A.W. Gotink (Anniek W.); S. Willemsen (Sp); M. Gijtenbeek (M.); J. Dudink (Jeroen); A.T.J.I. Go (Attie T. J. I.); I.K.M. Reiss (Irwin); E.A.P. Steegers (Eric); R.P.M. Steegers-Theunissen (Régine)

    2015-01-01

    textabstractWe aimed to investigate whether periconceptional maternal folate status affects human embryonic cerebellar size and growth trajectories. In a prospective periconceptional cohort participants filled out questionnaires and received weekly transvaginal 3D-ultrasounds between 7+0 and 12+6

  20. NANOG reporter cell lines generated by gene targeting in human embryonic stem cells

    DEFF Research Database (Denmark)

    Fischer, Yvonne; Ganic, Elvira; Ameri, Jacqueline

    2010-01-01

    Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role...

  1. Procedures for Derivation and Characterisation of Human Embryonic Stem Cells from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Kassem, Moustapha

    2012-01-01

    In 1998, a development occurred in stem cell biology with the fi rst report of the derivation of a human embryonic stem cell (hESC) line. Since then a number of techniques have been used to derive and characterise hESCs. Here, we describe the derivation methods used by our laboratory for isolation...

  2. Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.

    NARCIS (Netherlands)

    Graichen, R.; Xu, X.; Braam, S.R.; Balakrishnan, T.; Norfiza, S.; Sieh, S.; Soo, S.Y.; Tham, S.C.; Mummery, C.L.; Colman, A.; Zweigerdt, R.; Davidson, B.P.

    2008-01-01

    Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by t

  3. Genomic instability of human embryonic stem cell lines using different passaging culture methods.

    Science.gov (United States)

    Tosca, Lucie; Feraud, Olivier; Magniez, Aurélie; Bas, Cécile; Griscelli, Frank; Bennaceur-Griscelli, Annelise; Tachdjian, Gérard

    2015-01-01

    Human embryonic stem cells exhibit genomic instability that can be related to culture duration or to the passaging methods used for cell dissociation. In order to study the impact of cell dissociation techniques on human embryonic stem cells genomic instability, we cultured H1 and H9 human embryonic stem cells lines using mechanical/manual or enzymatic/collagenase-IV dissociation methods. Genomic instability was evaluated at early (p60) passages by using oligonucleotide based array-comparative genomic hybridization 105 K with a mean resolution of 50 Kb. DNA variations were mainly located on subtelomeric and pericentromeric regions with sizes <100 Kb. In this study, 9 recurrent genomic variations were acquired during culture including the well known duplication 20q11.21. When comparing cell dissociation methods, we found no significant differences between DNA variations number and size, DNA gain or DNA loss frequencies, homozygous loss frequencies and no significant difference on the content of genes involved in development, cell cycle tumorigenesis and syndrome disease. In addition, we have never found any malignant tissue in 4 different teratoma representative of the two independent stem cell lines. These results show that the occurrence of genomic instability in human embryonic stem cells is similar using mechanical or collagenase IV-based enzymatic cell culture dissociation methods. All the observed genomic variations have no impact on the development of malignancy.

  4. Oncogenic KRAS activates an embryonic stem cell-like program in human colon cancer initiation.

    Science.gov (United States)

    Le Rolle, Anne-France; Chiu, Thang K; Zeng, Zhaoshi; Shia, Jinru; Weiser, Martin R; Paty, Philip B; Chiu, Vi K

    2016-01-19

    Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer.

  5. Developing Human Embryonic Stem Cells for Grafting in Parkinson’s Disease

    Science.gov (United States)

    2007-03-01

    markers Oligodendrocyte markers Gap43, Nfh, Eno2 Th, Aadc, Dat Ngf Hoxc6 S100β, Cd68 Gfap, Tapa1 Mag, Mobp, Omg V. Distinct markers Normal...Derivation, growth and applications of human embryonic stem cells. Reproduction 128:259-267. Y. Takagi, J. Takahashi, H. Saiki, A. Morizane, T

  6. Asynchronous replication and autosome-pair non-equivalence in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Devkanya Dutta

    Full Text Available A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells.

  7. Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Pørneki, Ann Dorte Storm; Floridon, Charlotte

    2007-01-01

    Human embryonic stem cells (hESCs) have the potential to provide alternative sources for pancreatic islet grafts. In the present study we have investigated the influence of Activin A and Activin B on the expression of the pancreas marker gene Pdx1 in hESCs differentiated as embryoid bodies (EBs...

  8. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic...

  9. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

    NARCIS (Netherlands)

    Amps, Katherine; Andrews, Peter W.; Anyfantis, George; Armstrong, Lyle; Avery, Stuart; Baharvand, Hossein; Baker, Julie; Baker, Duncan; Munoz, Maria B.; Beil, Stephen; Benvenisty, Nissim; Ben-Yosef, Dalit; Biancotti, Juan-Carlos; Bosman, Alexis; Brena, Romulo Martin; Brison, Daniel; Caisander, Gunilla; Camarasa, Maria V.; Chen, Jieming; Chiao, Eric; Choi, Young Min; Choo, Andre B. H.; Collins, Daniel; Colman, Alan; Crook, Jeremy M.; Daley, George Q.; Dalton, Anne; De Sousa, Paul A.; Denning, Chris; Downie, Janet; Dvorak, Petr; Montgomery, Karen D.; Feki, Anis; Ford, Angela; Fox, Victoria; Fraga, Ana M.; Frumkin, Tzvia; Ge, Lin; Gokhale, Paul J.; Golan-Lev, Tamar; Gourabi, Hamid; Gropp, Michal; Lu Guangxiu, [No Value; Hampl, Ales; Harron, Katie; Healy, Lyn; Herath, Wishva; Holm, Frida; Hovatta, Outi; Hyllner, Johan; Inamdar, Maneesha S.; Irwanto, Astrid Kresentia; Ishii, Tetsuya; Jaconi, Marisa; Jin, Ying; Kimber, Susan; Kiselev, Sergey; Knowles, Barbara B.; Kopper, Oded; Kukharenko, Valeri; Kuliev, Anver; Lagarkova, Maria A.; Laird, Peter W.; Lako, Majlinda; Laslett, Andrew L.; Lavon, Neta; Lee, Dong Ryul; Lee, Jeoung Eun; Li, Chunliang; Lim, Linda S.; Ludwig, Tenneille E.; Ma, Yu; Maltby, Edna; Mateizel, Ileana; Mayshar, Yoav; Mileikovsky, Maria; Minger, Stephen L.; Miyazaki, Takamichi; Moon, Shin Yong; Moore, Harry; Mummery, Christine; Nagy, Andras; Nakatsuji, Norio; Narwani, Kavita; Oh, Steve K. W.; Oh, Sun Kyung; Olson, Cia; Otonkoski, Timo; Pan, Fei; Park, In-Hyun; Pells, Steve; Pera, Martin F.; Pereira, Lygia V.; Qi, Ouyang; Raj, Grace Selva; Reubinoff, Benjamin; Robins, Alan; Robson, Paul; Rossant, Janet; Salekdeh, Ghasem H.; Schulz, Thomas C.; Sermon, Karen; Mohamed, Jameelah Sheik; Shen, Hui; Sherrer, Eric; Sidhu, Kuldip; Sivarajah, Shirani; Skottman, Heli; Spits, Claudia; Stacey, Glyn N.; Strehl, Raimund; Strelchenko, Nick; Suemori, Hirofumi; Sun, Bowen; Suuronen, Riitta; Takahashi, Kazutoshi; Tuuri, Timo; Venu, Parvathy; Verlinsky, Yuri; Ward-van Oostwaard, Dorien; Weisenberger, Daniel J.; Wu, Yue; Yamanaka, Shinya; Young, Lorraine; Zhou, Qi

    2011-01-01

    The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphi

  10. Palate Shape and Depth: A Shape-Matching and Machine Learning Method for Estimating Ancestry from Human Skeletal Remains.

    Science.gov (United States)

    Maier, Christopher A; Zhang, Kang; Manhein, Mary H; Li, Xin

    2015-09-01

    In the past, assessing ancestry relied on the naked eye and observer experience; however, replicability has become an important aspect of such analysis through the application of metric techniques. This study examines palate shape and assesses ancestry quantitatively using a 3D digitizer and shape-matching and machine learning methods. Palate curves and depths were recorded, processed, and tested for 376 individuals. Palate shape was an accurate indicator of ancestry in 58% of cases. Cluster analysis revealed that the parabolic, hyperbolic, and elliptical shapes are discrete from one another. Preliminary results indicate that palate depth in Hispanic individuals is greatest. Palate shape appears to be a useful indicator of ancestry, particularly when assessed by a computer. However, these data suggest that palate shape is not useful for assessing ancestry in Hispanic individuals. Although ancestry may be determined from palate shape, the use of multiple features is recommended and more reliable.

  11. Observation of the human fetal corpses with maxillofacial malformations. 1. CT and MRI examinations of the fetal cleft lip and/or palate

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Chikara; Nakano, Yoko; Shigematsu, Shiro [Tokyo Dental Coll., Chiba (Japan)] (and others)

    1999-06-01

    Of the various types of congenital malformations, the cleft lip and/or palate is one of the most frequent. Observation of human fetal corpses exhibiting cleft lip and palate is very important to research on its onset of its mechanism and development. In recent years, some of researchers have performed clinical studies on prenatal diagnosis and surgical treatment for the entirey. However, there have hardly been any reports on detailed observations of the maxillofacial structure of a fetus with cleft lip and palate. We seized an opportunity of observing the maxillofacial structure of fetuses with cleft lip and/or palate using three-dimensional CT (3D-CT) and MR imaging as non-disjunctive methods. In the present study, nine fetal corpses having cleft lip and/or palate were examined. The results were as follows: CT and MRI were useful for non-invasive observation of the maxillofacial structure, including soft tissues. Because the osseous tissues of young fetus tissue is not fully mature, observation of bone structures was slightly difficult. When corpses were immersed in formalin for a long time, osseous tissue was decalcified, thus making it difficult to obtain clear images. We could observe the details of the maxillofacial structures such as the alveolar process, the hard palate, the maxillary sinus, the nasal cavity, the nasal bone, and the vomer, in some of the cases. 3D-CT and MR findings observed in the fetuses with cleft lip and/or palate should provide some basement of the imaging diagnosis of congenital disorder. (author)

  12. Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

    Directory of Open Access Journals (Sweden)

    Chih-Hong Lou

    2016-06-01

    Full Text Available Nonsense-mediated RNA decay (NMD is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

  13. Derivation of the human embryonic stem cell line RCe008-A (RC-4

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe008-A (RC-4 was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is available.

  14. Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

    Science.gov (United States)

    Kunze, M. E.

    1985-01-01

    A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

  15. Generation of KCL033 clinical grade human embryonic stem cell line

    OpenAIRE

    2016-01-01

    The KCL033 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility...

  16. Derivation of the human embryonic stem cell line RCe012-A (RC-8)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Collins, D. M.; Greenshields, A.; H. Bradburn; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyoty...

  17. Derivation of the human embryonic stem cell line RCe006-A (RC-2)

    OpenAIRE

    P.A. De Sousa; B. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe006-A (RC-2) was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12). Mic...

  18. Derivation of the human embryonic stem cell line RCe011-A (RC-7)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Collins, D. M.; Bruce, K.; P. Dand; RUSSELL, G.; H. Bradburn; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe011-A (RC-7) was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group t...

  19. Derivation of the human embryonic stem cell line RCe007-A (RC-3

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe007-A (RC-3 was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available.

  20. Derivation of the human embryonic stem cell line RCe007-A (RC-3)

    OpenAIRE

    P.A. De Sousa; B. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe007-A (RC-3) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available.

  1. Derivation of the human embryonic stem cell line RCe010-A (RC-6)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Collins, D. M.; H. Bradburn; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group...

  2. Derivation of the human embryonic stem cell line RCe014-A (RC-10)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Collins, D. M.; Greenshields, A.; H. Bradburn; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group ty...

  3. Derivation of the human embryonic stem cell line RCe008-A (RC-4)

    OpenAIRE

    P.A. De Sousa; B. Tye; Bruce, K.; P. Dand; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe008-A (RC-4) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is av...

  4. Human embryonic stem cells reveal recurrent genomic instability at 20q11.21.

    Science.gov (United States)

    Lefort, Nathalie; Feyeux, Maxime; Bas, Cécile; Féraud, Olivier; Bennaceur-Griscelli, Annelise; Tachdjian, Gerard; Peschanski, Marc; Perrier, Anselme L

    2008-12-01

    By analyzing five human embryonic stem (hES) cell lines over long-term culture, we identified a recurrent genomic instability in the human genome. An amplification of 2.5-4.6 Mb at 20q11.21, encompassing approximately 23 genes in common, was detected in four cell lines of different origins. This amplification, which has been associated with oncogenic transformation, may provide a selective advantage to hES cells in culture.

  5. Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells

    OpenAIRE

    Khalid, Omar; Kim, Jeffrey J.; Kim, Hyun-Sung; Hoang, Michael; Tu, Thanh G.; Elie, Omid; Lee, Connie; Vu, Catherine; Horvath, Steve; Spigelman, Igor; Kim, Yong

    2014-01-01

    Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs ...

  6. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    Science.gov (United States)

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  7. Human embryonic stem cells and respect for life

    OpenAIRE

    Meyer, J.(CERN, Geneva, Switzerland)

    2000-01-01

    The purpose of this essay is to stimulate academic discussion about the ethical justification of using human primordial stem cells for tissue transplantation, cell replacement, and gene therapy. There are intriguing alternatives to using embryos obtained from elective abortions and in vitro fertilisation to reconstitute damaged or dysfunctional human organs. These include the expansion and transplantation of latent adult progenitor cells.

  8. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

    2011-01-01

    We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  9. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chan-Jung Chang

    Full Text Available We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  10. Overlapping expression of microRNAs in human embryonic colon and colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    MicroRNAs (miRNAs) are essential for regulating cell differentiation and maintaining the pluripotent state of stem cells. Although dysregulation of specific miRNAs has been associated with certain types of cancer, to date no evidence has linked miRNA expression in embryonic and tumor tissues. We assessed the expression of mature miRNAs in human embryonic colon tissue, and in colorectal cancer and paired normal colon tissue. Overlapping miRNA expression was detected between embryonic colonic mucosa and colorectal cancer. We have found that the miR-17-92 cluster and its target, E2F1, exhibit a similar pattern of expression in human colon development and colonic carcinogenesis, regulating cell proliferation in both cases. In situ hybridization confirmed the high level of expression of miR-17-5p in the crypt progenitor compartment. We conclude that miRNA pathways play a major role in both embryonic development and neoplastic transformation of the colonic epithelium.

  11. Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Naoya; Yamada, Shigeru [Division of Pharmacology, National Institute of Health Sciences (Japan); Asanagi, Miki [Division of Pharmacology, National Institute of Health Sciences (Japan); Faculty of Engineering, Department of Materials Science and Engineering, Yokohama National University (Japan); Sekino, Yuko [Division of Pharmacology, National Institute of Health Sciences (Japan); Kanda, Yasunari, E-mail: kanda@nihs.go.jp [Division of Pharmacology, National Institute of Health Sciences (Japan)

    2016-02-05

    Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 μM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 μM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.

  12. Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions.

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    2006-01-01

    The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.

  13. Self-Organization of Spatial Patterning in Human Embryonic Stem Cells.

    Science.gov (United States)

    Deglincerti, Alessia; Etoc, Fred; Ozair, M Zeeshan; Brivanlou, Ali H

    2016-01-01

    The developing embryo is a remarkable example of self-organization, where functional units are created in a complex spatiotemporal choreography. Recently, human embryonic stem cells (ESCs) have been used to recapitulate in vitro the self-organization programs that are executed in the embryo in vivo. This represents an unique opportunity to address self-organization in humans that is otherwise not addressable with current technologies. In this chapter, we review the recent literature on self-organization of human ESCs, with a particular focus on two examples: formation of embryonic germ layers and neural rosettes. Intriguingly, both activation and elimination of TGFβ signaling can initiate self-organization, albeit with different molecular underpinnings. We discuss the mechanisms underlying the formation of these structures in vitro and explore future challenges in the field.

  14. Human embryonic stem cell research: implications from an ethical and legal standpoint.

    Science.gov (United States)

    Trepagnier, D M

    2000-12-01

    The purpose of this paper is to discuss the ethical and legal implications of one of the newest and most controversial medical breakthroughs. Stem cell research has been performed on mice for many years, but human embryonic stem cells are believed by scientists to be the basis for possible treatments and/or cures to many diseases affecting millions of people around the world. In order to perform research on human embryonic stem cells, numerous ethical issues must be addressed. Guidelines and protocols can be established in order to allow scientists to pursue new medical advances while maintaining the highest ethical standards in the use of human embryos. An alternative to using embryos is adult stem cells which have recently proven to be more versatile than previously believed. Opposing views will always be encountered when facing new science technologies. Where should the ethical line be drawn?

  15. The Lin28/Let-7 system in early human embryonic tissue and ectopic pregnancy.

    Directory of Open Access Journals (Sweden)

    Teresa Lozoya

    Full Text Available Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs, in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7-9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤ 6-weeks and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤ 6-weeks of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (deregulated pathways in the pathophysiology of ectopic pregnancy in humans.

  16. The Lin28/Let-7 system in early human embryonic tissue and ectopic pregnancy.

    Science.gov (United States)

    Lozoya, Teresa; Domínguez, Francisco; Romero-Ruiz, Antonio; Steffani, Liliana; Martínez, Sebastián; Monterde, Mercedes; Ferri, Blanca; Núñez, Maria Jose; AinhoaRomero-Espinós; Zamora, Omar; Gurrea, Marta; Sangiao-Alvarellos, Susana; Vega, Olivia; Simón, Carlos; Pellicer, Antonio; Tena-Sempere, Manuel

    2014-01-01

    Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7-9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤ 6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤ 6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.

  17. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

    Science.gov (United States)

    Postovit, Lynne-Marie; Margaryan, Naira V; Seftor, Elisabeth A; Kirschmann, Dawn A; Lipavsky, Alina; Wheaton, William W; Abbott, Daniel E; Seftor, Richard E B; Hendrix, Mary J C

    2008-03-18

    Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.

  18. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

  19. Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Lingping Kong; Lingzhi Wu; Jie Zhang; Yaping Liao; Huaqiao Wang

    2009-01-01

    BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown.OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (Aβ25-35).DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008.MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human cortical neurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies (Lab Vision,USA),and mouse anti-hTERT monoclonal antibody (Epitomics,USA),were used in this study.METHODS:(1) Recombinant adenovirus vectors,encoding hTERT (Ad-hTERT) and green fluorescent protein (Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days.Likewise,human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic cortical neurons in the control group were cultured as normal.(2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 μmol/L Aβ25-35 for 24 hours.Normal human embryonic cortical neurons treated with 10 μmol/L Aβ25-35 for 24 hours served as a model group.Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35.MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay

  20. Ethical questions concerning research on human embryos, embryonic stem cells and chimeras.

    Science.gov (United States)

    Bobbert, Monika

    2006-12-01

    Research using human embryos and embryonic stem cells is viewed as important for various reasons. Apart from questions concerning legal regulations, numerous ethical objections are raised pertaining to the use of surplus embryos from reproductive medicine as well as the creation of embryos and stem cells through cloning. In the hopes of avoiding ethical problems, alternatives have been proposed including the extraction of egg cells from "dead" embryos derived from in vitro fertilization procedures, the extraction of pluripotent stem cells from blastocysts, technologies such as "altered nuclear transfer" (ANT) and "oocyte-assisted reprogramming" (ANT-OAR) as well as parthenogenesis. Initial ethical assessments show that certain questions pertaining to such strategies have remained unanswered. Furthermore, with the help of new or more differentiated biotechnological procedures, it is possible to create chimeras and hybrids in which human and non-human cells are combined. Human-animal chimeras, in which gametes or embryonic tissue have been mixed with embryonic or adult stem cells, demonstrate a different "quality" and "degree of penetration" from those produced in previous experiments. Not only does this have consequences regarding questions of patentability, this situation also raises fundamental questions concerning the human being's self image, the concept of person, identity and species and the moral rights and duties that are connected with such concepts. There is a need for legal regulation, on the national as well as the international level.

  1. Cleft palate caused by congenital teratoma.

    Science.gov (United States)

    Veyssière, Alexis; Streit, Libor; Traoré, Hamady; Bénateau, Hervé

    2017-02-01

    A cleft palate results from incomplete fusion of the lateral palatine processes, the median nasal septum and the median palatine process. This case report describes a rare case of congenital teratoma originating from the nasal septum that may have interfered with the fusion of the palatal shelves during embryonic development, resulting in a cleft palate. An infant girl was born at 40 weeks of gestation weighing 3020 g with a complete cleft palate associated with a large central nasopharyngeal tumour. Computed tomography (CT) of the head showed a well defined mass of mixed density. The tumour was attached to the nasal septum in direct contact with the cleft palate. A biopsy confirmed the teratoma. Tumour resection was performed at 5 months, soft palate reconstruction at 7 months and hard palate closure at 14 months. There was no sign of local recurrence 1 year later. Most teratomas are benign and the prognosis is usually good. However, recurrence is not rare if germ cell carcinomatous foci are present within the teratoma. For these reasons, we advocate the use of a two-stage procedure in which closure of the cleft palate is postponed until histological examination confirms complete excision of the teratoma.

  2. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

    Science.gov (United States)

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.

    2011-01-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  3. Sequence characterization of a human embryonic craniofacial cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Padanilam, B.J.; Barsel, S.; Solursh, M. [and others

    1994-09-01

    Broad-based sequencing approaches for the characterization of human cDNA libraries have proven successful in identifying large numbers of novel genes of specific tissue or developmental stages. To pursue our interests in human craniofacial development, stages. To pursue our interests in human craniofacial development, we have made use of both subtracted and unsubtracted cDNA libraries constructed from embryonic craniofacial tissue obtained from pooled samples at 42-54 days gestation. Single-pass sequencing was carried out using an ABI automated sequencer and T3 or T7 primers. Sequences were characterized using BLAST and GRAIL, and the identified homologous sequences grouped according to gene class and family. Four genes have been mapped using repeat sequence elements identified in the clones. Using primers developed from sequence data, other genes are being mapped using a panel of somatic cell hybrids. To date, a total of 786 sequences have been returned with 35% identifying no homologies, and 35% with strong homologies to previously identified genes. A number of genes previously identified to play a role in human embryonic development have been returned from the sequence comparisons providing evidence that the library is representative of this tissue and stage of development. Previous characterization of the library has also identified a number of novel embryonically expressed human homeobox genes. Genes felt to be of special relevance based on their homology to characterized genes known to play a role in development or that are members of novel classes but with high scores on GRAIL searches are being characterized using whole mount in situ hybridization with mouse embryos. Characterization of the library with respect to chromosomal mapping, gene types and make-up, and embryonic expression patterns will be presented.

  4. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  5. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  6. Teratogen screening using transcriptome profiling of differentiating human embryonic stem cells.

    Science.gov (United States)

    Mayshar, Yoav; Yanuka, Ofra; Benvenisty, Nissim

    2011-06-01

    Teratogens are substances that may cause defects in normal embryonic development while not necessarily being toxic in adults. Identification of possible teratogenic compounds has been historically beset by the species-specific nature of the teratogen response. To examine teratogenic effects on early human development we performed non-biased expression profiling of differentiating human embryonic and induced pluripotent stem cells treated with several drugs--ethanol, lithium, retinoic acid (RA), caffeine and thalidomide, which is known to be highly species specific. Our results point to the potency of specific teratogens and their affected tissues and pathways. Specifically, we could show that ethanol caused dramatic increase in endodermal differentiation, RA caused misregulation of neural development and thalidomide affected both these processes. We thus propose this method as a valuable addition to currently available animal screening approaches.

  7. Embryonic origins of human vascular smooth muscle cells: implications for in vitro modeling and clinical application.

    Science.gov (United States)

    Sinha, Sanjay; Iyer, Dharini; Granata, Alessandra

    2014-06-01

    Vascular smooth muscle cells (SMCs) arise from multiple origins during development, raising the possibility that differences in embryological origins between SMCs could contribute to site-specific localization of vascular diseases. In this review, we first examine the developmental pathways and embryological origins of vascular SMCs and then discuss in vitro strategies for deriving SMCs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We then review in detail the potential for vascular disease modeling using iPSC-derived SMCs and consider the pathological implications of heterogeneous embryonic origins. Finally, we touch upon the role of human ESC-derived SMCs in therapeutic revascularization and the challenges remaining before regenerative medicine using ESC- or iPSC-derived cells comes of age.

  8. Dielectrophoretic study of human embryonic stem cells and their differentiated progeny

    OpenAIRE

    Velugotla, Srinivas

    2013-01-01

    This thesis describes for the first time, how the membrane capacitance of pluripotent human embryonic stem cells (H1, H9, RCM1) increases with their differentiation (H1-MSC, H9-MSC, RCM1-trophoblast) based on the literature review. The method used to determine membrane capacitance was dielectrophoresis (DEP), which is an electrokinetic technique capable of characterising and sorting cells without the need for antibody-based cell surface markers, magnetic beads, or other chemica...

  9. Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases

    Directory of Open Access Journals (Sweden)

    Piya Prajumwongs

    2016-01-01

    Full Text Available Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation.

  10. Generation of KCL033 clinical grade human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Liani Devito

    2016-03-01

    Full Text Available The KCL033 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility and specific and non-specific human pathogens.

  11. Ablation of the Sox11 Gene Results in Clefting of the Secondary Palate Resembling the Pierre Robin Sequence.

    Science.gov (United States)

    Huang, Huarong; Yang, Xiaojuan; Bao, Meiling; Cao, Huanhuan; Miao, Xiaoping; Zhang, Xiaoyun; Gan, Lin; Qiu, Mengsheng; Zhang, Zunyi

    2016-03-25

    Mouse gene inactivation has shown that the transcription factor Sox11 is required for mouse palatogenesis. However, whether Sox11 is primarily involved in the regulation of palatogenesis still remains elusive. In this study, we explored the role ofSox11in palatogenesis by analyzing the developmental mechanism in cleft palate formation in mutants deficient in Sox11. Sox11 is expressed both in the developing palatal shelf and in the surrounding structures, including the mandible. We found that cleft palate occurs only in the mutant in which Sox11is directly deleted. As in the wild type, the palatal shelves in the Sox11 mutant undergo outgrowth in a downward direction and exhibit potential for fusion and elevation. However, mutant palatal shelves encounter clefting, which is associated with a malpositioned tongue that results in physical obstruction of palatal shelf elevation at embryonic day 14.5 (E14.5). We found that loss of Sox11led to reduced cell proliferation in the developing mandibular mesenchyme via Cyclin D1, leading to mandibular hypoplasia, which blocks tongue descent. Extensive analyses of gene expression inSox11 deficiency identified FGF9 as a potential candidate target of Sox11 in the modulation of cell proliferation both in the mandible and the palatal shelf between E12.5 and E13.5. Finally we show, using in vitro assays, that Sox11 directly regulates the expression of Fgf9 and that application of FGF9 protein to Sox11-deficient palatal shelves restores the rate of BrdU incorporation. Taken together, the palate defects presented in the Sox11 loss mutant mimic the clefting in the Pierre Robin sequence in humans.

  12. The culture of human embryonic stem cells in microchannel perfusion bioreactors

    Science.gov (United States)

    Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit

    2007-12-01

    The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.

  13. Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells*

    Institute of Scientific and Technical Information of China (English)

    Yan Zhang; Junmei Zhou; Zhenfu Fang; Manxi Jiang; Xuejin Chen

    2013-01-01

    The difference between Noggin and basic fibroblast growth factor for the neural precursor differen-tiation from human embryonic stem cel s has not been studied. In this study, 100 µg/L Noggin or 20 µg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen-tiate human embryonic stem cel s H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro-scope. Immunofluorescence staining revealed expression levels of Nestin,β-III Tubulin and Sox-1 were higher in the induced cel s and reverse-transcription PCR showed induced cel s expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cel differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in-creases the differentiation of neural precursors.

  14. Integration of immunological aspects in the European Human Embryonic Stem Cell Registry.

    Science.gov (United States)

    Borstlap, Joeri; Kurtz, Andreas

    2008-05-01

    The immunological properties of stem cells are of increasing importance in regenerative medicine. Immunomodulatory mechanisms seem to play an important role not only with respect to the understanding of underlying mechanisms of autologous versus allogenic therapeutic approaches, but also for endogeneous tissue regeneration. The newly established European human embryonic stem cell registry (hESCreg) offers an international database for the registration, documentation and characterisation of human embryonic stem cells (hESC) and their use. By doing so, hESCreg aims to develop a model procedure for further standardisation efforts in the field of stem cell research and regenerative medicine, and eventually the registry may lead to a repository of therapy-related information. Currently the stem cell characterisation data acquired by the registry are divided into several categories such as cell derivation, culture conditions, genetic constitution, stem cell marker expression and degree of modification. This article describes immunological aspects of stem cell characterisation and explores the layout and relevance of a possible additional section to the hESCreg repository to include immunological characteristics of human embryonic stem cells.

  15. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  16. Three-dimensional morphology of the human embryonic brain

    Directory of Open Access Journals (Sweden)

    N. Shiraishi

    2015-09-01

    Full Text Available The morphogenesis of the cerebral vesicles and ventricles was visualized in 3D movies using images derived from human embryo specimens between Carnegie stage 13 and 23 from the Kyoto Collection. These images were acquired with a magnetic resonance microscope equipped with a 2.35-T superconducting magnet. Three-dimensional images using the same scale demonstrated brain development and growth effectively. The non-uniform thickness of the brain tissue, which may indicate brain differentiation, was visualized with thickness-based surface color mapping. A closer view was obtained of the unique and complicated differentiation of the rhombencephalon, especially with regard to the internal view and thickening of the brain tissue. The present data contribute to a better understanding of brain and cerebral ventricle development.

  17. Developmental epigenetics of the murine secondary palate.

    Science.gov (United States)

    Seelan, Ratnam S; Mukhopadhyay, Partha; Pisano, M Michele; Greene, Robert M

    2012-01-01

    Orofacial clefts occur with a frequency of 1 to 2 per 1000 live births. Cleft palate, which accounts for 30% of orofacial clefts, is caused by the failure of the secondary palatal processes--medially directed, oral projections of the paired embryonic maxillary processes--to fuse. Both gene mutations and environmental effects contribute to the complex etiology of this disorder. Although much progress has been made in identifying genes whose mutations are associated with cleft palate, little is known about the mechanisms by which the environment adversely influences gene expression during secondary palate development. An increasing body of evidence, however, implicates epigenetic processes as playing a role in adversely influencing orofacial development. Epigenetics refers to inherited changes in phenotype or gene expression caused by processes other than changes in the underlying DNA sequence. Such processes include, but are not limited to, DNA methylation, microRNA effects, and histone modifications that alter chromatin conformation. In this review, we describe our current understanding of the possible role epigenetics may play during development of the secondary palate. Specifically, we present the salient features of the embryonic palatal methylome and profile the expression of numerous microRNAs that regulate protein-encoding genes crucial to normal orofacial ontogeny.

  18. Shrink-film configurable multiscale wrinkles for functional alignment of human embryonic stem cells and their cardiac derivatives.

    Science.gov (United States)

    Chen, Aaron; Lieu, Deborah K; Freschauf, Lauren; Lew, Valerie; Sharma, Himanshu; Wang, Jiaxian; Nguyen, Diep; Karakikes, Ioannis; Hajjar, Roger J; Gopinathan, Ajay; Botvinick, Elliot; Fowlkes, Charless C; Li, Ronald A; Khine, Michelle

    2011-12-22

    A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.

  19. In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis.

    Science.gov (United States)

    Vanhee, Stijn; De Mulder, Katrien; Van Caeneghem, Yasmine; Verstichel, Greet; Van Roy, Nadine; Menten, Björn; Velghe, Imke; Philippé, Jan; De Bleser, Dominique; Lambrecht, Bart N; Taghon, Tom; Leclercq, Georges; Kerre, Tessa; Vandekerckhove, Bart

    2015-02-01

    Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.

  20. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors

    DEFF Research Database (Denmark)

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have...... analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors....

  1. Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

  2. Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells.

    Science.gov (United States)

    Dingwall, Steve; Lee, Jung Bok; Guezguez, Borhane; Fiebig, Aline; McNicol, Jamie; Boreham, Douglas; Collins, Tony J; Bhatia, Mick

    2015-09-08

    Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs.

  3. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

    Science.gov (United States)

    Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

    2010-08-06

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

  4. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    Science.gov (United States)

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

  5. Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells.

    Science.gov (United States)

    Meganathan, Kesavan; Jagtap, Smita; Wagh, Vilas; Winkler, Johannes; Gaspar, John Antonydas; Hildebrand, Diana; Trusch, Maria; Lehmann, Karola; Hescheler, Jürgen; Schlüter, Hartmut; Sachinidis, Agapios

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

  6. Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kesavan Meganathan

    Full Text Available Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs. Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE coupled with Tandem Mass spectrometry to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s. Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3 after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2, that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

  7. Osteopontin, osteocalcin, and osteoprotegerin expression in human tissue affected by cleft lip and palate

    Directory of Open Access Journals (Sweden)

    Smane L.

    2016-01-01

    Full Text Available Cleft lip and palate (CLP is a common congenital anomaly with a complex etiology which has not been elucidated yet. This study investigated whether expression of osteopontin (OPN, osteoprotegerin (OPG, and osteocalcin (OC, which are essential for the normal craniofacial bone remodelling, is not regulated in children with CLP. Alveolar bone tissue samples were obtained from patients with complete bilateral (CB CLP (n = 14 during corrective plastic surgery and unaffected control subjects (n = 9. OPN, OPG, and OC expression was assessed by immunohistochemistry, and data were analyzed with the Mann-Whitney test. OPN expression was observed only sporadically in the alveolar bone of 3 patients, in contrast to the control group (z = −2.962; P < 0.003. The number of OPG-positive bone cells varied from occasional to moderate, in contrast to the control group (z = −2.247; P = 0.025. OC-positive osteocytes were present in moderate to numerous numbers in both patients and controls, with no significant difference between them (z = −1.356; P < 0.175. The prominent expression of OC characteristic for CBCLP affected hard tissue indicates a high potential of bone mineralization. Few OPG-positive osteocytes in the bone tissue implicate the disregulation of osteoclast differentiation, maturation, and activity, but few OPN-containing cells may prove the common disregulation of bone remodelling during cleft morphopathogenesis.

  8. Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.

    Science.gov (United States)

    Braam, Stefan R; Denning, Chris; Matsa, Elena; Young, Lorraine E; Passier, Robert; Mummery, Christine L

    2008-01-01

    Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fibroblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.

  9. Dopaminergic Differentiation of Human Embryonic Stem Cells on PA6-Derived Adipocytes.

    Science.gov (United States)

    Guloglu, M Oktar; Larsen, Anna

    2016-01-01

    Human embryonic stem cells (hESCs) are a promising source for cell replacement therapies. Parkinson's disease is one of the candidate diseases for the cell replacement therapy since the motor manifestations of the disease are associated with the loss of dopaminergic neurons in the substantia nigra pars compacta. Stromal cell-derived inducing activity (SDIA) is the most commonly used method for the dopaminergic differentiation of hESCs. This chapter describes a simple, reliable, and scalable dopaminergic induction method of hESCs using PA6-derived adipocytes. Coculturing hESCs with PA6-derived adipocytes markedly reduces the variable outcomes among experiments. Moreover, the colony differentiation step of this method can also be used for the dopaminergic induction of mouse embryonic stem cells and NTERA2 cells as well.

  10. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

    Institute of Scientific and Technical Information of China (English)

    YING CHEN; QING ZHANG YANG; DA YUAN CHEN; MIN KANG WANG; JIN SONG LI; SHAO LIANG HUANG; XIANG YIN KONG; YAO ZHOU SHI; ZHI QIANG WANG; JIA HUI XIA; ZHI GAO LONG; ZHI XU HE; ZHI GANG XUE; WEN XIANG DING; HUI ZHEN SHENG; AILIAN LIU; KAI WANG; WEN WEI MAO; JIAN XIN CHU; YONG LU; ZHENG FU FANG; YING TANG SHI

    2003-01-01

    To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

  11. Derivation of the human embryonic stem cell line RCe006-A (RC-2

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

  12. Derivation of the human embryonic stem cell line RCe014-A (RC-10

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  13. Derivation of the human embryonic stem cell line RCe012-A (RC-8

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  14. Differentiation of the nuclear groups in the posterior horn of the human embryonic spinal cord.

    Science.gov (United States)

    Pytel, A; Bruska, M; Woźniak, W

    2011-11-01

    The formation of nuclear groups in the posterior horns of the human embryonic spinal cord was traced in serial sections of embryos of developmental stages 13 to 23 (32 to 56 postovulatory days). The following observations, new for the human, are presented: 1. The differentiation of the neural tube into 3 zones (germinal, mantle and marginal) is detected in the middle of the 5(th) week. 2. The primordia of the posterior horns are marked at stage 14 (33 days). 3. In the middle of the 7(th) week the nucleus proprius and substantia gelatinosa are discerned. 4. Differentiation of the nuclei within the posterior horns proceeds in the ventrodorsal and rostrocaudal gradients.

  15. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

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    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  16. Derivation of the human embryonic stem cell line RCe011-A (RC-7

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe011-A (RC-7 was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  17. Derivation of the human embryonic stem cell line RCe010-A (RC-6

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  18. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1–60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  19. Derivation of Trisomy 21 affected human embryonic stem cell line Genea053

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea053 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analysis demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology and expressed pluripotent cell markers including 83% Nanog positive, 87% Oct4, 88% Tra1-60 and 98% SSEA4. The cell line was negative for Mycoplasma and visible contamination.

  20. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...... collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.......Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts...... to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned...

  1. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells.

    Science.gov (United States)

    Akopian, Veronika; Andrews, Peter W; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B; Ludwig, Tenneille E; McKay, Ronald D G; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K W; Pera, Martin F; Rossant, Janet; Stacey, Glyn N; Suemori, Hirofumi

    2010-04-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.

  2. Molecular Mechanisms Regulating Impaired Neurogenesis of Fragile X Syndrome Human Embryonic Stem Cells

    Science.gov (United States)

    Telias, Michael; Mayshar, Yoav; Amit, Ami

    2015-01-01

    Fragile X syndrome (FXS) is the most common form of inherited cognitive impairment. It is caused by developmental inactivation of the FMR1 gene and the absence of its encoded protein FMRP, which plays pivotal roles in brain development and function. In FXS embryos with full FMR1 mutation, FMRP is expressed during early embryogenesis and is gradually downregulated at the third trimester of pregnancy. FX-human embryonic stem cells (FX-hESCs), derived from FX human blastocysts, demonstrate the same pattern of developmentally regulated FMR1 inactivation when subjected to in vitro neural differentiation (IVND). In this study, we used this in vitro human platform to explore the molecular mechanisms downstream to FMRP in the context of early human embryonic neurogenesis. Our results show a novel role for the SOX superfamily of transcription factors, specifically for SOX2 and SOX9, which could explain the reduced and delayed neurogenesis observed in FX cells. In addition, we assess in this study the “GSK3β theory of FXS” for the first time in a human-based model. We found no evidence for a pathological increase in GSK3β protein levels upon cellular loss of FMRP, in contrast to what was found in the brain of Fmr1 knockout mice. Our study adds novel data on potential downstream targets of FMRP and highlights the importance of the FX-hESC IVND system. PMID:26393806

  3. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    Science.gov (United States)

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  4. Ethical consideration of experimentation using living human embryos: the Catholic Church's position on human embryonic stem cell research and human cloning.

    Science.gov (United States)

    Ohara, N

    2003-01-01

    Although the potential applications of human embryonic stem cells and therapeutic cloning hold promise for the alleged medical benefits, these technologies have posed profound ethical issues because they necessitate the destruction of human embryos. A fundamental point in the issues is the concept of the moral status of human embryos. The Catholic Church has held that human life begins at the moment of conception and therefore, has defended the dignity, inviolable right to life and integrity of human embryos. The Catholic Church has opposed human embryonic stem cell research and any kind of human cloning because they are contrary to the dignity of procreation, of conjugal union and of human embryos. Moreover, these techniques have the risk of creating a sub-category of human beings that are destined basically for the convenience of others. In conclusion, science and technology can never be independent of the criterion of morality, since technology exists for man and must respect his finality.

  5. The Effect of Low Level Laser Irradiation on Human Embryonic Stem Cells

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    Hossein Baharvand

    2005-01-01

    Full Text Available Introduction: Different effects of low level laser irradiation (LLLI on various cell types have already been demonstrated. However, its effects on embryonic stem cells have not yet been shown. The present study evaluates the morphological and immunocytochemical effects of LLLI on human embryonic stem cell (hESC colonies. Material and Methods: Equal-sized pieces of hESC line (Royan H1 were irradiated with a single dose of 830-nm Ga-Al-As diode laser (3, 5, and 8 jcm-2, 30mW and cultured on mouse embryonic fibroblasts. The morphology of the colonies was evaluated qualitatively by observation under an inverted microscope (grades A, B, C, and D exhibited 0-30%, 30-50%, 50-80%, and 80-100% differentiation, respectively. The stemness area was assessed by expression of surface antigens using anti-Tra-1-60 and anti-Tra-1-81. Results: Our data demonstrated a dose-dependent stimulatory effect of LLLI on hESC differentiation. Two doses of 5 and 8jcm-2 induced statistically significant differentiation (grades C and D. Conclusions: These data showed that LLLI influenced hESC differentiation, which might be used for cell therapy after transplantation

  6. Derivation and characterization of human embryonic germ cells: serum-free culture and differentiation potential.

    Science.gov (United States)

    Hua, Jinlian; Yu, Haisheng; Liu, Sheng; Dou, Zhongying; Sun, Yadong; Jing, Xiaoqi; Yang, Chunrong; Lei, Anmin; Wang, Huayan; Gao, Zhimin

    2009-08-01

    This study examined the effects of a chemically defined culture medium supplement, knock-out serum replacement (KSR), on the growth and differentiation of human embryonic germ cells (hEgc) and found that the efficiency of the initial establishment of hEGC lines in KSR medium was significantly higher than in fetal calf serum (FCS) medium. The percentage of undifferentiated hEGC colonies growing in KSR medium was significantly higher than in FCS-based medium (P embryonic germ cell-like morphology. They showed normal and stable diploid karyotype and expressed alkaline phosphatase (AP), stage-specific embryonic antigens (SSEA) and other specific markers of pluripotent cells. In addition, hEGC could form simple and cystic embryoid bodies (EB) that consisted of various cell types including neural, epithelial and rhythmically beating cardiac cells, even sperm-like and oocyte-like cells. Tumour-like outgrowths were formed in nude mice and found to contain a variety of cell types, including uterine epithelium, adipocytes, squamous tissue and skin structures. In conclusion, an appropriate serum-free culture system has been developed for the establishment of hEGC lines. This may provide an in-vitro model to study differentiation and can be used as a potential source of therapy for infertility and regenerative medicine.

  7. Increased expression of the matrix metalloproteinase 2 in differentiating Tera 2 human embryonal carcinoma cells.

    Science.gov (United States)

    Tienari, J; Pertovaara, L; Saksela, O; Lehtonen, E; Vartio, T

    1994-01-15

    Secretion of proteolytic enzymes by cells has been implicated in tissue remodeling during embryonic development as well as in invasive neoplastic diseases. We studied the regulation of type-IV-collagenase activity in Tera 2 human embryonal carcinoma cells, which in the undifferentiated state proliferate rapidly and are tumorigenic. The undifferentiated cells produced relatively low levels of matrix-metalloproteinase-2 (MMP-2) activity. This activity was not markedly affected by exogenous basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though the plasminogen activator activity of the cells was increased by these agents. Tera 2 cells can be induced by retinoic acid to differentiate into quiescent cells, of which many express neuronal characteristics. The type-IV-collagenase activity of the cells increased markedly during the differentiation. This increase was mainly due to increased expression of MMP-2. Expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was not markedly affected by the differentiation of Tera 2 cells. The results show that in the Tera 2 cell system, increased expression of MMP-2 is characteristic of the differentiated derivatives. This is in contrast with many other model systems, where increased type-IV-collagenase activity is associated with the malignant phenotype. This pattern of regulation may reflect the facts that Tera 2 cells resemble early embryonic cells and that their differentiation mimics related cell-differentiation processes in the developing embryo.

  8. Derivation of Neural Precursor Cells from Human Embryonic Stem Cells for DNA Methylomic Analysis.

    Science.gov (United States)

    Roubal, Ivan; Park, Sun Joo; Kim, Yong

    2016-01-01

    Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application.

  9. Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2015-01-01

    Full Text Available Human embryonic stem cells (hESCs are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs. hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

  10. A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells.

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    Shelley R Hough

    Full Text Available BACKGROUND: Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment. METHODOLOGY/PRINCIPAL FINDINGS: We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3. SIGNIFICANCE: These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency.

  11. Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

    Institute of Scientific and Technical Information of China (English)

    Qiangzhe Zhang; Li Chen; Tian Tian; Xin Wang; Pu Li; Jurgen Hescheler; Guangju Ji; Yue Ma; Junjie Jiang; Pengcheng Han; Qi Yuan; Jing Zhang; Xiaoqian Zhang; Yanyan Xu; Henghua Cao; Qingzhang Meng

    2011-01-01

    Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs)are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the panretinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, hut not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiologicai studies indicated that with 64.7 ± 0.88% (mean ± s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca2+ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin

  12. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    Science.gov (United States)

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  13. Molecular characterisation of stromal populations derived from human embryonic stem cells

    DEFF Research Database (Denmark)

    Harkness, L.; Twine, N. A.; Abu Dawud, R.;

    2015-01-01

    Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide an un......ESC-stromal cells can thus be considered as a possible alternative candidate cells for hMSC, to be employed in regenerative medicine protocols.......Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide...... an unlimited source of clinical grade cells for therapy. We have generated MSC-like cells from hESC (called here hESC-stromal) that exhibit surface markers and differentiate to osteoblasts and adipocytes, similar to BM-hMSC. In the present study, we used microarray analysis to compare the molecular phenotype...

  14. An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.

    Science.gov (United States)

    Zhou, Jun-Mei; Chu, Jian-Xin; Chen, Xue-Jin

    2008-01-01

    Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.

  15. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency

    Directory of Open Access Journals (Sweden)

    A. Fontán-Lozano

    2016-05-01

    Full Text Available Little is known about the functions of downstream regulatory element antagonist modulator (DREAM in embryonic stem cells (ESCs. However, DREAM interacts with cAMP response element-binding protein (CREB in a Ca2+-dependent manner, preventing CREB binding protein (CBP recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs, human adipose-derived stromal cells (hASCs, human bone marrow-derived stromal cells (hBMSCs, and human newborn foreskin fibroblasts (hFFs, and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

  16. Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

    Science.gov (United States)

    Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

    2010-10-01

    Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.

  17. Two human homeobox genes, c1 and c8: structure analysis and expression in embryonic development.

    Science.gov (United States)

    Simeone, A; Mavilio, F; Acampora, D; Giampaolo, A; Faiella, A; Zappavigna, V; D'Esposito, M; Pannese, M; Russo, G; Boncinelli, E

    1987-07-01

    Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hydridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embryos and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.

  18. Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells

    Science.gov (United States)

    Panula, Sarita; Reda, Ahmed; Stukenborg, Jan-Bernd; Ramathal, Cyril; Sukhwani, Meena; Albalushi, Halima; Edsgärd, Daniel; Nakamura, Michiko; Söder, Olle; Orwig, Kyle E.; Yamanaka, Shinya; Reijo Pera, Renee A.; Hovatta, Outi

    2016-01-01

    The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. PMID:27768780

  19. Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

    Science.gov (United States)

    2014-10-01

    SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making Improve...to determine whether Fetal Mammary Stem Cell (fMaSC) signatures correlate with response to chemotherapy and metastasis in different breast cancer...positioned to achieve its aims. 15. SUBJECT TERMS Breast Cancer Prognosis, Mammary Stem Cells, Embryonic Development, Single Cell Transcriptomics 16

  20. Site-Specific Expression of Gelatinolytic Activity during Morphogenesis of the Secondary Palate in the Mouse Embryo

    Science.gov (United States)

    Gkantidis, Nikolaos; Blumer, Susan; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx. PMID:23091646

  1. Site-specific expression of gelatinolytic activity during morphogenesis of the secondary palate in the mouse embryo.

    Directory of Open Access Journals (Sweden)

    Nikolaos Gkantidis

    Full Text Available Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs. As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5, we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP, but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx.

  2. Laser-induced fusion of human embryonic stem cells with optical tweezers

    Energy Technology Data Exchange (ETDEWEB)

    Chen Shuxun; Wang Xiaolin; Sun Dong [Department of Mechanical and Biomedical Engineering, City University of Hong Kong (Hong Kong); Cheng Jinping; Han Cheng, Shuk [Department of Biology and Chemistry, City University of Hong Kong (Hong Kong); Kong, Chi-Wing [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Li, Ronald A. [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Center of Cardiovascular Research, Mount Sinai School of Medicine, New York, New York 10029 (United States)

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  3. Derivation of the clinical grade human embryonic stem cell line RCe013-A (RC-9

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe013-A (RC-9 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following informed consent. RCe013-A (RC-9 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  4. Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe016-A (RC-12 was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12 shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15, which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

  5. Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe019-A (RC-15 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  6. Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe015-A (RC-11 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  7. Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe021-A (RC-17 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  8. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  9. Laser-induced fusion of human embryonic stem cells with optical tweezers

    Science.gov (United States)

    Chen, Shuxun; Cheng, Jinping; Kong, Chi-Wing; Wang, Xiaolin; Han Cheng, Shuk; Li, Ronald A.; Sun, Dong

    2013-07-01

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  10. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    Science.gov (United States)

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.

  11. Therapeutic potential of human embryonic stem cell transplantation in patients with cerebral palsy

    OpenAIRE

    Shroff, Geeta; Gupta, Anupama; Barthakur, Jitender Kumar

    2014-01-01

    Background The present study evaluated the efficacy and safety of human embryonic stem cell (hESC) therapy in patients with CP. Materials and methods This analysis included patients (30 days-18 yr) with documented diagnosis of CP. The study consisted of four treatment phases (T1, T2, T3, T4) separated by gap phases. Efficacy of hESC therapy was evaluated based on Gross Motor Function Classification Scores Expanded and Revised (GMFCS-E & R; 1-good to 5-bad). Results Ninety one patients were in...

  12. Early embryonic chromosome instability results in stable mosaic pattern in human tissues.

    Directory of Open Access Journals (Sweden)

    Hasmik Mkrtchyan

    Full Text Available The discovery of copy number variations (CNV in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.

  13. HLA class I homologous transcripts in the human embryonal carcinoma cell line Tera-2.

    Science.gov (United States)

    Rinke de Wit, T F; Struyk, L; Vloemans, S; Glazebrook, J; Boyle, J M; Stern, P L; van den Elsen, P J

    1990-01-01

    We have used the human teratocarcinoma-derived embryonal carcinoma cell line Tera-2 cl. 13 to explore the putative expression of novel HLA class I(-like) genes. Serological analyses revealed that Tera-2 cells do not express polymorphic HLA class I (-A, -B, -C) specificities, but do express HLA class I-like antigens. These phenotypic properties parallel those of certain mouse embryonal carcinoma cells. To study the expression of HLA class I(-like) genes in the Tera-2 cells two different approaches were used. Screening of a Tera-2 cDNA library with a full-length HLA class I cDNA probe under conditions that would allow for the identification of relatively distinct HLA class I-like sequences yielded 27 positive clones, all of which were of the regular HLA-A, -B, -C type. Reverse northern hybridizations of the restriction enzyme-digested Tlab region comprising cosmids with Tera-2 cDNA as the probe resulted in the identification of several putative human genes whose equivalents map within the mouse Tla region. However, none of these genes appeared to be structurally related to HLA class I. A putative H3.3 histone gene was identified in the proximal Tla region of the C57BL/10 mouse. It is concluded that no structural homologues of mouse Qa/Tla genes are expressed in the human developmental cell line Tera-2.

  14. Future Implications of Human Embryonic Testing and Modification: Great Medicine or GATTACA?

    Science.gov (United States)

    Brezina, Paul R; Marinelli, Enrico; Bailey, Amelia P

    2016-01-01

    The past several decades have seen tremendous advances in the field of medical genetics. Currently, the application of genetic testing on human embryos determines if embryos harbor a lethal condition or a serious genetic disease. The purpose of this sort of testing is not to "improve" the offspring of a couple. Rather, current testing strategies focus on helping couples to have a healthy family in an efficient manner. Newly emerging technologies have opened the door to test embryos for an exponentially growing number of traits. Additionally, recent reports describe the actual modification of human embryonic DNA. The implications from the application of this technology are many and have the potential to fundamentally change the social paradigm of the human experience. Embryonic testing and modification does have the potential to accomplish good and is not inherently amoral. However, thoughtful consideration should be given by scientists, legislators, and the general population on how to apply this technology in a manner that is both appropriate and equitable and does not result in further social stratification and polarization, both within individual nations and the global community.

  15. Ethical aspects of human embryonic stem cell research in the islamic world: positions and reflections.

    Science.gov (United States)

    Ilkilic, Ilhan; Ertin, Hakan

    2010-06-01

    Rapid technological developments in human embryonic stem cell research are holding promises of future new medical treatment for a range of currently incurable chronic diseases. At the same time, stem cell research using human embryos raises radically new, previously unimaginable ethical issues posing a dramatic challenge to humankind. By analysing the discourses on these ethical issues we can show that the cultural values and religious convictions of all stakeholders involved play a decisive role in formulating ethical positions. In the Islamic world, too, stem cell research using human embryos provokes new discussions about the moral status of the embryo according to Islamic ethical norms. In our paper we describe the theological and philosophical criteria used in this debate and discuss some ethical positions vis-à-vis embryonic stem cell research formulated in the Islamic world, including official regulations existing in some Muslim countries. While most of the existing literature in this field is primarily descriptive, the present paper endeavours to examine not only the arguments and their historical conditions as such; in addition, we will for the first time provide a critical reflection on the methodology underlying commonly held positions. In our view, this reflection is of paramount importance in establishing a straightforward constructive dialogue between different cultures and academic disciplines.

  16. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.

    Science.gov (United States)

    Mummery, Christine L; Zhang, Jianhua; Ng, Elizabeth S; Elliott, David A; Elefanty, Andrew G; Kamp, Timothy J

    2012-07-20

    Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

  17. Embryonic atrazine exposure alters zebrafish and human miRNAs associated with angiogenesis, cancer, and neurodevelopment.

    Science.gov (United States)

    Wirbisky, Sara E; Weber, Gregory J; Schlotman, Kelly E; Sepúlveda, Maria S; Freeman, Jennifer L

    2016-12-01

    MicroRNAs (miRNAs) are short, single-stranded RNA that regulate post-transcriptional control of mRNA translation. Knowledge on the role of these critical regulators in toxicological responses in increasing, but is still limited. Atrazine is a herbicide used throughout the Midwestern US that is reported to frequently contaminate potable water supplies above the maximum contaminant level of 3 parts per billion. Atrazine is a suspected endocrine disrupting chemical and studies have begun to investigate the genetic mechanisms of toxicity; however, studies investigating epigenetic mechanisms are limited. In this study both zebrafish and human miRNAs were significantly altered in response to an embryonic atrazine exposure of 0.3, 3, or 30 ppb in zebrafish. Altered miRNAs are known to play a role in angiogenesis, cancer, or neuronal development, differentiation, and maturation. Targeted analysis of altered human miRNAs with genes previously identified to be altered by atrazine exposure revealed several targets linked to cell cycle and cell signaling. Further analysis of hsa-miRNA-126-3p, which had altered expression in all three atrazine treatments at 72 hpf, revealed alterations also occurred at 60 hpf in the 30 ppb treatment group. Results from this study indicate miRNA deregulation in zebrafish and human miRNAs following an embryonic atrazine exposure in zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  19. Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng; SHEN Huan; JIANG Wei; SONG Zhi-hua; WANG Cheng-yan; WEI Li-hui

    2011-01-01

    Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background,which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use,especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.Methods Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.Results We generated a new Chinese human embryonic stem cells line,CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines:normal morphology,karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.Conclusions This newly established Chinese cell line,CH1,which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells,will provide

  20. Global expression profile of highly enriched cardiomyocytes derived from human embryonic stem cells.

    Science.gov (United States)

    Xu, Xiu Qin; Soo, Set Yen; Sun, William; Zweigerdt, Robert

    2009-09-01

    Human embryonic stem cells (hESC), with their ability to differentiate into cardiomyocytes in culture, hold great potential for cell replacement therapies and provide an in vitro model of human heart development. A genomewide characterization of the molecular phenotype of hESC-derived cardiomyocytes is important for their envisioned applications. We have employed a lineage selection strategy to generate a pure population of cardiomyocytes (>99%) from transgenic hESC lines. Global gene expression profiling showed that these cardiomyocytes are distinct from pluripotent and differentiated hESC cultures. Pure cardiomyocytes displayed similarities with heart tissue, but in many aspects presented an individual transcriptome pattern. A subset of 1,311 cardiac-enriched transcripts was identified, which were significantly overpresented (p human heart development.

  1. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Jared Carlson-Stevermer

    2016-01-01

    Full Text Available CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  2. Human life, media and market: a sociotechnical perspective of research with embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Júlio Cesar de Almeida Nobre

    2014-04-01

    Full Text Available New biotechnologies of reproduction emerge nowadays as radical destabilizing agents of what we understand as human life. Within this context, research involving stem cells are on the agenda of several academic articles and major newspapers and magazines around the world. The arguments vary from a sort of pride about the emancipatory power of technology to a fear about the absence of limits to restrict undesirable advances. Amid such controversies, our proposal is, based on Actor Network Theory, to draw a map of networks that produce what we understand as human life, insofar as it seems to be unstabled by the new biotechnologies of reproduction. We followed different mediators along their deviations, in order to produce a report based on the analysis of current bioethical controversies involving, specifically, embryonic stem cell research. These controversies led to a strong connection between media, new biotechnologies of human reproduction and a market logic.

  3. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

    Science.gov (United States)

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-12

    CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  4. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  5. The evolution of lineage-specific regulatory activities in the human embryonic limb.

    Science.gov (United States)

    Cotney, Justin; Leng, Jing; Yin, Jun; Reilly, Steven K; DeMare, Laura E; Emera, Deena; Ayoub, Albert E; Rakic, Pasko; Noonan, James P

    2013-07-03

    The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution.

  6. Cis-regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs.

    Science.gov (United States)

    Abbasi, Amir A; Minhas, Rashid; Schmidt, Ansgar; Koch, Sabine; Grzeschik, Karl-Heinz

    2013-10-01

    The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs.

  7. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  8. The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts.

    Science.gov (United States)

    Tzukerman, Maty; Rosenberg, Tzur; Reiter, Irena; Ben-Eliezer, Shoshana; Denkberg, Galit; Coleman, Raymond; Reiter, Yoram; Skorecki, Karl

    2006-04-01

    The awareness of the important role that the surrounding tissue microenvironment and stromal response play in the process of tumorigenesis has grown as a result of in vivo models of tumor xenograft growth in immunocompromised mice. In the current study, we used human embryonic stem cells in order to study the interactions of tumor cells with the surrounding microenvironment of differentiated human cell tissues and structures. Several cancer cell types stably expressing an H2A-green fluorescence protein fusion protein, which allowed tracking of tumor cells, were injected into mature teratomas and developed into tumors. The salient findings were: (a) the observation of growth of tumor cells with high proliferative capacity within the differentiated microenvironment of the teratoma, (b) the identification of invasion by tumor cells into surrounding differentiated teratoma structures, and (c) the identification of blood vessels of human teratoma origin, growing adjacent to and within the cancer cell-derived tumor. Mouse embryonic stem cell-derived teratomas also supported cancer cell growth, but provided a less suitable model for human tumorigenesis studies. Anticancer immunotherapy treatment directed against A431 epidermoid carcinoma cell-related epitopes induced the complete regression of A431-derived tumor xenografts following direct i.m. injection in immunocompromised mice, as opposed to corresponding tumors growing within a human embryonic stem cell-derived microenvironment, wherein remnant foci of viable tumor cells were detected and resulted in tumor recurrence. We propose using this novel experimental model as a preclinical platform for investigating and manipulating the stromal response in tumor cell growth as an additional tool in cancer research.

  9. Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.

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    Xionghao Liu

    Full Text Available BACKGROUND: Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs but poorly in hESCs. METHODOLOGY/PRINCIPAL FINDINGS: Human ribosomal DNA (rDNA repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50% and homologous recombination frequency (>10(-5 were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. CONCLUSION/SIGNIFICANCE: This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs.

  10. Evaluating the first-in-human clinical trial of a human embryonic stem cell-based therapy.

    Science.gov (United States)

    Chapman, Audrey R; Scala, Courtney C

    2012-09-01

    Phase I clinical trials generally raise greater ethical and human protection challenges than later stage clinical trials, suggesting a need to proceed cautiously. This is particularly the case for Phase I trials with a novel therapy being tested in humans for the first time, usually termed first-in-human (FIH) trials. In January 2009, the Food and Drug Administration approved the Investigational New Drug application of Geron Corporation, a small California-based biopharmaceutical company, to initiate a clinical trial to assess GRNOPC1, a human embryonic stem cell-derived candidate therapy for severe spinal cord injuries. This article evaluates the ethical and human subject protection issues raised by the Geron FIH trial. It identifies problems with the approval process and with the conduct of the trial, and then recommends ways to improve review of future proposed trials with novel and high-risk therapies.

  11. ISOLATION AND EXPANSION OF HUMAN EMBRYONIC NEURAL STEM/PROGENITOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective: To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method. Method:The cerebral cortex cells were isolated from aborted embryos (11~13 weeks)by mechanical dissociation, and cultured in DMEM/F12 culture medium supplemented with N2 and growth factors for proliferation. Upon passaging, the neurospheres were pipetted gentlely to separate them into several cell masses and then grown in growth medium. The cells were grown in DMEM/F12 medium with serum (without growth factors) to induce differentiation. The stem cell, neuron, astrocyte and oligodendrocyte were identified by immunocytochemistry with antibodies to vimentin, MAP2, GFAP and GalC, respectively. Results:The primary cells grew together and formed neurospheres at 5th~7th day. They were all vimentin positive and could be passaged for at least 8passages. After passaging, the cell masses grew up and formed new neurospheres rapidly. These cells could differentiated into MAP2 ( + ), GFAP( + ) or GalC( + ) cells. Conclusion: The neural stem cells from human embryonic cerebral cortex have the capacity of proliferation and multi - differentiation in vitro. The passaging methods we used in this experiment were practical and convenient.

  12. Early embryonic failure: Expression and imprinted status of candidate genes on human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Sherman, L.S.; Bennett, P.R.; Moore, G.E. [Queen Charlotte`s and Chelsea Hospital, London (United Kingdom)

    1994-09-01

    Two cases of maternal uniparental (hetero)disomy for human chromosome 21 (mUPD21) have been identified in a systematic search for UPD in 23 cases of early embryonic failure (EEF). Bi-parental origin of the other chromosome pairs was confirmed using specific VNTR probes or dinucleotide repeat analysis. Both maternally and paternally derived isochromosomes 21q have previously been identified in two individuals with normal phenotypes. Full UPD21 has a different mechanism of origin than uniparental isochromosome 21q and its effect on imprinted genes and phenotypic outcome will therefore not necessarily be the same. EEF associated with mUPD21 suggests that developmentally important genes on HSA 21 may be imprinted such that they are only expressed from either the maternally or paternally derived alleles. We have searched for monoallelic expression of candidate genes on HSA 21 in human pregnancy (CBS, IFNAR, COL6A1) using intragenic DNA polymorphisms. These genes were chosen either because their murine homologues lie in imprinted regions or because they are potentially important in embryogenesis. Once imprinted candidate genes have been identified, their methylation status and expression in normal, early embryonic failure and uniparental disomy 21 pregnancies will be studied. At the same time, a larger number of cases of EEF are being examined to further investigate the incidence of UPD21 in this group.

  13. Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

    Science.gov (United States)

    Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

    2010-04-01

    The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

  14. Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

    Science.gov (United States)

    Gokhale, Paul J; Au-Young, Janice K; Dadi, SriVidya; Keys, David N; Harrison, Neil J; Jones, Mark; Soneji, Shamit; Enver, Tariq; Sherlock, Jon K; Andrews, Peter W

    2015-01-01

    We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

  15. Comparison of the efficacy of hard palate grafts with acellular human dermis grafts in lower eyelid surgery.

    Science.gov (United States)

    Li, Tina G; Shorr, Norman; Goldberg, Robert A

    2005-09-01

    The Madame Butterfly procedure of lower eyelid reconstruction addresses lower eyelid retraction after blepharoplasty caused by middle lamellar tethering. After scar lysis and subperiosteal midface lift, the surgical procedure uses a lower eyelid spacer graft to prevent further postoperative contraction of the middle and posterior lamellae. Different graft materials have been used, including autogenous hard palate and AlloDerm. The preoperative and postoperative photographs of consecutive patients who had undergone the Madame Butterfly procedure were analyzed retrospectively. Thirty-five patients undergoing AlloDerm grafting were compared with 25 patients undergoing hard palate grafting. Their lower eyelid heights were digitally measured and analyzed statistically for differences between the two groups. No statistically significant difference was found in the amount of eyelid elevation between the AlloDerm and hard palate groups, although a trend was seen that hard palate grafts resulted in both better elevation (p = 0.121) and a lower failure rate (p = 0.092). Female patients in both groups were found to experience significantly greater eyelid elevation than male patients (p = 0.018). Both AlloDerm and hard palate were found to be successful grafting materials for lower eyelid elevation in patients with postblepharoplasty lower eyelid retraction. Each has advantages and disadvantages, and surgeons should tailor their choices according to each patient.

  16. Cleft Lip and Cleft Palate

    Science.gov (United States)

    ... health conditions > Cleft lip and cleft palate Cleft lip and cleft palate E-mail to a friend Please fill ... repair cleft lip and palate. What are cleft lip and cleft palate? Cleft lip is a birth defect in ...

  17. Spontaneously differentiated GATA6-positive human embryonic stem cells represent an important cellular step in human embryonic development; they are not just an artifact of in vitro culture.

    Science.gov (United States)

    Lee, Jun Ho; Hong, Ki Sung; Mantel, Charlie; Broxmeyer, Hal E; Lee, Man Ryul; Kim, Kye-Seong

    2013-10-15

    In this study, we isolated and characterized spontaneously differentiated human embryonic stem cells (SD-hESCs) found in hESC colonies in comparison to the morphologically premature ESCs in the colonies to investigate the potential role of SD-hESCs in embryogenesis. SD-hESCs were distinguished from undifferentiated hESCs by their higher expression of GATA6, a marker for primitive endoderm and transthyretin, a marker visceral endoderm in embryoid bodies (EBs). SD-hESCs expressed OCT4 and NANOG, markers for pluripotent stem cells, at significantly lower levels than undifferentiated hESCs. EBs derived from isolated SD-hESCs were morphologically distinct from cells directly derived from the undifferentiated hESCs; they contained higher number of cysts compared to EBs from undifferentiated hESC-derived EBs (42% vs. 20%). Furthermore, the extracellular signal molecule, BMP2/4, induced a higher GATA4/6 expression and cystic EB formation than control and noggin-treated EBs. Since cystic formation in EBs play a role in primitive endoderm formation during embryogenesis, the SD-hESC may be a relevant cell type equipped to differentiate into primitive endoderm. Our results suggest that SD-ESCs generated during routine hESC culture are not just an artifact of in vitro culture and these cells could serve as a useful model to study the process of embryogenesis.

  18. Generation of a constitutively expressing Tetracycline repressor (TetR human embryonic stem cell line BJNhem20-TetR

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    Ronak Shetty

    2016-03-01

    Full Text Available Human embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator sequence to generate doxycycline based inducible line. For example, in human embryonic stem cells, the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al., 2009. Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells.

  19. A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro

    Directory of Open Access Journals (Sweden)

    Lewis Fiona C

    2012-08-01

    Full Text Available Abstract Background Human embryonic stem cells (hESCs represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF feeder-layers and exogenous protein media supplementation. Results These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP, which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages. Conclusions PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

  20. Gene targeting in a HUES line of human embryonic stem cells via electroporation.

    Science.gov (United States)

    Ruby, Katherine M; Zheng, Binhai

    2009-07-01

    Genetic modification is critical for achieving the full potential of human embryonic stem (ES) cells as a tool for therapeutic development and for basic research. Targeted modifications in human ES cells have met with limited success because of the unique culture conditions for many human ES cell lines. The HUES lines of human ES cells were developed for ease of manipulation and are gaining increased utility in stem cell research. We tested conditions for gene targeting via electroporation in the HUES-9 human ES cell line and demonstrate here successful gene targeting at the gene encoding Fezf2 (also known as Fezl), a transcription factor involved in corticospinal neuron development. With a targeting strategy involving positive and negative selection that is applicable to all genes, we observed a gene targeting frequency of approximately 1.5% for Fezf2, a gene not expressed in human ES cells. We found that conditions developed for gene targeting in mouse ES cells can be readily adapted to HUES cells with few key modifications. HUES-9 cells exhibit an intrinsically high efficiency of clonal expansion and sustain electroporation-based gene targeting procedures without any significant loss of pluripotency marker expression or karyotypic stability. Thus, human ES cell lines adapted for enzymatic passage and efficient clonal expansion can be highly amenable to genetic modifications, which will facilitate their application in basic science and clinical development.

  1. Concise review: animal substance-free human embryonic stem cells aiming at clinical applications.

    Science.gov (United States)

    Hovatta, Outi; Rodin, Sergey; Antonsson, Liselotte; Tryggvason, Karl

    2014-11-01

    Human embryonic stem cells have been considered the gold standard as a cell source for regenerative medicine since they were first cultured in 1998. They are pluripotent and can form principally all the cells types in the body. They are obtained from supernumerary human in vitro fertilization embryos that cannot be used for infertility treatment. Following studies on factors regulating pluripotency and differentiation, we now have techniques to establish and effectively expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos in chemically defined feeder-free cultures. The genetic stability and absence of tumorigenic mutations can be determined. There are satisfactory animal tests for functionality and safety. The first clinical trials are ongoing for two indications: age-related macular degeneration and spinal cord injury. ©AlphaMed Press.

  2. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021.

    Science.gov (United States)

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Main, Heather; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1-60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  3. Integration of human model neurons (NT2) into embryonic chick nervous system.

    Science.gov (United States)

    Podrygajlo, Grzegorz; Wiegreffe, Christoph; Scaal, Martin; Bicker, Gerd

    2010-02-01

    Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. Approximately a third of the differentiated neurons expressed cell markers related to cholinergic neurotransmission. To examine whether this human cell model system can be directed toward a motoneuronal fate, postmitotic neurons were co-cultured with mouse myotubes. Outgrowing neuronal processes established close contact with the myotubes and formed neuromuscular junction-like structures that bound alpha-bungarotoxin. To determine how grafted precursor cells and neurons respond to embryonic nerve tissue, NT2 cells at different stages of neural development were injected into chick embryo neural tube and brain. Grafted NT2 neurons populated both parts of the nervous system, sometimes migrating away from the site of injection. The neural tube appeared to be more permissive for neurite extensions than the brain. Moreover, extending neurites of spinal grafts were approaching the ventral roots, thus resembling motoneuronal projections.

  4. Generation of red blood cells from human embryonic/induced pluripotent stem cells for blood transfusion.

    Science.gov (United States)

    Ebihara, Yasuhiro; Ma, Feng; Tsuji, Kohichiro

    2012-06-01

    Red blood cell (RBC) transfusion is necessary for many patients with emergency or hematological disorders. However, to date the supply of RBCs remains labile and dependent on voluntary donations. In addition, the transmission of infectious disease via blood transfusion from unspecified donors remains a risk. Establishing a large quantity of safe RBCs would help to address this issue. Human embryonic stem (hES) cells and the recently established human induced pluripotent stem (hiPS) cells represent potentially unlimited sources of donor-free RBCs for blood transfusion, as they can proliferate indefinitely in vitro. Extensive research has been done to efficiently generate transfusable RBCs from hES/iPS cells. Nevertheless, a number of challenges must be overcome before the clinical usage of hES/iPS cell-derived RBCs can become a reality.

  5. Engineered human embryonic stem cell-derived lymphocytes to study in vivo trafficking and immunotherapy.

    Science.gov (United States)

    Knorr, David A; Bock, Allison; Brentjens, Renier J; Kaufman, Dan S

    2013-07-01

    Human embryonic stem cell (hESC)-derived natural killer (NK) cells are a promising source of antitumor lymphocytes for immunotherapeutics. They also provide a genetically tractable platform well suited for the study of antitumor immunotherapies in preclinical models. We have previously demonstrated the potency of hESC-derived NK cells in vivo. Here we use both bioluminescent and fluorescent imaging to demonstrate trafficking of hESC-derived NK cells to tumors in vivo. Our dual-imaging approach allowed us to more specifically define the kinetics of NK cell trafficking to tumor sites. NK cell persistence and trafficking were further evaluated by flow cytometry and immunohistochemistry. This integrated approach provides a unique system to apply the use of human pluripotent stem cells to study the kinetics and biodistribution of adoptively transferred lymphocytes, advances broadly applicable to the field of immunotherapy.

  6. Derivation of NEM2 affected human embryonic stem cell line Genea080

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea080 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male allele pattern. The hESC line had pluripotent cell morphology, 90% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 99% SSEA4 and gave a PluriTest Pluripotency score of 32.08, Novelty of 1.3. The cell line was negative for Mycoplasma and visible contamination.

  7. Derivation of NEM2 affected human embryonic stem cell line Genea079

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

  8. Derivation of NEM2 affected human embryonic stem cell line Genea078

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea078 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 76% of cells expressed Nanog, 93% Oct4, 67% Tra1–60 and 97% SSEA4 and gave a Pluritest Pluripotency score of 42.18, Novelty of 1.37. The cell line was negative for Mycoplasma and visible contamination.

  9. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  10. Generation of OCIAD1 inducible overexpression human embryonic stem cell line: BJNhem20-OCIAD1-Tet-On

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    Deeti K. Shetty

    2016-03-01

    Full Text Available Human embryonic stem cell line BJNhem20-OCIAD1-Tet-On was generated using non-viral method. The constructs pCAG-Tet-On and pTRE-Tight vector driving OCIAD1 expression were transfected using microporation procedure. pCAG-Tet-On cells can be used for inducible expression of any coding sequence cloned into pTRE-Tight vector. For example, in human embryonic stem cells, Tet-On system has been used to generate SOX2 overexpression cell line (Adachi et al., 2010.

  11. Abnormalities in centrosome number in human embryos and embryonic stem cells.

    Science.gov (United States)

    Gu, Yi-Fan; OuYang, Qi; Dai, Can; Lu, Chang-Fu; Lin, Ge; Gong, Fei; Lu, Guang-Xiu

    2016-05-01

    Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc.

  12. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Institute of Scientific and Technical Information of China (English)

    Li-Wei Zheng; Logan Linthicum; Pamela K DenBesten; Yan Zhang

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCI) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which ,vas also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  13. Transcriptional analysis of early lineage commitment in human embryonic stem cells

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    Wormald Sam

    2007-03-01

    Full Text Available Abstract Background The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. Results We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. Conclusion These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.

  14. Transcriptional analysis of early lineage commitment in human embryonic stem cells

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    Laslett, Andrew L; Grimmond, Sean; Gardiner, Brooke; Stamp, Lincon; Lin, Adelia; Hawes, Susan M; Wormald, Sam; Nikolic-Paterson, David; Haylock, David; Pera, Martin F

    2007-01-01

    Background The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. Results We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. Conclusion These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo. PMID:17335568

  15. Genetic manipulation of human embryonic stem cells in serum and feeder-free media.

    Science.gov (United States)

    Braam, Stefan R; Denning, Chris; Mummery, Christine L

    2010-01-01

    Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these conditions, plasmid transfection, virus infection, and siRNA transfection are highly effective. Stable genetically modified hESC lines can be generated with plasmid transfection, viral infection, or electroporation without loss of pluripotency or differentiation potential. The majority of lines generated in this system display a normal karyotype.

  16. Effect of VEGF on Neural Differentiation of Human Embryonic Stem Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Shujie JIAO; Huifang XU; Jie XU; Yanqiang ZHAN; Suming ZHANG

    2009-01-01

    The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines,TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups: group A, routine induction; group B, routine induction+10 ng/mL VEGF; group C, routine in-duction+10 ng/mL VEGF+10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de-tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence.The percentage of Nestin positive cells in group B was significantly higher than in groups A and C,while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C (P0.05). It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

  17. A review of the emerging potential therapy for neurological disorders: human embryonic stem cell therapy.

    Science.gov (United States)

    Shroff, Geeta; Dhanda Titus, Jyoti; Shroff, Rhea

    2017-01-01

    The first human embryonic stem cell (hESC) line was developed in the late nineties. hESCs are capable of proliferating indefinitely and differentiate into all the three embryonic germ layers. Further, the differentiation of hESC lines into neural precursor cells and neurons, astrocytes and oligodendrocytes showed their potential in treating several incurable neurological disorders such as spinal cord injury (SCI), cerebral palsy (CP), Parkinson's disease (PD). In this review, we will discuss the global scenario of research and therapeutic use of hESCs in the treatment of neurological disorders. Following this, we will discuss the development of a unique hESC line, how it differs from the other available hESC lines and its use in the treatment of neurological disorders. hESCs were isolated from mixture of neuronal and non-neuronal progenitor cells in their pre progenitor state in a Good Laboratory Practices, Good Tissue Practices and Good Manufacturing Practices compliant laboratory. Blastomere cells have served as a source to derive the hESCs and the xeno-free culture was demonstrated to be more safe and effective in clinical therapeutic application of hESCs. All the patients showed a remarkable improvement in their conditions and no serious adverse events were reported. This study concluded that hESC lines could be scalable and used in the treatment of various neurological disorders such as SCI, CP, and PD.

  18. Human parthenogenetic embryonic stem cells:one potential resource for cell therapy

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem(iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies.However,the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells.Embryonic stem cells(ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy.Recent studies on human parthenogenetic embryonic stem cells(hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics,but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions.To generate various pluripotent stem cells,diverse reprogramming techniques and approaches will be developed and integrated.This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology,and ultimately benefit cell therapy and regenerative medicine.

  19. A distinct microRNA signature for definitive endoderm derived from human embryonic stem cells.

    Science.gov (United States)

    Hinton, Andrew; Afrikanova, Ivka; Wilson, Mike; King, Charles C; Maurer, Brian; Yeo, Gene W; Hayek, Alberto; Pasquinelli, Amy E

    2010-06-01

    Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.

  20. Three key variables involved in feeder preparation for the maintenance of human embryonic stem cells.

    Science.gov (United States)

    Zhou, Di; Liu, Tiancheng; Zhou, Xiaoying; Lu, Guangxiu

    2009-07-01

    Although the development of a feeder-free culture system for future applications of human embryonic stem cells (hESCs), at present the regular culture system uses mitotically inactivated mouse embryonic fibroblasts (mEFs) as feeder cells for maintaining undifferentiated hESCs. Mitomycin C (MMC) is used to inactivate mEFs, but this causes DNA damage, and it is unclear whether MMC remains in the culture system after several washes. Three variables have been evaluated with respect to feeder preparation and MMC involvement, including mEF exposure to MMC, density of feeder cells, and different wash steps during the preparation of feeder cells. These variables are critical to the subsequent planting of hESCs because remnants of MMC would be unsafe with respect to long-term culture of hESCs The novel data here evaluates the remnant amounts of MMC in a hESCs culture system using HPLC/MS/MS. The ultimate objective of this study is the control of MMC within a safe range.

  1. In Vitro Differentiation and Maturation of Human Embryonic Stem Cell into Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Amer Mahmood

    2011-01-01

    Full Text Available Human embryonic stem cells (hESCs, which have the potential to generate virtually any differentiated progeny, are an attractive cell source for transplantation therapy, regenerative medicine, and tissue engineering. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Basic science in the field of embryonic development, stem cell differentiation, and tissue engineering has offered important insights into key pathways and scaffolds that regulate hESC differentiation, which have produced advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, ectoderm, and many of their downstream derivatives. These findings have lead to identification of several pathways controlling the differentiation of hESCs into mesodermal derivatives such as myoblasts, mesenchymal cells, osteoblasts, chondrocytes, adipocytes, as well as hemangioblastic derivatives. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of bone and vascular diseases.

  2. Human parthenogenetic embryonic stem cells: one potential resource for cell therapy

    Institute of Scientific and Technical Information of China (English)

    HAO Jie; HU WanWan; SHENG Chao; YU Yang; ZHOU Qi

    2009-01-01

    Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or in duced pluripotent stem (iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies. However, the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells. Embryonic stem cells (ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy. Recent studies on human parthenogenetic embryonic stem cells (hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics, but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions. To generate various pluripotent stem cells, diverse reprogramming techniques and approaches will be developed and integrated. This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology, and ultimately benefit cell therapy and regenerative medicine.

  3. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Marica Grskovic

    2007-08-01

    Full Text Available Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.

  4. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

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    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  5. Cleft Palate Foundation

    Science.gov (United States)

    ... available in English , Spanish , and Mandarin ! Information on Cleft Lip and Palate Our booklets and factsheets address a variety of issues related to cleft lip and palate, such as speech, hearing, genetics, and what to ...

  6. Cleft Lip and Palate

    Science.gov (United States)

    Cleft lip and cleft palate are birth defects that occur when a baby's lip or mouth do not form properly. They ... during pregnancy. A baby can have a cleft lip, a cleft palate, or both. A cleft lip happens if ...

  7. Human embryonic stem cell-derived mesenchymal stem cells as cellular delivery vehicles for prodrug gene therapy of glioblastoma.

    Science.gov (United States)

    Bak, Xiao Ying; Lam, Dang Hoang; Yang, Jingye; Ye, Kai; Wei, Esther Lee Xing; Lim, Sai Kiang; Wang, Shu

    2011-11-01

    Mesenchymal stem cells (MSCs) possess tumor-tropic properties and consequently have been used to deliver therapeutic agents for cancer treatment. Their potential in cancer therapy highlights the need for a consistent and renewable source for the production of uniform human MSCs suitable for clinical applications. In this study, we seek to investigate whether human embryonic stem cells can be used as a cell source to fulfill this goal. We generated MSC-like cells from two human embryonic stem cell lines, HuES9 and H1, and observed that MSC-like cells derived from human embryonic stem cells were able to migrate into human glioma intracranial xenografts after being injected into the cerebral hemisphere contralateral to the tumor inoculation site. We engineered these cells with baculoviral and lentiviral vectors, respectively, for transient and stable expression of the herpes simplex virus thymidine kinase gene. In tumor-bearing mice the engineered MSC-like cells were capable of inhibiting tumor growth and prolonging survival in the presence of ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere. Our findings suggest that human embryonic stem cell-derived MSCs may be a viable and attractive alternative for large-scale derivation of targeting vehicles for cancer therapy.

  8. A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture.

    Science.gov (United States)

    Meng, Guoliang; Liu, Shiying; Krawetz, Roman; Chan, Michael; Chernos, Judy; Rancourt, Derrick E

    2008-06-01

    Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.

  9. Adult, embryonic and fetal hemoglobin are expressed in human glioblastoma cells.

    Science.gov (United States)

    Emara, Marwan; Turner, A Robert; Allalunis-Turner, Joan

    2014-02-01

    Hemoglobin is a hemoprotein, produced mainly in erythrocytes circulating in the blood. However, non-erythroid hemoglobins have been previously reported in other cell types including human and rodent neurons of embryonic and adult brain, but not astrocytes and oligodendrocytes. Human glioblastoma multiforme (GBM) is the most aggressive tumor among gliomas. However, despite extensive basic and clinical research studies on GBM cells, little is known about glial defence mechanisms that allow these cells to survive and resist various types of treatment. We have shown previously that the newest members of vertebrate globin family, neuroglobin (Ngb) and cytoglobin (Cygb), are expressed in human GBM cells. In this study, we sought to determine whether hemoglobin is also expressed in GBM cells. Conventional RT-PCR, DNA sequencing, western blot analysis, mass spectrometry and fluorescence microscopy were used to investigate globin expression in GBM cell lines (M006x, M059J, M059K, M010b, U87R and U87T) that have unique characteristics in terms of tumor invasion and response to radiotherapy and hypoxia. The data showed that α, β, γ, δ, ζ and ε globins are expressed in all tested GBM cell lines. To our knowledge, we are the first to report expression of fetal, embryonic and adult hemoglobin in GBM cells under normal physiological conditions that may suggest an undefined function of those expressed hemoglobins. Together with our previous reports on globins (Ngb and Cygb) expression in GBM cells, the expression of different hemoglobins may constitute a part of series of active defence mechanisms supporting these cells to resist various types of treatments including chemotherapy and radiotherapy.

  10. Selective microRNA-Offset RNA expression in human embryonic stem cells.

    Science.gov (United States)

    Asikainen, Suvi; Heikkinen, Liisa; Juhila, Juuso; Holm, Frida; Weltner, Jere; Trokovic, Ras; Mikkola, Milla; Toivonen, Sanna; Balboa, Diego; Lampela, Riina; Icay, Katherine; Tuuri, Timo; Otonkoski, Timo; Wong, Garry; Hovatta, Outi

    2015-01-01

    Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.

  11. Selective microRNA-Offset RNA expression in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Suvi Asikainen

    Full Text Available Small RNA molecules, including microRNAs (miRNAs, play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs are similar in length to miRNAs, align to miRNA precursor (pre-miRNA loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.

  12. Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems.

    Science.gov (United States)

    Desai, Nina; Rambhia, Pooja; Gishto, Arsela

    2015-02-22

    Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.

  13. Pathway Analysis and Modeling of the Differentiation of Human Embryonic Stem Cells into Hepatocyte-like Cells

    Science.gov (United States)

    Daskalaki, Andriani; Jozefczuk, Justyna; Lehrach, Hans; Adjaye, James; Wierling, Christoph

    2011-06-01

    A more detailed understanding of the differentiation of human embryonic and induced pluripotent stem cells into hepatocyte-like cells can help to improve therapies for liver diseases, like steatohepatitis. In this work we used microarray-based expression data to analyze the in vitro differentiation of human embryonic stem cells into hepatocytes. Pathway analysis has been carried out on gene expression data of different stages of the differentiation process from embryonic stem cells into hepatocyte-like cells via definitive endoderm and hepatic endoderm. Based on pathway analysis we identified signaling pathways, like the GPCR signaling pathway as well as FOXA2 regulatory networks. Based on these highly enriched pathways we constructed a model prototype to better understand and study the differentiation of stem cells into hepatocytes.

  14. Second heart field and the development of the outflow tract in human embryonic heart.

    Science.gov (United States)

    Yang, Yan-Ping; Li, Hai-Rong; Cao, Xi-Mei; Wang, Qin-Xue; Qiao, Cong-Jin; Ya, Jing

    2013-04-01

    The second heart field (SHF) is indicated to contribute to the embryonic heart development. However, less knowledge is available about SHF development of human embryo due to the difficulty of collecting embryos. In this study, serial sections of human embryos from Carnegie stage 10 (CS10) to CS16 were stained with antibodies against Islet-1 (Isl-1), Nkx2.5, GATA4, myosin heavy chain (MHC) and α-smooth muscle actin (α-SMA) to observe spatiotemporal distribution of SHF and its contribution to the development of the arterial pole of cardiac tube. Our findings suggest that during CS10 to CS12, SHF of the human embryo is composed of the bilateral pharyngeal mesenchyme, the central mesenchyme of the branchial arch and splanchnic mesoderm of the pericardial cavity dorsal wall. With development, SHF translocates and consists of ventral pharyngeal mesenchyme and dorsal wall of the pericardial cavity. Hence, the SHF of human embryo shows a dynamic spatiotemporal distribution pattern. The formation of the Isl-1 positive condense cell prongs provides an explanation for the saddle structure formation at the distal pole of the outflow tract. In human embryo, the Isl-1 positive cells of SHF may contribute to the formation of myocardial outflow tract (OFT) and the septum during different development stages.

  15. Discovery and characterization of novel microRNAs during endothelial differentiation of human embryonic stem cells.

    Science.gov (United States)

    Yoo, Jung Ki; Kim, Jumi; Choi, Seong-Jun; Noh, Hye Min; Kwon, Young Do; Yoo, Hanna; Yi, Hyo Seon; Chung, Hyung Min; Kim, Jin Kyeoung

    2012-07-20

    MicroRNAs (miRNAs) are small RNAs that participate in the regulation of genes associated with the differentiation and proliferation. In this study, 5 novel miRNAs were identified from human mesenchymal stem cells and characterized using various analyses. To investigate the potential functions associated with the regulation of cell differentiation, the differences in miRNA expression were examined in undifferentiated and differentiated human embryonic stem (ES) cells using reverse transcription (RT)-PCR analysis. Specifically, 3 miRNAs exhibited decreased expression levels in human umbilical vein endothelial cells (HUVECs) and endothelial cells derived from human ES cells. Putative target genes related to differentiation or maturation of endothelial cells were predicted by seed sequences of 2 novel miRNAs and analyzed for their expression via miRNA-mediated regulation using a luciferase assay. In HUVECs, CDH5 gene expression was directly repressed by hsa-miR-6086. Similarly, hsa-miR-6087 significantly downregulated endoglin expression. Therefore, the roles of these 2 miRNAs may be to directly suppress their target genes, popularly known as endothelial cell markers. Taken together, our results demonstrate that several novel miRNAs perform critical roles in human endothelial cell development.

  16. Computational Analysis of Transcriptional Circuitries in Human Embryonic Stem Cells Reveals Multiple and Independent Networks

    Directory of Open Access Journals (Sweden)

    Xiaosheng Wang

    2014-01-01

    Full Text Available It has been known that three core transcription factors (TFs, NANOG, OCT4, and SOX2, collaborate to form a transcriptional circuitry to regulate pluripotency and self-renewal of human embryonic stem (ES cells. Similarly, MYC also plays an important role in regulating pluripotency and self-renewal of human ES cells. However, the precise mechanism by which the transcriptional regulatory networks control the activity of ES cells remains unclear. In this study, we reanalyzed an extended core network, which includes the set of genes that are cobound by the three core TFs and additional TFs that also bind to these cobound genes. Our results show that beyond the core transcriptional network, additional transcriptional networks are potentially important in the regulation of the fate of human ES cells. Several gene families that encode TFs play a key role in the transcriptional circuitry of ES cells. We also demonstrate that MYC acts independently of the core module in the regulation of the fate of human ES cells, consistent with the established argument. We find that TP53 is a key connecting molecule between the core-centered and MYC-centered modules. This study provides additional insights into the underlying regulatory mechanisms involved in the fate determination of human ES cells.

  17. Short hard palate in prenatal trisomy 21

    DEFF Research Database (Denmark)

    Lauridsen, H; Hansen, Birgit; Reintoft, I;

    2005-01-01

    and palatine parts) in trisomy 21 fetuses, and to compare the results to normal standards. Design - Material from 31 human fetuses with genetically verified trisomy 21 was studied. The fetuses were derived from legally induced or spontaneous abortions. Palates were, after sectioning, radiographed in lateral...... of the palatal components in trisomy 21 was compared to normal standards. Results - For CRL 150 mm and CRL 170 mm it appears that all three palatal lengths, total length, maxillary length, and palatinal length are significantly shorter in fetuses with trisomy 21. Conclusion - The main conclusion of our study...... is that the total palatal length in prenatal trisomy 21 is shorter than normal and that this is due both to a shortness of the maxillary and the palatine components of the hard palate....

  18. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    Science.gov (United States)

    Kawamura, Kazuhiro; Chen, Yuan; Shu, Yimin; Cheng, Yuan; Qiao, Jie; Behr, Barry; Pera, Renee A Reijo; Hsueh, Aaron J W

    2012-01-01

    Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility

  19. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Kawamura

    Full Text Available Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for

  20. Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Hepatocyte transplantation and bioartificial liver(BAL)as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders.Unfortunately,the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies.Embryonic stem cells (ESCs),which could be isolated directly from the blastocyst inner cell mass,have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords.However,differentiation of hESCS into hepatocytes with significant numbers remains a challenge.This review updates our current understanding of differentiation of ESCs into hepatic lineage cells,their future therapeutic uses and problems in liver regeneration.

  1. Stimulation of human embryonic stem cell-derived cardiomyocytes on thin-film microelectrodes.

    Science.gov (United States)

    Viitanen, Jouko; Heimala, Päivi; Hokkanen, Ari; Iljin, Kristiina; Kerkelä, Erja; Kolari, Kai; Kattelus, Hannu

    2011-05-01

    We describe successful long-term stimulation of human embryonic stem cell-derived cardiomyocyte clusters on thin-film microelectrode structures in vitro. Interdigitated electrode structures were constructed using plain titanium on glass as the electrode material. Titanium rapidly oxidizes in atmospheric conditions to produce an insulating TiO(χ) layer with high relative permittivity. Capacitive coupling to the incubation medium and to the cells adherent to the electrodes was still efficient, and the dielectric layer prevented electrolysis, allowing a wider window of possible stimulation amplitudes to be used, relative to conducting surfaces. A common hypothesis suggests that to achieve proper differentiation of electroactive cells from the stem cells electrical stimuli are also needed. Spontaneously beating cardiomyocyte clusters were seeded on the glass-electrode surfaces, and we successfully altered and resynchronized a clearly different beat interval. The new pace was reliably maintained for extended periods of several tens of minutes.

  2. Regional differences in expression of specific markers for human embryonic stem cells

    DEFF Research Database (Denmark)

    Laursen, Steen B; Møllgård, Kjeld; Olesen, Christian

    2007-01-01

    Characterization of human embryonic stem cell (hESC) lines derived from the inner cell masses of blastocysts generally includes expression analysis of markers such as OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. Expression is usually detected by immunocytochemical staining of entire colonies...... staining to weak or absent NANOG staining, and vice versa. SSEA4 staining was only observed in small clusters or single cells and not confined to the TRA territory. Co-expression of all markers was only detected in small areas. SSEA1 expression was found exclusively outside the TRA territory. In conclusion......, pronounced regional differences in the expression of markers considered specific for undifferentiated hESC may suggest the existence of different cell populations....

  3. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  4. Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

    Science.gov (United States)

    Roberts, R Michael; Loh, Kyle M; Amita, Mitsuyoshi; Bernardo, Andreia S; Adachi, Katsuyuki; Alexenko, Andrei P; Schust, Danny J; Schulz, Laura C; Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Pedersen, Roger A

    2014-05-01

    It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

  5. Art and human embryonic stem cells: from the bench to the high street.

    Science.gov (United States)

    Duprat, Sebastien

    2009-03-01

    ESTOOLS, a project funded by the European Commission (FP6), gathers expertise on human embryonic stem cells in 10 countries of the European Research Area. The ESTOOLS outreach program uses Art extensively as the only universal cross-cultural and cross-religion means of communication. The Smile of a Stem Cell photo exhibition, a major component of this program, aims to fill a missing link between public dissemination of science and science-illiterate citizens. Scientists are also engaged to stand at a distance from their work and observe it with an outsider's perspective, which enhances their competency to communicate science. The photo exhibition, by its situation upstream of scientific education, makes itself open to interest and enthusiasm among a public with no prerequired scientific knowledge or abilities.

  6. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    Science.gov (United States)

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-09-02

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

  7. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

    2015-01-01

    producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human...... embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed...... by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid...

  8. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T.G.; Prokhorova, Tatyana; Akimov, Vyacheslav

    2011-01-01

    To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned...... by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those...... accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis...

  9. Soft tissue chondroma of hard palate associated with cleft palate

    OpenAIRE

    Rajendra Nehete; Anita Nehete; Sandeep Singla; Sudhir Sankalecha

    2012-01-01

    Soft tissue chondroma of palate is very rare. It has never been reported in a cleft palate patient. We report a case of 22-year-old male who came with asymptomatic swelling on the palate since birth, along with complete cleft of secondary palate. He had symptoms related to cleft palate only, i.e., nasal regurgitation and speech abnormalities. Swelling was excised and the cleft palate was repaired. Histopathological examination revealed chondroma of the palate. The patient had no recurrence af...

  10. The dynamics of polycomb group proteins in early embryonic nervous system in mouse and human.

    Science.gov (United States)

    Qi, Lu; Cao, Jing-Li; Hu, Yi; Yang, Ji-Gao; Ji, Yuan; Huang, Jing; Zhang, Yi; Sun, Da-Guang; Xia, Hong-Fei; Ma, Xu

    2013-11-01

    Polycomb group (PcG) proteins are transcription regulatory proteins that control the expression of a variety of genes and the antero-posterior neural patterning from early embryogenesis. Although expression of PcG genes in the nervous system has been noticed, but the expression pattern of PcG proteins in early embryonic nervous system is still unclear. In this study, we analyzed the expression pattern of PRC1 complex members (BMI-1 and RING1B) and PRC2 complex members (EED, SUZ12 and EZH2) in early embryonic nervous system in mouse and human by Western blot and Immunohistochemistry. The results of Western blot showed that EED protein was significantly up-regulated with the increase of the day of pregnancy during the early embryogenesis in mouse. BMI-1 protein level was significantly increased from the day 10 of pregnancy, when compared with the day 9 of pregnancy. But the SUZ12, EZH2 and RING1B protein level did not change significantly. From the results of Immunohistochemistry, we found that the four PcG proteins were all expressed in the fetal brain and fetal spinal cord in mouse. In human, the expression of EED, SUZ12, and EZH2 was not significantly different in cerebral cortex and sacral spinal cord, but BMI-1 and RING1B expression was enhanced with the development of embryos in early pregnancy. Collectively, our findings showed that PRC1 and PRC2 were spatiotemporally expressed in brain and spinal cord of early embryos.

  11. Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience.

    Science.gov (United States)

    Menasché, Philippe; Vanneaux, Valérie; Fabreguettes, Jean-Roch; Bel, Alain; Tosca, Lucie; Garcia, Sylvie; Bellamy, Valérie; Farouz, Yohan; Pouly, Julia; Damour, Odile; Périer, Marie-Cécile; Desnos, Michel; Hagège, Albert; Agbulut, Onnik; Bruneval, Patrick; Tachdjian, Gérard; Trouvin, Jean-Hugues; Larghero, Jérôme

    2015-03-21

    There is now compelling evidence that cells committed to a cardiac lineage are most effective for improving the function of infarcted hearts. This has been confirmed by our pre-clinical studies entailing transplantation of human embryonic stem cell (hESC)-derived cardiac progenitors in rat and non-human primate models of myocardial infarction. These data have paved the way for a translational programme aimed at a phase I clinical trial. The main steps of this programme have included (i) the expansion of a clone of pluripotent hESC to generate a master cell bank under good manufacturing practice conditions (GMP); (ii) a growth factor-induced cardiac specification; (iii) the purification of committed cells by immunomagnetic sorting to yield a stage-specific embryonic antigen (SSEA)-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (iv) the incorporation of these cells into a fibrin scaffold; (v) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (vi) an extensive cytogenetic and viral testing; and (vii) the characterization of the final cell product and its release criteria. The data collected throughout this process have led to approval by the French regulatory authorities for a first-in-man clinical trial of transplantation of these SSEA-1(+) progenitors in patients with severely impaired cardiac function. Although several facets of this manufacturing process still need to be improved, these data may yet provide a useful platform for the production of hESC-derived cardiac progenitor cells under safe and cost-effective GMP conditions. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  12. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells.

    Science.gov (United States)

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-10-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer.

  13. Lefty Glycoproteins in Human Embryonic Stem Cells: Extracellular Delivery Route and Posttranslational Modification in Differentiation.

    Science.gov (United States)

    Khalkhali-Ellis, Zhila; Galat, Vasiliy; Galat, Yekaterina; Gilgur, Alina; Seftor, Elisabeth A; Hendrix, Mary J C

    2016-09-19

    Lefty is a member of transforming growth factor-beta (TGF-β) superfamily and a potent antagonist of the TGF-β/Nodal/Activin signaling pathway. Lefty is critical in sustaining self-renewal/pluripotency status, and implicated in the differentiation of embryonic stem cells (ESCs). However, emerging studies depict Lefty as a multifaceted protein involved in myriad cellular events. Lefty proteins (human Lefty A and B) are secreted glycoproteins, but their mode of secretion and the significance of their "glycan" moiety remain mostly unexplored. By employing an in vitro system of human ESCs (hESCs), we observed that Lefty protein(s) are encased in exosomes for extracellular release. The exosomal- and cell-associated Lefty diverge in their proteolytic processing, and possess N-glycan structures of high mannose and complex nature. Differentiation of hESCs to mesenchymal cells (MSCs) or neuronal progenitor cells (NPCs) entails distinct changes in the Lefty A/Lefty B gene(s), and protein expression. Specifically, the proteolytic cleavage and N-glycan composition of the cell-associated and exosomal Lefty differ in the differentiated progenies. These modifications affected Lefty's inhibitory effect on Nodal signaling in aggressive melanoma cells. The microheterogeneity in the processing and glycosylation of Lefty protein(s) between hESCs, MSCs, and NPCs could present efficient means of diversifying the endogenous functions of Lefty. Whether Lefty's diverse functions in embryonic patterning, as well as its diffusion range in the extracellular environment, are similarly affected remains to be determined. Our studies underscore the potential relevance of Lefty-packaged exosomes for combating debilitating diseases such as cancer.

  14. Flow in the human upper airway: work of breathing and the compliant soft palate and tongue

    Science.gov (United States)

    Jermy, Mark; Adams, Cletus; Aplin, Jonathan; Buchajczyk, Marcin; van Hove, Sibylle; Kabaliuk, Natalia; Geoghegan, Patrick; Cater, John

    2016-11-01

    The human upper airway (nasal cavity, pharynx and trachea) filters, heats and humidifies inspired air. Its pressure drop affects the work of breathing (WOB, energy expended to inspire and expire) to a degree which varies from person to person, and which is altered by breathing therapy devices. We report experimental studies using 3D printed models of the upper airway based on CT scans of single individuals (adult and paediatric), and average geometries based on PCA analysis of 150 individuals. Particle Image Velocimetry (PIV), gas concentration and pressure measurements, coupled with CFD simulation. These reveal the details of the washout of CO2 rich exhaled gas, the direction-dependent time-varying pressure drop, and the effect of high-flow nasal therapy (HFNT) on these phenomena. A 1D multi-compartment model is used to estimate the work of breathing. For the first time, soft (compliant) elements have been included in the model airways and show that the assumption of rigid tissue is acceptable for unassisted breathing, but unrealistic for therapy-assisted flows.

  15. A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

    Science.gov (United States)

    Yao, Zizhen; Mich, John K; Ku, Sherman; Menon, Vilas; Krostag, Anne-Rachel; Martinez, Refugio A; Furchtgott, Leon; Mulholland, Heather; Bort, Susan; Fuqua, Margaret A; Gregor, Ben W; Hodge, Rebecca D; Jayabalu, Anu; May, Ryan C; Melton, Samuel; Nelson, Angelique M; Ngo, N Kiet; Shapovalova, Nadiya V; Shehata, Soraya I; Smith, Michael W; Tait, Leah J; Thompson, Carol L; Thomsen, Elliot R; Ye, Chaoyang; Glass, Ian A; Kaykas, Ajamete; Yao, Shuyuan; Phillips, John W; Grimley, Joshua S; Levi, Boaz P; Wang, Yanling; Ramanathan, Sharad

    2017-01-05

    During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Self-contained induction of neurons from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Okuno

    Full Text Available BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM. However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

  17. Human Embryonic Stem Cell Research: Ethical Views of Buddhist, Hindu and Catholic Leaders in Malaysia.

    Science.gov (United States)

    Sivaraman, Mathana Amaris Fiona; Noor, Siti Nurani Mohd

    2016-04-01

    Embryonic Stem Cell Research (ESCR) raises ethical issues. In the process of research, embryos may be destroyed and, to some, such an act entails the 'killing of human life'. Past studies have sought the views of scientists and the general public on the ethics of ESCR. This study, however, explores multi-faith ethical viewpoints, in particular, those of Buddhists, Hindus and Catholics in Malaysia, on ESCR. Responses were gathered via semi-structured, face-to-face interviews. Three main ethical quandaries emerged from the data: (1) sanctity of life, (2) do no harm, and (3) 'intention' of the research. Concerns regarding the sanctity of life are directed at particular research protocols which interfere with religious notions of human ensoulment and early consciousness. The principle of 'do no harm' which is closely related to ahimsa prohibits all acts of violence. Responses obtained indicate that respondents either discourage research that inflicts harm on living entities or allow ESCR with reservations. 'Intention' of the research seems to be an interesting and viable rationale that would permit ESCR for the Buddhists and Hindus. Research that is intended for the purpose of alleviating human suffering is seen as being ethical. This study also notes that Catholics oppose ESCR on the basis of the inviolability of human life.

  18. Derivation and characterization of human embryonic stem cell lines from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhao Wu; Huimin Dai; Lei Qian; Qing Tian; Lei Xiao; Xiaojun Tan; Hui Li; Lingjun Rao; Lixiazi He; Lei Bao; Jing Liao; Chun Cui; Zhenyu Zuo; Qiao Li

    2011-01-01

    Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

  19. Stable genetic modification of human embryonic stem cells by lentiviral vectors.

    Science.gov (United States)

    Gropp, Michal; Itsykson, Pavel; Singer, Orna; Ben-Hur, Tamir; Reinhartz, Etti; Galun, Eithan; Reubinoff, Benjamin E

    2003-02-01

    Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.

  20. Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells.

    Science.gov (United States)

    Soman, Pranav; Tobe, Brian T D; Lee, Jin Woo; Winquist, Alicia A M; Singec, Ilyas; Vecchio, Kenneth S; Snyder, Evan Y; Chen, Shaochen

    2012-10-01

    Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

  1. Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

    Science.gov (United States)

    Zdravkovic, Tamara; Nazor, Kristopher L; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C; Loring, Jeanne F; Fisher, Susan J

    2015-12-01

    Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

  2. Feeder-free monolayer cultures of human embryonic stem cells express an epithelial plasma membrane protein profile.

    NARCIS (Netherlands)

    van Hoof, D.; Braam, S.R.; Dormeyer, W.; Ward-van Oostwaard, D.; Heck, A.; Krijgsveld, J.; Mummery, C.L.

    2008-01-01

    Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contr

  3. Functional Characterization of Single-Nucleotide Polymorphisms in the Human Undifferentiated Embryonic-Cell Transcription Factor 1 Gene

    NARCIS (Netherlands)

    Thummer, Rajkumar P.; Drenth-Diephuis, Loes J.; Carney, Karen E.; Eggen, Bart J. L.

    2010-01-01

    Single-nucleotide polymorphisms (SNPs) are single-nucleotide sequence variations between individuals. Two missense SNPs are present in the human undifferentiated embryonic-cell transcription factor 1 (UTF1) gene and their consequences for UTF1 function are investigated in this study. Expression of t

  4. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    NARCIS (Netherlands)

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.

    2015-01-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecu

  5. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    NARCIS (Netherlands)

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the

  6. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    Science.gov (United States)

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

  7. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    NARCIS (Netherlands)

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.

    2015-01-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecu

  8. Patenting human embryonic stem cells in peril: the decision of the Enlarged Board of Appeal in G 2/06

    NARCIS (Netherlands)

    S.J.R. Bostyn

    2009-01-01

    The Enlarged Board of Appeal has recently decided one of the most sensitive cases it has ever had on its hands, but probably not the last. The referral in the G 2/06 case related to the patentability of human embryonic stem cells (hESC). The main question was whether inventions pertaining to the hES

  9. The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

    Science.gov (United States)

    Murdoch, Alison; Braude, Peter; Courtney, Aidan; Brison, Daniel; Hunt, Charles; Lawford-Davies, James; Moore, Harry; Stacey, Glyn; Sethe, Sebastian

    2012-03-01

    The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

  10. WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Huiming Xu; Weicheng Wang; Chunliang Li; Hongyao Yu; Acong Yang; Beibei Wang; Ying Jin

    2009-01-01

    POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem (ES) cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin (Ub)-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu-man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en-dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference (RNAi), suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.

  11. Dynamics of the transcriptome response of cultured human embryonic stem cells to ionizing radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Sokolov, Mykyta V., E-mail: sokolovm@mail.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Panyutin, Irina V., E-mail: ipanyutinv@mail.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Panyutin, Igor G., E-mail: igorp@helix.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Neumann, Ronald D., E-mail: rneumann@mail.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States)

    2011-05-10

    One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively, molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1 Gy of gamma-radiation at 2 h and 16 h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel, the cell growth, DDR kinetics, and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2 h post-IR showed almost an exclusively p53-dependent, predominantly pro-apoptotic, signature with a total of only 30 up-regulated genes. In contrast, the gene expression patterns at 16 h post-IR showed 354 differentially expressed genes, mostly involved in pro-survival pathways, such as increased expression of metallothioneins, ubiquitin cycle, and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time, avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells.

  12. ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ- AND MURINE EMBRYONIC εy-GLOBIN GENES

    Institute of Scientific and Technical Information of China (English)

    张俊武; 张雪青; 陈平

    2001-01-01

    Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globingene expression and to test cell toxicity of the drug.Methods. ME L cells were transfected with the recombinant construct μLCRAγψβδβ3 and the stable transfor-mants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rab-bit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection as-say. The hematologdcal toxicity and electrolyte toxicity of the drug were tested.Results. An inducible and dose-dependent expression of the human γγ-, ββ- and mouse Aa-globin gene was ob-served in the transfected MEL cells. The induction of the human γ-globin gene is significant stronger than that ofthe ββ-globin gene. With 2.5 ~ 5 mmol/L isobutyramide, the induction of the human γ-globin gene is even moreeffective than that of mouse aa-globin gene. After a 15-day injection under the doses of 500 ~ 900mg * kg- 1 * d-1,the level of the mouse embryonic εy-globin mRNA could be significantly induced up to 3 ~ 4 fold of that of uninject-ed controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected withdifferent doses of isobutyramide at the interval of 24 hours for 2 ~4 weeks, were generally within the normalrange. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+,Na +, C1- and CO.2 were all within normal range and serum ionic osmotic pressure remained stable as well.Conclusion. Our results suggested that isobutyramide is a weak inducer of cell differentiation, but it canselectively activate transcription of human γy-globin gene at a certain degree, and it can act on early stages of ery-throid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have nohematological toxicity. Our results have further proved the potential value of

  13. Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice.

    Science.gov (United States)

    Drury-Stewart, Danielle; Song, Mingke; Mohamad, Osama; Guo, Ying; Gu, Xiaohuan; Chen, Dongdong; Wei, Ling

    2013-08-08

    Ischemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model. Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention. After 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals. Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition

  14. Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells

    Science.gov (United States)

    Garcia-Perez, Jose L.; Morell, Maria; Scheys, Joshua O.; Kulpa, Deanna A.; Morell, Santiago; Carter, Christoph C.; Hammer, Gary D.; Collins, Kathleen L.; O’Shea, K. Sue; Menendez, Pablo; Moran, John V.

    2010-01-01

    Long INterspersed Element-1 (LINE-1 or L1) retrotransposition continues to impact human genome evolution1,2. L1s can retrotranspose in the germline, during early development, and in select somatic cells3,4,5,6,7,8; however, the host response to L1 retrotransposition remains largely unexplored. Here, we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors (IHDACs) rapidly reverses this silencing, and chromatin immunoprecipitation (ChIP) experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing also was observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukemia virus (MMLV) or human immunodeficiency virus (HIV), suggesting these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, per se, is not sufficient to reactivate previously silenced reporter genes. Thus, our data suggest that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition. PMID:20686575

  15. Consensus comparative analysis of human embryonic stem cell-derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Shaohong Zhang

    Full Text Available Global transcriptional analyses have been performed with human embryonic stem cells (hESC derived cardiomyocytes (CMs to identify molecules and pathways important for human CM differentiation, but variations in culture and profiling conditions have led to greatly divergent results among different studies. Consensus investigation to identify genes and gene sets enriched in multiple studies is important for revealing differential gene expression intrinsic to human CM differentiation independent of the above variables, but reliable methods of conducting such comparison are lacking. We examined differential gene expression between hESC and hESC-CMs from multiple microarray studies. For single gene analysis, we identified genes that were expressed at increased levels in hESC-CMs in seven datasets and which have not been previously highlighted. For gene set analysis, we developed a new algorithm, consensus comparative analysis (CSSCMP, capable of evaluating enrichment of gene sets from heterogeneous data sources. Based on both theoretical analysis and experimental validation, CSSCMP is more efficient and less susceptible to experimental variations than traditional methods. We applied CSSCMP to hESC-CM microarray data and revealed novel gene set enrichment (e.g., glucocorticoid stimulus, and also identified genes that might mediate this response. Our results provide important molecular information intrinsic to hESC-CM differentiation. Data and Matlab codes can be downloaded from S1 Data.

  16. Efficient Generation of Corticofugal Projection Neurons from Human Embryonic Stem Cells.

    Science.gov (United States)

    Zhu, Xiaoqing; Ai, Zongyong; Hu, Xintian; Li, Tianqing

    2016-06-27

    Efforts to study development and function of corticofugal projection neurons (CfuPNs) in the human cerebral cortex for health and disease have been limited by the unavailability of highly enriched CfuPNs. Here, we develop a robust, two-step process for generating CfuPNs from human embryonic stem cells (hESCs): directed induction of neuroepithelial stem cells (NESCs) from hESCs and efficient differentiation of NESCs to about 80% of CfuPNs. NESCs or a NESC faithfully maintain unlimitedly self-renewal and self-organized abilities to develop into miniature neural tube-like structures. NESCs retain a stable propensity toward neuronal differentiation over culture as fate-restricted progenitors of CfuPNs and interneurons. When grafted into mouse brains, NESCs successfully integrate into the host brains, differentiate into CfuPNs and effectively reestablish specific patterns of subcortical projections and synapse structures. Efficient generation of CfuPNs in vitro and in vivo will facilitate human cortex development and offer sufficient CfuPNs for cell therapy.

  17. Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

    Science.gov (United States)

    Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

    2009-03-01

    Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

  18. Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members.

    Directory of Open Access Journals (Sweden)

    David T Madden

    Full Text Available Two of the greatest challenges in regenerative medicine today remain (1 the ability to culture human embryonic stem cells (hESCs at a scale sufficient to satisfy clinical demand and (2 the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.

  19. The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells.

    Science.gov (United States)

    Boisvert, Erin M; Engle, Sandra J; Hallowell, Shawn E; Liu, Ping; Wang, Zhao-Wen; Li, Xue-Jun

    2015-11-19

    Nociceptive neurons play an essential role in pain sensation by transmitting painful stimuli to the central nervous system. However, investigations of nociceptive neuron biology have been hampered by the lack of accessibility of human nociceptive neurons. Here, we describe a system for efficiently guiding human embryonic stem cells into nociceptive neurons by first inducing these cells to the neural lineage. Subsequent addition of retinoic acid and BMP4 at specific time points and concentrations yielded a high population of neural crest progenitor cells (AP2α(+), P75(+)), which further differentiated into nociceptive neurons (TRKA(+), Nav1.7(+), P2X3(+)). The overexpression of Neurogenin 1 (Neurog1) promoted the neurons to express genes related to sensory neurons (Peripherin, TrkA) and to further mature into TRPV1(+) nociceptive neurons. Importantly, the overexpression of Neurog1 increased the response of these neurons to capsaicin stimulation, a hallmark of mature functional nociceptive neurons. Taken together, this study reveals the important role that Neurog1 plays in generating functional human nociceptive neurons.

  20. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  1. Differentiation of human embryonic stem cells after transplantation in immune-deficient mice.

    Science.gov (United States)

    Przyborski, Stefan A

    2005-10-01

    Our current knowledge of how human tissues grow and develop is limited. We need to increase our understanding of tissue formation if we are to fully realize the potential of stem cells as a source of material for research into health and disease and possible therapeutic applications. Transplanted pluripotent human embryonic stem cells (hESCs) provide a potential system to model and investigate cell differentiation in humans. hESCs transplanted into immune-deficient mice form complex teratomas consisting of a range of differentiated somatic tissues, some of which appear highly organized and resemble structures normally identified in the embryo and adult. Analysis of such tumors may provide a unique opportunity to study organogenesis and lead to novel approaches in bioengineering and the growth of functioning structures composed of a range of alternative cell types. However, little has been done to characterize the developmental potential of hESCs after transplantation. This concise review presents evidence for the ability of hESCs to differentiate in vivo and highlights some of the prominent questions that need to be addressed if transplantation is to be used as a research tool to study hESC differentiation.

  2. Human embryonic and induced pluripotent stem cell research trends: complementation and diversification of the field.

    Science.gov (United States)

    Kobold, Sabine; Guhr, Anke; Kurtz, Andreas; Löser, Peter

    2015-05-12

    Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a "gold standard," but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Human Embryonic and Induced Pluripotent Stem Cell Research Trends: Complementation and Diversification of the Field

    Directory of Open Access Journals (Sweden)

    Sabine Kobold

    2015-05-01

    Full Text Available Research in human induced pluripotent stem cells (hiPSCs is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs, the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a “gold standard,” but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas.

  4. Endogenous APOBEC3B restricts LINE-1 retrotransposition in transformed cells and human embryonic stem cells.

    Science.gov (United States)

    Wissing, Silke; Montano, Mauricio; Garcia-Perez, Jose Luis; Moran, John V; Greene, Warner C

    2011-10-21

    Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.

  5. Human very small embryonic-like cells generate skeletal structures, in vivo.

    Science.gov (United States)

    Havens, Aaron M; Shiozawa, Yusuke; Jung, Younghun; Sun, Hongli; Wang, Jincheng; McGee, Samantha; Mishra, Anjali; Taichman, L Susan; Danciu, Theodora; Jiang, Yajuan; Yavanian, Gregory; Leary, Elizabeth; Krebsbach, Paul H; Rodgerson, Denis; Taichman, Russell S

    2013-02-15

    Human very small embryonic-like (hVSEL) cells are a resident population of multipotent stem cells in the bone marrow involved in the turnover and regeneration of tissues. The levels of VSEL cells in blood are greatly increased in response to injury, and they have been shown to repair injured tissues. Adult hVSEL cells, SSEA-4(+)/CD133(+)/CXCR4(+)/Lin(-)/CD45(-), express the pluripotency markers (Oct-4 and Nanog) and may be able to differentiate into cells from all 3 germ lineages. hVSEL cells isolated from blood by apheresis following granulocyte-colony-stimulating factor mobilization were fractionated and enriched by elutriation and fluorescence activated cell sorting. Collagen sponge scaffolds containing 2,000-30,000 hVSEL cells were implanted into cranial defects generated in SCID mice. Analysis by microcomputed tomography showed that a cell population containing VSEL cells produced mineralized tissue within the cranial defects compared with controls at 3 months. Histologic studies showed significant bone formation and cellular organization within the defects compared with cellular or scaffold controls alone. Antibodies to human leukocyte antigens demonstrated that the newly generated tissues were of human origin. Moreover, human osteocalcin was identified circulating in the peripheral blood. There was evidence that some level of hVSEL cells migrated away from the defect site, using quantitative real-time polymerase chain reaction to detect for human-specific Alu sequences. This study demonstrates that hVSEL cells are able to generate human bone tissue in a mouse model of skeletal repair. These studies lay the foundation for future cell-based regenerative therapies for osseous and connective tissue disorders, including trauma and degenerative conditions, such as osteoporosis, fracture repair, and neoplastic repair.

  6. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

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    Armin Arshi, Yasuhiro Nakashima, Haruko Nakano, Sarayoot Eaimkhong, Denis Evseenko, Jason Reed, Adam Z Stieg, James K Gimzewski and Atsushi Nakano

    2013-01-01

    Full Text Available While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

  7. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

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    Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

    2013-04-01

    While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

  8. Variations of X chromosome inactivation occur in early passages of female human embryonic stem cells.

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    Tamar Dvash

    Full Text Available X chromosome inactivation (XCI is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs derived from inner cell mass (ICM of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1 cells in a pre-XCI state, 2 cells that already exhibit XCI, or 3 cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs.

  9. Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

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    János Pálóczi

    2016-01-01

    Full Text Available Background and Aims. Human embryonic stem cell- (hESC- derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days and then on more differentiated cardiomyocytes (6 + 24 days, both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP (10−7, 10−6, and 10−5 M, BNP (10−9, 10−8, and 10−7 M, and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10−5 M. Results. SNAP (10−6, 10−5 M significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10−6 M also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms.

  10. Changes in glycosphingolipid composition during differentiation of human embryonic stem cells to ectodermal or endodermal lineages.

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    Liang, Yuh-Jin; Yang, Bei-Chia; Chen, Jin-Mei; Lin, Yu-Hsing; Huang, Chia-Lin; Cheng, Yuan-Yuan; Hsu, Chi-Yen; Khoo, Kay-Hooi; Shen, Chia-Ning; Yu, John

    2011-12-01

    Glycosphingolipids (GSLs) are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction and can possibly be used as cell type-specific markers. Our previous study indicated that there was a striking switch in the core structures of GSLs during differentiation of human embryonic stem cells (hESCs) into embryoid body (EB), suggesting a close association of GSLs with cell differentiation. In this study, to further clarify if alterations in GSL patterns are correlated with lineage-specific differentiation of hESCs, we analyzed changes in GSLs as hESCs were differentiated into neural progenitors or endodermal cells by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) analyses. During hESC differentiation into neural progenitor cells, we found that the core structures of GSLs switched from globo- and lacto- to mostly ganglio-series dominated by GD3. On the other hand, when hESCs were differentiated into endodermal cells, patterns of GSLs totally differed from those observed in EB outgrowth and neural progenitors. The most prominent GSL identified by the MALDI-MS and MS/MS analysis was Gb(4) Ceramide, with no appreciable amount of stage-specific embryonic antigens 3 or 4, or GD3, in endodermal cells. These changes in GSL profiling were accompanied by alterations in the biosynthetic pathways of expressions of key glycosyltransferases. Our findings suggest that changes in GSLs are closely associated with lineage specificity and differentiation of hESCs.

  11. Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells

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    Carolina Paola García

    2014-03-01

    Full Text Available Embryonic stem cells (ESCs need to maintain their genomic integrity in response to DNA damage to safeguard the integrity of the organism. DNA double strand breaks (DSBs are one of the most lethal forms of DNA damage and, if not repaired correctly, they can lead to cell death, genomic instability and cancer. How human ESCs (hESCs maintain genomic integrity in response to agents that cause DSBs is relatively unclear. In the present study we aim to determine the hESC response to the DSB inducing agent camptothecin (CPT. We find that hESCs are hypersensitive to CPT, as evidenced by high levels of apoptosis. CPT treatment leads to DNA-damage sensor kinase (ATM and DNA-PKcs phosphorylation on serine 1981 and serine 2056, respectively. Activation of ATM and DNA-PKcs was followed by histone H2AX phosphorylation on Ser 139, a sensitive reporter of DNA damage. Nuclear accumulation and ATM-dependent phosphorylation of p53 on serine 15 were also observed. Remarkably, hESC viability was further decreased when ATM or DNA-PKcs kinase activity was impaired by the use of specific inhibitors. The hypersensitivity to CPT treatment was markedly reduced by blocking p53 translocation to mitochondria with pifithrin-μ. Importantly, programmed cell death was achieved in the absence of the cyclin dependent kinase inhibitor, p21Waf1, a bona fide p53 target gene. Conversely, differentiated hESCs were no longer highly sensitive to CPT. This attenuated apoptotic response was accompanied by changes in cell cycle profile and by the presence of p21Waf1. The results presented here suggest that p53 has a key involvement in preventing the propagation of damaged hESCs when genome is threatened. As a whole, our findings support the concept that the phenomenon of apoptosis is a prominent player in normal embryonic development.

  12. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions.

    Science.gov (United States)

    Klimchenko, Olena; Di Stefano, Antonio; Geoerger, Birgit; Hamidi, Sofiane; Opolon, Paule; Robert, Thomas; Routhier, Mélanie; El-Benna, Jamel; Delezoide, Anne-Lise; Boukour, Siham; Lescure, Bernadette; Solary, Eric; Vainchenker, William; Norol, Françoise

    2011-03-17

    The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

  13. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension.

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    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E; Qvortrup, Klaus; Baar, Keith; Svensson, René B; Magnusson, S Peter; Krogsgaard, Michael; Koch, Manuel; Kjaer, Michael

    2010-06-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon. (c) 2010 Elsevier Ltd. All rights reserved.

  14. Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

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    Levenstein, Mark E; Berggren, W Travis; Lee, Ji Eun; Conard, Kevin R; Llanas, Rachel A; Wagner, Ryan J; Smith, Lloyd M; Thomson, James A

    2008-12-01

    Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

  15. Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells

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    Malik Mohammed T

    2005-01-01

    Full Text Available Abstract Background Pituitary tumor transforming gene1 (PTTG1 is a novel oncogene that is expressed in most tumors. It encodes a protein that is primarily involved in the regulation of sister chromatid separation during cell division. The oncogenic potential of PTTG1 has been well characterized in the mouse, particularly mouse fibroblast (NIH3T3 cells, in which it induces cell proliferation, promotes tumor formation and angiogenesis. Human tumorigenesis is a complex and a multistep process often requiring concordant expression of a number of genes. Also due to differences between rodent and human cell biology it is difficult to extrapolate results from mouse models to humans. To determine if PTTG1 functions similarly as an oncogene in humans, we have characterized its effects on human embryonic kidney (HEK293 cells. Results We report that introduction of human PTTG1 into HEK293 cells through transfection with PTTG1 cDNA resulted in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Pathologic analysis revealed that these tumors were poorly differentiated. Both analysis of HEK293 cells transiently transfected with PTTG1 cDNA and analysis of tumors developed on injection of HEK293 cells that had been stably transfected with PTTG1 cDNA indicated significantly higher levels of secretion and expression of bFGF, VEGF and IL-8 compared to HEK293 cells transfected with pcDNA3.1 vector or uninvolved tissues collected from the mice. Mutation of the proline-rich motifs at the C-terminal of PTTG1 abolished its oncogenic properties. Mice injected with this mutated PTTG1 either did not form tumors or formed very small tumors. Taken together our results suggest that PTTG1 is a human oncogene that possesses the ability to promote tumorigenesis in human cells at least in part through the regulation of expression or secretion of bFGF, VEGF and IL-8. Conclusions Our results

  16. Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells

    Science.gov (United States)

    Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in...

  17. Gene expression profiling of embryonic human neural stem cells and dopaminergic neurons from adult human substantia nigra.

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    Hany E S Marei

    Full Text Available Neural stem cells (NSC with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat neurodegenerative insults such as Parkinson's disease. We used Agilent's and Illumina Whole Human Genome Oligonucleotide Microarray to compare the genomic profiles of human embryonic NSC at a single time point in culture, and a multicellular tissue from postmortem adult substantia nigra (SN which are rich in dopaminergic (DA neurons. We identified 13525 up-regulated genes in both cell types of which 3737 (27.6% genes were up-regulated in the hENSC, 4116 (30.4% genes were up-regulated in the human substantia nigra dopaminergic cells, and 5672 (41.93% were significantly up-regulated in both cell population. Careful analysis of the data that emerged using DAVID has permitted us to distinguish several genes and pathways that are involved in dopaminergic (DA differentiation, and to identify the crucial signaling pathways that direct the process of differentiation. The set of genes expressed more highly at hENSC is enriched in molecules known or predicted to be involved in the M phase of the mitotic cell cycle. On the other hand, the genes enriched in SN cells include a different set of functional categories, namely synaptic transmission, central nervous system development, structural constituents of the myelin sheath, the internode region of axons, myelination, cell projection, cell somata, ion transport, and the voltage-gated ion channel complex. Our results were also compared with data from various databases, and between different types of arrays, Agilent versus Illumina. This approach has allowed us to confirm the consistency of our obtained results for a large number of genes that delineate the phenotypical differences of embryonic NSCs, and SN cells.

  18. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

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    Heema Hewitson

    2016-03-01

    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  19. Limited Gene Expression Variation in Human Embryonic Stem Cell and Induced Pluripotent Stem Cell Derived Endothelial Cells

    OpenAIRE

    2013-01-01

    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based ...

  20. Is stage-specific embryonic antigen 4 a marker for human ductal stem/progenitor cells?

    Science.gov (United States)

    Afrikanova, Ivka; Kayali, Ayse; Lopez, Ana; Hayek, Alberto

    2012-08-01

    The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4(+) cells scattered in the ducts do not show a ductal cell phenotype. SSEA4(+)-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4(+) cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells.

  1. Is Stage-Specific Embryonic Antigen 4 a Marker for Human Ductal Stem/Progenitor Cells?

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    Kayali, Ayse; Lopez, Ana; Hayek, Alberto

    2012-01-01

    Abstract The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4+ cells scattered in the ducts do not show a ductal cell phenotype. SSEA4+-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4+ cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells. PMID:23515456

  2. Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

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    Stachelscheid, H; Wulf-Goldenberg, A; Eckert, K; Jensen, J; Edsbagge, J; Björquist, P; Rivero, M; Strehl, R; Jozefczuk, J; Prigione, A; Adjaye, J; Urbaniak, T; Bussmann, P; Zeilinger, K; Gerlach, J C

    2013-09-01

    Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.

  3. NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

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    Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

    2014-08-01

    Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

  4. New glimpses of caveolin-1 functions in embryonic development and human diseases

    Institute of Scientific and Technical Information of China (English)

    Saijun MO; Shengli YANG; Zongbin CUI

    2011-01-01

    Caveolin-1 (Cav-1) isoforms,including Cav-1α and Cav-1β,were identified as integral membrane proteins and the major components of caveolae.Cav-1 proteins are highly conserved during evolution from Caenorhabditis elegans to human and are capable of interacting with many signaling molecules through their caveolin scaffolding domains to regulate the activities of multiple signaling pathways.Thus,Cav-1 plays crucial roles in the regulation of cellular proliferation,differentiation and apoptosis in a cell-specific and contextual manner.In addition,Cav-1 is essential for embryonic development of vertebrates owing to its regulation of BMP,Wnt,TGF-β and other key signaling molecules.Moreover,Cav-1 is mainly expressed in terminally differentiated cells and its abnormal expression is often associated with human diseases,such as tumor progression,cardiovascular diseases,fibrosis,lung regeneration,and diseases related to virus.In this review,we will further discuss the potential of Cav-1 as a target for disease therapy and multiple drug resistance.

  5. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

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    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  6. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

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    Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui

    2009-07-31

    The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  7. Derivation of high purity neuronal progenitors from human embryonic stem cells.

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    Gabriel Nistor

    Full Text Available The availability of human neuronal progenitors (hNPs in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use.

  8. SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

    Science.gov (United States)

    Toscano, Miguel G; Navarro-Montero, Oscar; Ayllon, Veronica; Ramos-Mejia, Veronica; Guerrero-Carreno, Xiomara; Bueno, Clara; Romero, Tamara; Lamolda, Mar; Cobo, Marien; Martin, Francisco; Menendez, Pablo; Real, Pedro J

    2015-01-01

    Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

  9. Nanotopographical control of human embryonic stem cell differentiation into definitive endoderm.

    Science.gov (United States)

    Ghanian, Mohammad Hossein; Farzaneh, Zahra; Barzin, Jalal; Zandi, Mojgan; Kazemi-Ashtiani, Mohammad; Alikhani, Mehdi; Ehsani, Morteza; Baharvand, Hossein

    2015-11-01

    Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regenerative medicine for endoderm-derived organs such as the pancreas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to promote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentiation. Here, we have developed a simple method to precisely fabricate electrospun poly(ε-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the optimal topography leading to the promoted differentiation compared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE-specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell-matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in prospective tissue engineering applications.

  10. A large-scale proteomic analysis of human embryonic stem cells

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    Sherrer Eric

    2007-12-01

    Full Text Available Abstract Background Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. Results Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. Conclusion Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.

  11. In vitro induction of human embryonic stem cells into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Human embryonic stem cells (hES cells) are pluripotent and provide a unique, unlimited resource for human hepatocytes, which can serve as a novel cell source for cell transplantation and bioartificial liver (BAL). Here, we have developed a procedure by which hES cells can differentiate into hepatocyte-like cells. After being cultured in suspension in bacteriological petri dishes for 7 d, hES cells developed into cystic embryoid bodies (EBs). The EBs were then cultured in conditional medium containing dexamethasone and insulin in collagen type I-coated tissue culture dishes for two weeks. The hES cell-derived hepatocyte-like cells (HLCs) displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced HLCs expressed several hepatocyte specific genes including AFP, ALB, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells executed a range of hepatocyte functions, such as ICG uptake/excretion, glycogen deposits, albumin production and ammonium metabolism. Taken together, our results show that HLCs exhibit similar morphologic, phenotypic, and functional characteristics to hepatocytes.

  12. Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development.

    Science.gov (United States)

    Parent, Audrey V; Russ, Holger A; Khan, Imran S; LaFlam, Taylor N; Metzger, Todd C; Anderson, Mark S; Hebrok, Matthias

    2013-08-01

    Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into thymic epithelial progenitors (TEPs) by precise regulation of TGFβ, BMP4, RA, Wnt, Shh, and FGF signaling. The hESC-derived TEPs further mature into functional TECs that support T cell development upon transplantation into thymus-deficient mice. Importantly, the engrafted TEPs produce T cells capable of in vitro proliferation as well as in vivo immune responses. Thus, hESC-derived TEP grafts may have broad applications for enhancing engraftment in cell-based therapies as well as restoring age- and stress-related thymic decline.

  13. MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells.

    Science.gov (United States)

    Lakshmipathy, Uma; Love, Brad; Goff, Loyal A; Jörnsten, Rebecka; Graichen, Ralph; Hart, Ronald P; Chesnut, Jonathan D

    2007-12-01

    Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.

  14. Efficient engineering of vascularized ectopic bone from human embryonic stem cell-derived mesenchymal stem cells.

    Science.gov (United States)

    Domev, Hagit; Amit, Michal; Laevsky, Ilana; Dar, Ayelet; Itskovitz-Eldor, Joseph

    2012-11-01

    Human mesenchymal stem cells (hMSCs) can be derived from various adult and fetal tissues. However, the quality of tissues for the isolation of adult and fetal hMSCs is donor dependent with a nonreproducible yield. In addition, tissue engineering and cell therapy require large-scale production of a pure population of lineage-restricted stem cells that can be easily induced to differentiate into a specific cell type. Therefore, human embryonic stem cells (hESCs) can provide an alternative, plentiful source for generation of reproducible hMSCs. We have developed efficient differentiation protocols for derivation of hMSCs from hESCs, including coculture with murine OP9 stromal cells and feeder layer-free system. Our protocols have resulted in the generation of up to 49% of hMSCs, which expressed CD105, CD90, CD29, and CD44. The hMSCs exhibited high adipogenic, chondrocytic, and osteogenic differentiation in vitro. The latter correlated with osteocalcin secretion and vascular endothelial growth factor (VEGF) production by the differentiating hMSCs. hMSC-derived osteoblasts further differentiated and formed ectopic bone in vivo, and induced the formation of blood vessels in Matrigel implants. Our protocol enables generation of a purified population of hESC-derived MSCs, with the potential of differentiating into several mesodermal lineages, and particularly into vasculogenesis-inducing osteoblasts, which can contribute to the development of bone repair protocols.

  15. Myocardial regeneration strategies using human embryonic stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Capi, Oren; Gepstein, Lior

    2006-11-28

    Regenerative medicine is a new biomedicine discipline that takes advantage of the recent advancements in the fields of stem cell biology, molecular biology, and tissue engineering to derive tissue substitutes, in an attempt to replace or modify the function of diseased organs. The heart represents an attractive candidate for these emerging technologies since adult cardiac tissue has limited regenerative capacity. Consequentially, myocardial cell replacement therapy has emerged as a novel therapeutic paradigm for restoration of the myocardial electromechanical function. This innovative strategy has been significantly hampered, however, by the paucity of cell sources for human cardiomyocytes. The recent establishment of the human embryonic stem cell (hESC) lines may provide a possible solution for this cell-sourcing problem. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. This review will describe the hESC system, their differentiation into cardiomyocytes, and the structural and functional characterization of these cardiac lineage derivatives. The potential applications of this unique differentiating system in several research areas will be discussed with special emphasis on the steps required to fully harness their unique potential in the emerging field of cardiovascular regenerative medicine.

  16. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    Science.gov (United States)

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

  17. GADD45β Determines Chemoresistance and Invasive Growth of Side Population Cells of Human Embryonic Carcinoma

    Directory of Open Access Journals (Sweden)

    Toshihiko Inowa

    2010-01-01

    Full Text Available Side population (SP cells are an enriched population of stem, and the existence of SP cells has been reported in human cancer cell lines. In this study, we performed an SP analysis using 11 human cancer cell lines and confirmed the presence of SP cells in an embryonic carcinoma cell line, NEC8. NEC8 SP cells showed characteristics of cancer stem cells, such as high growth rate, chemoresistance and high invasiveness. To further characterize the NEC8 SP cells, we used DNA microarrays. Among 38,500 genes, we identified 12 genes that were over-expressed in SP cells and 1 gene that was over-expressed in non-SP cells. Among these 13 genes, we focused on GADD45b. GADD45b was over-expressed in non-SP cells, but the inhibition of GADD45b had no effect on non-SP cells. Paradoxically, the inhibition of GADD45b significantly reduced the viability of NEC8 SP cells. The inhibition of ABCG2, which determines the SP phenotype, had no effect on the invasiveness of NEC8 SP cells, but the inhibition of GADD45b significantly reduced invasiveness. These results suggest that GADD45b, but not ABCG2, might determine the cancer stem cell-like phenotype, such as chemoresistance and the high invasiveness of NEC8 SP cells, and might be a good therapeutic target.

  18. The regulation of self-renewal in human embryonic stem cells.

    Science.gov (United States)

    Avery, Stuart; Inniss, Katie; Moore, Harry

    2006-10-01

    Human embryonic stem (hES) cells have the ability to self-renew while maintaining their pluripotency. The ability of stem cells to self-renew expansively is essential in both development and maintenance of adult tissues. ES cell lines were first generated from mouse blastocysts, these lines provided much needed information regarding ES cell propagation, growth factor dependence, and marker expression. However, the application potential of murine models is restricted in value because many differences between mouse and human ES cells have since been identified. The process of hES cells self-renewal appears to be regulated by many different pathways; however, the molecular mechanisms enabling this process are not fully characterized. Further defining these mechanisms will enable growth of hES cells under defined conditions and aid controlled differentiation of cells into specified lineages, in turn providing cells suitable for therapeutic applications. This review provides a summary of the mechanisms known to control self-renewal and pluripotency in hES cells.

  19. CXCR4 activation promotes differentiation of human embryonic stem cells to neural stem cells.

    Science.gov (United States)

    Zhang, Lijun; Hua, Qiuhong; Tang, Kaiyi; Shi, Changjie; Xie, Xin; Zhang, Ru

    2016-11-19

    G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth, death, movement, transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study, we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol, we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially, the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted, whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4, knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together, our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine.

  20. Techniques of Human Embryonic Stem Cell and Induced Pluripotent Stem Cell Derivation.

    Science.gov (United States)

    Lewandowski, Jarosław; Kurpisz, Maciej

    2016-10-01

    Developing procedures for the derivation of human pluripotent stem cells (PSCs) gave rise to novel pathways into regenerative medicine research. For many years, stem cells have attracted attention as a potentially unlimited cell source for cellular therapy in neurodegenerative disorders, cardiovascular diseases, and spinal cord injuries, for example. In these studies, adult stem cells were insufficient; therefore, many attempts were made to obtain PSCs by other means. This review discusses key issues concerning the techniques of pluripotent cell acquisition. Technical and ethical issues hindered the medical use of somatic cell nuclear transfer and embryonic stem cells. Therefore, induced PSCs (iPSCs) emerged as a powerful technique with great potential for clinical applications, patient-specific disease modelling and pharmaceutical studies. The replacement of viral vectors or the administration of analogous proteins or chemical compounds during cell reprogramming are modifications designed to reduce tumorigenesis risk and to augment the procedure efficiency. Intensified analysis of new PSC lines revealed other barriers to overcome, such as epigenetic memory, disparity between human and mouse pluripotency, and variable response to differentiation of some iPSC lines. Thus, multidimensional verification must be conducted to fulfil strict clinical-grade requirements. Nevertheless, the first clinical trials in patients with spinal cord injury and macular dystrophy were recently carried out with differentiated iPSCs, encouraging alternative strategies for potential autologous cellular therapies.

  1. Embryonic stem cells as an ectodermal cellular model of human p63-related dysplasia syndromes.

    NARCIS (Netherlands)

    Rostagno, P.; Wolchinsky, Z.; Vigano, A.M.; Shivtiel, S.; Zhou, H.; Bokhoven, J.H.L.M. van; Ferone, G.; Missero, C.; Mantovani, R.; Aberdam, D.; Virolle, T.

    2010-01-01

    Heterozygous mutations in the TP63 transcription factor underlie the molecular basis of several similar autosomal dominant ectodermal dysplasia (ED) syndromes. Here we provide a novel cellular model derived from embryonic stem (ES) cells that recapitulates in vitro the main steps of embryonic skin d

  2. Evolutionary emergence of microRNAs in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Hong Cao

    Full Text Available Human embryonic stem (hES cells have unique abilities to divide indefinitely without differentiating and potential to differentiate into more than 200 cell types. These properties make hES cells an ideal model system for understanding early human development and for regenerative medicine. Molecular mechanisms including cellular signaling and transcriptional regulation play important roles in hES cell differentiation. However, very little information is available on posttranscriptional regulation of hES cell pluripotency, self-renewal, and early decisions about cell fate. microRNAs (miRNAs, 22-nt long non-coding small RNAs found in plants and animals, regulate gene expression by targeting mRNAs for cleavage or translation repression. In hES cells we found that 276 miRNAs were expressed; of these, a set of 30 miRNAs had significantly changed expression during differentiation. Using a representative example, miR-302b, we show that miRNAs in human ES cells assemble into a bona fide RISC that contains Ago2 and can specifically cleave perfectly matched target RNA. Our results demonstrate that human ES cell differentiation is accompanied by changes in the expression of a unique set of miRNAs, providing a glimpse of a new molecular circuitry that may regulate early development in humans. Chromosomes 19 and X contained 98 and 40 miRNA genes, respectively, indicating that majority of miRNA genes in hES cells were expressed from these two chromosomes. Strikingly, distribution analysis of miRNA gene loci across six species including dog, rat, mouse, rhesus, chimpanzee, and human showed that miRNA genes encoded in chromosome 19 were drastically increased in chimpanzees and humans while miRNA gene loci on other chromosomes were decreased as compared with dog, rat, and mouse. Comparative genomic studies showed 99% conservation of chromosome 19 miRNA genes between chimpanzees and humans. Together, these findings reveal the evolutionary emergence, approximately

  3. A gene expression signature shared by human mature oocytes and embryonic stem cells

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    Pantesco Véronique

    2009-01-01

    Full Text Available Abstract Background The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation. Results Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5, 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1. Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome. Conclusion These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.

  4. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.

    Science.gov (United States)

    Finkbeiner, Stacy R; Freeman, Jennifer J; Wieck, Minna M; El-Nachef, Wael; Altheim, Christopher H; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S; Grikscheit, Tracy C; Teitelbaum, Daniel H; Spence, Jason R

    2015-10-12

    Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue.

  5. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids

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    Stacy R. Finkbeiner

    2015-11-01

    Full Text Available Short bowel syndrome (SBS is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs or induced pluripotent stem cells (iPSCs, called human intestinal organoids (HIOs, have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue.

  6. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun; Wang, Jing [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Chen, Guian [Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Reproductive Medical Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Fan, Dongsheng, E-mail: dsfan@yahoo.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Deng, Min, E-mail: dengmin1706@yahoo.com.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China)

    2011-01-14

    Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  7. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

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    Zhumur Ghosh

    Full Text Available Human induced pluripotent stem cells (hiPSCs generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs, as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

  8. Periconception Maternal Folate Status and Human Embryonic Cerebellum Growth Trajectories: The Rotterdam Predict Study.

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    Irene V Koning

    Full Text Available We aimed to investigate whether periconceptional maternal folate status affects human embryonic cerebellar size and growth trajectories. In a prospective periconceptional cohort participants filled out questionnaires and received weekly transvaginal 3D-ultrasounds between 7+0 and 12+6 weeks gestational age (GA. Viable non-malformed singleton pregnancies were selected for cerebellar measurements; transcerebellar diameter, (TCD, left and right cerebellar diameters (LCD, RCD. Linear mixed models were performed to estimate associations between questionnaire data on the timing of maternal folic acid supplement initiation and longitudinal cerebellar measurements as a function of crown-rump length (CRL and GA. Maternal red blood cell folate concentrations were analysed before 8 weeks GA to validate the associations. A total of 263 serial high quality three-dimensional ultrasound scans of 135 pregnancies were studied. Preconceptional compared to postconceptional initiation of folic acid use was associated with slightly larger cerebellar diameters per millimetre increase of CRL (TCD: β = 0.260mm, 95%CI = 0.023-0.491, p<0.05; LCD: β = 0.171mm, 95%CI = 0.038-0.305, p<0.05; RCD: β = 0.156mm, 95%CI = 0.032-0.280, p<0.05 and with proportional cerebellar growth (TCD/CRL:β = 0.015mm/mm, 95%CI = 0.005-0.024, p<0.01; LCD/CRL:β = 0.012mm/mm, 95%CI = 0.005-0.018, p<0.01; RCD/CRL:β = 0.011mm/mm, 95%CI = 0.005-0.017, p<0.01. Cerebellar growth was significantly highest in the third quartile of maternal red blood cell folate levels (1538-1813 nmol/L. These first findings show that periconceptional maternal folate status is associated with human embryonic cerebellar development. Implications of these small but significant variations for fetal cerebellar growth trajectories and the child's neurodevelopmental outcome are yet unknown and warrant further investigation.

  9. Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Rossner, Pavel, E-mail: prossner@biomed.cas.cz; Rossnerova, Andrea; Beskid, Olena; Tabashidze, Nana; Libalova, Helena; Uhlirova, Katerina; Topinka, Jan; Sram, Radim J.

    2014-05-15

    Highlights: • We analyzed the effect of air pollutants on NHEJ and chromosome aberrations. • In HEL12469 cells B[a]P and extractable organic matter induced DSBs. • The compounds induced XRCC4 expression and a weak Ku70/80 response. • We found increased frequency of aberrations of chromosomes 1, 2, 4, 5, 7 and 17. • The tested compounds preferentially affected chromosome 7. - Abstract: In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24 h with the following concentrations of tested chemicals: B[a]P: 1 μM, 10 μM, 25 μM; EOMs: 1 μg/ml, 10 μg/ml, 25 μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25 μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and

  10. Human BCAS3 expression in embryonic stem cells and vascular precursors suggests a role in human embryogenesis and tumor angiogenesis.

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    Kavitha Siva

    Full Text Available Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3 is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker.

  11. Definitive Endoderm Differentiation of Human Embryonic Stem Cells Combined with Selective Elimination of Undifferentiated Cells by Methionine Deprivation.

    Science.gov (United States)

    Tsuyama, Tomonori; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Human embryonic stem cells (ESCs) show a characteristic feature in that they are highly dependent on methionine metabolism. Undifferentiated human ESCs cannot survive under condition that methionine is deprived from culture medium. We describe here a procedure for definitive endoderm differentiation from human ESCs, in which human ESCs are subject to 10 days' (d) differentiation combined with methionine deprivation between differentiation days (d) 8 to (d) 10. Methionine deprivation results in elimination of undifferentiated cells from the culture with no significant loss of definitive endoderm cells, as compared to those cultured under complete condition throughout the whole culture period.

  12. Perspectives on palatalization

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    Martin Krämer

    2016-08-01

    Full Text Available In this article we provide a discussion of the definition of palatalization as a ­phonological ­phenomenon, its crosslinguistic variation, phonetic or functional grounding and phonetic (unnaturalness of palatalization, and theoretical approaches to palatalization patterns. After ­providing this background to the collection of articles in this special issue of 'Glossa' we will give an overview of the contributions collected here.

  13. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research.

    Science.gov (United States)

    Manninen, Bertha Alvarez

    2008-01-31

    One argument used by detractors of human embryonic stem cell research (hESCR) invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event) as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical operation. Finally, I will end the

  14. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research

    Directory of Open Access Journals (Sweden)

    Manninen Bertha

    2008-01-01

    Full Text Available Abstract One argument used by detractors of human embryonic stem cell research (hESCR invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical

  15. New President, New Human Embryonic Stem Cell Research Policy: Comparative International Perspectives and Embryonic Stem Cell Research Laws in France*

    Science.gov (United States)

    Drabiak-Syed, By Katherine

    2013-12-01

    This article provides an overview of French legislative history, Parliamentary debates, and recent amendments in hESC research policy, as well as additional comparisons with laws across the European Union. Unlike policy discussions in the U.S., French dialogue on hESC research generally rejects the arbitrary division between the status of the embryo and hESCs, recognizing that hESC research necessarily requires the destruction of human embryos. Accordingly, French discourse debates the competing interests of science with secular ethical and civic considerations relating to the symbolic status of the embryo and society's duty to moderate what constitutes appropriate boundaries on research. Parliament recently amended France's hESC research laws to explicitly permit hESC research, signaling the beginning of reform efforts under President Hollande's new power structure, but the inclusion of secular moral considerations in the policy debate will likely restrain the extent of any future changes.

  16. Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

    Science.gov (United States)

    Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

    2014-01-01

    In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

  17. Knockdown of E2F2 inhibits tumorigenicity, but preserves stemness of human embryonic stem cells.

    Science.gov (United States)

    Suzuki, Daniela Emi; Nakahata, Adriana Miti; Okamoto, Oswaldo Keith

    2014-06-01

    Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear, however, whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells, among which is E2F2, a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1, in addition to proliferation of hESC, indicated by a higher proportion of cells in G1, fewer cells in G2/M phase, and a reduced capacity to generate hESC colonies in vitro. Nonetheless, E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly, E2F2 knockdown in hESC significantly inhibited tumor growth in vivo, which was considerably smaller than tumors generated from control hESC, although displaying typical teratoma traits, a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness, providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells.

  18. Induction of human embryonic and induced pluripotent stem cells into urothelium.

    Science.gov (United States)

    Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

    2014-05-01

    In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

  19. Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway

    Science.gov (United States)

    Bosnjak, Zeljko J.; Yan, Yasheng; Canfield, Scott; Muravyeva, Maria Y.; Kikuchi, Chika; Wells, Clive; Corbett, John; Bai, Xiaowen

    2013-01-01

    Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies. PMID:22873495

  20. Development and morphogenesis of human wrist joint during embryonic and early fetal period.

    Science.gov (United States)

    Hita-Contreras, Fidel; Martínez-Amat, Antonio; Ortiz, Raúl; Caba, Octavio; Alvarez, Pablo; Prados, José C; Lomas-Vega, Rafael; Aránega, Antonia; Sánchez-Montesinos, Indalecio; Mérida-Velasco, Juan A

    2012-06-01

    The development of the human wrist joint has been studied widely, with the main focus on carpal chondrogenesis, ligaments and triangular fibrocartilage. However, there are some discrepancies concerning the origin and morphogenetic time-table of these structures, including nerves, muscles and vascular elements. For this study we used serial sections of 57 human embryonic (n = 30) and fetal (n = 27) specimens from O'Rahilly stages 17-23 and 9-14 weeks, respectively. The following phases in carpal morphogenesis have been established: undifferentiated mesenchyme (stage 17), condensated mesenchyme (stages 18 and 19), pre-chondrogenic (stages 19 and 20) and chondrogenic (stages 21 and over). Carpal chondrification and osteogenic processes are similar, starting with capitate and hamate (stage 19) and ending with pisiform (stage 22). In week 14, a vascular bud penetrates into the lunate cartilaginous mold, early sign of the osteogenic process that will be completed after birth. In stage 18, median, ulnar and radial nerves and thenar eminence appear in the hand plate. In stage 21, there are indications of the interosseous muscles, and in stage 22 flexor digitorum superficialis, flexor digitorum profundus and lumbrical muscles, transverse carpal ligament and collateral ligaments emerge. In stage 23, the articular disc, radiocarpal and ulnocarpal ligaments and deep palmar arterial arch become visible. Radiate carpal and interosseous ligaments appear in week 9, and in week 10, dorsal radiocarpal ligament and articular capsule are evident. Finally, synovial membrane is observed in week 13. We have performed a complete analysis of the morphogenesis of the structures of the human wrist joint. Our results present new data on nervous and arterial elements and provide the basis for further investigations on anatomical pathology, comparative morphology and evolutionary anthropology. © 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.

  1. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development.

    Science.gov (United States)

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-12-05

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes.

  2. Profiling TRA-1-81 antigen distribution on a human embryonic stem cell.

    Science.gov (United States)

    Qiu, Dengli; Xiang, Jialing; Li, Zhaoxia; Krishnamoorthy, Aparna; Chen, Liaohai; Wang, Rong

    2008-05-02

    Human embryonic stem (hES) cells hold great promise in regenerative medicine. Although hES cells have unlimited self-renewal potential, they tend to differentiate spontaneously in culture. TRA-1-81 is a biomarker of undifferentiated hES cells. Quantitative characterization of TRA-1-81 expression level in a single cell helps capture the "turn-on" signal and understand the mechanism of early differentiation. Here, we report on our examination of TRA-1-81 distribution and association on a hES cell membrane using an atomic force microscope (AFM). Our results suggest that aggregated distribution of TRA-1-81 antigen is characteristic for undifferentiated hES cells. We also evaluated the TRA-1-81 expression level at approximately 17,800 epitopes and approximately 700 epitopes per cell on an undifferentiated cell and a spontaneously differentiated cell, respectively. The method in this study can be adapted in examining other surface proteins on various cell types, thus providing a general tool for investigating protein distribution and association at the single cell level.

  3. Basic fibroblast growth factor and its receptors in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Ales Hampl

    2005-12-01

    Full Text Available Human embryonic stem cells (hESCs are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs, which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.

  4. A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells

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    Zengrong Zhu

    2015-06-01

    Full Text Available The development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs, including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.

  5. Functional and molecular characterization of the role of CTCF in human embryonic stem cell biology.

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    Sri Kripa Balakrishnan

    Full Text Available The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC biology. We demonstrate that CTCF associates with several important pluripotency genes, including NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADPribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.

  6. Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

    Science.gov (United States)

    Serra, Margarida; Correia, Cláudia; Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J T; Alves, Paula M

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

  7. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

    Science.gov (United States)

    Birket, Matthew J; Orr, Adam L; Gerencser, Akos A; Madden, David T; Vitelli, Cathy; Swistowski, Andrzej; Brand, Martin D; Zeng, Xianmin

    2011-02-01

    Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1β and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

  8. Folate antagonist, methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina.

    Science.gov (United States)

    Hara, Akira; Taguchi, Ayako; Aoki, Hitomi; Hatano, Yuichiro; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro

    2010-06-25

    Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-D-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.

  9. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

    Directory of Open Access Journals (Sweden)

    Nathan Salomonis

    2009-11-01

    Full Text Available The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org, we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

  10. CBARA1 plays a role in stemness and proliferation of human embryonic stem cells.

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    Kevin Chen

    Full Text Available Human embryonic stem cells (hESCs are capable of unlimited self-renewal and can generate almost all of the cells in the body. Although some pluripotency factors have been identified, much remains unclear regarding the molecules and mechanisms that regulate hESC self-renewal and pluripotency. In this study, we identified a mitochondrial gene, CBARA1, that is expressed in undifferentiated hESCs and that is down-regulated rapidly after cellular differentiation. To study its role in hESCs, endogenous CBARA1 expression was knocked down using shRNA. CBARA1 knockdown in hESCs resulted in down-regulation of Oct4 and Nanog expression, attenuated cell growth, and G0/G1 phase cell cycle arrest; however, knockdown did not noticeably affect apoptosis. Taken together, these results suggest that CBARA1 is a marker for undifferentiated hESCs that plays a role in maintaining stemness, cell cycle progression, and proliferation.

  11. Morphogenesis and three-dimensional movement of the stomach during the human embryonic period.

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    Kaigai, N; Nako, A; Yamada, S; Uwabe, C; Kose, K; Takakuwa, T

    2014-05-01

    The stomach develops as the local widening of the foregut after Carnegie stage (CS) 13 that moves in a dramatic and dynamic manner during the embryonic period. Using the magnetic resonance images of 377 human embryos, we present the morphology, morphometry, and three-dimensional movement of the stomach during CS16 and CS23. The stomach morphology revealed stage-specific features. The angular incisura and the cardia were formed at CS18. The change in the angular incisura angle was approximately 90° during CS19 and CS20, and was stomach revealed that the stomach gradually becomes "deflected" during development. The stomach may appear to move to the left laterally and caudally due to its deflection and differential growth. The track of the reference points in the stomach may reflect the visual three-dimensional movement. The movement of point M, representing the movement of the greater curvature, was different from that of points C (cardia) and P (pyloric antrum). The P and C were located just around the midsagittal plane in all the stages observed. Point M moved in the caudal-left lateral direction until CS22. Moreover, the vector CP does not rotate around the dorsoventral axis, as widely believed, but around the transverse axis. The plane CPM rotated mainly around the longitudinal axis. The data obtained will be useful for prenatal diagnosis in the near future.

  12. Differential impact of science policy on subfields of human embryonic stem cell research.

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    Moon, Seongwuk; Cho, Seong Beom

    2014-01-01

    In this research, we examine how restrictive policy influenced performance in human embryonic stem cell research (hESC) between 1998 and 2008. In previous research, researchers argued whether restrictive policy decreased the performance of stem cell research in some nations, especially in the US. Here, we hypothesize that this policy influenced specific subfields of the hESC research. To investigate the selective policy effects, we categorize hESC research publications into three subfields-derivation, differentiation, and medical application research. Our analysis shows that restrictive policy had different effects on different subfields. In general, the US outperformed in overall hESC research throughout these periods. In the derivation of hESC, however, the US almost lost its competence under restrictive policy. Interestingly, the US scientific community showed prominent resilience in hESC research through international collaboration. We concluded that the US resilience and performance stemmed from the wide breadth of research portfolio of US scientists across the hESC subfields, combined with their strategic efforts to collaborate internationally on derivation research.

  13. Red Ginseng Extract Facilitates the Early Differentiation of Human Embryonic Stem Cells into Mesendoderm Lineage

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    Yoon Young Kim

    2011-01-01

    Full Text Available Human embryonic stem cells (hESCs have capacities to self-renew and differentiate into all cell types in vitro. Red ginseng (RG is known to have a wide range of pharmacological effects in vivo; however, the reports on its effects on hESCs are few. In this paper, we tried to demonstrate the effects of RG on the proliferation and differentiation of hESCs. Undifferentiated hESCs, embryoid bodies (EBs, and hESC-derived cardiac progenitors (CPs were treated with RG extract at 0.125, 0.25, and 0.5 mg/mL. After treatment of undifferentiated hESCs from day 2 to day 6 of culture, BrdU labeling showed that RG treatment increased the proliferation of hESCs, and the expression of Oct4 and Nanog was increased in RG-treated group. To find out the effects of RG on early differentiation stage cells, EBs were treated with RG extract for 10 days and attached for further differentiation. Immunostaining for three germ layer markers showed that RG treatment increased the expressions of Brachyury and HNF3β on EBs. Also, RG treatment increased the expression of Brachyury in early-stage and of Nkx2.5 in late-stage hESC-derived CPs. These results demonstrate facilitating effects of RG extract on the proliferation and early differentiation of hESC.

  14. Analysis of Mitochondrial Function and Localisation during Human Embryonic Stem Cell Differentiation In Vitro

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    Prowse, Andrew B. J.; Chong, Fenny; Elliott, David A.; Elefanty, Andrew G.; Stanley, Edouard G.; Gray, Peter P.; Munro, Trent P.; Osborne, Geoffrey W.

    2012-01-01

    Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag. PMID:23284940

  15. Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

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    Andrew B J Prowse

    Full Text Available Human embryonic stem cell (hESC derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

  16. Toxic effects of decabromodiphenyl ether (BDE-209 on human embryonic kidney cells

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    Min eLi

    2014-05-01

    Full Text Available Polybrominated diphenyl ethers (PBDEs are widely used as flame-retardant additives in consumer and household products and can escape into the environment over time. PBDEs have become a global environmental organic pollutant due to the properties of persistence, toxicity, and bioaccumulation. The well-studied toxic effects of PBDEs mainly include thyroid hormone disruption and neurotoxicity. There is no consistent conclusions on the carcinogenic potential of PBDEs to date. Here, we explored the toxic effects of BDE-209 on human embryonic kidney cells (HEK293T. The comparison of the gene expression profiles of HEK293T cells with BDE-209 treatment and the negative control found that BDE-209 exposure may alter nucleosome organization through significantly changing the expression of histone gene clusters. The remodeled chromatin structure could further disturb systemic lupus erythematosus as one of the toxic effects of BDE-209. Additionally, gene sets of different cancer modules are positively correlated with BDE-209 exposure. This suggests that BDE-209 has carcinogenic potential for a variety of tumors. Collectively, BDE-209 has a broader toxicity not limited to disruption of thyroid hormone-related biological processes. Notably, the toxic effects of BDE-209 dissolved in dimethyl sulfoxide (DMSO is not the simply additive effects of BDE-209 and DMSO alone.

  17. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

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    Karina Needham

    2014-01-01

    Full Text Available In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps, with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31–56 days and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

  18. Flow cytometry of human embryonic kidney cells: A light scattering approach

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    Kunze, M. E.; Goolsby, C. L.; Todd, P. W.; Morrison, D. R.; Lewis, M. L.

    1985-01-01

    The mammalian kidney contains cells that transport water, convert vitamin D to active forms, synthesize hormones such a renin and erythropoietin, and produce enzymes such as urokinase, a plasminogen activator. Several of these functions are maintained by human embryonic kidney cells (HEK) cultivated in vitro. Biochemical study of these functions in their individual cell types in vitro requires purified populations of cells. Light-scattering activated cell sorting (LACS) was explored as a means of achieving such purifications. It was found that HEK cells at the first 1 to 5 passages in culture were heterogeneous with respect to 2-parameter light scattering intensity distribution, in which combined measurements included forward angle scattering (2.5 to 19 deg), 90 deg scattering, and time-of-flight size measurements. Size was measured at a resolution of 0.15 microns/channel in 256 channels using pulse-height independent pulse-width measurements. Two-parameter distributions combining these measurements were obtained for HEK cell subpopulations that had been purified by microgravity electrophoresis and subsequently propagated in culture. These distributions contained at least 3 subpopulations in all purified fractions, and results of experiments with prepurified cultured HEK cells indicated that subpopulations of living cells that were high in plasminogen-activator activity also contained the highest per cent of cells with high 90 deg light scatter intensity.

  19. Dynamics of single human embryonic stem cells and their pairs: a quantitative analysis

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    Wadkin, L E; Neganova, I; Parker, N G; Chichagova, V; Swan, G; Laude, A; Lako, M; Shukurov, A

    2016-01-01

    Numerous biological approaches are available to characterise the mechanisms which govern the formation of human embryonic stem cell (hESC) colonies. To understand how the kinematics of single and pairs of hESCs impact colony formation, we study their mobility characteristics using time-lapse imaging. We perform a detailed statistical analysis of their speed, survival, directionality, distance travelled and diffusivity. We confirm that single and pairs of cells migrate as a diffusive random walk. Moreover, we show that the presence of Cell Tracer significantly reduces hESC mobility. Our results open the path to employ the theoretical framework of the diffusive random walk for the prognostic modelling and optimisation of the growth of hESC colonies. Indeed, we employ this random walk model to estimate the seeding density required to minimise the occurrence of hESC colonies arising from more than one founder cell and the minimal cell number needed for successful colony formation. We believe that our prognostic m...

  20. Epigenetic Reprogramming of Human Embryonic Stem Cells into Skeletal Muscle Cells and Generation of Contractile Myospheres

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    Sonia Albini

    2013-03-01

    Full Text Available Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB, but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3 confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for “disease in a dish” models of muscular physiology and dysfunction.