Generation of stomach tissue from mouse embryonic stem cells.

Science.gov (United States)

Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

2015-08-01

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

The promise of human embryonic stem cells in aging-associated diseases

Science.gov (United States)

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

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...

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

2013-01-01

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. PMID:23255130

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....

The ethics of patenting human embryonic stem cells.

Science.gov (United States)

Chapman, Audrey R

2009-09-01

Just as human embryonic stem cell research has generated controversy about the uses of human embryos for research and therapeutic applications, human embryonic stem cell patents raise fundamental ethical issues. The United States Patent and Trademark Office has granted foundational patents, including a composition of matter (or product) patent to the Wisconsin Alumni Research Foundation (WARF), the University of Wisconsin-Madison's intellectual property office. In contrast, the European Patent Office rejected the same WARF patent application for ethical reasons. This article assesses the appropriateness of these patents placing the discussion in the context of the deontological and consequentialist ethical issues related to human embryonic stem cell patenting. It advocates for a patent system that explicitly takes ethical factors into account and explores options for new types of intellectual property arrangements consistent with ethical concerns.

Maternal transfer of methimazole and effects on thyroid hormone availability in embryonic tissues.

Science.gov (United States)

Van Herck, Stijn L J; Geysens, Stijn; Bald, Edward; Chwatko, Grazyna; Delezie, Evelyne; Dianati, Elham; Ahmed, R G; Darras, Veerle M

2013-07-01

Methimazole (MMI) is an anti-thyroid drug used in the treatment of chronic hyperthyroidism. There is, however, some debate about its use during pregnancy as MMI is known to cross the mammalian placenta and reach the developing foetus. A similar problem occurs in birds, where MMI is deposited in the egg and taken up by the developing embryo. To investigate whether maternally derived MMI can have detrimental effects on embryonic development, we treated laying hens with MMI (0.03% in drinking water) and measured total and reduced MMI contents in the tissues of hens and embryos at different stages of development. In hens, MMI was selectively increased in the thyroid gland, while its levels in the liver and especially brain remained relatively low. Long-term MMI treatment induced a pronounced goitre with a decrease in thyroxine (T₄) content but an increase in thyroidal 3,5,3'-triiodothyronine (T₃) content. This resulted in normal T₃ levels in tissues except in the brain. In chicken embryos, MMI levels were similar in the liver and brain. They gradually decreased during development but always remained above those in the corresponding maternal tissues. Contrary to the situation in hens, T₄ availability was only moderately affected in embryos. Peripheral T₃ levels were reduced in 14-day-old embryos but normal in 18-day-old embryos, while brain T₃ content was decreased at all embryonic stages tested. We conclude that all embryonic tissues are exposed to relatively high doses of MMI and its oxidised metabolites. The effect of maternal MMI treatment on embryonic thyroid hormone availability is most pronounced for brain T₃ content, which is reduced throughout the embryonic development period.

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3.

Science.gov (United States)

Kapacee, Zoher; Yeung, Ching-Yan Chloé; Lu, Yinhui; Crabtree, David; Holmes, David F; Kadler, Karl E

2010-10-01

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days. Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Immunohistochemical Study of Expression of Sohlh1 and Sohlh2 in Normal Adult Human Tissues.

Directory of Open Access Journals (Sweden)

Xiaoli Zhang

Full Text Available The expression pattern of Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix 1 and Sohlh2 in mice has been reported in previous studies. Sohlh1 and Sohlh2 are specifically expressed in spermatogonia, prespermatogonia in male mice and oocytes of primordial and primary follicles in female mice. In this report, we studied the expression pattern of Sohlh1 and Sohlh2 in human adult tissues. Immunohistochemical staining of Sohlh1 and Sohlh2 was performed in 5 samples of normal ovaries and testes, respectively. The results revealed that Sohlh genes are not only expressed in oocytes and spermatogonia, but also in granular cells, theca cells, Sertoli cells and Leydig cells, and in smooth muscles of blood vessel walls. To further investigate the expression of Sohlh genes in other adult human tissues, we collected representative normal adult tissues developed from three embryonic germ layers. Compared with the expression in mice, Sohlhs exhibited a much more extensive expression pattern in human tissues. Sohlhs were detected in testis, ovary and epithelia developed from embryonic endoderm, ectoderm and tissues developed from embryonic mesoderm. Sohlh signals were found in spermatogonia, Sertoli cells and also Leydig cells in testis, while in ovary, the expression was mainly in oocytes of primordial and primary follicles, granular cells and theca cells of secondary follicles. Compared with Sohlh2, the expression of Sohlh1 was stronger and more extensive. Our study explored the expression of Sohlh genes in human tissues and might provide insights for functional studies of Sohlh genes.

Teratoma Formation by Human Embryonic Stem Cells is site-dependent and enhanced by the presence of Matrigel

DEFF Research Database (Denmark)

Prokhorova, Tatyana A; Harkness, Linda M; Frandsen, Ulrik

2008-01-01

When implanted into immunodeficient mice, human embryonic stem cells (hESC) give rise to teratoma, tumour-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data...

Dimethadione embryotoxicity in the rat is neither correlated with maternal systemic drug concentrations nor embryonic tissue levels

Energy Technology Data Exchange (ETDEWEB)

Ozolinš, Terence R.S., E-mail: ozolinst@queensu.ca [Department of Biomedical and Molecular Sciences, Program in Pharmacology and Toxicology, Queen’s University, Botterell Hall, Kingston, ON K7L 3N6 (Canada); Weston, Andrea D. [Currently at Applied Biotechnology/Lead Discovery, Bristol-Myers Squibb, 5 Research Pkwy Wallingford, CT 06492-1996 (United States); Perretta, Anthony [Currently at Pfizer Research and Development, Eastern Point Road, Groton, CT 06340 (United States); Thomson, Jason J. [Currently at Yale Stem Cell Center, Yale School of Medicine, PO Box 208073, New Haven, CT 06520-8073 (United States); Brown, Nigel A. [Division of Basic Medical Sciences, St. George’s University of London, UK SW17 0RE (United Kingdom)

2015-11-15

Pregnant rats treated with dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, produce offspring having a 74% incidence of congenital heart defects (CHD); however, the incidence of CHD has high inter-litter variability (40–100%) that presents a challenge when studying the initiating events prior to the presentation of an abnormal phenotype. We hypothesized that the variability in CHD incidence was the result of differences in maternal systemic concentrations or embryonic tissue concentrations of DMO. To test this hypothesis, dams were administered 300 mg/kg DMO every 12 h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses). Maternal serum levels of DMO were assessed on GD 11, 12, 13, 14, 15, 18 and 21. Embryonic tissue concentrations of DMO were assessed on GD 11, 12, 13 and 14. In a separate cohort of GD 12 embryos, DMO concentrations and parameters of growth and development were assessed to determine if tissue levels of DMO were correlated with these endpoints. Embryos were exposed directly to different concentrations of DMO with whole embryo culture (WEC) and their growth and development assessed. Key findings were that neither maternal systemic concentrations nor tissue concentrations of DMO identified embryos that were sensitive or resistant to DMO in vivo. Direct exposure of embryos to DMO via WEC also failed to show correlations between embryonic concentrations of DMO with developmental outcomes in vitro. We conclude that neither maternal serum nor embryonic tissue concentrations of DMO predict embryonic outcome. - Highlights: • Dimethadione (DMO) induces septation defects (VSD) in rat offspring. • Despite high rate of VSD defects inter-litter variability is 40–100%. • Maternal and embryonic concentrations of DMO were assessed. • Neither serum nor tissue levels of DMO were correlated with embryotoxicity.

Dimethadione embryotoxicity in the rat is neither correlated with maternal systemic drug concentrations nor embryonic tissue levels

International Nuclear Information System (INIS)

Ozolinš, Terence R.S.; Weston, Andrea D.; Perretta, Anthony; Thomson, Jason J.; Brown, Nigel A.

2015-01-01

Pregnant rats treated with dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, produce offspring having a 74% incidence of congenital heart defects (CHD); however, the incidence of CHD has high inter-litter variability (40–100%) that presents a challenge when studying the initiating events prior to the presentation of an abnormal phenotype. We hypothesized that the variability in CHD incidence was the result of differences in maternal systemic concentrations or embryonic tissue concentrations of DMO. To test this hypothesis, dams were administered 300 mg/kg DMO every 12 h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses). Maternal serum levels of DMO were assessed on GD 11, 12, 13, 14, 15, 18 and 21. Embryonic tissue concentrations of DMO were assessed on GD 11, 12, 13 and 14. In a separate cohort of GD 12 embryos, DMO concentrations and parameters of growth and development were assessed to determine if tissue levels of DMO were correlated with these endpoints. Embryos were exposed directly to different concentrations of DMO with whole embryo culture (WEC) and their growth and development assessed. Key findings were that neither maternal systemic concentrations nor tissue concentrations of DMO identified embryos that were sensitive or resistant to DMO in vivo. Direct exposure of embryos to DMO via WEC also failed to show correlations between embryonic concentrations of DMO with developmental outcomes in vitro. We conclude that neither maternal serum nor embryonic tissue concentrations of DMO predict embryonic outcome. - Highlights: • Dimethadione (DMO) induces septation defects (VSD) in rat offspring. • Despite high rate of VSD defects inter-litter variability is 40–100%. • Maternal and embryonic concentrations of DMO were assessed. • Neither serum nor tissue levels of DMO were correlated with embryotoxicity.

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

Are there factors preventing cancer development during embryonic life

International Nuclear Information System (INIS)

Einhorn, L.

1983-01-01

On the basis of the following literature observations, a hypothesis is advanced that the development of cancer is actively inhibited during embryonic life. Although the processes of cell differentiation and proliferation are - without comparison - most pronounced during embryonic life, cancer is rarely found in the newborn and is seldom a cause of neonatal death or spontaneous abortion. Attempts to induce cancer in early-stage animal embryos by irradiation or by transplacental chemical carcinogenesis have been unsuccessful, even when exposed animals have been observed throughout their lifetime. After the period of major organogenesis, however, the embryos become susceptible to carcinogenesis. In humans, the most common embryonic tumors arise in tissues which have an unusually late ongoing development and are still partly immature at or shortly before birth. For many human embryonic tumors the survival rates are higher, and spontaneous regression more frequent, in younger children, i.e. prognosis is age-dependent. Thus, although cancer generally appears in tissues capable of proliferation and differentiation, induction of malignancy in the developmentally most active tissues seems to be beset with difficulty. One possible explanation for this paradox could be that cancer is controlled by the regulators influencing development, regulators that are most active during embryonic life. (Auth.)

Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

NARCIS (Netherlands)

Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

2017-01-01

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

NARCIS (Netherlands)

Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

Mechanical control of notochord morphogenesis by extra-embryonic tissues in mouse embryos.

Science.gov (United States)

Imuta, Yu; Koyama, Hiroshi; Shi, Dongbo; Eiraku, Mototsugu; Fujimori, Toshihiko; Sasaki, Hiroshi

2014-05-01

Mammalian embryos develop in coordination with extraembryonic tissues, which support embryonic development by implanting embryos into the uterus, supplying nutrition, providing a confined niche, and also providing patterning signals to embryos. Here, we show that in mouse embryos, the expansion of the amniotic cavity (AC), which is formed between embryonic and extraembryonic tissues, provides the mechanical forces required for a type of morphogenetic movement of the notochord known as convergent extension (CE) in which the cells converge to the midline and the tissue elongates along the antero-posterior (AP) axis. The notochord is stretched along the AP axis, and the expansion of the AC is required for CE. Both mathematical modeling and physical simulation showed that a rectangular morphology of the early notochord caused the application of anisotropic force along the AP axis to the notochord through the isotropic expansion of the AC. AC expansion acts upstream of planar cell polarity (PCP) signaling, which regulates CE movement. Our results highlight the importance of extraembryonic tissues as a source of the forces that control the morphogenesis of embryos. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

Directory of Open Access Journals (Sweden)

Ruchi Sharma

2010-01-01

Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development

OpenAIRE

Parent, Audrey V.; Russ, Holger A.; Khan, Imran S.; LaFlam, Taylor N.; Metzger, Todd C.; Anderson, Mark S.; Hebrok, Matthias

2013-01-01

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 th...

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

Science.gov (United States)

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Human Embryonic Stem Cell Therapy in Crohn’s Disease: A Case Report

Science.gov (United States)

Shroff, Geeta

2016-01-01

Patient: Male, 21 Final Diagnosis: Crohn’s disease Symptoms: Intolerance to specific foods • abdominal pain and diarrhea Medication: Human embryonic stem cell therapy Clinical Procedure: Human embryonic stem cell transplantation Specialty: Gastroenterology Objective: Unusual or unexpected effect of treatment Background: Crohn’s disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565 000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn’s disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn’s disease. Case Report: A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Conclusions: Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn’s disease. PMID:26923312

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......We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h......NSSC + HUVEC cultures. Our data suggest that organotypic cultures can be employed to test the differentiation potential of stem cells and demonstrate the importance of stem cell interaction with 3D-intact tissue microenvironment for their differentiation....

Guidelines for human embryonic stem cell research

National Research Council Canada - National Science Library

Committee on Guidelines for Human Embryonic Stem Cell Research, National Research Council

2005-01-01

Since 1998, the volume of research being conducted using human embryonic stem (hES) cells has expanded primarily using private funds because of restrictions on the use of federal funds for such research...

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

Science.gov (United States)

Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known abo...

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.

Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

Directory of Open Access Journals (Sweden)

Jonathan Göke

2011-12-01

Full Text Available Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas "gene regulatory hotspots" which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.

Data for human cell spheroid model of embryonic tissue fusion in vitro.

Data.gov (United States)

U.S. Environmental Protection Agency — Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube...

Human Embryonic Stem Cell Therapy in Crohn's Disease: A Case Report.

Science.gov (United States)

Shroff, Geeta

2016-02-29

Crohn's disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565,000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn's disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn's disease. A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn's disease.

REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

Directory of Open Access Journals (Sweden)

Kaushali Thakore-Shah

Full Text Available REST (RE1 silencing transcription factor, also known as NRSF (neuron-restrictive silencer factor, is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells.

Mechanobiology of embryonic limb development.

Science.gov (United States)

Nowlan, Niamh C; Murphy, Paula; Prendergast, Patrick J

2007-04-01

Considerable evidence exists to support the hypothesis that mechanical forces have an essential role in healthy embryonic skeletal development. Clinical observations and experimental data indicate the importance of muscle contractions for limb development. However, the influence of these forces is seldom referred to in biological descriptions of bone development, and perhaps this is due to the fact that the hypothesis that mechanical forces are essential for normal embryonic skeletal development is difficult to test and elaborate experimentally in vivo, particularly in humans. Computational modeling has the potential to address this issue by simulating embryonic growth under a range of loading conditions but the potential of such models has yet to be fully exploited. In this article, we review the literature on mechanobiology of limb development in three main sections: (a) experimental alteration of the mechanical environment, (b) mechanical properties of embryonic tissues, and (c) the use of computational models. Then we analyze the main issues, and suggest how experimental and computational fields could work closer together to enhance our understanding of mechanobiology of the embryonic skeleton.

Adipose tissue-derived stem cells in oral mucosa tissue engineering ...

African Journals Online (AJOL)

Jane

2011-10-10

Oct 10, 2011 ... stem cells (ADSCs) may play an important role in this field. In this research ..... Adipose tissue is derived from embryonic mesodermal precursors and .... Clonogenic multipotent stem cells in human adipose tissue differentiate ...

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

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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.

Demonstration of β-adrenergic receptors and catecholamine-mediated effects on cell proliferation in embryonic palatal tissue

International Nuclear Information System (INIS)

Pisano, M.M.

1986-01-01

The ability of catecholamines to modulate cell proliferation, differentiation and morphogenesis in other systems, and modulate adenylate cyclase activity in the developing palate during the period of cellular differentiation, made it of interest to determine their involvement in palatal ontogenesis. Catecholamines exert their physiologic effects via interaction with distinct membrane-bound receptors, one class being the B-adrenergic receptors which are coupled to stimulation of adenylate cyclase and the generation of cAMP. A direct radioligand binding technique utilizing the B-adrenergic antagonist [ 3 H]-dihydroalprenolol ([ 3 H]-DHA) was employed in the identification of B-adrenergic receptors in the developing murine secondary palate. Specific binding of [ 3 H]-DHA in embryonic (day 13) palatal tissue homogenates was saturable and of high affinity. The functionality of B-adrenergic receptor binding sites was assessed from the ability of embryonic palate mesenchmyal cells in vitro to respond to catecholamines with elevations of cAMP. Embryonic palate mesenchymal cells responded to various B-adrenergic catecholamine agonists with significant, dose-dependent accumulations of intracellular cAMP. Embryonic (day 13) maxillary tissue homogenates were analyzed for the presence of catecholamines by high performance liquid chromatography and radioenzymatic assay. Since normal palatal and craniofacial morphogenesis depends on proper temporal and spatial patterns of growth, the effect of B-adrenergic catecholamines on embryonic palate mesenchymal cell proliferation was investigated

Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

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Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

2011-08-01

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Iodine-enhanced micro-CT imaging: methodological refinements for the study of the soft-tissue anatomy of post-embryonic vertebrates.

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Gignac, Paul M; Kley, Nathan J

2014-05-01

The now widespread use of non-destructive X-ray computed tomography (CT) and micro-CT (µCT) has greatly augmented our ability to comprehensively detail and quantify the internal hard-tissue anatomy of vertebrates. However, the utility of X-ray imaging for gaining similar insights into vertebrate soft-tissue anatomy has yet to be fully realized due to the naturally low X-ray absorption of non-mineralized tissues. In this study, we show how a wide diversity of soft-tissue structures within the vertebrate head-including muscles, glands, fat deposits, perichondria, dural venous sinuses, white and gray matter of the brain, as well as cranial nerves and associated ganglia-can be rapidly visualized in their natural relationships with extraordinary levels of detail using iodine-enhanced (i-e) µCT imaging. To date, Lugol's iodine solution (I2 KI) has been used as a contrast agent for µCT imaging of small invertebrates, vertebrate embryos, and certain isolated parts of larger, post-embryonic vertebrates. These previous studies have all yielded promising results, but visualization of soft tissues in smaller invertebrate and embryonic vertebrate specimens has generally been more complete than that for larger, post-embryonic vertebrates. Our research builds on these previous studies by using high-energy µCT together with more highly concentrated I2 KI solutions and longer staining times to optimize the imaging and differentiation of soft tissues within the heads of post-embryonic archosaurs (Alligator mississippiensis and Dromaius novaehollandiae). We systematically quantify the intensities of tissue staining, demonstrate the range of anatomical structures that can be visualized, and generate a partial three-dimensional reconstruction of alligator cephalic soft-tissue anatomy. © 2014 Wiley Periodicals, Inc.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells.

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Shin, Dong-Hyuk; Lee, Jeoung-Eun; Eum, Jin Hee; Chung, Young Gie; Lee, Hoon Taek; Lee, Dong Ryul

2017-12-01

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

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Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

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 majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both...... 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...... foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates....

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

Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting

International Nuclear Information System (INIS)

Snow, Grace E; Kasper, Allison C; Busch, Alexander M; Schwarz, Elisabeth; Ewings, Katherine E; Bee, Thomas; Spinella, Michael J; Dmitrovsky, Ethan; Freemantle, Sarah J

2009-01-01

Testicular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency. Human embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-trans retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes. Canonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833

Notch signaling activation in human embryonic stem cells is required for embryonic but not trophoblastic lineage commitment

OpenAIRE

Yu, Xiaobing; Zou, Jizhong; Ye, Zhaohui; Hammond, Holly; Chen, Guibin; Tokunaga, Akinori; Mali, Prashant; Li, Yue-Ming; Civin, Curt; Gaiano, Nicholas; Cheng, Linzhao

2008-01-01

The Notch signaling pathway plays important roles in cell fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell fate choices in human embryonic stem (hES) cells. Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hES cell lines. We report here that activation of Notch signaling is required for undifferentiated hES cells to form the pr...

Immunofluorescent histological studies of the role of fibronectin in the expression of the associative preferences of embryonic tissues.

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Armstrong, P B; Armstrong, M T

1981-08-01

The identity of the chemical factors controlling the spreading behaviour of sheets of cells was examined in organ culture. When aggregates of two dissimilar tissues are apposed in organ culture, one tissue spreads reproducibly over the surface of the second. The present study employed indirect immunofluorescent localization techniques to evaluate the hypothesis that the spreading behaviour of chick embryonic heart tissue in culture is dominated by the presence or absence of the cell-surface and extracellular matrix protein fibronectin in the surface layers of the aggregates. Specifically, the hypothesis proposes that aggregates that display surface fibronectin earlier after culturing and/or in higher quantities segregate internally to aggregates that are slower to develop a surface layer of fibronectin or in which this layer contains reduced amounts of fibronectin. The hypothesis has been supported for 3 categories of behaviour of chick embryo heart tissue: (1) myocyte aggregates spread over myocyte aggregates containing a 20% admixture of heart fibroblasts, which in turn spread over heart fibroblast aggregates; (2) 5-day embryonic ventricle-tissue fragments maintained in culture for 0.5 days spread over ventricle fragments cultured for 2.5 days; and (3) 2-day embryonic ventricle spreads over 5-day ventricle. In all these situations, the aggregate type that segregates to an internal position displays more fibronectin at its surface than aggregate types that spread to occupy an external position. Evidence is presented that the fibronectin in heart tissue aggregates is elaborated by heart fibroblasts.

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

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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.

Self-organization of spatial patterning in human embryonic stem cells

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

2017-01-01

The developing embryo is a remarkable example of self-organization, where functional units are created in a complex spatio-temporal 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 a unique opportunity to address self-organization in humans that is otherwise not addressable with current technologies. In this essay, 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. PMID:26970615

Pluripotent hybrid cells contribute to extraembryonic as well as embryonic tissues.

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Do, Jeong Tae; Choi, Hyun Woo; Choi, Youngsok; Schöler, Hans R

2011-06-01

The restricted gene expression of a differentiated cell can be reversed by forming hybrid with embryonic stem cells (ESCs). The resulting hybrid cells showed not only an ESC-specific marker expression but also a differentiation potential similar to the pluripotent fusion partner. Here, we evaluated whether the tetraploid fusion hybrid cells have a unique differentiation potential compared with diploid pluripotent cells. The first Oct4-GFP-positive cells were observed at day 2 following fusion between ESCs and neurosphere cells (OG2(+/-)/ROSA26(+/-)). Reprogramming efficiency was as high as 94.5% at passage 5 and 96.4% at passage 13. We have found that the tetraploid hybrid cells could form chimera with contribution to placenta after blastocyst injection. This result indicates that the tetraploid pluripotent fusion hybrid cells have wide range of differentiation potential. Therefore, we suggest that once the somatic cells are reprogrammed by fusion with ESCs, the tetraploid hybrid cells contributed to the extraembryonic as well as embryonic tissues.

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...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

Embryonic miRNA profiles of normal and ectopic pregnancies.

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Francisco Dominguez

Full Text Available Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs and controlled abortions (voluntary termination of pregnancy; VTOP. Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223 in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans.

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. © 2016 Elsevier Inc. All rights reserved.

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

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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.

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. Copyright © 2012 John Wiley & Sons, Ltd.

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

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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.

Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

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Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

2009-07-01

Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

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

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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.

Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

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Ryutaro Hirasawa

Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

On the genesis of articular cartilage. Embryonic joint development and gene expression - implications for tissue engineering

NARCIS (Netherlands)

Jenner, F

2013-01-01

Articular chondrocytes descend from a distinct cohort of progenitor cells located in the embryonic joint anlagen, termed interzones. Their unique lineage might explain some of the problems encountered using chondrocytes of different lineages for articular cartilage tissue engineering. While it is

3D bio-etching of a complex composite-like embryonic tissue.

Science.gov (United States)

Hazar, Melis; Kim, Yong Tae; Song, Jiho; LeDuc, Philip R; Davidson, Lance A; Messner, William C

2015-08-21

Morphogenesis involves a complex series of cell signaling, migration and differentiation events that are coordinated as tissues self-assemble during embryonic development. Collective cell movements such as those that occur during morphogenesis have typically been studied in 2D with single layers of cultured cells adhering to rigid substrates such as glass or plastic. In vivo, the intricacies of the 3D microenvironment and complex 3D responses are pivotal in the formation of functional tissues. To study such processes as collective cell movements within 3D multilayered tissues, we developed a microfluidic technique capable of producing complex 3D laminar multicellular structures. We call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. We use a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues. These tissues were previously isolated by microsurgical excision from embryos of the African claw-toed frog, Xenopus laevis. The ability to shape the 3D form of multicellular tissues and to control 3D stimulation will have a high impact on tissue engineering and regeneration applications in bioengineering and medicine as well as provide significant improvements in the synthesis of highly complex 3D integrated multicellular biosystems.

Transplantation of Human Embryonic Stem Cells in Patients with Multiple Sclerosis and Lyme Disease

OpenAIRE

Shroff, Geeta

2016-01-01

Case series Patient: Male, 42 ? Female, 30 Final Diagnosis: Human embryonic stem cells showed good therapeutic potential for treatment of multiple sclerosis with lyme disease Symptoms: Fatigue ? weakness in limbs Medication: ? Clinical Procedure: Human embryonic stem cells transplantation Specialty: Transplantology Objective: Rare disease Background: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease in which the myelin sheath of nerve cells is damaged. It can cause dela...

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

Science.gov (United States)

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

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.

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2013-02-28

... Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research... Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research. OMB No. 0925-0601-- Expiration Date... and Use of Information Collection: The form is used by applicants to request that human embryonic stem...

Derivation of Stromal (Skeletal and Mesenchymal) Stem-Like Cells from Human Embryonic Stem Cells

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Harkness, Linda; Abdallah, Basem M.; Elsafadi, Mona; Al-Nbaheen, May S.; Aldahmash, Abdullah; Kassem, Moustapha

2012-01-01

Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

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Chen Qian

Full Text Available Platelet-derived growth factor receptor alpha (PDGFRα is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures.To address the temporal requirement of Pdgfra in embryonic development.We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies.Current study showed that (i conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5 resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives.Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b if mutations / sequence variations of these regulatory elements cause these anomalies.

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

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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.

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

The influence of IVF/ICSI treatment on human embryonic growth trajectories.

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Eindhoven, S C; van Uitert, E M; Laven, J S E; Willemsen, S P; Koning, A H J; Eilers, P H C; Exalto, N; Steegers, E A P; Steegers-Theunissen, R P M

2014-12-01

groups (βIVF/ICSI = 6 g; P = 0.36 and βIVF/ICSI = 80 g; P = 0.24, respectively). Variations in embryonic growth trajectories of spontaneously conceived pregnancies with reliable pregnancy dating may partially be a result of less precise pregnancy dating and differences in endometrium receptivity compared with IVF/ICSI pregnancies. The absence of a significant difference in embryonic and fetal growth trajectories suggests safety of IVF/ICSI treatment with regard to early embryonic growth. However, further research is warranted to ascertain the influence of IVF/ICSI treatments in a larger study population, and to estimate the impact of the underlying causes of the subfertility and other periconceptional exposures on human embryonic and fetal growth trajectories. This study was supported by the Department of Obstetrics and Gynaecology of the Erasmus MC, University Medical Centre. No competing interests are declared. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells

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Langlois, Thierry; da Costa Reis Monte Mor, Barbara; Lenglet, Gaëlle

2014-01-01

. Here, we show that TET2 expression is low in human embryonic stem (ES) cell lines and increases during hematopoietic differentiation. ShRNA-mediated TET2 knockdown had no effect on the pluripotency of various ES cells. However, it skewed their differentiation into neuroectoderm at the expense...... profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm...... and hematopoietic differentiation. Stem Cells 2014....

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.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

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Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

Alternative splicing events identified in human embryonic stem cells and neural progenitors.

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Gene W Yeo

2007-10-01

Full Text Available Human embryonic stem cells (hESCs and neural progenitor (NP cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders. While much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hESC to NP, very little work has been performed to understand post-transcriptional changes during neuronal differentiation. Alternative RNA splicing (AS, a major form of post-transcriptional gene regulation, is important in mammalian development and neuronal function. Human ESC, hESC-derived NP, and human central nervous system stem cells were compared using Affymetrix exon arrays. We introduced an outlier detection approach, REAP (Regression-based Exon Array Protocol, to identify 1,737 internal exons that are predicted to undergo AS in NP compared to hESC. Experimental validation of REAP-predicted AS events indicated a threshold-dependent sensitivity ranging from 56% to 69%, at a specificity of 77% to 96%. REAP predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved AS events. Our results also reveal that focusing on differentially expressed genes between hESC and NP will overlook 14% of potential AS genes. In addition, we found that REAP predictions are enriched in genes encoding serine/threonine kinase and helicase activities. An example is a REAP-predicted alternative exon in the SLK (serine/threonine kinase 2 gene that is differentially included in hESC, but skipped in NP as well as in other differentiated tissues. Lastly, comparative sequence analysis revealed conserved intronic cis-regulatory elements such as the FOX1/2 binding site GCAUG as being proximal to candidate AS exons, suggesting that FOX1/2 may participate in the regulation of AS in NP and hESC. In summary, a new methodology for exon array analysis was introduced

Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

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Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

2015-01-01

Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin...... and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation....... 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...

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

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2013-04-29

...; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH... Embryonic Stem Cell Line to be Approved for Use in NIH-Funded Research, 0925-0601, Expiration Date 04/30... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be...

Agonist-induced desensitization of human β3-adrenoceptors expressed in human embryonic kidney cells

NARCIS (Netherlands)

Michel-Reher, Martina B.; Michel, Martin C.

2013-01-01

β3-Adrenoceptors are resistant to agonist-induced desensitization in some cell types but susceptible in others including transfected human embryonic kidney (HEK) cells. Therefore, we have studied cellular and molecular changes involved in agonist-induced β3-adrenoceptor desensitization in HEK cells.

Human embryonic stem cells handbook

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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....

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

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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

2016-05-01

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? Serial first trimester head circumference (HC) and head volume (HV) measurements were used to create reliable growth trajectories of the embryonic head, which were significantly associated with fetal head size and periconceptional maternal smoking, age and ITALIC! in vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) treatment. Fetal growth is influenced by periconceptional maternal conditions. We selected 149 singleton pregnancies with a live born non-malformed fetus from the Rotterdam periconception cohort. Bi-parietal diameter and occipital frontal diameter to calculate HC, HV and crown-rump length (CRL) were measured weekly between 9 + 0 and 12 + 6 weeks gestational age (GA) using 3D-US and VR. Fetal HC was obtained from second trimester structural anomaly scans. Growth trajectories of the embryonic head were created with general additive models and linear mixed models were used to estimate associations with maternal periconceptional conditions as a function of GA and CRL, respectively. A total of 303 3D-US images of 149 pregnancies were eligible for embryonic head measurements (intra-class correlation coefficients >0.99). Associations were found between embryonic HC and fetal HC ( ITALIC! ρ = 0.617, ITALIC! P head measured by HC and HV (All ITALIC! P head may be of benefit in future early antenatal care. This study was funded by the Department of Obstetrics and Gynaecology, Erasmus MC University Medical Centre and Sophia Foundation for Medical Research, Rotterdam, The Netherlands (SSWO grant number 644). No competing interests are declared. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email

[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

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Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

2008-10-14

To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

Proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian infectious bronchitis coronavirus

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Kong Xiangang

2011-03-01

Full Text Available Abstract Background Avian infectious bronchitis (IB is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV. Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection in ovo. In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS. Results 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection. Conclusions To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.

Human embryonic stem cells and microenvironment

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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

Embryonic stem cell-like cells derived from adult human testis

NARCIS (Netherlands)

Mizrak, S. C.; Chikhovskaya, J. V.; Sadri-Ardekani, H.; van Daalen, S.; Korver, C. M.; Hovingh, S. E.; Roepers-Gajadien, H. L.; Raya, A.; Fluiter, K.; de Reijke, Th M.; de la Rosette, J. J. M. C. H.; Knegt, A. C.; Belmonte, J. C.; van der Veen, F.; de rooij, D. G.; Repping, S.; van Pelt, A. M. M.

2010-01-01

Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the

A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.

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Du, Vicard; Luciani, Nathalie; Richard, Sophie; Mary, Gaëtan; Gay, Cyprien; Mazuel, François; Reffay, Myriam; Menasché, Philippe; Agbulut, Onnik; Wilhelm, Claire

2017-09-12

The ability to create a 3D tissue structure from individual cells and then to stimulate it at will is a major goal for both the biophysics and regenerative medicine communities. Here we show an integrated set of magnetic techniques that meet this challenge using embryonic stem cells (ESCs). We assessed the impact of magnetic nanoparticles internalization on ESCs viability, proliferation, pluripotency and differentiation profiles. We developed magnetic attractors capable of aggregating the cells remotely into a 3D embryoid body. This magnetic approach to embryoid body formation has no discernible impact on ESC differentiation pathways, as compared to the hanging drop method. It is also the base of the final magnetic device, composed of opposing magnetic attractors in order to form embryoid bodies in situ, then stretch them, and mechanically stimulate them at will. These stretched and cyclic purely mechanical stimulations were sufficient to drive ESCs differentiation towards the mesodermal cardiac pathway.The development of embryoid bodies that are responsive to external stimuli is of great interest in tissue engineering. Here, the authors culture embryonic stem cells with magnetic nanoparticles and show that the presence of magnetic fields could affect their aggregation and differentiation.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

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Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

2017-11-28

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

KAUST Repository

Parrotta, Elvira

2017-11-28

Background: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Methods: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Results: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm–1, which is enriched in human induced pluripotent stem cells. Conclusions: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

KAUST Repository

Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo M.; Cuda, Giovanni

2017-01-01

Background: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Methods: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Results: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm–1, which is enriched in human induced pluripotent stem cells. Conclusions: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

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...

Regenerative Medicine, Disease Modelling, and Drug Discovery in Human Pluripotent Stem Cell-Derived Kidney Tissue

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Navin Gupta

2017-08-01

Full Text Available The multitude of research clarifying critical factors in embryonic organ development has been instrumental in human stem cell research. Mammalian organogenesis serves as the archetype for directed differentiation protocols, subdividing the process into a series of distinct intermediate stages that can be chemically induced and monitored for the expression of stage-specific markers. Significant advances over the past few years include established directed differentiation protocols of human embryonic stem cells and human induced pluripotent stem cells (hiPSC into human kidney organoids in vitro. Human kidney tissue in vitro simulates the in vivo response when subjected to nephrotoxins, providing a novel screening platform during drug discovery to facilitate identification of lead candidates, reduce developmental expenditures, and reduce future rates of drug-induced acute kidney injury. Patient-derived hiPSC, which bear naturally occurring DNA mutations, may allow for modelling of human genetic diseases to enable determination of pathological mechanisms and screening for novel therapeutics. In addition, recent advances in genome editing with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 enable the generation of specific mutations to study genetic disease, with non-mutated lines serving as an ideal isogenic control. The growing population of patients with end-stage kidney disease is a worldwide healthcare problem, with high morbidity and mortality rates, that warrants the discovery of novel forms of renal replacement therapy. Coupling the outlined advances in hiPSC research with innovative bioengineering techniques, such as decellularised kidney and three-dimensional printed scaffolds, may contribute to the development of bioengineered transplantable human kidney tissue as a means of renal replacement therapy.

The cellular distribution of histone H5 in embryonic and adult tissues of Xenopus laevis and chicken

NARCIS (Netherlands)

Moorman, A. F.; de Boer, P. A.; Lamers, W. H.; Charles, R.

1986-01-01

The cellular distribution of histone H5 in embryonic and adult tissues of Xenopus laevis and chicken has been established with monoclonal antibodies to histone H5. Both in Xenopus and in chicken, the protein has presumably a more widespread cellular distribution than hitherto expected but is absent

Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology.

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Sebastian Schaaf

Full Text Available Human embryonic stem cell (hESC progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz and strong (mean 100 µN contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research.

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

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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.

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

International Nuclear Information System (INIS)

Hirata, Naoya; Yamada, Shigeru; Asanagi, Miki; Sekino, Yuko; Kanda, Yasunari

2016-01-01

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.

Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography.

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Iwata, Kyoko; Mio, Yasuyuki

2016-07-01

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.

[Embryonic stem cells. Future perspectives].

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Groebner, M; David, R; Franz, W M

2006-05-01

Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.

The periconception maternal cardiovascular risk profile influences human embryonic growth trajectories in IVF/ICSI pregnancies.

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Wijnands, K P J; van Uitert, E M; Roeters van Lennep, J E; Koning, A H J; Mulders, A G M G J; Laven, J S E; Steegers, E A P; Steegers-Theunissen, R P M

2016-06-01

associated with embryonic growth (-0.03√mm; P = 0.291). Stratified by mode of conception, the CV risk score was inversely and significantly associated with embryonic growth (β = -0.04√mm; P = 0.025, adjusted for possible confounders) in the IVF/ICSI group. Compared with the first quartile, embryos in the upper quartile were 10.4% smaller at 6(+0) weeks (4.4 versus 4.9 mm) and 3.1% smaller at 12(+0) weeks (56.5 versus 58.4 mm) of gestation. Although the CV risk score was slightly, but significantly, higher in women conceiving spontaneously compared with those undergoing IVF/ICSI treatment [CV risk score = 2.06 (SD: 1.23) and 1.60 (SD: 1.15), respectively], no association was established with embryonic growth in that particular group. Participants included in the present cohort are women with a singleton ongoing pregnancy without any pre-existing disease and selected from a tertiary hospital. Hence, they represent a selected group of women. Larger and population-based periconception birth cohort studies are recommended to demonstrate external validity. Differences in embryonic growth between pregnancies conceived spontaneously and after IVF/ICSI treatment in relation with CV risk factors substantiate the importance of more investigation into differences in sensitivity of endometrial, endothelial, placental and embryonic tissues. Funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam, the Netherlands. The authors declare no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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...

Left-Right Asymmetry of Maturation Rates in Human Embryonic Neural Development.

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de Kovel, Carolien G F; Lisgo, Steven; Karlebach, Guy; Ju, Jia; Cheng, Gang; Fisher, Simon E; Francks, Clyde

2017-08-01

Left-right asymmetry is a fundamental organizing feature of the human brain, and neuropsychiatric disorders such as schizophrenia sometimes involve alterations of brain asymmetry. As early as 8 weeks postconception, the majority of human fetuses move their right arms more than their left arms, but because nerve fiber tracts are still descending from the forebrain at this stage, spinal-muscular asymmetries are likely to play an important developmental role. We used RNA sequencing to measure gene expression levels in the left and right spinal cords, and the left and right hindbrains, of 18 postmortem human embryos aged 4 to 8 weeks postconception. Genes showing embryonic lateralization were tested for an enrichment of signals in genome-wide association data for schizophrenia. The left side of the embryonic spinal cord was found to mature faster than the right side. Both sides transitioned from transcriptional profiles associated with cell division and proliferation at earlier stages to neuronal differentiation and function at later stages, but the two sides were not in synchrony (p = 2.2 E-161). The hindbrain showed a left-right mirrored pattern compared with the spinal cord, consistent with the well-known crossing over of function between these two structures. Genes that showed lateralization in the embryonic spinal cord were enriched for association signals with schizophrenia (p = 4.3 E-05). These are the earliest stage left-right differences of human neural development ever reported. Disruption of the lateralized developmental program may play a role in the genetic susceptibility to schizophrenia. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Storing live embryonic and adult human cartilage grafts for transplantation using a joint simulating device.

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Cohen, I; Robinson, D; Cohen, N; Nevo, Z

2000-11-01

Cartilage transplantation as a means to replace damaged articular surfaces is of interest. A major obstacle is the long-term preservation of cartilage grafts. The commonly used technique of freezing the grafts inevitably leads to cellular death. The current study compares the technique to an innovative approach using a pulsed-pressure perfusion system termed a joint simulating device (JSD), intended to simulate intra-articular mechanical forces. Human articular cartilage explants were harvested from both embryonic epiphyseal tissue and femoral heads of elderly women (over 70 years of age) undergoing a partial joint replacement (hemi-arthroplasty) and were divided in two groups: half of the samples were incubated in the JSD while the remaining half were grown in static culture within tissue culture plates. After 10 days all samples were evaluated for: (a) cell vitality as assessed by image analysis and XTT assay; (b) biosynthetic activity as expressed by radioactive sulfate incorporation into glycosaminoglycans (GAG's); and (c) proteoglycan content as assessed by alcian blue staining intensity. A 10-fold increase in sulfate incorporation in samples held in the JSD compared to the static culture group was observed in embryonic cartilage. In adult cartilage culture in the JSD elevated sulfate incorporation by threefold as compared to static culture. Central necrosis was observed in specimens grown in the static culture plates, while it did not occur in the samples held in the JSD. Cell vitality as assessed by XTT assay was significantly better in the JSD group as compared to static culture. The difference was more pronounced in the embryonic specimens as compared to adult cartilage. The specimens cultured within the JSD retained proteoglycans significantly better than those cultured in static culture. Maintenance of cartilage specimens in a JSD was highly effective in keeping the vitality of cartilage explants in vitro over a 10-day period. A possible future

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

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Lund, Raymond D; Wang, Shaomei; Klimanskaya, Irina; Holmes, Toby; Ramos-Kelsey, Rebeca; Lu, Bin; Girman, Sergej; Bischoff, N; Sauvé, Yves; Lanza, Robert

2006-01-01

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

Human Embryonic Stem Cells Suffer from Centrosomal Amplification

Czech Academy of Sciences Publication Activity Database

Holubcová, Z.; Matula, P.; Sedláčková, M.; Vinarský, Vladimír; Doležalová, Dáša; Bárta, Tomáš; Dvořák, Petr; Hampl, Aleš

2011-01-01

Roč. 29, č. 1 (2011), s. 46-56 ISSN 1066-5099 R&D Projects: GA ČR GA204/09/2044 Grant - others:GA MŠk(CZ) 1M0538; GA MŠk(CZ) 2B06052; EU FP6 project ESTOOLS(XE) LSHG-CT-2006-018739 Program:1M Institutional research plan: CEZ:AV0Z50390703 Keywords : human embryonic stem cells * centrosome * chromosome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.781, year: 2011

Microgravity, Stem Cells, and Embryonic Development: Challenges and Opportunities for 3D Tissue Generation

International Nuclear Information System (INIS)

Andreazzoli, Massimiliano; Angeloni, Debora; Broccoli, Vania; Demontis, Gian C.

2017-01-01

Space is a challenging environment for the human body, due to the combined effects of reduced gravity (microgravity) and cosmic radiation. Known effects of microgravity range from the blood redistribution that affects the cardiovascular system and the eye to muscle wasting, bone loss, anemia, and immune depression. About cosmic radiation, the shielding provided by the spaceship hull is far less efficient than that afforded at ground level by the combined effects of the Earth atmosphere and magnetic field. The eye and its nervous layer (the retina) are affected by both microgravity and heavy ions exposure. Considering the importance of sight for long-term manned flights, visual research aimed at devising measures to protect the eye from environmental conditions of the outer space represents a special challenge to meet. In this review we focus on the impact of microgravity on embryonic development, discussing the roles of mechanical forces in the context of the neutral buoyancy the embryo experiences in the womb. At variance with its adverse effects on the adult human body, simulated microgravity may provide a unique tool for understanding the biomechanical events involved in the development and assembly in vitro of three-dimensional (3D) ocular tissues. Prospective benefits are the development of novel safety measures to protect the human eye from cosmic radiation in microgravity during long-term manned spaceflights in the outer space, as well as the generation of human 3D-retinas with its supporting structures to develop innovative and effective therapeutic options for degenerative eye diseases.

Microgravity, Stem Cells, and Embryonic Development: Challenges and Opportunities for 3D Tissue Generation

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Andreazzoli, Massimiliano [Department of Biology, University of Pisa, Pisa (Italy); Angeloni, Debora [Institute of Life Sciences, Scuola Superiore Sant' Anna, Pisa (Italy); Broccoli, Vania [National Research Council, Institute of Neuroscience, Milan (Italy); Stem Cells and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan (Italy); Demontis, Gian C., E-mail: giancarlo.demontis@farm.unipi.it [Department of Pharmacy and Centro D' Ateneo “E. Piaggio”, University of Pisa, Pisa (Italy)

2017-04-25

Space is a challenging environment for the human body, due to the combined effects of reduced gravity (microgravity) and cosmic radiation. Known effects of microgravity range from the blood redistribution that affects the cardiovascular system and the eye to muscle wasting, bone loss, anemia, and immune depression. About cosmic radiation, the shielding provided by the spaceship hull is far less efficient than that afforded at ground level by the combined effects of the Earth atmosphere and magnetic field. The eye and its nervous layer (the retina) are affected by both microgravity and heavy ions exposure. Considering the importance of sight for long-term manned flights, visual research aimed at devising measures to protect the eye from environmental conditions of the outer space represents a special challenge to meet. In this review we focus on the impact of microgravity on embryonic development, discussing the roles of mechanical forces in the context of the neutral buoyancy the embryo experiences in the womb. At variance with its adverse effects on the adult human body, simulated microgravity may provide a unique tool for understanding the biomechanical events involved in the development and assembly in vitro of three-dimensional (3D) ocular tissues. Prospective benefits are the development of novel safety measures to protect the human eye from cosmic radiation in microgravity during long-term manned spaceflights in the outer space, as well as the generation of human 3D-retinas with its supporting structures to develop innovative and effective therapeutic options for degenerative eye diseases.

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

Directory of Open Access Journals (Sweden)

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.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

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Toyoda, Hidenao; Nagai, Yuko; Kojima, Aya; Kinoshita-Toyoda, Akiko

2017-04-01

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro.

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Sathananthan, Henry; Selvaraj, Kamala; Clark, Joan

2011-08-01

The fine structure of the three germ layers in human ectopic embryos (stage 7) have been documented by digital light and electron microscopy. The formation of ectoderm, endoderm and mesoderm and notochordal cells, and also the extraembryonic membranes, amnion and yolk sac, are imaged. The germ layers give rise to all the cells and tissues of the human body. Possible clues to the early differentiation of embryonic stem cells (ESC) in vitro were obtained, since these events are more or less mimicked in cultures of ESC derived from the inner cell mass of human blastocysts. The findings are discussed with reference to previous studies on the fine structure of ESC using the same technique. Copyright © 2011. Published by Elsevier Ltd.

L1TD1 Is a Marker for Undifferentiated Human Embryonic Stem Cells

OpenAIRE

Wong, Raymond Ching-Bong; Ibrahim, Abel; Fong, Helen; Thompson, Noelle; Lock, Leslie F.; Donovan, Peter J.

2011-01-01

Background Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream ...

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

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Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

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 mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

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Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Efficient and Fast Differentiation of Human Neural Stem Cells from Human Embryonic Stem Cells for Cell Therapy

Directory of Open Access Journals (Sweden)

Xinxin Han

2017-01-01

Full Text Available Stem cell-based therapies have been used for repairing damaged brain tissue and helping functional recovery after brain injury. Aberrance neurogenesis is related with brain injury, and multipotential neural stem cells from human embryonic stem (hES cells provide a great promise for cell replacement therapies. Optimized protocols for neural differentiation are necessary to produce functional human neural stem cells (hNSCs for cell therapy. However, the qualified procedure is scarce and detailed features of hNSCs originated from hES cells are still unclear. In this study, we developed a method to obtain hNSCs from hES cells, by which we could harvest abundant hNSCs in a relatively short time. Then, we examined the expression of pluripotent and multipotent marker genes through immunostaining and confirmed differentiation potential of the differentiated hNSCs. Furthermore, we analyzed the mitotic activity of these hNSCs. In this report, we provided comprehensive features of hNSCs and delivered the knowledge about how to obtain more high-quality hNSCs from hES cells which may help to accelerate the NSC-based therapies in brain injury treatment.

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells.

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West, Michael D; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-30

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1 , encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro -derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

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Nakagawa, Kimie; Sawada, Natsumi; Hirota, Yoshihisa; Uchino, Yuri; Suhara, Yoshitomo; Hasegawa, Tomoka; Amizuka, Norio; Okamoto, Tadashi; Tsugawa, Naoko; Kamao, Maya; Funahashi, Nobuaki; Okano, Toshio

2014-01-01

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

Generation of branching ureteric bud tissues from human pluripotent stem cells.

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Mae, Shin-Ichi; Ryosaka, Makoto; Toyoda, Taro; Matsuse, Kyoko; Oshima, Yoichi; Tsujimoto, Hiraku; Okumura, Shiori; Shibasaki, Aya; Osafune, Kenji

2018-01-01

Recent progress in kidney regeneration research is noteworthy. However, the selective and robust differentiation of the ureteric bud (UB), an embryonic renal progenitor, from human pluripotent stem cells (hPSCs) remains to be established. The present study aimed to establish a robust induction method for branching UB tissue from hPSCs towards the creation of renal disease models. Here, we found that anterior intermediate mesoderm (IM) differentiates from anterior primitive streak, which allowed us to successfully develop an efficient two-dimensional differentiation method of hPSCs into Wolffian duct (WD) cells. We also established a simplified procedure to generate three-dimensional WD epithelial structures that can form branching UB tissues. This system may contribute to hPSC-based regenerative therapies and disease models for intractable disorders arising in the kidney and lower urinary tract. Copyright © 2017 Elsevier Inc. All rights reserved.

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

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Oberlin, Estelle; Fleury, Maud; Clay, Denis; Petit-Cocault, Laurence; Candelier, Jean-Jacques; Mennesson, Benoît; Jaffredo, Thierry; Souyri, Michèle

2010-11-25

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Restoration of heart functions using human embryonic stem cells derived heart muscle cells.

Science.gov (United States)

Gepstein, Lior; Kehat, Izhak

2005-02-01

Extract: Recent advances in molecular and cellular biology and specifically in the areas of stem cell biology and tissue engineering have paved the way for the development of a new field in biomedicine, regenerative medicine. This exciting approach seeks to develop new biological solutions, using the mobilization of endogenous stem cells or delivery of exogenous cells to replace or modify the function of diseased, absent, or malfunctioning tissue. The adult heart represents an attractive candidate for these emerging technologies, since adult cardiomyocytes have limited regenerative capacity. Thus, any significant heart cell loss or dysfunction, such as occurs during heart attack, is mostly irreversible and may lead to the development of progressive heart failure, one of the leading causes of world-wide morbidity and mortality. Similarly, dysfunction of the specialized electrical conduction system within the heart may result in inefficient rhythm initiation or impulse conduction, leading to significant slowing of the heart rate, usually requiring the implantation of a permanent electronic pacemaker. Replacement of the dysfunctional myocardium (heart muscle) by implantation of external heart muscle cells is emerging as a novel paradigm for restoration of the myocardial electromechanical properties, but has been significantly hampered by the paucity of cell sources for human heart cells and by the relatively limited evidence for functional integration between grafted and host cells. The recently described human embryonic stem cell (hESC) lines may provide a possible solution for the aforementioned cell sourcing problem.

Self-organization of human embryonic stem cells on micropatterns

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Deglincerti, Alessia; Etoc, Fred; Guerra, M. Cecilia; Martyn, Iain; Metzger, Jakob; Ruzo, Albert; Simunovic, Mijo; Yoney, Anna; Brivanlou, Ali H.; Siggia, Eric; Warmflash, Aryeh

2018-01-01

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate to specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. While embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, these methods do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach where human embryonic stem cells are confined to disk-shaped, sub-millimeter colonies. After 42 hours of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 days; it uses commercial microfabricated slides (CYTOO), human laminin-521 (LN-521) as extra-cellular matrix coating, and either conditioned or chemically-defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. PMID:27735934

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

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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.

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

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Roche, E; Sepulcre, M P; Enseñat-Waser, R; Maestre, I; Reig, J A; Soria, B

2003-07-01

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

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

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

2008-01-01

cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...

Derivation of the human embryonic stem cell line RCM1

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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.

Human embryonic stem cell-encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering

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Tang, Minghui; Chen, Wenchuan; Weir, Michael D.; Thein-Han, Wahwah; Xu, Hockin H. K.

2012-01-01

Human embryonic stem cells (hESCs) are exciting for regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there has been no report on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESCs were cultured to form embryoid bodies (EBs), and the MSCs were then migrated out of the EBs. hESCd-MSCs had surface markers characteristic of MSCs, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSCs were encapsulated in alginate at a density of 1 million cells/mL, with an average microbead size of 207 µm. CPC contained mannitol porogen to create a porosity of 64% and macropores with size of 218 µm, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSCs encapsulated in microbeads in CPC had good viability from 1 to 21 d. ALP gene expression at 21 d was 25-fold that at 1 d. Osteocalcin (OC) at 21 d was two orders of magnitude of that at 1 d. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21 d was 5-fold that at 1 d. Mineral synthesis by the encapsulated hESCd-MSCs at 21 d was 7-fold that at 1 d. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSCs were encapsulated in alginate microbeads in macroporous CPC showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications. PMID:22633970

Human Embryonic Stem Cell Responses to Ionizing Radiation Exposures: Current State of Knowledge and Future Challenges

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Mykyta V. Sokolov

2012-01-01

Full Text Available Human embryonic stem cells, which are derived from the inner cell mass of the blastocyst, have become an object of intense study over the last decade. They possess two unique properties that distinguish them from many other cell types: (i the ability to self-renew indefinitely in culture under permissive conditions, and (ii the pluripotency, defined as the capability of giving rise to all cell types of embryonic lineage under the guidance of the appropriate developmental cues. The focus of many recent efforts has been on the elucidating the signaling pathways and molecular networks operating in human embryonic stem cells. These cells hold great promise in cell-based regenerative therapies, disease modeling, drug screening and testing, assessing genotoxic and mutagenic risks associated with exposures to a variety of environmental factors, and so forth. Ionizing radiation is ubiquitous in nature, and it is widely used in diagnostic and therapeutic procedures in medicine. In this paper, our goal is to summarize the recent progress in understanding how human embryonic stem cells respond to ionizing radiation exposures, using novel methodologies based on “omics” approaches, and to provide a critical discussion of what remains unknown; thus proposing a roadmap for the future research in this area.

Real-time Visualization of Tissue Dynamics during Embryonic Development and Malignant Transformation

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Yamada, Kenneth

Tissues undergo dramatic changes in organization during embryonic development, as well as during cancer progression and invasion. Recent advances in microscopy now allow us to visualize and track directly the dynamic movements of tissues, their constituent cells, and cellular substructures. This behavior can now be visualized not only in regular tissue culture on flat surfaces (`2D' environments), but also in a variety of 3D environments that may provide physiological cues relevant to understanding dynamics within living organisms. Acquisition of imaging data using various microscopy modalities will provide rich opportunities for determining the roles of physical factors and for computational modeling of complex processes in living tissues. Direct visualization of real-time motility is providing insight into biology spanning multiple spatio-temporal scales. Many cells in our body are known to be in contact with connective tissue and other forms of extracellular matrix. They do so through microscopic cellular adhesions that bind to matrix proteins. In particular, fluorescence microscopy has revealed that cells dynamically probe and bend the matrix at the sites of cell adhesions, and that 3D matrix architecture, stiffness, and elasticity can each regulate migration of the cells. Conversely, cells remodel their local matrix as organs form or tumors invade. Cancer cells can invade tissues using microscopic protrusions that degrade the surrounding matrix; in this case, the local matrix protein concentration is more important for inducing the micro-invasive protrusions than stiffness. On the length scales of tissues, transiently high rates of individual cell movement appear to help establish organ architecture. In fact, isolated cells can self-organize to form tissue structures. In all of these cases, in-depth real-time visualization will ultimately provide the extensive data needed for computer modeling and for testing hypotheses in which physical forces interact

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

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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

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

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Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

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

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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.

Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.

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Säljö, Karin; Barone, Angela; Vizlin-Hodzic, Dzeneta; Johansson, Bengt R; Breimer, Michael E; Funa, Keiko; Teneberg, Susann

2017-04-01

High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

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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.

MSX-1 gene expression and regulation in embryonic palatal tissue.

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Nugent, P; Greene, R M

1998-01-01

The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

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Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

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Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

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Shelly E Tannenbaum

Full Text Available Clinically compliant human embryonic stem cells (hESCs should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

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Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

2016-08-01

Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. © The Author(s) 2016.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

DEFF Research Database (Denmark)

Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

2017-01-01

Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

Expression of embryonic stem cell markers in keloid-associated lymphoid tissue.

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Grant, Chelsea; Chudakova, Daria A; Itinteang, Tinte; Chibnall, Alice M; Brasch, Helen D; Davis, Paul F; Tan, Swee T

2016-07-01

To identify, characterise and localise the population of primitive cells in keloid scars (KS). 5-µm-thick formalin-fixed paraffin-embedded sections of KS samples from 10 patients underwent immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers OCT4, SOX2, pSTAT3 and NANOG, and keloid-associated lymphoid tissue (KALT) markers CD4 and CD20. NanoString gene expression analysis and in situ hybridisation (ISH) were used to determine the abundance and localisation of the mRNA for these ESC markers. IHC staining revealed the expression of the ESC markers OCT4, SOX2, pSTAT3 and NANOG by a population of cells within KS tissue. These are localised to the endothelium of the microvessels within the KALTs. NanoString gene expression analysis confirmed the abundance of the transcriptional expression of the same ESC markers. ISH localised the expression of the ESC transcripts to the primitive endothelium in KS tissue. This report demonstrates the expression of ESC markers OCT4, SOX2, pSTAT3 and NANOG by the endothelium of the microvessels within the KALTs. These findings show a unique niche of primitive cells within KS, expressing ESC markers, revealing a potential therapeutic target in the treatment of KS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

[Yes to research, no to utilization? Medical, pharmacological and toxicological utilization of human embryonic stem cells from an ethical point of view].

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Kress, H

2008-09-01

In exceptional cases, the German Stem Cell Act allows research on human embryonic stem cells. However, it does not allow the implementation of the research results if this in turn requires the use of further embryonic stem cell lines. It has, in the meantime, transpired that such research results could be of concrete use. Thus, in the distant future, it could be used in the clinical treatment of patients. Already in the nearer future the use of human embryonic stem cell lines can be envisaged for both the development and testing of medicines as well as in the field of toxicology. To this end, research concerning embryo toxicity and neurotoxicity is ground-breaking. The toxicological and pharmacological use of human embryonic stem cell lines should serve the protection of human health as well as the safe and reliable use of medicines. In addition, animal experiments could be reduced, which is desirable from a point of view of animal protection ethics. Since research on human embryonic stem cell lines is actually permitted in Germany, the use of the respective research results should be allowed all the more. This follows from the basic human right to health protection and health care. Legal ambiguities, which still exist in this respect, should be removed.

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.

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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.

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...

Embryonic kidney function in a chronic renal failure model in rodents.

Science.gov (United States)

Fujimoto, Eisuke; Yamanaka, Shuichiro; Kurihara, Sho; Tajiri, Susumu; Izuhara, Luna; Katsuoka, Yuichi; Yokote, Shinya; Matsumoto, Kei; Kobayashi, Eiji; Okano, Hirotaka James; Chikaraishi, Tatsuya; Yokoo, Takashi

2017-08-01

Rapid advancements have been made in alternative treatments for renal diseases. Our goal for renal regeneration is to establish a kidney graft derived from human embryonic tissues. In this study, we investigated the effects of host renal failure on the structure and activity of transplanted embryonic kidney and bladder, and found that diuretics effectively induced urine production in the transplanted kidney. Uremic conditions were reproduced using a 5/6 renal infarction rat model. An embryonic kidney plus bladder (embryonic day 15) was isolated from a pregnant Lewis rat and transplanted into the para-aortic area of a 5/6 renal-infarcted Lewis rat. Following growth, the embryonic bladder was successfully anastomosed to the host ureter. We assessed graft function in terms of survival rates and found no differences between normal (n = 5) and renal failure (n = 8) groups (median survival: 70.5 vs 74.5 h; p = 0.331) in terms of survival, indicating that the grafts prolonged rat survival, even under renal failure conditions. Furosemide (n = 9) significantly increased urine volume compared with saline-treated controls (n = 7; p < 0.05), confirming that the grafts were functional. We also demonstrated the possibilities of an in vivo imaging system for determining the viability of transplanted embryonic kidney with bladder. The results of this study demonstrate that transplanted embryonic kidney and bladder can grow and function effectively, even under uremic conditions.

PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function

NARCIS (Netherlands)

Birket, Matthew J.; Casini, Simona; Kosmidis, Georgios; Elliott, David A.; Gerencser, Akos A.; Baartscheer, Antonius; Schumacher, Cees; Mastroberardino, Pier G.; Elefanty, Andrew G.; Stanley, Ed G.; Mummery, Christine L.

2013-01-01

Diminished 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, which is

Tension (re)builds: Biophysical mechanisms of embryonic wound repair.

Science.gov (United States)

Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo

2017-04-01

Embryonic tissues display an outstanding ability to rapidly repair wounds. Epithelia, in particular, serve as protective layers that line internal organs and form the skin. Thus, maintenance of epithelial integrity is of utmost importance for animal survival, particularly at embryonic stages, when an immune system has not yet fully developed. Rapid embryonic repair of epithelial tissues is conserved across species, and involves the collective migration of the cells around the wound. The migratory cell behaviours associated with wound repair require the generation and transmission of mechanical forces, not only for the cells to move, but also to coordinate their movements. Here, we review the forces involved in embryonic wound repair. We discuss how different force-generating structures are assembled at the molecular level, and the mechanisms that maintain the balance between force-generating structures as wounds close. Finally, we describe the mechanisms that cells use to coordinate the generation of mechanical forces around the wound. Collective cell movements and their misregulation have been associated with defective tissue repair, developmental abnormalities and cancer metastasis. Thus, we propose that understanding the role of mechanical forces during embryonic wound closure will be crucial to develop therapeutic interventions that promote or prevent collective cell movements under pathological conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

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...

Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

OpenAIRE

Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2012-01-01

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for a...

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

OpenAIRE

Romain Barbet; Isabelle Peiffer; Antoinette Hatzfeld; Pierre Charbord; Jacques A. Hatzfeld

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs ...

Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

International Nuclear Information System (INIS)

Carlberg, Bjoern; Liu, Johan; Axell, Mathilda Zetterstroem; Kuhn, H Georg; Nannmark, Ulf

2009-01-01

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150 μm, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1 μm. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between cells

Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Carlberg, Bjoern; Liu, Johan [BioNano Systems Laboratory, Department of Microtechnology and Nanoscience, Chalmers University of Technology, Goeteborg, SE-412 96 (Sweden); Axell, Mathilda Zetterstroem; Kuhn, H Georg [Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Goeteborg, SE-413 45 (Sweden); Nannmark, Ulf, E-mail: bjorn.carlberg@chalmers.s, E-mail: mathilda.zetterstrom@neuro.gu.s, E-mail: georg.kuhn@neuro.gu.s, E-mail: ulf.nannmark@anatcell.gu.s, E-mail: jliu@chalmers.s [Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Goeteborg, SE-405 30 (Sweden)

2009-08-15

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150{mu}m, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1{mu}m. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between

YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues.

Science.gov (United States)

Liu, Ming; Zhao, Shuangyun; Lin, Qingjie; Wang, Xiu-Ping

2015-04-01

Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

Science.gov (United States)

Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

2016-07-01

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. © 2016. Published by The Company of Biologists Ltd.

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

International Nuclear Information System (INIS)

Varga, Nóra; Veréb, Zoltán; Rajnavölgyi, Éva; Német, Katalin; Uher, Ferenc; Sarkadi, Balázs; Apáti, Ágota

2011-01-01

Highlights: ► MSC like cells were derived from hESC by a simple and reproducible method. ► Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. ► 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.

Deciphering the Epigenetic Code in Embryonic and Dental Pulp Stem Cells

Science.gov (United States)

Bayarsaihan, Dashzeveg

2016-01-01

A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors. PMID:28018144

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.

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 isolatio...

Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death.

Directory of Open Access Journals (Sweden)

Magalie Vatin

Full Text Available Recurrent spontaneous abortion (RSA is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (∼30%. Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic.

Science.gov (United States)

Whiting, Paul; Kerby, Julie; Coffey, Peter; da Cruz, Lyndon; McKernan, Ruth

2015-10-19

Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. Here, with a focus upon retinal disease, we discuss the current status of the development of such therapies. We also highlight some of our own experiences of progressing a retinal pigment epithelium cell replacement therapy towards the clinic. © 2015 The Author(s).

Somatic genomic variations in extra-embryonic tissues

Energy Technology Data Exchange (ETDEWEB)

Weier, Jingly F.; Ferlatte, Christy; Weier, Heinz-Ulli G.

2010-05-21

In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism.

Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

Directory of Open Access Journals (Sweden)

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.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

Science.gov (United States)

Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

2017-03-15

Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

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. 2010 Elsevier Inc. All rights reserved.

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.

Comprehensive quantitative comparison of the membrane proteome and PTM-ome of human embryonic stem cells and neural stem cells

DEFF Research Database (Denmark)

Braga, Marcella Nunes de Melo; Schulz, Melanie; Jakobsen, Lene

Introduction: Human embryonic stem cells (hESCs) can differentiate into all three germ layers and self-renew. Due to its ability to differentiate in vitro into human neural stem cells (hNSCs), which can further be differentiated into motor neurons and dopaminergic neurons, these cells are potential...... identified phosphorylated and SA glycosylated proteins, respectively. This study allowed us to identify several significantly regulated proteins during the differentiation process, including proteins involved in the early embryonic development as well as in the neural development. In the latter group...... of proteins we could identify a number of proteins associated with synaptic vesicles, which are vesicles that store neurotransmitters in the nerve-terminals. An example of an upregulated protein in hESCs is the gap junction alpha 1 (GJA1), a phosphorylated protein which plays a crucial role in embryonic...

Systematically profiling and annotating long intergenic non-coding RNAs in human embryonic stem cell.

Science.gov (United States)

Tang, Xing; Hou, Mei; Ding, Yang; Li, Zhaohui; Ren, Lichen; Gao, Ge

2013-01-01

While more and more long intergenic non-coding RNAs (lincRNAs) were identified to take important roles in both maintaining pluripotency and regulating differentiation, how these lincRNAs may define and drive cell fate decisions on a global scale are still mostly elusive. Systematical profiling and comprehensive annotation of embryonic stem cells lincRNAs may not only bring a clearer big picture of these novel regulators but also shed light on their functionalities. Based on multiple RNA-Seq datasets, we systematically identified 300 human embryonic stem cell lincRNAs (hES lincRNAs). Of which, one forth (78 out of 300) hES lincRNAs were further identified to be biasedly expressed in human ES cells. Functional analysis showed that they were preferentially involved in several early-development related biological processes. Comparative genomics analysis further suggested that around half of the identified hES lincRNAs were conserved in mouse. To facilitate further investigation of these hES lincRNAs, we constructed an online portal for biologists to access all their sequences and annotations interactively. In addition to navigation through a genome browse interface, users can also locate lincRNAs through an advanced query interface based on both keywords and expression profiles, and analyze results through multiple tools. By integrating multiple RNA-Seq datasets, we systematically characterized and annotated 300 hES lincRNAs. A full functional web portal is available freely at http://scbrowse.cbi.pku.edu.cn. As the first global profiling and annotating of human embryonic stem cell lincRNAs, this work aims to provide a valuable resource for both experimental biologists and bioinformaticians.

Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

Science.gov (United States)

Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

2006-07-01

Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

Directory of Open Access Journals (Sweden)

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.

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.

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.

[The legal and ethical aspects of nerve tissue transplantation].

Science.gov (United States)

Sramka, M; Rattaj, M

1992-01-01

The authors have specified the following criteria for the withdrawal of embryonal tissue at their department: 1) only tissue from dead fetus is allowed to be used in neurotransplantation; 2) embryonal tissue is to be obtained after spontaneous abortions from volunteers or from women asking for artificial abortion; 3) the women should be informed about the curative purposes of embryonal tissue voluntary donorship and they must give a written consent; 4) decision on abortion should be separated from the use of embryonal tissue; 5) women should not know recipients; no payments should be made for tissue; 6) the donor is not permitted to impregnate in order to use embryos for research or clinical purposes; 7) sampling of BWR, HBsAG, anti-HIV, cytomegalovirus, herpes I and II is to be made for serologic examinations and that from the cervix for cultivation and sensitivity, as well as ultrasound verification of a germinal age is done in potential donors; 8) consent should be signed to embryonal brain transplantation by recipient or his legitimate deputy if the recipient is certifiable. The above criteria should protect both the donor and the recipient. The use of embryonal tissue cultures seems to be promising. In addition to legal and ethic problems, immunological problems and problems concerning the aseptic withdrawal of embryonal tissue are falling off.

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

Science.gov (United States)

Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

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.

Modeling the behavior of human body tissues on penetration

Science.gov (United States)

Conci, A.; Brazil, A. L.; Popovici, D.; Jiga, G.; Lebon, F.

2018-02-01

Several procedures in medicine (such as anesthesia, injections, biopsies and percutaneous treatments) involve a needle insertion. Such procedures operate without vision of the internal involved areas. Physicians and anesthetists rely on manual (force and tactile) feedback to guide their movements, so a number of medical practice is strongly based on manual skill. In order to be expert in the execution of such procedures the medical students must practice a number of times, but before practice in a real patient they must be trained in some place and a virtual environment, using Virtual Reality (VR) or Augmented Reality (AR) is the best possible solution for such training. In a virtual environment the success of user practices is improved by the addition of force output using haptic device to improve the manual sensations in the interactions between user and computer. Haptic devices enable simulate the physical restriction of the diverse tissues and force reactions to movements of operator hands. The trainees can effectively "feel" the reactions to theirs movements and receive immediate feedback from the actions executed by them in the implemented environment. However, in order to implement such systems, the tissue reaction to penetration and cutting must be modeled. A proper model must emulate the physical sensations of the needle action in the skin, fat, muscle, and so one, as if it really done in a patient that is as they are holding a real needle and feeling each tissue resistance when inserting it through the body. For example an average force value for human skin puncture is 6.0 N, it is 2.0 N for subcutaneous fat tissue and 4.4 N for muscles: this difference of sensations to penetration of each layers trespassed by the needle makes possible to suppose the correct position inside the body. This work presents a model for tissues before and after the cutting that with proper assumptions of proprieties can model any part of human body. It was based on experiments

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

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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.

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.

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

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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.

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

Directory of Open Access Journals (Sweden)

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.

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.

Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

Science.gov (United States)

Goodwin, T. J.; McCarthy, M.; Lin, Y-H

2006-01-01

In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

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

embryonic and fetal pancreas anlage in humans. Pdx1(+) cells are found in cell clusters also expressing Serpina1 and FABP1, suggesting activation of intestinal/liver developmental programs. Moreover, Activin B up-regulates Sonic Hedgehog (Shh) and its target Gli1, which during normal development...

Ethical tissue: a not-for-profit model for human tissue supply.

Science.gov (United States)

Adams, Kevin; Martin, Sandie

2011-02-01

Following legislative changes in 2004 and the establishment of the Human Tissue Authority, access to human tissues for biomedical research became a more onerous and tightly regulated process. Ethical Tissue was established to meet the growing demand for human tissues, using a process that provided ease of access by researchers whilst maintaining the highest ethical and regulatory standards. The establishment of a licensed research tissue bank entailed several key criteria covering ethical, legal, financial and logistical issues being met. A wide range of stakeholders, including the HTA, University of Bradford, flagged LREC, hospital trusts and clinical groups were also integral to the process.

The use of animal tissues alongside human tissue: Cultural and ethical considerations.

Science.gov (United States)

Kaw, Anu; Jones, D Gareth; Zhang, Ming

2016-01-01

Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display. © 2015 Wiley Periodicals, Inc.

CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

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Xianglian Ge

2016-01-01

Full Text Available Clustered interspaced short palindromic repeats (CRISPR/CRISPR-associated protein 9 (Cas9-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

Subretinal Implantation of Retinal Pigment Epithelial Cells Derived From Human Embryonic Stem Cells: Improved Survival When Implanted as a Monolayer

Science.gov (United States)

Diniz, Bruno; Thomas, Padmaja; Thomas, Biju; Ribeiro, Ramiro; Hu, Yuntao; Brant, Rodrigo; Ahuja, Ashish; Zhu, Danhong; Liu, Laura; Koss, Michael; Maia, Mauricio; Chader, Gerald; Hinton, David R.; Humayun, Mark S.

2013-01-01

Purpose. To evaluate cell survival and tumorigenicity of human embryonic stem cell–derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). Methods. Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). Results. The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. Conclusions. hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. PMID:23833067

Tracking the mechanical dynamics of human embryonic stem cell chromatin

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Hinde Elizabeth

2012-12-01

Full Text Available Abstract Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM. Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

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.

Human embryonic stem cell-derived pancreatic endoderm alleviates diabetic pathology and improves reproductive outcome in C57BL/KsJ-Lep(db/+) gestational diabetes mellitus mice.

Science.gov (United States)

Xing, Baoheng; Wang, Lili; Li, Qin; Cao, Yalei; Dong, Xiujuan; Liang, Jun; Wu, Xiaohua

2015-07-01

Gestational diabetes mellitus is a condition commonly encountered during mid to late pregnancy with pathologic manifestations including hyperglycemia, hyperinsulinemia, insulin resistance, and fetal maldevelopment. The cause of gestational diabetes mellitus can be attributed to both genetic and environmental factors, hence complicating its diagnosis and treatment. Pancreatic progenitors derived from human embryonic stem cells were shown to be able to effectively treat diabetes in mice. In this study, we have developed a system of treating diabetes using human embryonic stem cell-derived pancreatic endoderm in a mouse model of gestational diabetes mellitus. Human embryonic stem cells were differentiated in vitro into pancreatic endoderm, which were then transplanted into db/+ mice suffering from gestational diabetes mellitus. The transplant greatly improved glucose metabolism and reproductive outcome of the females compared with the control groups. Our findings support the feasibility of using differentiated human embryonic stem cells for treating gestational diabetes mellitus patients. Copyright © 2015 Elsevier Inc. All rights reserved.

Derivation of novel genetically diverse human embryonic stem cell lines.

Science.gov (United States)

Stefanova, Valentina T; Grifo, James A; Hansis, Christoph

2012-06-10

Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery, and toxicity testing. A multitude of hESC lines have been derived worldwide since the first 5 lines by Thomson et al. 13 years ago, but many of these are poorly characterized, unavailable, or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived 12 new hESC lines at New York University from discarded genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not been previously reported, for example, mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology, and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

NARCIS (Netherlands)

Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G. J.; Mummery, Christine L.; Casini, Simona

2015-01-01

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes

Nuclear vlimata and aneuploidy in embryonic cells is caused by meiosis. Behaviour and properties of meiotic cells

OpenAIRE

Logothetou-Rella, H.

1995-01-01

This study demonstrates that human embryonic cells divide by meiosis. The use of trophoblastic tissue cells (early embryo) and amniotic cells (late embryo) exhibited the following characteristic events of meiosis: nuclear (NVs) and nucleolar (NuVs) vlimata formation; NV invasion in host cells; extrusion of chromosomes; nuclear fusion; metaphase fusion; hybrid cell formation; nuclear, nucleolar and cytoplasmic bridges, chromosomal transfer, variablesized nuc...

Differential bone-forming capacity of osteogenic cells from either embryonic stem cells or bone marrow-derived mesenchymal stem cells

NARCIS (Netherlands)

Both, Sanne Karijn; van Apeldoorn, Aart A.; Jukes, J.M.; Englund, Mikael C.O.; Hyllner, Johan; van Blitterswijk, Clemens; de Boer, Jan

2011-01-01

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue-engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in

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

Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells.

NARCIS (Netherlands)

Freund, C.M.A.H.; Ward-van Oostwaard, D.; Monshouwer-Kloots, J.; van den Brink, S.; van Rooijen, M.A.; Xu, X.; Zweigerdt, R.; Mummery, C.L.; Passier, R.

2008-01-01

Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/insulin-like

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.

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

International Nuclear Information System (INIS)

Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

2010-01-01

Research highlights: → Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. → hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. → 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.

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.

Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. III. An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system

NARCIS (Netherlands)

Wessels, A.; Vermeulen, J. L.; Verbeek, F. J.; Virágh, S.; Kálmán, F.; Lamers, W. H.; Moorman, A. F.

1992-01-01

A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated G1N2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i.e., 4 1/2 weeks of development) these

Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

DEFF Research Database (Denmark)

Norrman, Karin; Strömbeck, Anna; Semb, Henrik

2013-01-01

for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize...... of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were...

Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells.

Science.gov (United States)

Saito, Taku; Yano, Fumiko; Mori, Daisuke; Kawata, Manabu; Hoshi, Kazuto; Takato, Tsuyoshi; Masaki, Hideki; Otsu, Makoto; Eto, Koji; Nakauchi, Hiromitsu; Chung, Ung-il; Tanaka, Sakae

2015-01-01

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

Induction of anchorage-independent growth of human embryonic fibroblasts with a deletion in the short arm of chromosome 11 by human papillomavirus type 16 DNA

International Nuclear Information System (INIS)

Smits, H.L.; Raadsheer, E.; Rood, I.; Mehendale, S.; Slater, R.M.; van der Noordaa, J.; Ter Schegget, J.

1988-01-01

Human embryonic fibroblasts with a large deletion (11p11.11p15.1) in the short arm of one chromosome 11 (del-11 cells) appeared to be susceptible to transformation by early human papillomavirus type 16 (HPV-16) DNA, whereas diploid human embryonic fibroblasts were not. This difference in susceptibility might be explained by the absence of a tumor suppressor gene located within the deleted part on the short arm of chromosome 11. The presence of abundant viral early-gene transcripts in transformed cells suggests that transformation was induced by an elevated level of an HPV-16 early-gene product(s). The low transcriptional activity of HPV-16 in diploid cells may indicate that cellular genes affect viral transcription. Interruption of the HPV-16 E2 early open reading frame is probably required for high-level HPV-16 early-gene expression driven from the homologous enhancer-promoter region

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

Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

Science.gov (United States)

Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

2010-04-29

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

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

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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.

Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency

NARCIS (Netherlands)

Brinkhof, Bas; van Tol, Helena T A; Groot Koerkamp, Marian J A; Wubbolts, Richard W; Haagsman, Henk P; Roelen, Bernard A J

2017-01-01

In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture.

Science.gov (United States)

Ratcliffe, Elizabeth; Hourd, Paul; Guijarro-Leach, Juan; Rayment, Erin; Williams, David J; Thomas, Robert J

2013-01-01

Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes.

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

Science.gov (United States)

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Distribution of epidermal growth factor receptors in rat tissues during embryonic skin development, hair formation, and the adult hair growth cycle

DEFF Research Database (Denmark)

Green, M R; Couchman, J R

1984-01-01

on the binding distribution of [125I]EGF, representing the tissue localization of available EGF receptors, during embryonic rat skin development including hair follicle formation and the adult hair growth cycle. At 16 days embryonic development a relatively low receptor density is seen over all the epidermal...... condensates marking the first stage of hair follicle development. This restricted and temporary loss of EGF receptors above these specialized mesenchymal condensates implies a role for the EGF receptor and possibly EGF or an EGF-like ligand in stimulating the epithelial downgrowth required for hair follicle...... development. In the anagen hair bulb, receptors for EGF are detected over the outer root sheath and the epithelial cell layers at the base of the follicle and show a correlation with the areas of epithelial proliferation in the hair bulb. During the catagen and telogen phases of the hair cycle, receptors...

Embryonic development of human lice: rearing conditions and susceptibility to spinosad

Directory of Open Access Journals (Sweden)

Gastón Mougabure Cueto

2006-05-01

Full Text Available The embryonic development of human lice was evaluated according to the changes in the morphology of the embryo observed through the transparent chorion. Based on ocular and appendage development, three stages of embryogenesis were established: early, medium, and late. Influence of temperature and relative humidity (RH on the laboratory rearing of Pediculus humanus capitis eggs was assessed. The optimal ranges for temperature and RH were 27-31°C and 45-75%. The susceptibility of human louse eggs to insecticide spinosad (a macrocyclic lactone was assessed by immersion method. The results showed similar susceptibility to spinosad in early, medium, and late stages of head lice eggs. In addition, this study showed similar susceptibility of head and body lice eggs to spinosad, an insecticide that has not been used as pediculicide in Argentina (lethal concentration 50: 0.01%.

Derivation and characterization of the NYSCFe003-A human embryonic stem cell line

Directory of Open Access Journals (Sweden)

Ana Sevilla

2017-12-01

Full Text Available The human embryonic stem cell line NYSCFe003-A was derived from a day 5 to day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe003-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

Science.gov (United States)

Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

2012-04-10

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

[Expression of embryonic markers in pterygium derived mesenchymal cells].

Science.gov (United States)

Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

2010-12-01

Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

DEFF Research Database (Denmark)

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A

2012-01-01

oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted......The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high...... YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3ß, PDX1, CD34, p63, nestin, PAX6) markers. Double...

MRI diagnosis of embryonal tumors in the spinal canal

International Nuclear Information System (INIS)

Sun Jilin; Zhang Xinchuan; Zhang Huaning; Liu Lianxiang; Wu Yujin

1997-01-01

To evaluate MRI diagnostic value of the embryonal tumors in the spinal canal. Materials and methods: The MRI appearances of 15 cases of histologically confirmed embryonal tumors in the spinal canal were analyzed. (1) Lipoma (3 cases) had characteristic MRI appearance, demonstrating high signal intensity on T 1 WI, and moderately high signal on T 2 WI. High signal intensity of the lipoma was turned into low signal intensity by fat suppression technique. (2) Dermoids (2 cases) and epidermoid (7 cases) exhibiting low or iso-low signal on T 1 WI and high or iso-high signal on T 2 WI. All had an iso-intense capsule on T 1 WI. However, the two tumors could not be distinguished from each other. (3) Teratoma (3 cases) appeared as a mass of inhomo-generous signals in the spinal canal including soft tissue, fatty tissue and calcification within the same tumor. The diagnosis of embryonal tumors in the spinal canal mainly depend on their MRI appearances, specific tumor location and patient's age

Physicochemical processes in embryonic plant tissue during the transition to the state of cold anabiosis and storage at liquid nitrogen temperature

Science.gov (United States)

Khodko, A. T.; Lysak, Yu. S.

2017-10-01

Critical opalescence phenomenon was observed in the cytoplasm of garlic embryonic tissue—meristem—upon cooling in liquid nitrogen vapor, indicating liquid-liquid phase transition in the system. It was established that cells of the meristem tissue survive the cooling-thawing cycle. We suggest that the transition of meristem tissue to the state of anabiosis is mainly due to a drastic slowing of the diffusion in the cytoplasm caused by the passage of the solution through the critical point, followed by the formation of a dispersed system—a highly concentrated emulsion—as a result of a liquid-liquid phase transition. This macrophase separation is characteristic of polymer-solvent systems. We established the regime of cooling down to liquid nitrogen temperature and subsequent thawing in the cryopreservation cycle for the biological object under study, which ensures the preservation of tissue viability.

The role of nanotechnology in induced pluripotent and embryonic stem cells research.

Science.gov (United States)

Chen, Lukui; Qiu, Rong; Li, Lushen

2014-12-01

This paper reviews the recent studies on development of nanotechnology in the field of induced pluripotent and embryonic stem cells. Stem cell therapy is a promising therapy that can improve the quality of life for patients with refractory diseases. However, this option is limited by the scarcity of tissues, ethical problem, and tumorigenicity. Nanotechnology is another promising therapy that can be used to mimic the extracellular matrix, label the implanted cells, and also can be applied in the tissue engineering. In this review, we briefly introduce implementation of nanotechnology in induced pluripotent and embryonic stem cells research. Finally, the potential application of nanotechnology in tissue engineering and regenerative medicine is also discussed.

Testicular Embryonic Rhabdomyosarcoma, Case report with brief ...

African Journals Online (AJOL)

Testicular Embryonic Rhabdomyosarcoma, Case report with brief literature review. AM Adam, MMAM Ibnouf, IAF Allah. Abstract. Background: Rhabdomyosarcoma (RMS) is a malignant solid tumour arising from mesenchymal tissues which normally differentiate to form striated muscle. It can occur in a wide variety of sites.

Bioenergetic Changes during Differentiation of Human Embryonic Stem Cells along the Hepatic Lineage

DEFF Research Database (Denmark)

Hopkinson, Branden M; Madsen, Claus Desler; Kalisz, Mark

2017-01-01

Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless, a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells...... of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction...

Plasticity of Calcium Signaling Cascades in Human Embryonic Stem Cell-Derived Neural Precursors

Czech Academy of Sciences Publication Activity Database

Forostyak, Oksana; Romanyuk, Nataliya; Verkhratsky, A.; Syková, Eva; Dayanithi, Govindan

2013-01-01

Roč. 22, č. 10 (2013), s. 1506-1521 ISSN 1547-3287 R&D Projects: GA ČR GAP304/11/2373; GA ČR(CZ) GBP304/12/G069 Grant - others:FP7(XE) PITN-GA-2008-214003 project AXREGEN; FP7(XE) PITN-GA-2009-237956 project EdU-GLIA Institutional support: RVO:68378041 Keywords : human embryonic stem cells * voltage-operated Ca2+ channels * spontaneous Ca2+ oscillations Subject RIV: FH - Neurology Impact factor: 4.202, year: 2013

Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

Science.gov (United States)

Liu, Yarong; Fox, Victoria; Lei, Yuning; Hu, Biliang; Joo, Kye-Il; Wang, Pin

2014-07-01

The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications. © 2013 Wiley Periodicals, Inc.

GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

Science.gov (United States)

Krtolica, Ana; Larocque, Nick; Genbacev, Olga; Ilic, Dusko; Coppe, Jean-Philippe; Patil, Christopher K.; Zdravkovic, Tamara; McMaster, Michael; Campisi, Judith; Fisher, Susan J.

2012-01-01

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ~6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical–basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical–basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation—βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell–cell contacts can trigger adoption of a neuronal fate. PMID:21396766

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.

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

International Nuclear Information System (INIS)

Simeone, A.; Mavilio, F.; Acampora, D.

1987-01-01

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 homeodomains identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hybridization 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

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

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Alvaro Plaza Reyes

2016-01-01

Full Text Available Human embryonic stem cell (hESC-derived retinal pigment epithelial (RPE cells could replace lost tissue in geographic atrophy (GA but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

Directory of Open Access Journals (Sweden)

Marjorie Pick

Full Text Available BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs towards the megakaryocyte (Mk lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Ethical and regulatory aspects of embryonic stem cell research.

Science.gov (United States)

Jain, Kewal K

2002-12-01

Ethical and regulatory issues concerning embryonic stem (ES) cell research are reviewed here a year after the controversy became a public and political issue in the US. The background of various issues are examined and the current regulations in various countries are reviewed. In the US, the debate is linked with abortion, as well as the status of a fetus as a human being, and is politically driven. Obtaining stem cells from embryonic tissues involves destruction of the embryo, to which objections are raised. Religious beliefs are examined and no serious impediments to ES cell research could be identified. Regulations vary from one country to another and it is unlikely that there will ever be any universally uniform ethical and regulatory standards for ES cell research. Currently, the most liberal and favourable environments for ES cell research are in the UK, Singapore, Sweden, India, Israel and China. Unless the US liberalises ES cell research, it may lose its lead in ES cell research and investments in this area may drift to countries with better environments for research. Suggestions are offered in this review to improve the ethical environment for ES cell research.

Transcriptomics resources of human tissues and organs

DEFF Research Database (Denmark)

Uhlén, Mathias; Hallström, Björn M.; Lindskog, Cecilia

2016-01-01

a framework for defining the molecular constituents of the human body as well as for generating comprehensive lists of proteins expressed across tissues or in a tissue-restricted manner. Here, we review publicly available human transcriptome resources and discuss body-wide data from independent genome......Quantifying the differential expression of genes in various human organs, tissues, and cell types is vital to understand human physiology and disease. Recently, several large-scale transcriptomics studies have analyzed the expression of protein-coding genes across tissues. These datasets provide...

Tissue-based map of the human proteome

DEFF Research Database (Denmark)

Uhlén, Mathias; Fagerberg, Linn; Hallström, Björn M.

2015-01-01

Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transc...

Chapter 8. Ionisation radiation and human organism. Radioactivity of human tissues

International Nuclear Information System (INIS)

Toelgyessy, J.; Harangozo, M.

2000-01-01

This is a chapter of textbook of radioecology for university students. In this chapter authors deal with ionisation radiation and human organism as well as with radioactivity of human tissues. Chapter consists of next parts: (1) Radiation stress of human organism; (2) Radioactivity of human tissues and the factors influencing radioactive contamination; (3) Possibilities of decreasing of radiation stress

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...

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...... of hESC, using one colony for each individual marker. Four newly established hESC lines showed the expected expression pattern and were capable of differentiating into the three germ layers in vitro. Neighbouring sections of entire colonies grown for 4, 11, 21 and 28 days respectively were stained...

Embryonal rhabdomyosarcoma in an immature Baird's tapir (Tapirus bairdii).

Science.gov (United States)

Bonar, Christopher J; Lewandowski, Albert H; Skowronek, Anthony J

2007-03-01

An immature Baird's tapir (Tapirus bairdii) with a history of seizure-like episodes developed signs of respiratory disease. The initial clinical diagnosis was pneumonia, and antibiotic therapy was started. The animal failed to improve after 14 days of therapy and developed unilateral, bloody nasal discharge. Endoscopic examination and radiography revealed a soft tissue mass in the nasopharynx depressing the soft palate. The tapir died 32 days after initial presentation. Histologic examination of the mass demonstrated a mesenchymal tumor composed of spindle cells with elongate nuclei forming densely packed fascicles. The neoplastic spindle cells showed prominent cross-striations. Immunohistochemistry revealed the cells to be positive for desmin and myoglobin, but negative for smooth muscle actin, confirming diagnosis of rhabdomyosarcoma. Embryonal rhabdomyosarcoma is the most common nasopharyngeal soft tissue tumor of humans, and it has been reported infrequently in dogs, horses, and pigs. Neoplasia should be a differential diagnosis in cases of unilateral nasal discharge and inspiratory stridor, even in young animals.

Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells

International Nuclear Information System (INIS)

Miyazaki, Takamichi; Futaki, Sugiko; Hasegawa, Kouichi; Kawasaki, Miwa; Sanzen, Noriko; Hayashi, Maria; Kawase, Eihachiro; Sekiguchi, Kiyotoshi; Nakatsuji, Norio; Suemori, Hirofumi

2008-01-01

Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs. We first determined the major integrins expressed on the hESCs to reveal the preference of the hESCs for rhLMs, and found that the hESCs mainly expressed integrin α6β1, which binds predominantly to laminin-111, -332 and -511/-521. When the hESCs were seeded onto rhLMs, the cells indeed adhered markedly to rhLM-332, and to rhLM-511 and rhLM-111 to a lesser extent. The hESCs proliferated on these three rhLMs for several passages while preserving their pluripotency. These results show that rhLM-111, -332, and -511 are good substrates to expand undifferentiated hESCs due to their high affinity to integrin α6β1 expressed on hESCs

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

OpenAIRE

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil; Butler, Peter C.

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in ...

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.

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

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Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

2017-02-01

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study. Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as being a promising source of cells for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. In the current study, we have fabricated cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials and demonstrated that supplementation of mesenchymal niche cells as well as provision of mechanical loading particularly stretching have significantly promoted the maturation of the cardiomyocytes and hence improved the mechanical functional characteristics of the tissue strips

Regulations of enzymes in animals: effects of developmental processes, cancer, and radiation. Final report. [Analysis of enzymes in human cancer tissue

Energy Technology Data Exchange (ETDEWEB)

Knox, W.E.

1978-09-01

Low grade tumors of various origins are chemically very different. High grade tumors, whatever their origin, are chemically very similar to one another and to embryonic tissues. Analyses of human tumor tissues and sera from cancer patients were conducted for two new groups of enzymes expected to be informative about the physiological state of the tissue. The enzymes measured in tumors and sera were chosen because they were characteristic of fetal tissues and high grade neoplasms in rats, and could, therefore, be expected to exist in human cancers (and fetuses) and to predominate more in those of higher grade malignancies. Results indicated that the classification of enzymes (or isozymes) as fetal or adult types in the rat could be extended to man. Human cancers do contain most of the enzymes expected, and lack others, as expected. Analyses of the same enzymes in sera gave less clear results. It was recognized at the outset that no simple proportionality existed between tissue and serum levels. The tendency existed in cancer patients to have in serum elevated amounts of those enzymes characteristic of undifferentiated tissues. The abnormalities in a specific patient are conditioned by his physiological state, by the grade of his tumor, and by the mass of tumor present. The tumor mass had a very significant effect, so that monitoring this tumor burden by chemical means should be quite possible. The latest work focused on particular enzymes that have not previously been measured in cancer patients. These studies concentrated on pyrroline-5-carboxylate (P-5-C) reductase and its inhibition and on lysosomal glucosidases and phosphatases. Both groups are relatively high in fetal and neoplastic tissues.

Random lasing in human tissues

International Nuclear Information System (INIS)

Polson, Randal C.; Vardeny, Z. Valy

2004-01-01

A random collection of scatterers in a gain medium can produce coherent laser emission lines dubbed 'random lasing'. We show that biological tissues, including human tissues, can support coherent random lasing when infiltrated with a concentrated laser dye solution. To extract a typical random resonator size within the tissue we average the power Fourier transform of random laser spectra collected from many excitation locations in the tissue; we verified this procedure by a computer simulation. Surprisingly, we found that malignant tissues show many more laser lines compared to healthy tissues taken from the same organ. Consequently, the obtained typical random resonator was found to be different for healthy and cancerous tissues, and this may lead to a technique for separating malignant from healthy tissues for diagnostic imaging

Graphene for enhanced embryonic stem cell photo-transfection efficiency

CSIR Research Space (South Africa)

Mthunzi, P

2013-04-01

Full Text Available Due to their pluripotency properties, embryonic stem (ES) cells possess great potential in regenerative therapy. Since reported a promising tissue engineering scaffold material, here, graphene is demonstrated to significantly improve the ES cell...

A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells

OpenAIRE

Geng, Yijie; Feng, Bradley

2016-01-01

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the ...

Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

Directory of Open Access Journals (Sweden)

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.

The Postischemic Environment Differentially Impacts Teratoma or Tumor Formation After Transplantation of Human Embryonic Stem Cell-Derived Neural Progenitors

Czech Academy of Sciences Publication Activity Database

Seminatore, CH.; Polentes, J.; Ellman, D.; Kozubenko, Nataliya; Itier, V.; Tine, S.; Tritschler, L.; Brenot, M.; Guidou, E.; Blondeau, J.; Lhuillier, M.; Bugi, A.; Aubry, L.; Jendelová, Pavla; Syková, Eva; Perrier, A. L.; Finsen, B.; Onteniente, B.

2010-01-01

Roč. 41, č. 1 (2010), s. 153-159 ISSN 0039-2499 Institutional research plan: CEZ:AV0Z50390703 Keywords : brain transplantation * human embryonic stem cells * neural differentiation Subject RIV: FH - Neurology Impact factor: 5.756, year: 2010

Embryonic stem cell therapy of heart failure in genetic cardiomyopathy.

Science.gov (United States)

Yamada, Satsuki; Nelson, Timothy J; Crespo-Diaz, Ruben J; Perez-Terzic, Carmen; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Behfar, Atta; Terzic, Andre

2008-10-01

Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K(+) (K(ATP)) channel subunits. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional K(ATP) channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. Disclosure of potential conflicts of interest is

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.

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development

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Silvia Mazzotta

2016-10-01

Full Text Available Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

GATM, the human ortholog of the mouse imprinted Gatm gene, escapes genomic imprinting in placenta

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Toshinobu Miyamoto

2005-03-01

Full Text Available The GATM gene encodes L-arginine:glycine amidinotransferase, which catalyzes the conversion of L-arginine into guanidinoacetate, the rate-limiting step in the synthesis of creatine. Since, deficiencies in creatine synthesis and transport lead to certain forms of mental retardation in human, the human GATM gene appears to be involved in brain development. Recently it has been demonstrated that the mouse Gatm is expressed during development and is imprinted with maternal expression in the placenta and yolk sac, but not in embryonic tissues. We investigated the imprinting status of the human GATM by analyzing its expression in four human placentas. GATM was biallelically expressed, thus suggesting that this gene escapes genomic imprinting in placentas, differently from what has been reported in mouse extra-embryonic tissues.

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.

Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

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Zhenqiang Zhao

2016-12-01

Full Text Available Mouse embryonic fibroblasts (MEFs and human foreskin fibroblasts (HFFs are used for the culture of human embryonic stem cells (hESCs. MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs =1:1 and HFFs feeder respectively, and then were differentiated into DA neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR (qRT-PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of TH positive cells and expressed higher levels of FOXA2, PITX3, NURR1 and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.

Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

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Ruixin Hao

Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

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.

High-content screening of small compounds on human embryonic stem cells.

Science.gov (United States)

Barbaric, Ivana; Gokhale, Paul J; Andrews, Peter W

2010-08-01

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

Modeling Niemann Pick type C1 using human embryonic and induced pluripotent stem cells.

Science.gov (United States)

Ordoñez, M Paulina; Steele, John W

2017-02-01

Data generated in Niemann Pick type C1 (NPC1) human embryonic and human induced pluripotent stem cell derived neurons complement on-going studies in animal models and provide the first example, in disease-relevant human cells, of processes that underlie preferential neuronal defects in a NPC1. Our work and that of other investigators in human neurons derived from stem cells highlight the importance of performing rigorous mechanistic studies in relevant cell types to guide drug discovery and therapeutic development, alongside of existing animal models. Through the use of human stem cell-derived models of disease, we can identify and discover or repurpose drugs that revert early events that lead to neuronal failure in NPC1. Together with the study of disease pathogenesis and efficacy of therapies in animal models, these strategies will fulfill the promise of stem cell technology in the development of new treatments for human diseases. This article is part of a Special Issue entitled SI: Exploiting human neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

In vitro generation of functional insulin-producing cells from lipoaspirated human adipose tissue-derived stem cells.

Science.gov (United States)

Mohamad Buang, Mohamad Lizan; Seng, Heng Kien; Chung, Lee Han; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

2012-01-01

Tissue engineering strategy has been considered as an alternative treatment for diabetes mellitus due to lack of permanent pharmaceutical treatment and islet donors for transplantation. Various cell lines have been used to generate functional insulin-producing cells (IPCs) including progenitor pancreatic cell lines, embryonic stem cells (ESCs), umbilical cord blood stem cells (UCB-SCs), adult bone marrow stem cells (BMSCs), and adipose tissue-derived stem cells (ADSCs). Human ADSCs from lipoaspirated abdominal fat tissue was differentiated into IPCs following a two-step induction protocol based on a combination of alternating high and low glucose, nicotinamide, activin A and glucagon-like peptide 1 (GLP-1) for a duration of 3 weeks. During differentiation, histomorphological changes of the stem cells towards pancreatic β-islet characteristics were observed via light microscope and transmission electron microscope (TEM). Dithizone (DTZ) staining, which is selective towards IPCs, was used to stain the new islet-like cells. Production of insulin hormone by the cells was analyzed via enzyme-linked immunosorbent assay (ELISA), whereas its hormonal regulation was tested via a glucose challenge test. Histomorphological changes of the differentiated cells were noted to resemble pancreatic β-cells, whereas DTZ staining positively stained the cells. The differentiated cells significantly produced human insulin as compared to the undifferentiated ADSCs, and its production was increased with an increase of glucose concentration in the culture medium. These initial data indicate that human lipoaspirated ADSCs have the potential to differentiate into functional IPCs, and could be used as a therapy to treat diabetes mellitus in the future. Copyright © 2012 IMSS. Published by Elsevier Inc. All rights reserved.

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

Directory of Open Access Journals (Sweden)

Thomas C Schulz

Full Text Available Development of a human embryonic stem cell (hESC-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Generation of human embryonic stem cells from abnormal blastocyst diagnosed with albinism.

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Sun, Yi; Zhou, Xiaoying; Chen, Jing; Du, Juan; Lu, Guangxiu; Lin, Ge; Ouyang, Qi

2016-11-01

Human embryonic stem cell (hESC) line chHES-478 was derived from abnormal blastocyst diagnosed with albinism after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-478 cell line carried a compound heterozygous mutation, c.896G>A(p.Arg299His) and c.929_930insC(p.Pro310Glnfs*9), of TYR gene. Characteristic tests proved that the chHES-478 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

Self-contained induction of neurons from human embryonic stem cells.

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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.

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

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Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

2013-01-01

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

Directed Differentiation of Human Embryonic Stem Cells into Prostate Organoids In Vitro and its Perturbation by Low-Dose Bisphenol A Exposure.

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Esther L Calderon-Gierszal

Full Text Available Studies using rodent and adult human prostate stem-progenitor cell models suggest that developmental exposure to the endocrine disruptor Bisphenol-A (BPA can predispose to prostate carcinogenesis with aging. Unknown at present is whether the embryonic human prostate is equally susceptible to BPA during its natural developmental window. To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA. The directed differentiation of human embryonic stem cells (hESC into prostatic organoids in a spatial system was accomplished with precise temporal control of growth factors and steroids. Activin-induced definitive endoderm was driven to prostate specification by combined exposure to WNT10B and FGF10. Matrigel culture for 20-30 days in medium containing R-Spondin-1, Noggin, EGF, retinoic acid and testosterone was sufficient for mature prostate organoid development. Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate. Exposure to 1 nM or 10 nM BPA throughout differentiation culture disturbed early morphogenesis in a dose-dependent manner with 1 nM BPA increasing and 10 nM BPA reducing the number of branched structures formed. While differentiation of branched structures to mature organoids seemed largely unaffected by BPA exposure, the stem-like cell population increased, appearing as focal stem cell nests that have not properly entered lineage commitment rather than the rare isolated stem cells found in normally differentiated structures. These findings provide the first direct evidence that low-dose BPA exposure targets hESC and perturbs morphogenesis as the embryonic cells differentiate towards human prostate organoids, suggesting that the developing human prostate may be susceptible to disruption by in utero BPA exposures.

Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

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Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

2017-03-09

Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.

NARCIS (Netherlands)

Braam, S.R.; Zeinstra, L.M.; Litjens, S.H.M.; Ward-van Oostwaard, D.; van den Brink, S.; van Laake, L.W.; Lebrin, F.; Kats, P.; Hochstenbach, R.; Passier, R.; Sonnenberg, A.; Mummery, C.L.

2008-01-01

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing

Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

DEFF Research Database (Denmark)

Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue

2014-01-01

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

Cloning and characterization of a novel human zinc finger gene, hKid3, from a C2H2-ZNF enriched human embryonic cDNA library

International Nuclear Information System (INIS)

Gao Li; Sun Chong; Qiu Hongling; Liu Hui; Shao Huanjie; Wang Jun; Li Wenxin

2004-01-01

To investigate the zinc finger genes involved in human embryonic development, we constructed a C 2 H 2 -ZNF enriched human embryonic cDNA library, from which a novel human gene named hKid3 was identified. The hKid3 cDNA encodes a 554 amino acid protein with an amino-terminal KRAB domain and 11 carboxyl-terminal C 2 H 2 zinc finger motifs. Northern blot analysis indicates that two hKid3 transcripts of 6 and 8.5 kb express in human fetal brain and kidney. The 6 kb transcript can also be detected in human adult brain, heart, and skeletal muscle while the 8.5 kb transcript appears to be embryo-specific. GFP-fused hKid3 protein is localized to nuclei and the ZF domain is necessary and sufficient for nuclear localization. To explore the DNA-binding specificity of hKid3, an oligonucleotide library was selected by GST fusion protein of hKid3 ZF domain, and the consensus core sequence 5'-CCAC-3' was evaluated by competitive electrophoretic mobility shift assay. Moreover, The KRAB domain of hKid3 exhibits transcription repressor activity when tested in GAL4 fusion protein assay. These results indicate that hKid3 may function as a transcription repressor with regulated expression pattern during human development of brain and kidney

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

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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. Copyright 2010 Elsevier Inc. All rights reserved.

A Humanized Mouse Model Generated Using Surplus Neonatal Tissue

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Matthew E. Brown

2018-04-01

Full Text Available Summary: Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs. Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue. : Corresponding author William Burlingham and colleagues created a humanized mouse model called the NeoThy. The NeoThy uses human neonatal, rather than fetal, tissue sources for generating a human immune system within immunocompromised mouse hosts. NeoThy mice are an attractive alternative to conventional humanized mouse models, as they enable robust and reproducible iPSC immunogenicity experiments in vivo. Keywords: NeoThy, humanized mouse, iPSC, PSC, immunogenicity, transplantation, immunology, hematopoietic stem cells, induced pluripotent stem cells, thymus

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.

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Bruin, Jennifer E; Rezania, Alireza; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

2013-09-01

Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

Delta-like protein (DLK) is a novel immunohistochemical marker for human hepatoblastomas

DEFF Research Database (Denmark)

Dezso, Katalin; Halász, Judit; Bisgaard, Hanne Cathrine

2008-01-01

Delta-like protein (DLK) is a membrane protein with mostly unknown function. It is expressed by several embryonic tissues among others by the hepatoblasts of rodent and human fetal livers. We have investigated in the present study if this protein is expressed in human hepatoblastomas. The presenc...

Generation of hematopoietic lineage cells from embryonic like cells

Directory of Open Access Journals (Sweden)

Gholam Reza Khamisipour

2014-10-01

Full Text Available Background: Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells. A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Material and Methods: Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Results: Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. Conclusion: These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

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Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

2004-03-01

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

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Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

DEFF Research Database (Denmark)

Mahmood, Amer; Harkness, Linda; Abdallah, Basem

2012-01-01

EBs using BMP2 (bone morphogenic protein 2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold......Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...

Adipogenesis and epicardial adipose tissue: a novel fate of the epicardium induced by mesenchymal transformation and PPARγ activation.

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Yamaguchi, Yukiko; Cavallero, Susana; Patterson, Michaela; Shen, Hua; Xu, Jian; Kumar, S Ram; Sucov, Henry M

2015-02-17

The hearts of many mammalian species are surrounded by an extensive layer of fat called epicardial adipose tissue (EAT). The lineage origins and determinative mechanisms of EAT development are unclear, in part because mice and other experimentally tractable model organisms are thought to not have this tissue. In this study, we show that mouse hearts have EAT, localized to a specific region in the atrial-ventricular groove. Lineage analysis indicates that this adipose tissue originates from the epicardium, a multipotent epithelium that until now is only established to normally generate cardiac fibroblasts and coronary smooth muscle cells. We show that adoption of the adipocyte fate in vivo requires activation of the peroxisome proliferator activated receptor gamma (PPARγ) pathway, and that this fate can be ectopically induced in mouse ventricular epicardium, either in embryonic or adult stages, by expression and activation of PPARγ at times of epicardium-mesenchymal transformation. Human embryonic ventricular epicardial cells natively express PPARγ, which explains the abundant presence of fat seen in human hearts at birth and throughout life.

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks.

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Sorkio, Anni; Koch, Lothar; Koivusalo, Laura; Deiwick, Andrea; Miettinen, Susanna; Chichkov, Boris; Skottman, Heli

2018-07-01

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

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Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

2017-06-01

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

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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.

Patently controversial: markets, morals, and the President's proposal for embryonic stem cell research.

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Fins, Joseph J; Schachter, Madeleine

2002-09-01

This essay considers the implications of President George W. Bush's proposal for human embryonic stem cell research. Through the perspective of patent law, privacy, and informed consent, we elucidate the ongoing controversy about the moral standing of human embryonic stem cells and their derivatives and consider how the inconsistencies in the president's proposal will affect clinical practice and research.

Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

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Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi

2011-09-01

Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).

Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

Science.gov (United States)

Tian, Xinghui; Kaufman, Dan S

2008-07-01

Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

The role of allofibroblasts transplantation in cartilaginous tissue regeneration process

OpenAIRE

Khadjibaev Аbdukhakim Muminovich; Tilyakov Akbar Buriyevich; Magrupov Bokhodir Asadullaevich; Urazmetova Maisa Dmitriyevna; Ubaydullaev Bobur Sabirovich

2017-01-01

Aim of investigation. Ground of embryonal allofibroblasts in the process of cartilaginous tissue regeneration. Material and methods. Investigation is based on the study the results of stimulation cartilaginous tissue regeneration process in the conditions of embryonal allofibroblasts application in 24 experimental sexually mature rabbits in which the model of symphysis pubis rupture with its following recovery have been used. Pieces of cartilaginous tissue have been fixed in 10% neutral forma...

Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells.

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Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios

2016-12-30

Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.

Three-dimensional morphology of the human embryonic brain

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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.

Polarized spectral features of human breast tissues through wavelet ...

Indian Academy of Sciences (India)

Abstract. Fluorescence characteristics of human breast tissues are investigated through wavelet transform and principal component analysis (PCA). Wavelet transform of polar- ized fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate different tissue types.

Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells

Czech Academy of Sciences Publication Activity Database

Dvořák, Petr; Dvořáková, D.; Košková, S.; Vidinská, M.; Najvirtová, M.; Krekáč, D.; Hampl, Aleš

2005-01-01

Roč. 23, č. 8 (2005), s. 1200-1211 ISSN 1066-5099 R&D Projects: GA ČR(CZ) GA301/03/1122; GA ČR(CZ) GA305/05/0434; GA MŠk(CZ) LN00A065 Institutional research plan: CEZ:AV0Z50390512 Keywords : growth factor * human embryonic stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.094, year: 2005

Effects of oxidative stress on human embryonic stem cells; global gene expression, advanced glycation end products and NEDD1 levels

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Barandalla Sobrados, M.

2017-01-01

A number of unfavorable conditions can affect the development of the early embryo inducing oxidative stress both in vivo, for instance in gestational diabetes, and in vitro, when embryos are derived from Assisted Reproductive Technologies (ART). Human Embryonic Stem Cells (hESCs) potentially offer a

Mechanized syringe homogenization of human and animal tissues.

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Kurien, Biji T; Porter, Andrew C; Patel, Nisha C; Kurono, Sadamu; Matsumoto, Hiroyuki; Scofield, R Hal

2004-06-01

Tissue homogenization is a prerequisite to any fractionation schedule. A plethora of hands-on methods are available to homogenize tissues. Here we report a mechanized method for homogenizing animal and human tissues rapidly and easily. The Bio-Mixer 1200 (manufactured by Innovative Products, Inc., Oklahoma City, OK) utilizes the back-and-forth movement of two motor-driven disposable syringes, connected to each other through a three-way stopcock, to homogenize animal or human tissue. Using this method, we were able to homogenize human or mouse tissues (brain, liver, heart, and salivary glands) in 5 min. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric enzyme assay for prolidase, we have found that the homogenates obtained were as good or even better than that obtained used a manual glass-on-Teflon (DuPont, Wilmington, DE) homogenization protocol (all-glass tube and Teflon pestle). Use of the Bio-Mixer 1200 to homogenize animal or human tissue precludes the need to stay in the cold room as is the case with the other hands-on homogenization methods available, in addition to freeing up time for other experiments.

Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

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Wada, Tamaki; Honda, Makoto; Minami, Itsunari; Tooi, Norie; Amagai, Yuji; Nakatsuji, Norio; Aiba, Kazuhiro

2009-01-01

Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved. PMID:19701462

Anti-human tissue factor antibody ameliorated intestinal ischemia reperfusion-induced acute lung injury in human tissue factor knock-in mice.

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Xiaolin He

Full Text Available BACKGROUND: Interaction between the coagulation and inflammation systems plays an important role in the development of acute respiratory distress syndrome (ARDS. Anti-coagulation is an attractive option for ARDS treatment, and this has promoted development of new antibodies. However, preclinical trials for these antibodies are often limited by the high cost and availability of non-human primates. In the present study, we developed a novel alternative method to test the role of a humanized anti-tissue factor mAb in acute lung injury with transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: Human tissue factor knock-in (hTF-KI transgenic mice and a novel humanized anti-human tissue factor mAb (anti-hTF mAb, CNTO859 were developed. The hTF-KI mice showed a normal and functional expression of hTF. The anti-hTF mAb specifically blocked the pro-coagulation activity of brain extracts from the hTF-KI mice and human, but not from wild type mice. An extrapulmonary ARDS model was used by intestinal ischemia-reperfusion. Significant lung tissue damage in hTF-KI mice was observed after 2 h reperfusion. Administration of CNTO859 (5 mg/kg, i.v. attenuated the severity of lung tissue injury, decreased the total cell counts and protein concentration in bronchoalveolar lavage fluid, and reduced Evans blue leakage. In addition, the treatment significantly reduced alveolar fibrin deposition, and decreased tissue factor and plasminogen activator inhibitor-1 activity in the serum. This treatment also down-regulated cytokine expression and reduced cell death in the lung. CONCLUSIONS: This novel anti-hTF antibody showed beneficial effects on intestinal ischemia-reperfusion induced acute lung injury, which merits further investigation for clinical usage. In addition, the use of knock-in transgenic mice to test the efficacy of antibodies against human-specific proteins is a novel strategy for preclinical studies.

Function of FEZF1 during early neural differentiation of human embryonic stem cells.

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Liu, Xin; Su, Pei; Lu, Lisha; Feng, Zicen; Wang, Hongtao; Zhou, Jiaxi

2018-01-01

The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study, using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover, loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs, suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.

Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

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Cristian Miere

2016-03-01

Full Text Available The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele. 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.

Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

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Laureen Jacquet

2016-03-01

Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. 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 and in vivo assays.

Efficient differentiation of human embryonic stem cells to definitive endoderm.

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D'Amour, Kevin A; Agulnick, Alan D; Eliazer, Susan; Kelly, Olivia G; Kroon, Evert; Baetge, Emmanuel E

2005-12-01

The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

Isolation and characterization of true mesenchymal stem cells derived from human term decidua capable of multilineage differentiation into all 3 embryonic layers.

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Macias, Maria I; Grande, Jesús; Moreno, Ana; Domínguez, Irene; Bornstein, Rafael; Flores, Ana I

2010-11-01

The objective of the study was to isolate and characterize a population of mesenchymal stem cells (MSCs) from human term placental membranes. We isolated an adherent cell population from extraembryonic membranes. Morphology, phenotype, growth characteristics, karyotype, and immunological and differentiation properties were analyzed. The isolated placental MSCs were from maternal origin and named as decidua-derived mesenchymal stem cells (DMSCs). DMSCs differentiated into derivatives of all germ layers. It is the first report about placental MSC differentiation into alveolar type II cells. Clonally expanded DMSCs differentiated into all embryonic layers, including pulmonary cells. DMSCs showed higher life span than placental cells from fetal origin and proliferated without genomic instability. The data suggest that DMSCs are true multipotent MSCs, distinguishing them from other placental MSCs. DMSCs could be safely used in the mother as a potential source of MSCs for pelvic floor dysfunctions and immunological diseases. Additionally, frozen DMSCs can be stored for both autologous and allogeneic tissue regeneration. Copyright © 2010 Mosby, Inc. All rights reserved.

Embryonic vaccines against cancer: an early history.

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Brewer, Bradley G; Mitchell, Robert A; Harandi, Amir; Eaton, John W

2009-06-01

Almost 100 years have passed since the seminal observations of Schöne showing that vaccination of animals with fetal tissue would prevent the growth of transplantable tumors. Many subsequent reports have affirmed the general idea that immunologic rejection of transplantable tumors, as well as prevention of carcinogenesis, may be affected by vaccination with embryonic/fetal material. Following a decade of intense research on this phenomenon during approximately 1964-1974, interest appears to have waned. This earlier experimental work may be particularly pertinent in view of the rising interest in so-called cancer stem cells. We believe that further work - perhaps involving the use of embryonic stem cells as immunogens - is warranted and that the results reviewed herein support the concept that vaccination against the appearance of cancers of all kinds is a real possibility.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

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Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

2013-07-19

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

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Saurabh Singh

2005-01-01

Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

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Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

2003-09-01

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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Kathryn L McCabe

Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017

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Fatemeh Akyash

2017-09-01

Full Text Available This report explains briefly the minutes of a 1-day workshop entitled; “human embryonic stem cells (hESCs and good manufacturing practice (GMP” held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

Science.gov (United States)

Akyash, Fatemeh; Sadeghian-Nodoushan, Fatemeh; Tahajjodi, Somayyeh Sadat; Nikukar, Habib; Farashahi Yazd, Ehsan; Azimzadeh, Mostafa; D Moore, Harry; Aflatoonian, Behrouz

2017-05-01

This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27 th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

Cytomegalovirus induces abnormal chondrogenesis and osteogenesis during embryonic mandibular development

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Bringas Pablo

2008-03-01

Full Text Available Abstract Background Human clinical studies and mouse models clearly demonstrate that cytomegalovirus (CMV disrupts normal organ and tissue development. Although CMV is one of the most common causes of major birth defects in humans, little is presently known about the mechanism(s underlying CMV-induced congenital malformations. Our prior studies have demonstrated that CMV infection of first branchial arch derivatives (salivary glands and teeth induced severely abnormal phenotypes and that CMV has a particular tropism for neural crest-derived mesenchyme (NCM. Since early embryos are barely susceptible to CMV infection, and the extant evidence suggests that the differentiation program needs to be well underway for embryonic tissues to be susceptible to viral infection and viral-induced pathology, the aim of this study was to determine if first branchial arch NCM cells are susceptible to mCMV infection prior to differentiation of NCM derivatives. Results E11 mouse mandibular processes (MANs were infected with mouse CMV (mCMV for up to 16 days in vitro. mCMV infection of undifferentiated embryonic mouse MANs induced micrognathia consequent to decreased Meckel's cartilage chondrogenesis and mandibular osteogenesis. Specifically, mCMV infection resulted in aberrant stromal cellularity, a smaller, misshapen Meckel's cartilage, and mandibular bone and condylar dysmorphogenesis. Analysis of viral distribution indicates that mCMV primarily infects NCM cells and derivatives. Initial localization studies indicate that mCMV infection changed the cell-specific expression of FN, NF-κB2, RelA, RelB, and Shh and Smad7 proteins. Conclusion Our results indicate that mCMV dysregulation of key signaling pathways in primarily NCM cells and their derivatives severely disrupts mandibular morphogenesis and skeletogenesis. The pathogenesis appears to be centered around the canonical and noncanonical NF-κB pathways, and there is unusual juxtaposition of abnormal stromal

2010 Great Lakes Human Health Fish Tissue Study Fish Tissue Data Dictionary

Science.gov (United States)

The Office of Science and Technology (OST) is providing the fish tissue results from the 2010 Great Lakes Human Health Fish Tissue Study (GLHHFTS). This document includes the “data dictionary” for Mercury, PFC, PBDE and PCBs.

Cytotoxic assessment of silver nanoparticles in embryonic development and kidney tissue in pregnant mice

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Bagher seyedalipour

2015-10-01

Full Text Available Background and Aim: Regarding the widespread use of silver nanoparticles in medecine and lack of a detailed study of toxicity effects of these particles on fetus, this study was carried out to investigate histopathological changes of the kidneys and also embryonic development following exposure to silver nanoparticles. Materials and Methods: In this experimental study, thirty five female NMRI mice were randomly divided into five equal groups i.e. one control group and four experimental groups. The experimental groups intraperitoneally (IP received silver nanoparticles at concentrations of 50, 100, 200 and 400 mg/ kg . .every other day. On the 17th day  of pregnancy, the mice were dissected and  their kidneys and embryos tissues were separated and stained with hematoxylin and eosin for histopathological examinations. .Finally, the obtained data was fed into SPSS software (V:16 using statistical tests including Kolmogrof-Smearnof, one-way variance analysis, Dante, Mann-Whitney and Kruskal-Wallis and P<0.05 was taken as the significant level. Results: Histopathological assessment of kidney tissue following IP administration of silver nanoparticle indicated pathological changes including congestion, necrosis, inflammatory cell infiltration, vacuolar degeneration compared to the control group. Our findings showed that silver nanoparticles during the gestation period affects fetal organogenesis, evolution of neural structure, liver lobulation and fetal growth retardation. Mean number of somites in groups receiving doses of 200 and 400 mg kg, . significantly reduced compared to the control group (P<0.05. Conclusion: The obtained results suggest that  passing of silver nanoparticles through placenta is possible and damage caused by the particles  could lead to the deformity or developmental retardation of the fetus.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

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M.A. Sukoyan

2002-05-01

Full Text Available Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Engineering Human Neural Tissue by 3D Bioprinting.

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Gu, Qi; Tomaskovic-Crook, Eva; Wallace, Gordon G; Crook, Jeremy M

2018-01-01

Bioprinting provides an opportunity to produce three-dimensional (3D) tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a method for fabricating human neural tissue by 3D printing human neural stem cells with a bioink, and subsequent gelation of the bioink for cell encapsulation, support, and differentiation to functional neurons and supporting neuroglia. The bioink uniquely comprises the polysaccharides alginate, water-soluble carboxymethyl-chitosan, and agarose. Importantly, the method could be adapted to fabricate neural and nonneural tissues from other cell types, with the potential to be applied for both research and clinical product development.

Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

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Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

2016-02-01

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies. © 2015 AlphaMed Press.

Viscoelastic Properties of Human Tracheal Tissues.

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Safshekan, Farzaneh; Tafazzoli-Shadpour, Mohammad; Abdouss, Majid; Shadmehr, Mohammad B

2017-01-01

The physiological performance of trachea is highly dependent on its mechanical behavior, and therefore, the mechanical properties of its components. Mechanical characterization of trachea is key to succeed in new treatments such as tissue engineering, which requires the utilization of scaffolds which are mechanically compatible with the native human trachea. In this study, after isolating human trachea samples from brain-dead cases and proper storage, we assessed the viscoelastic properties of tracheal cartilage, smooth muscle, and connective tissue based on stress relaxation tests (at 5% and 10% strains for cartilage and 20%, 30%, and 40% for smooth muscle and connective tissue). After investigation of viscoelastic linearity, constitutive models including Prony series for linear viscoelasticity and quasi-linear viscoelastic, modified superposition, and Schapery models for nonlinear viscoelasticity were fitted to the experimental data to find the best model for each tissue. We also investigated the effect of age on the viscoelastic behavior of tracheal tissues. Based on the results, all three tissues exhibited a (nonsignificant) decrease in relaxation rate with increasing the strain, indicating viscoelastic nonlinearity which was most evident for cartilage and with the least effect for connective tissue. The three-term Prony model was selected for describing the linear viscoelasticity. Among different models, the modified superposition model was best able to capture the relaxation behavior of the three tracheal components. We observed a general (but not significant) stiffening of tracheal cartilage and connective tissue with aging. No change in the stress relaxation percentage with aging was observed. The results of this study may be useful in the design and fabrication of tracheal tissue engineering scaffolds.

Predicting Tissue-Specific Enhancers in the Human Genome

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Pennacchio, Len A.; Loots, Gabriela G.; Nobrega, Marcelo A.; Ovcharenko, Ivan

2006-07-01

Determining how transcriptional regulatory signals areencoded in vertebrate genomes is essential for understanding the originsof multi-cellular complexity; yet the genetic code of vertebrate generegulation remains poorly understood. In an attempt to elucidate thiscode, we synergistically combined genome-wide gene expression profiling,vertebrate genome comparisons, and transcription factor binding siteanalysis to define sequence signatures characteristic of candidatetissue-specific enhancers in the human genome. We applied this strategyto microarray-based gene expression profiles from 79 human tissues andidentified 7,187 candidate enhancers that defined their flanking geneexpression, the majority of which were located outside of knownpromoters. We cross-validated this method for its ability to de novopredict tissue-specific gene expression and confirmed its reliability in57 of the 79 available human tissues, with an average precision inenhancer recognition ranging from 32 percent to 63 percent, and asensitivity of 47 percent. We used the sequence signatures identified bythis approach to assign tissue-specific predictions to ~;328,000human-mouse conserved noncoding elements in the human genome. Byoverlapping these genome-wide predictions with a large in vivo dataset ofenhancers validated in transgenic mice, we confirmed our results with a28 percent sensitivity and 50 percent precision. These results indicatethe power of combining complementary genomic datasets as an initialcomputational foray into the global view of tissue-specific generegulation in vertebrates.

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...

Ethical Assessment of Human Embryonic Stem Cell Research According to Turkish Muslim Scholars: First Critical Analysis and Some Reflections.

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Karakaya, Ahmet; Ilkilic, Ilhan

2016-08-01

Turkey, with a Muslim population of officially over 99 %, is one of the few secular states in the Muslim world. Although state institutions are not based on Islamic juridical and ethical norms, the latter play a significant role in defining people's attitudes towards controversial issues in the modern world, especially when backed by opinions of Muslim scholars living in Turkey. Accordingly, opinions of Muslim scholars undoubtedly have an important effect on bioethical decisions made by institutions and individuals. To explore the ethical positions of Muslim scholars living in Turkey and their arguments used in the ethical assessment of embryonic stem cell research; to discuss the biological-moral tensions arising in medical research on human embryos. Qualitative study. Muslim scholars located in different parts of Turkey. Qualitative method, involving the collection of opinions of various scholars, by means of 15 individual semi-structured interviews, evaluated using thematic qualitative analysis. Positions regarding embryonic stem cell research differ among Muslim scholars in Turkey. On the other hand, even where positions are similar, they are often supported by different arguments. Despite the heterogeneity of the arguments presented, the dominant position considers embryonic stem cell research as morally acceptable.

Xanthine oxidase activity regulates human embryonic brain cells growth

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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.

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

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Miller-Pinsler, Lutfiya; Wells, Peter G

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (pcatalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (pcatalase is a determinant of risk for EtOH embryopathies. Copyright © 2015 Elsevier Inc. All rights reserved.

A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

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Assou, Said; Le Carrour, Tanguy; Tondeur, Sylvie; Ström, Susanne; Gabelle, Audrey; Marty, Sophie; Nadal, Laure; Pantesco, Véronique; Réme, Thierry; Hugnot, Jean-Philippe; Gasca, Stéphan; Hovatta, Outi; Hamamah, Samir; Klein, Bernard; De Vos, John

2007-04-01

Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.

Comparing independent microarray studies: the case of human embryonic stem cells

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Hemmati-Brivanlou Ali

2005-07-01

Full Text Available Abstract Background Microarray studies of the same phenomenon in different labs often appear at variance because the published lists of regulated transcripts have disproportionately small intersections. We demonstrate that comparing studies by intersecting lists in this manner is methodologically flawed by reanalyzing three studies of the molecular signature of "stemness" in human embryonic stem cells. There are only 7 genes common to all three published lists, suggesting disagreement. Results Carefully reanalyzing all three together from the raw data we detect 111 genes upregulated and 95 downregulated in all three studies. The upregulated list was subject to rtRTPCR analysis and 75% of the genes were confirmed. Conclusion Our findings show that the three studies have a substantial core of common genes, which is missed if only the published lists are examined. Combined analysis of multiple experiments can be a powerful way to distil coherent conclusions.

Tributyltin induces mitochondrial fission through NAD-IDH dependent mitofusin degradation in human embryonic carcinoma cells.

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Yamada, Shigeru; Kotake, Yaichiro; Nakano, Mizuho; Sekino, Yuko; Kanda, Yasunari

2015-08-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT acts at the nanomolar level through genomic pathways via the peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR). We recently reported that TBT inhibits cell growth and the ATP content in the human embryonic carcinoma cell line NT2/D1 via a non-genomic pathway involving NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which metabolizes isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial NAD-IDH and energy production. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusins 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess cytotoxicity associated with metal exposure.

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

International Nuclear Information System (INIS)

Malkinson, Mertyn; Winocour, Ernest

2005-01-01

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM. AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host

Directed neuronal differentiation of human embryonic stem cells

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Noggle Scott A

2003-10-01

Full Text Available Abstract Background We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs to neural precursors and neurons. HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII. Results A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. Conclusion This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

Can physics help to explain embryonic development? An overview.

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Fleury, V

2013-10-01

Recent technical advances including digital imaging and particle image velocimetry can be used to extract the full range of embryonic movements that constitute the instantaneous 'morphogenetic fields' of a developing animal. The final shape of the animal results from the sum over time (integral) of the movements that make up the velocity fields of all the tissue constituents. In vivo microscopy can be used to capture the details of vertebrate development at the earliest embryonic stages. The movements thus observed can be quantitatively compared to physical models that provide velocity fields based on simple hypotheses about the nature of living matter (a visco-elastic gel). This approach has cast new light on the interpretation of embryonic movement, folding, and organisation. It has established that several major discontinuities in development are simple physical changes in boundary conditions. In other words, with no change in biology, the physical consequences of collisions between folds largely explain the morphogenesis of the major structures (such as the head). Other discontinuities result from changes in physical conditions, such as bifurcations (changes in physical behaviour beyond specific yield points). For instance, beyond a certain level of stress, a tissue folds, without any new gene being involved. An understanding of the physical features of movement provides insights into the levers that drive evolution; the origin of animals is seen more clearly when viewed under the light of the fundamental physical laws (Newton's principle, action-reaction law, changes in symmetry breaking scale). This article describes the genesis of a vertebrate embryo from the shapeless stage (round mass of tissue) to the development of a small, elongated, bilaterally symmetric structure containing vertebral precursors, hip and shoulder enlarges, and a head. Copyright © 2013. Published by Elsevier Masson SAS.

Status quo of management of the human tissue banks in Taiwan.

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Chou, Ching-Pang; Chou, Szu-Cheng; Chen, Ying-Hua; Chen, Yu-Hsuan; Lee, Ming-Shin

2017-03-01

As the technologies associated with transplantation and biological tissue engineering continue to advance, human cells and tissues form an integral part to the practice of regenerative medicine. The patient's use of tissues entails the risk of introducing, transmitting and spreading communicable diseases. To prevent such risk and to ensure that the human organs, tissues and cells remain intact and functional after being handled and processed, the transplanted tissues must be subject to good management standards through all stages of collection, screening, processing, storage and distribution as the safety of the users is of the utmost importance. On February 2009, the government of Taiwan promulgated the Regulations for Administration on Human Organ Bank that requires all human tissues banks to adhere to the Good Tissue Practice for Human Organ, Tissue and Cell in terms of establishment and operation in order to cope with the international management trend and the development and management need of the domestic industry. Six years have passed since the law became effective. This article seeks to introduce the current management mechanism and status quo of management of human tissue banks in Taiwan. We also conducted statistical analysis of the data relating to the tissue banks to identify potential risks and the room for improvement. The study concludes that human tissue banks in Taiwan are on the right track with their management practice, leading to a state of steady development and progress.

Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

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Rao, Yang; Cui, Jihong; Yin, Lu; Liu, Wei; Liu, Wenguang; Sun, Mei; Yan, Xingrong; Wang, Ling; Chen, Fulin

2016-10-22

Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

Ear embryonic rabdomiosarcoma. A case report

International Nuclear Information System (INIS)

Cueto, L.; Canabal, A.; Blanco, A.; Sabate, J.

2002-01-01

A case of embryonic rabdomiosarcoma in the ear of a 5-year-old girl who initially shows clinical symptoms of otitis media. The CT reveals a dense lesion of soft tissue which shows up slightly in the right external auditory channel. Also of interest were osteolytic areas in the petrous, clivus and zygomatic arch. A hypointensive lesion with marked enhancement after Gd-DPTA injection is observed. Discussed are the imaging methods used in the diagnosis of this tumor. (Author) 10 refs

A structure-based extracellular matrix expansion mechanism of fibrous tissue growth.

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Kalson, Nicholas S; Lu, Yinhui; Taylor, Susan H; Starborg, Tobias; Holmes, David F; Kadler, Karl E

2015-05-20

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis.

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...

Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro.

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Gerlach, Jörg C; Hout, Mariah; Edsbagge, Josefina; Björquist, Petter; Lübberstedt, Marc; Miki, Toshio; Stachelscheid, Harald; Schmelzer, Eva; Schatten, Gerald; Zeilinger, Katrin

2010-02-01

Spontaneous in vitro differentiation of mouse embryonic stem cells (mESC) is promoted by a dynamic, three-dimensional (3D), tissue-density perfusion technique with continuous medium perfusion and exchange in a novel four-compartment, interwoven capillary bioreactor. We compared ectodermal, endodermal, and mesodermal immunoreactive tissue structures formed by mESC at culture day 10 with mouse fetal tissue development at gestational day E9.5. The results show that the bioreactor cultures more closely resemble mouse fetal tissue development at gestational day E9.5 than control mESC cultured in Petri dishes.

Advancing biomaterials of human origin for tissue engineering

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Chen, Fa-Ming; Liu, Xiaohua

2015-01-01

Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multi-component construction of native extracellular matrices (ECMs) for cell accommodation, the synthetic biomaterials produced today routinely incorporate biologically active components to define an artificial in vivo milieu with complex and dynamic interactions that foster and regulate stem cells, similar to the events occurring in a natural cellular microenvironment. The range and degree of biomaterial sophistication have also dramatically increased as more knowledge has accumulated through materials science, matrix biology and tissue engineering. However, achieving clinical translation and commercial success requires regenerative biomaterials to be not only efficacious and safe but also cost-effective and convenient for use and production. Utilizing biomaterials of human origin as building blocks for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural tissue with regard to its physical and chemical properties for the orchestration of wound healing and tissue regeneration. In addition to directly using tissue transfers and transplants for repair, new applications of human-derived biomaterials are now focusing on the use of naturally occurring biomacromolecules, decellularized ECM scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to either target acceleration/magnification of the body's own repair capacity or use nature's paradigms to create new tissues for

99Tcm-N(NOEt2 Uptake Kinetics Difference among KMB17 Human Embryonic Lung Diploid Fibroblast and Different Human Lung Cancer Cells

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Wei JIA

2010-04-01

Full Text Available Background and objective PET/CT imaging is expensive, so searching the tumor imaging agent for SPECT/CT is necessary. 99Tcm-N(NOEt2 [bis (N-ethoxy-N-ethyl dithiocarbamato nitrido99Tcm (V] can be uptaken by lung cancer cells and other cells alike. The aim of this study is to evaluate the distinctive value in lung tumor with 99Tcm-N(NOEt2, the difference in its uptake kinetics in human embryonic lung diploid fibroblasts KMB17 and several kinds of lung cancer cells lines. Methods Firstly, six different cell culture medium which contained YTMLC Gejiu human lung squamous carcinoma cell, SPC-A1 human lung adenocarcinoma cell, AGZY low metastatic human lung adenocarcinoma, 973 high metastatic human lung adenocarcinoma cell, GLC-82 Gejiu human lung adenocarcinoma cell, and KMB17 human embryonic lung diploid fibroblast, respectively with equal cell density of 1×106/mL and the same volume were prepared; secondly, the same radioactive dose of 99Tcm-N(NOEt2 was added into each sample and then 300 μL mixed sample was taken out respectively and cultured in 37 oC culture box; Finally, 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min after cultivation, centrifuged each cultured sample and determined the intracellular radiocounts of each sample, calculated each cell sample’s uptake rate of 99Tcm-N(NOEt2 at different time. Results Statistical difference was found among six cell samples, and the uptake rate sequence from high to low is 973 and SPC-A1>YTMLC>GLC-82>AGZY>KMB17 respectively; furthermore, 30 min-45 min after culture, the uptake rate reached stability, and the 45 min uptake rate of each sample was higher than its 96.7% uptake peak. Conclusion Based on the results above mentioned, it is supposed that there are discriminative clinical value when using 99Tcm-N(NOEt2 as a tumor targeting imaging agent, and 30 min or so after injection may be the best imaging time in the early imaging stage.

Variation in alternative splicing across human tissues

OpenAIRE

Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

2004-01-01

Background: Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results: Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most p...

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

Science.gov (United States)

Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

2015-01-01

We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

Directory of Open Access Journals (Sweden)

Silvina Epsztejn-Litman

Full Text Available We report on the derivation of a diploid 46(XX human embryonic stem cell (HESC line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19, monitoring the expression of two parentally imprinted genes (SNRPN and H19 and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

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Heema Hewitson

2016-03-01

Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson 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.

Intravenous Exposure of Pregnant Mice to Silver Nanoparticles: Silver Tissue Distribution and Effects in Maternal and Extra-Embryonic Tissues and Embryos

Science.gov (United States)

Austin, Carlye Anne

concentration in visceral yolk sac and low silver concentration in embryos, suggests that visceral yolk sac tissue mitigates AgNP transfer to embryos. No significant treatment-related effects on embryo morphology or tissue histology were detected. Chapter three constitutes an expanded study of silver distribution in pregnant mice and developing embryos, with the addition of 10 nm AgNP treatment groups and examination of fetuses at GD16. Very low concentrations of silver were measured in GD10 embryos and GD16 fetuses following 10 nm AgNP treatment or in GD16 fetuses following 50 nm AgNP treatment. Highest silver concentrations were measured in maternal liver, spleen, and visceral yolk sac. AgNP particle size (10 or 50 nm) did not consistently affect silver tissue distribution. At GD10, 50 nm AgNP treatment resulted in significantly higher silver concentrations than 10 nm AgNP treatment for liver, spleen, and visceral yolk sac only; at GD16, in visceral yolk sac only, 10 nm AgNP treatment resulted in a significantly higher silver concentration than 50 nm AgNP treatment. In liver, spleen, visceral yolk sac, and uterus, absolute silver concentrations following 10 nm AgNP treatment were significantly lower at GD16 compared to GD10; the patterns of silver tissue distribution were similar at both time points. Silver nitrate and 10 nm AgNP treatments resulted in similar tissue concentrations in GD10 tissues with the exception of visceral yolk sac, for which the silver concentration was significantly higher after silver nitrate treatment. Silver distribution patterns were generally similar between 10 nm AgNP and silver nitrate treatments. No histological abnormalities were noted in maternal tissues, extra-embryonic tissues, or embryos. A significantly increased incidence of developmentally young (for gestational age) GD10 embryos was seen following 10 nm AgNP treatment; no significant morphological effects were observed in embryos or maternal tissues. Further research will be needed to

Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells

DEFF Research Database (Denmark)

Mamsen, Linn; Lutterodt, M C; Andersen, Elisabeth Anne Wreford

2010-01-01

BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimeste......BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first......-trimester gonads in relation to maternal smoking. METHODS: The study includes 24 human first-trimester testes, aged 37-68 days post-conception, obtained from women undergoing legal termination of pregnancy. A questionnaire was used to obtain information about smoking and drinking habits during pregnancy. Validated...... confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS: Prenatal exposure to maternal cigarette smoke...

Enzyme-mediated hyaluronic acid-tyramine hydrogels for the propagation of human embryonic stem cells in 3D.

Science.gov (United States)

Xu, Keming; Narayanan, Karthikeyan; Lee, Fan; Bae, Ki Hyun; Gao, Shujun; Kurisawa, Motoichi

2015-09-01

The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA-Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentration, we prepared HA-Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA-Tyr hydrogel with a compressive modulus of ∼350Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA-Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo. In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system. The scope of this study is to provide a stable 3D cultivation system for the expansion of human embryonic stem cells (hESCs) towards clinical applications. We report an enzyme mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. Unlike other HA-based photo-crosslinked hydrogel systems reported, we investigated the effects of mechanical strength of hydrogels on the self-renewal properties of hESCs in 3D. Then, we characterized hESCs cultured in hydrogels with lower mechanical strength

Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads

DEFF Research Database (Denmark)

Mamsen, Linn S; Ernst, Emil H; Borup, Rehannah

2017-01-01

The precise timing and sequence of changes in expression of key genes and proteins during human sex-differentiation and onset of steroidogenesis was evaluated by whole-genome expression in 67 first trimester human embryonic and fetal ovaries and testis and confirmed by qPCR and immunohistochemistry...... (IHC). SRY/SOX9 expression initiated in testis around day 40 pc, followed by initiation of AMH and steroidogenic genes required for androgen production at day 53 pc. In ovaries, gene expression of RSPO1, LIN28, FOXL2, WNT2B, and ETV5, were significantly higher than in testis, whereas GLI1...... was significantly higher in testis than ovaries. Gene expression was confirmed by IHC for GAGE, SOX9, AMH, CYP17A1, LIN28, WNT2B, ETV5 and GLI1. Gene expression was not associated with the maternal smoking habits. Collectively, a precise temporal determination of changes in expression of key genes involved in human...

Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

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Isabelle Hue

Full Text Available In addition to nourishing the embryo, extra-embryonic tissues (EETs contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC, endoderm (bXEC, and mesoderm (bXMC cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify "cores" of cell-type-specific (and substrate-independent genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21-D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection.

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Sadaoka, Tomohiko; Schwartz, Cindi L; Rajbhandari, Labchan; Venkatesan, Arun; Cohen, Jeffrey I

2018-01-01

Varicella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo ; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human

Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4α Transduction

OpenAIRE

Takayama, Kazuo; Inamura, Mitsuru; Kawabata, Kenji; Katayama, Kazufumi; Higuchi, Maiko; Tashiro, Katsuhisa; Nonaka, Aki; Sakurai, Fuminori; Hayakawa, Takao; Kusuda Furue, Miho; Mizuguchi, Hiroyuki

2012-01-01

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is kn...

Carcino-embryonic antigen in monitoring the growth of human colon adenocarcinoma tumour cells SK-CO-1 and HT-29 in vitro and in nude mice

DEFF Research Database (Denmark)

Sölétormos, G; Fogh, J M; Sehested-Hansen, B

1997-01-01

A set of experimental model systems were designed to investigate (a) the inter-relationship between growth of two human cancer cell lines (SK-CO-1, HT-29) and carcino-embryonic antigen (CEA) kinetics; and (b) whether neoplastic growth or CEA concentration is modulated by human growth hormone (hGH...

Art and human embryonic stem cells: from the bench to the high street.

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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.

Predicting human developmental toxicity of pharmaceuticals using human embryonic stem cells and metabolomics

International Nuclear Information System (INIS)

West, Paul R.; Weir, April M.; Smith, Alan M.; Donley, Elizabeth L.R.; Cezar, Gabriela G.

2010-01-01

Teratogens, substances that may cause fetal abnormalities during development, are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here, we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity, leading to better prediction of teratogenicity. In particular, our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition, this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity, where it correctly predicted the teratogenicity for seven of the eight drugs. Thus, our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways.

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways.

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Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

2017-06-27

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

The role of the pupal determinant broad during embryonic development of a direct-developing insect

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Rynerson, Melody R.; Truman, James W.; Riddiford, Lynn M.

2010-01-01

Metamorphosis is one of the most common, yet dramatic of life history strategies. In insects, complete metamorphosis with morphologically distinct larval stages arose from hemimetabolous ancestors that were more direct developing. Over the past century, several ideas have emerged that suggest the holometabolous pupa is developmentally homologous to the embryonic stages of the hemimetabolous ancestor. Other theories consider the pupal stage to be a modification of a hemimetabolous nymph. To address this question, we have isolated an ortholog of the pupal determinant, broad (br), from the hemimetabolous milkweed bug and examined its role during embryonic development. We show that Oncopeltus fasciatus br (Of'br) is expressed in two phases. The first occurs during germ band invagination and segmentation when Of'br is expressed ubiquitously in the embryonic tissues. The second phase of Of'br expression appears during the pronymphal phase of embryogenesis and persists through nymphal differentiation to decline just before hatching. Knock-down of Of'br transcripts results in defects that range from posterior truncations in the least-affected phenotypes to completely fragmented embryonic tissues in the most severe cases. Analysis of the patterning genes engrailed and hunchback reveal loss of segments and a failure in neural differentiation after Of'br depletion. Finally, we show that br is constitutively expressed during embyrogenesis of the ametabolous firebrat, Thermobia domestica. This suggests that br expression is prominent during embryonic development of ametabolous and hemimetabolous insects but was lost with the emergence of the completely metamorphosing insects. PMID:20127251

How does blastomere removal affect embryonic development? : A time-lapse analysis

DEFF Research Database (Denmark)

Kirkegaard, Kirstine; Hindkjær, Johnny Juhl; Ingerslev, Hans Jakob

of the 6-10 cell embryo. It has been argued that blastomere removal does not affect embryonic development, but few studies have focussed on safety of the procedure. Recently, time-lapse studies on mice have suggested that blastomere removal affects embryonic development. The present study was conducted...... to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis. Materials and methods: Couples undergoing IVF treatment or PGD were requested permission to include embryos in the project. The diagnosis healthy/diseased was made by analysis of a single blastomere....... For PGD 56 human embryos were biopsied 68 hours after fertilisation, the majority at the eight cell stage. As controls 43 non-biopsied embryos at the 6-8 cell stage were selected. All embryos were cultured until 5 days after fertilisation in a time-lapse incubator (EmbryoScope™). Key events such as time...

AMP-activated protein kinase-mediated glucose transport as a novel target of tributyltin in human embryonic carcinoma cells.

Science.gov (United States)

Yamada, Shigeru; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2013-05-01

Organotin compounds such as tributyltin (TBT) are known to cause various forms of cytotoxicity, including developmental toxicity and neurotoxicity. However, the molecular target of the toxicity induced by nanomolar levels of TBT has not been identified. In the present study, we found that exposure to 100 nM TBT induced growth arrest in human pluripotent embryonic carcinoma cell line NT2/D1. Since glucose provides metabolic energy, we focused on the glycolytic system. We found that exposure to TBT reduced the levels of both glucose-6-phosphate and fructose-6-phosphate. To investigate the effect of TBT exposure on glycolysis, we examined glucose transporter (GLUT) activity. TBT exposure inhibited glucose uptake via a decrease in the level of cell surface-bound GLUT1. Furthermore, we examined the effect of AMP-activated protein kinase (AMPK), which is known to regulate glucose transport by facilitating GLUT translocation. Treatment with the potent AMPK activator, AICAR, restored the TBT-induced reduction in cell surface-bound GLUT1 and glucose uptake. In conclusion, these results suggest that exposure to nanomolar levels of TBT causes growth arrest by targeting glycolytic systems in human embryonic carcinoma cells. Thus, understanding the energy metabolism may provide new insights into the mechanisms of metal-induced cytotoxicity.

Generation of a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9

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So-Jung Kim

2017-03-01

Full Text Available Nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2 is a well-known transcription factor that regulates the expression of a large number of anti-oxidant genes in mammalian cells (J.H. Kim et al., 2014. Here, we generated a homozygous Nrf2 knockout human embryonic stem cell (hESC line, H9Nrf2KO-A13, using the CRISPR/Cas9 genome editing method. The Nrf2 homozygous knockout H9 cell line maintains pluripotency, differentiation potential into three germ layers, and a normal karyotype.

High glucose suppresses embryonic stem cell differentiation into neural lineage cells

OpenAIRE

Yang, Penghua; Shen, Wei-bin; Reece, E. Albert; Chen, Xi; Yang, Peixin

2016-01-01

Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model ...

Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions

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Agnete Kirkeby

2012-06-01

Full Text Available To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.

PTBP1 is required for embryonic development before gastrulation.

Science.gov (United States)

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

2011-02-17

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

Human natural killer cell development in secondary lymphoid tissues

Science.gov (United States)

Freud, Aharon G.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

For nearly a decade it has been appreciated that critical steps in human natural killer (NK) cell development likely occur outside of the bone marrow and potentially necessitate distinct microenvironments within extramedullary tissues. The latter include the liver and gravid uterus as well as secondary lymphoid tissues such as tonsils and lymph nodes. For as yet unknown reasons these tissues are naturally enriched with NK cell developmental intermediates (NKDI) that span a maturation continuum starting from an oligopotent CD34+CD45RA+ hematopoietic precursor cell to a cytolytic mature NK cell. Indeed despite the detection of NKDI within the aforementioned tissues, relatively little is known about how, why, and when these tissues may be most suited to support NK cell maturation and how this process fits in with other components of the human immune system. With the discovery of other innate lymphoid subsets whose immunophenotypes overlap with those of NKDI, there is also need to revisit and potentially re-characterize the basic immunophenotypes of the stages of the human NK cell developmental pathway in vivo. In this review, we provide an overview of human NK cell development in secondary lymphoid tissues and discuss the many questions that remain to be answered in this exciting field. PMID:24661538

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.

Preparation, characterization and toxicological investigation of copper loaded chitosan nanoparticles in human embryonic kidney HEK-293 cells

Energy Technology Data Exchange (ETDEWEB)

Arora, Divya [Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu (India); Formulation and Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Dhanwal, Vandna [Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Nayak, Debasis [Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu (India); Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Saneja, Ankit [Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu (India); Formulation and Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Amin, Hina [Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Rasool, Reyaz ur [Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu (India); Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Gupta, Prem Narayan [Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu (India); Formulation and Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India); Goswami, Anindya, E-mail: agoswami@iiim.ac.in [Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu (India); Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu (India)

2016-04-01

Metallic nanoparticles often attribute severe adverse effects to the various organs or tissues at the molecular level despite of their applications in medical, laboratory and industrial sectors. The present study highlights the preparation of copper adsorbed chitosan nanoparticles (CuCSNPs), its characterization and validation of cytotoxicity in human embryonic kidney HEK-293 cells. Particle size of the CuCSNPs was determined by using Zetasizer and the copper loading was quantified with the help of ICP/MS. Further characterization of CuCSNPs was carried out by FT-IR analysis to determine the formation of nanoparticles and SEM was conducted for the morphological analysis of the CuCSNPs. The CuCSNPs exhibited pronounced cytotoxic effects towards HEK-293 cells as analyzed by MTT assay. Moreover, the CuCSNPs inhibited the colony formation and induced nuclear damage at the dose of 100 μg/mL, much more effectively than the in built control copper sulfate (CuSO{sub 4}). At the molecular level, the CuCSNPs were found to be triggering reactive oxygen species (ROS), activating effector caspases and subsequent PARP cleavage to induce cell death in HEK-293 cells. - Highlights: • Subtoxic levels of CuCSNPs induce apoptosis in HEK-293 cells. • CuCSNPs mediate toxicity via nuclear cleavage and ROS generation. • CuCSNPs favor caspase activation and PARP cleavage to induce cell death.

Adenovirus 36 DNA in human adipose tissue.

Science.gov (United States)

Ponterio, E; Cangemi, R; Mariani, S; Casella, G; De Cesare, A; Trovato, F M; Garozzo, A; Gnessi, L

2015-12-01

Recent studies have suggested a possible correlation between obesity and adenovirus 36 (Adv36) infection in humans. As information on adenoviral DNA presence in human adipose tissue are limited, we evaluated the presence of Adv36 DNA in adipose tissue of 21 adult overweight or obese patients. Total DNA was extracted from adipose tissue biopsies. Virus detection was performed using PCR protocols with primers against specific Adv36 fiber protein and the viral oncogenic E4orf1 protein nucleotide sequences. Sequences were aligned with the NCBI database and phylogenetic analyses were carried out with MEGA6 software. Adv36 DNA was found in four samples (19%). This study indicates that some individuals carry Adv36 in the visceral adipose tissue. Further studies are needed to determine the specific effect of Adv36 infection on adipocytes, the prevalence of Adv36 infection and its relationship with obesity in the perspective of developing a vaccine that could potentially prevent or mitigate infection.

Albumin-associated lipids regulate human embryonic stem cell self-renewal.

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Francesc R Garcia-Gonzalo

Full Text Available BACKGROUND: Although human embryonic stem cells (hESCs hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect. CONCLUSIONS/SIGNIFICANCE: Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems.

Geometry Modeling Program Implementation of Human Hip Tissue

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WANG Mo-nan

2017-10-01

Full Text Available Abstract:Aiming to design a simulate software of human tissue modeling and analysis，Visual Studio 2010 is selected as a development tool to develop a 3 D reconstruction software of human tissue with language C++.It can be used alone. It also can be a module of the virtual surgery systems. The system includes medical image segmentation modules and 3 D reconstruction modules，and can realize the model visualization. This software system has been used to reconstruct hip muscles，femur and hip bone accurately. The results show these geometry models can simulate the structure of hip tissues.

Geometry Modeling Program Implementation of Human Hip Tissue

Directory of Open Access Journals (Sweden)

WANG Monan

2017-04-01

Full Text Available Aiming to design a simulate software of human tissue modeling and analysis，Visual Studio 2010 is selected as a development tool to develop a 3 D reconstruction software of human tissue with language C++.It can be used alone. It also can be a module of the virtual surgery systems. The system includes medical image segmentation modules and 3 D reconstruction modules，and can realize the model visualization. This software system has been used to reconstruct hip muscles，femur and hip bone accurately. The results show these geometry models can simulate the structure of hip tissues.

Cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung tumors

International Nuclear Information System (INIS)

Kolesnichenko, T.S.; Gor'kova, T.G.

1985-01-01

Local factors such as proliferative activity and the numerical ratio between epithelial and mesenchymal cells, and also the character of interaction between the tissue components in ontogeny may play an important role in the realization of sensitivity of mice of a particular line to the development of lung tumors. These characteristics of lung tissue in mice of lines A and C57BL are investigated under normal conditions and during induced carcinogenesis. Results are given of a comparative study of the relative numbers of epithelial and mesenchymal cells in organ cultures of embryonic lungs. 3 H-thymidine was added to the cultures on the 14th day of the experiment in a concentration of 1 microCi/m1 medium. An autoradiographic study of the cultures was performed

Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

International Nuclear Information System (INIS)

Mochalova, Larisa; Gambaryan, Alexandra; Romanova, Julia; Tuzikov, Alexander; Chinarev, Alexander; Katinger, Dietmar; Katinger, Herman; Egorov, Andrej; Bovin, Nicolai

2003-01-01

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac α2-6Galβ1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acα2-6Galβ1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses

Human tissue models in cancer research: looking beyond the mouse.

Science.gov (United States)

Jackson, Samuel J; Thomas, Gareth J

2017-08-01

Mouse models, including patient-derived xenograft mice, are widely used to address questions in cancer research. However, there are documented flaws in these models that can result in the misrepresentation of human tumour biology and limit the suitability of the model for translational research. A coordinated effort to promote the more widespread development and use of 'non-animal human tissue' models could provide a clinically relevant platform for many cancer studies, maximising the opportunities presented by human tissue resources such as biobanks. A number of key factors limit the wide adoption of non-animal human tissue models in cancer research, including deficiencies in the infrastructure and the technical tools required to collect, transport, store and maintain human tissue for lab use. Another obstacle is the long-standing cultural reliance on animal models, which can make researchers resistant to change, often because of concerns about historical data compatibility and losing ground in a competitive environment while new approaches are embedded in lab practice. There are a wide range of initiatives that aim to address these issues by facilitating data sharing and promoting collaborations between organisations and researchers who work with human tissue. The importance of coordinating biobanks and introducing quality standards is gaining momentum. There is an exciting opportunity to transform cancer drug discovery by optimising the use of human tissue and reducing the reliance on potentially less predictive animal models. © 2017. Published by The Company of Biologists Ltd.

Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

Science.gov (United States)

Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

The Evolutionary Economics of Embryonic-Sac Fluids in Squamate Reptiles.

Science.gov (United States)

Bonnet, Xavier; Naulleau, Guy; Shine, Richard

2017-03-01

The parchment-shelled eggs of squamate reptiles take up substantial water from the nest environment, enabling the conversion of yolk into neonatal tissue and buffering the embryo against the possibility of subsequent dry weather. During development, increasing amounts of water are stored in the embryonic sacs (i.e., membranes around the embryo: amnion, allantois, and chorion). The evolution of viviparity (prolonged uterine retention of developing embryos) means that embryonic-sac fluid storage now imposes a cost (increased maternal burdening), confers less benefit (because the mother buffers fetal water balance), and introduces a potential conflict among uterine siblings (for access to finite water supplies). Our data on nine species of squamate reptiles and published information on three species show that the embryonic-sac fluids comprise around 33% of neonatal mass in viviparous species versus 94% in full-term eggs of oviparous squamates. Data on parturition in 149 vipers (Vipera aspis, a viviparous species) show that larger offspring store more fluids in their fetal sacs and that an increase in litter size is associated with a decrease in fluid-sac mass per offspring. Overall, the evolutionary transition from oviparity to viviparity may have substantially altered selective forces on offspring packaging and created competition among offspring for access to water reserves during embryonic development.

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, Huiqing; 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

N-acetylcysteine-pretreated human embryonic mesenchymal stem cell administration protects against bleomycin-induced lung injury.

Science.gov (United States)

Wang, Qiao; Zhu, Hong; Zhou, Wu-Gang; Guo, Xiao-Can; Wu, Min-Juan; Xu, Zhen-Yu; Jiang, Jun-feng; Shen, Ce; Liu, Hou-Qi

2013-08-01

The transplantation of mesenchymal stem cells (MSCs) has been reported to be a promising approach in the treatment of acute lung injury. However, the poor efficacy of transplanted MSCs is one of the serious handicaps in the progress of MSC-based therapy. Therefore, the purpose of this study was to investigate whether the pretreatment of human embryonic MSCs (hMSCs) with an antioxidant, namely N-acetylcysteine (NAC), can improve the efficacy of hMSC transplantation in lung injury. In vitro, the antioxidant capacity of NAC-pretreated hMSCs was assessed using intracellular reactive oxygen species (ROS) and glutathione assays and cell adhesion and spreading assays. In vivo, the therapeutic potential of NAC-pretreated hMSCs was assessed in a bleomycin-induced model of lung injury in nude mice. The pretreatment of hMSCs with NAC improved antioxidant capacity to defend against redox imbalances through the elimination of cellular ROS, increasing cellular glutathione levels, and the enhancement of cell adhesion and spreading when exposed to oxidative stresses in vitro. In addition, the administration of NAC-pretreated hMSCs to nude mice with bleomycin-induced lung injury decreased the pathological grade of lung inflammation and fibrosis, hydroxyproline content and numbers of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid and apoptotic cells, while enhancing the retention and proliferation of hMSCs in injured lung tissue and improving the survival rate of mice compared with results from untreated hMSCs. The pretreatment of hMSCs with NAC could be a promising therapeutic approach to improving cell transplantation and, therefore, the treatment of lung injury.

Prolactin suppresses malonyl-CoA concentration in human adipose tissue

DEFF Research Database (Denmark)

Nilsson, L. A.; Roepstorff, Carsten; Kiens, Bente

2009-01-01

Prolactin is best known for its involvement in lactation, where it regulates mechanisms that supply nutrients for milk production. In individuals with pathological hyperprolactinemia, glucose and fat homeostasis have been reported to be negatively influenced. It is not previously known, however......, whether prolactin regulates lipogenesis in human adipose tissue. The aim of this study was to investigate the effect of prolactin on lipogenesis in human adipose tissue in vitro. Prolactin decreased the concentration of malonyl-CoA, the product of the first committed step in lipogenesis, to 77......+/-6% compared to control 100+/-5% (p=0.022) in cultured human adipose tissue. In addition, prolactin was found to decrease glucose transporter 4 ( GLUT4) mRNA expression, which may cause decreased glucose uptake. In conclusion, we propose that prolactin decreases lipogenesis in human adipose tissue...

Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis

NARCIS (Netherlands)

Le Guen, Ludovic; Karpanen, Terhi; Schulte, Dörte; Harris, Nicole C; Koltowska, Katarzyna; Roukens, Guy; Bower, Neil I; van Impel, Andreas; Stacker, Steven A; Achen, Marc G; Schulte-Merker, Stefan; Hogan, Benjamin M

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics

Directory of Open Access Journals (Sweden)

Nigel G. Kooreman

2017-08-01

Full Text Available There is growing interest in using embryonic stem cell (ESC and induced pluripotent stem cell (iPSC derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

Fucoidan promotes early step of cardiac differentiation from human embryonic stem cells and long-term maintenance of beating areas.

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Hamidi, Sofiane; Letourneur, Didier; Aid-Launais, Rachida; Di Stefano, Antonio; Vainchenker, William; Norol, Françoise; Le Visage, Catherine

2014-04-01

Somatic stem cells require specific niches and three-dimensional scaffolds provide ways to mimic this microenvironment. Here, we studied a scaffold based on Fucoidan, a sulfated polysaccharide known to influence morphogen gradients during embryonic development, to support human embryonic stem cells (hESCs) differentiation toward the cardiac lineage. A macroporous (pore 200 μm) Fucoidan scaffold was selected to support hESCs attachment and proliferation. Using a protocol based on the cardiogenic morphogen bone morphogenic protein 2 (BMP2) and transforming growth factor (TGFβ) followed by tumor necrosis factor (TNFα), an effector of cardiopoietic priming, we examined the cardiac differentiation in the scaffold compared to culture dishes and embryoid bodies (EBs). At day 8, Fucoidan scaffolds supported a significantly higher expression of the 3 genes encoding for transcription factors marking the early step of embryonic cardiac differentiation NKX2.5 (prelease TGFβ and TNFα was confirmed by Luminex technology. We also found that Fucoidan scaffolds supported the late stage of embryonic cardiac differentiation marked by a significantly higher atrial natriuretic factor (ANF) expression (pstress in the soft hydrogel impaired sarcomere formation, as confirmed by molecular analysis of the cardiac muscle myosin MYH6 and immunohistological staining of sarcomeric α-actinin. Nevertheless, Fucoidan scaffolds contributed to the development of thin filaments connecting beating areas through promotion of smooth muscle cells, thus enabling maintenance of beating areas for up to 6 months. In conclusion, Fucoidan scaffolds appear as a very promising biomaterial to control cardiac differentiation from hESCs that could be further combined with mechanical stress to promote sarcomere formation at terminal stages of differentiation.

Neonatal Desensitization Supports Long-Term Survival and Functional Integration of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in Rat Joint Cartilage Without Immunosuppression

Science.gov (United States)

Zhang, Shufang; Jiang, Yang Zi; Zhang, Wei; Chen, Longkun; Tong, Tong; Liu, Wanlu; Mu, Qin; Liu, Hua; Ji, Junfeng; Ouyang, Hong Wei

2013-01-01

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models. PMID:22788986

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

International Nuclear Information System (INIS)

Miller-Pinsler, Lutfiya; Wells, Peter G.

2015-01-01

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat b /J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • EtOH developmental

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

Energy Technology Data Exchange (ETDEWEB)

Miller-Pinsler, Lutfiya [Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario (Canada); Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada)

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

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.

Aberrant patterns of X chromosome inactivation in a new line of human embryonic stem cells established in physiological oxygen concentrations.

Science.gov (United States)

de Oliveira Georges, Juliana Andrea; Vergani, Naja; Fonseca, Simone Aparecida Siqueira; Fraga, Ana Maria; de Mello, Joana Carvalho Moreira; Albuquerque, Maria Cecília R Maciel; Fujihara, Litsuko Shimabukuro; Pereira, Lygia Veiga

2014-08-01

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation, whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans, or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs, suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci, characteristic of the inactive X. Moreover, analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs.

The Human Tissue Act 2004 and the child donor.

Science.gov (United States)

Baston, Jenny

2009-05-01

In 2001, the inquiry panel appointed to investigate the removal, retention and disposal of human organs and tissues at the Royal Liverpool Children's Hospital published its report. The panel's recommendations led to a new approach to consent for organ removal and storage under the new Human Tissue Act 2004. For child bone marrow donors, the new consent process requires all donor children or their parent to undergo a separate assessment before the bone marrow donation. They must be assessed by an accredited assessor who will submit a recommendation to the Human Tissue Authority for consideration. The unfortunate circumstances highlighted in the inquiry have led to changes to law, practice and culture that are benefiting other children and families.

Polychlorinated naphthalenes in human adipose tissue from New York, USA

International Nuclear Information System (INIS)

Kunisue, Tatsuya; Johnson-Restrepo, Boris; Hilker, David R.; Aldous, Kenneth M.; Kannan, Kurunthachalam

2009-01-01

Polychlorinated naphthalenes (PCNs) are persistent, bioaccumulative, and toxic contaminants. Prior to this study, the occurrence of PCNs in human adipose tissues from the USA has not been analyzed. Here, we have measured concentrations of PCNs in human adipose tissue samples collected in New York City during 2003-2005. Concentrations of PCNs were in the range of 61-2500 pg/g lipid wt. in males and 21-910 pg/g lipid wt. in females. PCN congeners 52/60 (1,2,3,5,7/1,2,4,6,7) and 66/67 (1,2,3,4,6,7/1,2,3,5,6,7) were predominant, collectively accounting for 66% of the total PCN concentrations. Concentrations of PCNs in human adipose tissues were 2-3 orders of magnitude lower than the previously reported concentrations of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). Concentrations of PCNs were not correlated with PCB concentrations. The contribution of PCNs to dioxin-like toxic equivalents (TEQs) in human adipose tissues was estimated to be <1% of the polychlorinated dibenzo-p-dioxin/dibenzofuran (PCDD/F)-TEQs. - Polychlorinated naphthalenes have been measured in human adipose tissues from the USA for the first time

NCI’s Cooperative Human Tissue Network

Science.gov (United States)

Quality biospecimens are a foundational resource for cancer research. One of NCI’s longest running biospecimen programs is the Cooperative Human Tissue Network, a resource mainly for basic discovery and early translational research.

Label-free separation of human embryonic stem cells (hESCs) and their cardiac derivatives using Raman spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Chan, J W; Lieu, D K; Huser, T R; Li, R A

2008-09-08

Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and non-recoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a non-destructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combining the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98% and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the non-invasive and label-free sorting of (i) high-quality hESCs for expansion, and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

Proteomic Analysis of Chicken Skeletal Muscle during Embryonic Development

Directory of Open Access Journals (Sweden)

Hongjia Ouyang

2017-05-01

Full Text Available Embryonic growth and development of skeletal muscle is a major determinant of muscle mass, and has a significant effect on meat production in chicken. To assess the protein expression profiles during embryonic skeletal muscle development, we performed a proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ in leg muscle tissues of female Xinghua chicken at embryonic age (E 11, E16, and 1-day post hatch (D1. We identified 3,240 proteins in chicken embryonic muscle and 491 of them were differentially expressed (fold change ≥ 1.5 or ≤ 0.666 and p < 0.05. There were 19 up- and 32 down-regulated proteins in E11 vs. E16 group, 238 up- and 227 down-regulated proteins in E11 vs. D1 group, and 13 up- and 5 down-regulated proteins in E16 vs. D1 group. Protein interaction network analyses indicated that these differentially expressed proteins were mainly involved in the pathway of protein synthesis, muscle contraction, and oxidative phosphorylation. Integrative analysis of proteome and our previous transcriptome data found 189 differentially expressed proteins that correlated with their mRNA level. The interactions between these proteins were also involved in muscle contraction and oxidative phosphorylation pathways. The lncRNA-protein interaction network found four proteins DMD, MYL3, TNNI2, and TNNT3 that are all involved in muscle contraction and may be lncRNA regulated. These results provide several candidate genes for further investigation into the molecular mechanisms of chicken embryonic muscle development, and enable us to better understanding their regulation networks and biochemical pathways.

Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

DEFF Research Database (Denmark)

Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T

2009-01-01

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...... embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers......: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell...

[Induced pluripotent stem cells: a new paradigm to study human tissues].

Science.gov (United States)

Sansac, Caroline; Assou, Said; Bouckenheimer, Julien; Lemaître, Jean-Marc; De Vos, John

2016-01-01

Induced pluripotent stem cells (iPSCs) are obtained by reprogramming differentiated cells through forced expression of four embryonic transcription factors. The discovery of this technology, able to transform a differentiated cell into a pluripotent cell, has profoundly shifted the paradigm of the concept of cell identity, since it is now possible to obtain in vitro any cell type from an initial sample of skin or blood cells from a healthy volunteer or patient. Applications of iPSCs are exceedingly large, and comprise the in vitro modeling of normal or pathological tissues, including for massive drug screening. They also open new therapeutic avenues in the field of regenerative medicine. © Société de Biologie, 2016.

Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals

Directory of Open Access Journals (Sweden)

Nian Shen

2017-07-01

Full Text Available Cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min, cyclic strain (5%, and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease.

Browning of Subcutaneous White Adipose Tissue in Humans

OpenAIRE

Sidossis, Labros S.; Porter, Craig; Saraf, Manish K.; Børsheim, Elisabet; Radhakrishnan, Ravi S.; Chao, Tony; Ali, Arham; Chondronikola, Maria; Mlcak, Ronald; Finnerty, Celeste C.; Hawkins, Hal K.; Toliver-Kinsky, Tracy; Herndon, David N.

2015-01-01

Since the presence of brown adipose tissue (BAT) was confirmed in adult humans, BAT has become a therapeutic target for obesity and insulin resistance. We examined whether human subcutaneous white adipose tissue (sWAT) can adopt a BAT-like phenotype using a clinical model of prolonged and severe adrenergic stress. sWAT samples were collected from severely burned and healthy individuals. A subset of burn victims were prospectively followed during their acute hospitalization. Browning of sWAT w...

Characterization of muscarinic receptor subtypes in human tissues

International Nuclear Information System (INIS)

Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

1988-01-01

The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with [ 3 H]Pirenzepine and [ 3 H]N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M 1 neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M 1 , the cardiac M 2 and the glandular M 3

The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

International Nuclear Information System (INIS)

Li Jianzhong; Chen Xia; Yang Hua; Wang Shuiliang; Guo Baoyu; Yu Long; Wang Zhugang; Fu Jiliang

2006-01-01

Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191 +/- mice are normal and fertile. Homozygous Zfp191 -/- embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191 -/- and Zfp191 +/- embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191 -/- cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191 +/- intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation

Patents on inventions related to human embryonic stem cells: the morality clause after Brüstle v. Greenpeace.

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Panis, Sarah

2013-09-01

This paper analyses the meaning of Article 6, para. 2, sub c of the Biotechnology Directive prohibiting patents on inventions using human embryos for industrial or commercial purposes. It first examines the evolution ofthe Court of Justice ofthe EU's interpretation of this provision (which is part of the morality clause) and focuses on its most recent decision, Brüstle v. Greenpeace. This is considered a landmark case for three reasons: firstly, because it defines for the first time the term "embryo" in patent law; secondly, because it is the Court of Justice (and not EPO) that ruled on patent law; the third reason is its very broad interpretation of the morality exclusion. The exclusion is no longer limited to embryos but is extended to (even banked) embryonic stem cells and all downstream products made with them. It then looks into the consequences for the patentability of inventions using cells derived from human embryonic stem cells, such as Brüstle's invention. The recent decision by Germany's Federal Court of Justice on the validity of Brüstle's patent emphasises the limited influence on the patentability of those inventions. After that, the paper addresses possible cuts in funding stem cell research and even legislative bans of this type of research. This is followed by an evaluation of the existence and content of the morality exclusion. After a comparative analysis with the US, which is lacking in such morality exclusion, the paper concludes that the morality clause as a whole paid its dues but the provision on the use of human embryos is questionable as there is no European consensus against the use of human embryos for industrial or commercial purposes.

Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions

DEFF Research Database (Denmark)

Awan, Aashir; Oliveri, Roberto S; Jensen, Pernille L

2010-01-01

onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell......This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging...

A family of hyperelastic models for human brain tissue

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Mihai, L. Angela; Budday, Silvia; Holzapfel, Gerhard A.; Kuhl, Ellen; Goriely, Alain

2017-09-01

Experiments on brain samples under multiaxial loading have shown that human brain tissue is both extremely soft when compared to other biological tissues and characterized by a peculiar elastic response under combined shear and compression/tension: there is a significant increase in shear stress with increasing axial compression compared to a moderate increase with increasing axial tension. Recent studies have revealed that many widely used constitutive models for soft biological tissues fail to capture this characteristic response. Here, guided by experiments of human brain tissue, we develop a family of modeling approaches that capture the elasticity of brain tissue under varying simple shear superposed on varying axial stretch by exploiting key observations about the behavior of the nonlinear shear modulus, which can be obtained directly from the experimental data.

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.

Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

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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.

Automated grouping of action potentials of human embryonic stem cell-derived cardiomyocytes.

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Gorospe, Giann; Zhu, Renjun; Millrod, Michal A; Zambidis, Elias T; Tung, Leslie; Vidal, Rene

2014-09-01

Methods for obtaining cardiomyocytes from human embryonic stem cells (hESCs) are improving at a significant rate. However, the characterization of these cardiomyocytes (CMs) is evolving at a relatively slower rate. In particular, there is still uncertainty in classifying the phenotype (ventricular-like, atrial-like, nodal-like, etc.) of an hESC-derived cardiomyocyte (hESC-CM). While previous studies identified the phenotype of a CM based on electrophysiological features of its action potential, the criteria for classification were typically subjective and differed across studies. In this paper, we use techniques from signal processing and machine learning to develop an automated approach to discriminate the electrophysiological differences between hESC-CMs. Specifically, we propose a spectral grouping-based algorithm to separate a population of CMs into distinct groups based on the similarity of their action potential shapes. We applied this method to a dataset of optical maps of cardiac cell clusters dissected from human embryoid bodies. While some of the nine cell clusters in the dataset are presented with just one phenotype, the majority of the cell clusters are presented with multiple phenotypes. The proposed algorithm is generally applicable to other action potential datasets and could prove useful in investigating the purification of specific types of CMs from an electrophysiological perspective.

Microwave non-contact imaging of subcutaneous human body tissues.

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Kletsov, Andrey; Chernokalov, Alexander; Khripkov, Alexander; Cho, Jaegeol; Druchinin, Sergey

2015-10-01

A small-size microwave sensor is developed for non-contact imaging of a human body structure in 2D, enabling fitness and health monitoring using mobile devices. A method for human body tissue structure imaging is developed and experimentally validated. Subcutaneous fat tissue reconstruction depth of up to 70 mm and maximum fat thickness measurement error below 2 mm are demonstrated by measurements with a human body phantom and human subjects. Electrically small antennas are developed for integration of the microwave sensor into a mobile device. Usability of the developed microwave sensor for fitness applications, healthcare, and body weight management is demonstrated.

Disruption of cardiogenesis in human embryonic stem cells exposed to trichloroethylene.

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Jiang, Yan; Wang, Dan; Zhang, Guoxing; Wang, Guoqing; Tong, Jian; Chen, Tao

2016-11-01

Trichloroethylene (TCE) is ubiquitous in our living environment, and prenatal exposure to TCE is reported to cause congenital heart disease in humans. Although multiple studies have been performed using animal models, they have limited value in predicting effects on humans due to the unknown species-specific toxicological effects. To test whether exposure to low doses of TCE induces developmental toxicity in humans, we investigated the effect of TCE on human embryonic stem cells (hESCs) and cardiomyocytes (derived from the hESCs). In the current study, hESCs cardiac differentiation was achieved by using differentiation medium consisting of StemPro-34. We examined the effects of TCE on cell viability by cell growth assay and cardiac inhibition by analysis of spontaneously beating cluster. The expression levels of genes associated with cardiac differentiation and Ca 2+ channel pathways were measured by immunofluorescence and qPCR. The overall data indicated the following: (1) significant cardiac inhibition, which was characterized by decreased beating clusters and beating rates, following treatment with low doses of TCE; (2) significant up-regulation of the Nkx2.5/Hand1 gene in cardiac progenitors and down regulation of the Mhc-7/cTnT gene in cardiac cells; and (3) significant interference with Ca 2+ channel pathways in cardiomyocytes, which contributes to the adverse effect of TCE on cardiac differentiation during early embryo development. Our results confirmed the involvement of Ca 2+ turnover network in TCE cardiotoxicity as reported in animal models, while the inhibition effect of TCE on the transition of cardiac progenitors to cardiomyocytes is unique to hESCs, indicating a species-specific effect of TCE on heart development. This study provides new insight into TCE biology in humans, which may help explain the development of congenital heart defects after TCE exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1372-1380, 2016. © 2015 Wiley

Human tissue models in cancer research: looking beyond the mouse

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Samuel J. Jackson

2017-08-01

Full Text Available Mouse models, including patient-derived xenograft mice, are widely used to address questions in cancer research. However, there are documented flaws in these models that can result in the misrepresentation of human tumour biology and limit the suitability of the model for translational research. A coordinated effort to promote the more widespread development and use of ‘non-animal human tissue’ models could provide a clinically relevant platform for many cancer studies, maximising the opportunities presented by human tissue resources such as biobanks. A number of key factors limit the wide adoption of non-animal human tissue models in cancer research, including deficiencies in the infrastructure and the technical tools required to collect, transport, store and maintain human tissue for lab use. Another obstacle is the long-standing cultural reliance on animal models, which can make researchers resistant to change, often because of concerns about historical data compatibility and losing ground in a competitive environment while new approaches are embedded in lab practice. There are a wide range of initiatives that aim to address these issues by facilitating data sharing and promoting collaborations between organisations and researchers who work with human tissue. The importance of coordinating biobanks and introducing quality standards is gaining momentum. There is an exciting opportunity to transform cancer drug discovery by optimising the use of human tissue and reducing the reliance on potentially less predictive animal models.

Cytokine signalling in embryonic stem cells

DEFF Research Database (Denmark)

Kristensen, David Møbjerg; Kalisz, Mark; Nielsen, Jens Høiriis

2006-01-01

Cytokines play a central role in maintaining self-renewal in mouse embryonic stem (ES) cells through a member of the interleukin-6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK-STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric...... pathways seem to converge on c-myc as a common target to promote self-renewal. Whereas LIF does not seem to stimulate self-renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling...... via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem...

Evidence for the ectopic synthesis of melanin in human adipose tissue.

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Randhawa, Manpreet; Huff, Tom; Valencia, Julio C; Younossi, Zobair; Chandhoke, Vikas; Hearing, Vincent J; Baranova, Ancha

2009-03-01

Melanin is a common pigment in animals. In humans, melanin is produced in melanocytes, in retinal pigment epithelium (RPE) cells, in the inner ear, and in the central nervous system. Previously, we noted that human adipose tissue expresses several melanogenesis-related genes. In the current study, we confirmed the expression of melanogenesis-related mRNAs and proteins in human adipose tissue using real-time polymerase chain reaction and immunohistochemical staining. TYR mRNA signals were also detected by in situ hybridization in visceral adipocytes. The presence of melanin in human adipose tissue was revealed both by Fontana-Masson staining and by permanganate degradation of melanin coupled with liquid chromatography/ultraviolet/mass spectrometry determination of the pyrrole-2,3,5-tricarboxylic acid (PTCA) derivative of melanin. We also compared melanogenic activities in adipose tissues and in other human tissues using the L-[U-(14)C] tyrosine assay. A marked heterogeneity in the melanogenic activities of individual adipose tissue extracts was noted. We hypothesize that the ectopic synthesis of melanin in obese adipose may serve as a compensatory mechanism that uses its anti-inflammatory and its oxidative damage-absorbing properties. In conclusion, our study demonstrates for the first time that the melanin biosynthesis pathway is functional in adipose tissue.

Comparative in silico profiling of epigenetic modifiers in human tissues.

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Son, Mi-Young; Jung, Cho-Rok; Kim, Dae-Soo; Cho, Hyun-Soo

2018-04-06

The technology of tissue differentiation from human pluripotent stem cells has attracted attention as a useful resource for regenerative medicine, disease modeling and drug development. Recent studies have suggested various key factors and specific culture methods to improve the successful tissue differentiation and efficient generation of human induced pluripotent stem cells. Among these methods, epigenetic regulation and epigenetic signatures are regarded as an important hurdle to overcome during reprogramming and differentiation. Thus, in this study, we developed an in silico epigenetic panel and performed a comparative analysis of epigenetic modifiers in the RNA-seq results of 32 human tissues. We demonstrated that an in silico epigenetic panel can identify epigenetic modifiers in order to overcome epigenetic barriers to tissue-specific differentiation.

Translational neuropharmacology: the use of human isolated gastrointestinal tissues.

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Sanger, G J; Broad, J; Kung, V; Knowles, C H

2013-01-01

Translational sciences increasingly emphasize the measurement of functions in native human tissues. However, such studies must confront variations in patient age, gender, genetic background and disease. Here, these are discussed with reference to neuromuscular and neurosecretory functions of the human gastrointestinal (GI) tract. Tissues are obtained after informed consent, in collaboration with surgeons (surgical techniques help minimize variables) and pathologists. Given the difficulties of directly recording from human myenteric neurones (embedded between muscle layers), enteric motor nerve functions are studied by measuring muscle contractions/relaxations evoked by electrical stimulation of intrinsic nerves; responses are regionally dependent, often involving cholinergic and nitrergic phenotypes. Enteric sensory functions can be studied by evoking the peristaltic reflex, involving enteric sensory and motor nerves, but this has rarely been achieved. As submucosal neurones are more accessible (after removing the mucosa), direct neuronal recordings are possible. Neurosecretory functions are studied by measuring changes in short-circuit current across the mucosa. For all experiments, basic questions must be addressed. Because tissues are from patients, what are the controls and the influence of disease? How long does it take before function fully recovers? What is the impact of age- and gender-related differences? What is the optimal sample size? Addressing these and other questions minimizes variability and raises the scientific credibility of human tissue research. Such studies also reduce animal use. Further, the many differences between animal and human GI functions also means that human tissue research must question the ethical validity of using strains of animals with unproved translational significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

ChIP-seq Accurately Predicts Tissue-Specific Activity of Enhancers

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Visel, Axel; Blow, Matthew J.; Li, Zirong; Zhang, Tao; Akiyama, Jennifer A.; Holt, Amy; Plajzer-Frick, Ingrid; Shoukry, Malak; Wright, Crystal; Chen, Feng; Afzal, Veena; Ren, Bing; Rubin, Edward M.; Pennacchio, Len A.

2009-02-01

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover since they are scattered amongst the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here, we performed chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively-parallel sequencing, to map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain, and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases revealed reproducible enhancer activity in those tissues predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities and suggest that such datasets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

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D'Amour, Kevin A; Bang, Anne G; Eliazer, Susan; Kelly, Olivia G; Agulnick, Alan D; Smart, Nora G; Moorman, Mark A; Kroon, Evert; Carpenter, Melissa K; Baetge, Emmanuel E

2006-11-01

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Elasticity of human embryonic stem cells as determined by atomic force microscopy.

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Kiss, Robert; Bock, Henry; Pells, Steve; Canetta, Elisabetta; Adya, Ashok K; Moore, Andrew J; De Sousa, Paul; Willoughby, Nicholas A

2011-10-01

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Young's modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Young's modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs.

Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin

Czech Academy of Sciences Publication Activity Database

Kubíková, I.; Konečná, H.; Šedo, O.; Zdráhal, Z.; Řehulka, Pavel; Hříbková, H.; Řehulková, Helena; Hampl, Aleš; Chmelík, Josef; Dvořák, Petr

2009-01-01

Roč. 11, č. 3 (2009), s. 330-340 ISSN 1465-3249 R&D Projects: GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z40310501; CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : human embryonic stem cell * hESC * proteomic profiling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.204, year: 2009

GLUT3 gene expression is critical for embryonic growth, brain development and survival.

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Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U

2014-04-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. Copyright © 2014 Elsevier Inc. All rights reserved.

Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

DEFF Research Database (Denmark)

Sørensen, Kristina Pilekær; Lutterodt, Melissa Catherine R; Mamsen, Linn S

2011-01-01

The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time- and labour-consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier...

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

Science.gov (United States)

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

DEFF Research Database (Denmark)

Wewer, U M; Mercurio, A M; Chung, S Y

1990-01-01

Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... was then isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages.

Science.gov (United States)

Gasimli, Leyla; Hickey, Anne Marie; Yang, Bo; Li, Guoyun; dela Rosa, Mitche; Nairn, Alison V; Kulik, Michael J; Dordick, Jonathan S; Moremen, Kelley W; Dalton, Stephen; Linhardt, Robert J

2014-06-01

Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Differentiation of embryonic stem cells markedly changes the proteoglycanome. The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. Copyright © 2013 Elsevier B.V. All rights reserved.

SMAD7 directly converts human embryonic stem cells to telencephalic fate by a default mechanism

Science.gov (United States)

Ozair, Mohammad Zeeshan; Noggle, Scott; Warmflash, Aryeh; Krzyspiak, Joanna Ela; Brivanlou, Ali H.

2013-01-01

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However, dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here we show that SMAD7, a cell-intrinsic inhibitor of TGFβ signaling, is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time-course gene expression revealed down-regulation of MAPK components, and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. FGF-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues, pluripotent cells simply revert to a program of neural conversion. Hence the “primed” state of hESCs requires inhibition of the “default” state of neural fate acquisition. This has parallels in amphibians, suggesting an evolutionarily conserved mechanism. PMID:23034881

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology.

Science.gov (United States)

Hu, Jianwen; Han, Jizhong; Li, Haoran; Zhang, Xian; Liu, Lan Lan; Chen, Fei; Zeng, Bin

2018-01-01

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications. © 2018 S. Karger AG, Basel.

Higher O-GlcNAc Levels Are Associated with Defects in Progenitor Proliferation and Premature Neuronal Differentiation during in-Vitro Human Embryonic Cortical Neurogenesis

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Shama Parween

2017-12-01

Full Text Available The nutrient responsive O-GlcNAcylation is a dynamic post-translational protein modification found on several nucleocytoplasmic proteins. Previous studies have suggested that hyperglycemia induces the levels of total O-GlcNAcylation inside the cells. Hyperglycemia mediated increase in protein O-GlcNAcylation has been shown to be responsible for various pathologies including insulin resistance and Alzheimer's disease. Since maternal hyperglycemia during pregnancy is associated with adverse neurodevelopmental outcomes in the offspring, it is intriguing to identify the effect of increased protein O-GlcNAcylation on embryonic neurogenesis. Herein using human embryonic stem cells (hESCs as model, we show that increased levels of total O-GlcNAc is associated with decreased neural progenitor proliferation and premature differentiation of cortical neurons, reduced AKT phosphorylation, increased apoptosis and defects in the expression of various regulators of embryonic corticogenesis. As defects in proliferation and differentiation during neurodevelopment are common features of various neurodevelopmental disorders, increased O-GlcNAcylation could be one mechanism responsible for defective neurodevelopmental outcomes in metabolically compromised pregnancies such as diabetes.

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

Science.gov (United States)

Sanz, Carmen; Blázquez, Enrique

2011-09-01

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

Science.gov (United States)

Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Zhang, Wei; Lanza, Robert; Lu, Shi-Jiang; Jonas, Jost B; Xu, Liang

2017-09-01

To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 μl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 μl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. By integrating into

Engineered human broncho-epithelial tissue-like assemblies

Science.gov (United States)

Goodwin, Thomas J. (Inventor)

2012-01-01

Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.

Preliminary Examination of X-ray Scattering from Human Tissues

International Nuclear Information System (INIS)

Desouky, O.S.; Wilkinson, S.; Hall, C.; Rogers, K.; Round, A.

2008-01-01

Small Angle x-ray scattering (SAXS) and wide angle x-ray scattering (WAXS) patterns have been recorded from different human soft tissues using x-ray synchrotron radiation.Pathological breast, normal kidney and lung tissues show SAXS peaks at q-values equal to 0.291 nm -1 and 0.481 nm -1 (d 21.6 nm and d =13. nm) which are the 3 r d and 5 t h order of the well known axial D-spacing of collagen fibrils. The diffraction is particularly intense in the meridional direction indicating some febrile alignment. In contrast, the normal tissue of brain, liver and heart shows diffuse scatter.The wide-angle coherent scattering from normal human tissues of brain, liver, heart, lung, and kidney is typical of that for amorphous materials. The scatter of the healthy adipose breast tissue shows a sharp peak at momentum transfer 1.24 nm -1 (d= 0.417 nm). The data of the other tissues appears to consist of a broad scattering peak. The two scattering regimes succeed in differentiating between the two major components of breast tissue, collagen and adipose tissue. The results of this study suggest that the soft tissues may have scattering patterns that are characteristics for the particular tissue types and tissue disease state. These results indicate that it may be possible use the coherent scattering as a diagnostic tool

Heads and tails of endoderm development and adult tissue homeostasis in zebrafish

NARCIS (Netherlands)

Faro, A.

2010-01-01

The regulatory signaling pathways crucial during embryonic development seem to play key roles in adult tissues homeostasis and are often deregulated in pathological conditions. The Wnt pathway plays a pivotal role in orchestrating cell fate decisions during embryonic development, organogenesis, and

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

Science.gov (United States)

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

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.

Innovative virtual reality measurements for embryonic growth and development

NARCIS (Netherlands)

C.M. Verwoerd-Dikkeboom (Christine); A.H.J. Koning (Anton); W.C.J. Hop (Wim); P.J. van der Spek (Peter); N. Exalto (Niek); R.P.M. Steegers-Theunissen (Régine)

2010-01-01

textabstractBackground Innovative imaging techniques, using up-to-date ultrasonic equipment, necessitate specific biometry. The aim of our study was to test the possibility of detailed human embryonic biometry using a virtual reality (VR) technique. Methods In a longitudinal study, three-dimensional

Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

International Nuclear Information System (INIS)

Sakai, T.; Kisiel, W.

1990-01-01

Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which 125 I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity

Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

Science.gov (United States)

Hubbard, Dallin; Enda, Michael; Bond, Tanner; Moghaddam, Seyyed Pouya Hadipour; Conarton, Josh; Scaife, Courtney; Volckmann, Eric; Ghandehari, Hamidreza

2015-11-02

Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

Advancing biomaterials of human origin for tissue engineering

OpenAIRE

Chen, Fa-Ming; Liu, Xiaohua

2015-01-01

Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking in...

Revocation of European patent for neural progenitors highlights patent challenges for inventions relating to human embryonic stem cells.

Science.gov (United States)

Rigby, Barbara

2013-11-01

Cells derived from human embryonic stem cells have great therapeutic potential. Patents are key to allowing companies that develop methods of generating such cells to recuperate their investment. However, in Europe, inventions relating to the use of human embryos for commercial purposes are excluded from patentability on moral grounds. The scope of this morality exclusion was recently tested before Germany's highest court and before the European Patent Office (EPO), with diverging results. The decision by the EPO's Opposition Division to revoke EP1040185 relating to neural precursors and methods for their generation has received a mixed reception. The decision has very recently been appealed, and the outcome of this Appeal should provide more definitive guidance on the scope of the morality exclusion.

Genetic effects on gene expression across human tissues

NARCIS (Netherlands)

Battle, Alexis; Brown, Christopher D.; Engelhardt, Barbara E.; Montgomery, Stephen B.; Aguet, François; Ardlie, Kristin G.; Cummings, Beryl B.; Gelfand, Ellen T.; Getz, Gad; Hadley, Kane; Handsaker, Robert E.; Huang, Katherine H.; Kashin, Seva; Karczewski, Konrad J.; Lek, Monkol; Li, Xiao; MacArthur, Daniel G.; Nedzel, Jared L.; Nguyen, Duyen T.; Noble, Michael S.; Segrè, Ayellet V.; Trowbridge, Casandra A.; Tukiainen, Taru; Abell, Nathan S.; Balliu, Brunilda; Barshir, Ruth; Basha, Omer; Bogu, Gireesh K.; Brown, Andrew; Castel, Stephane E.; Chen, Lin S.; Chiang, Colby; Conrad, Donald F.; Cox, Nancy J.; Damani, Farhan N.; Davis, Joe R.; Delaneau, Olivier; Dermitzakis, Emmanouil T.; Eskin, Eleazar; Ferreira, Pedro G.; Frésard, Laure; Gamazon, Eric R.; Garrido-Martín, Diego; Gewirtz, Ariel D. H.; Gliner, Genna; Gloudemans, Michael J.; Guigo, Roderic; Hall, Ira M.; Han, Buhm; He, Yuan

2017-01-01

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression

Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

Science.gov (United States)

Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

2016-08-01

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

Astrocyte calcium signal and gliotransmission in human brain tissue.

Science.gov (United States)

Navarrete, Marta; Perea, Gertrudis; Maglio, Laura; Pastor, Jesús; García de Sola, Rafael; Araque, Alfonso

2013-05-01

Brain function is recognized to rely on neuronal activity and signaling processes between neurons, whereas astrocytes are generally considered to play supportive roles for proper neuronal function. However, accumulating evidence indicates that astrocytes sense and control neuronal and synaptic activity, indicating that neuron and astrocytes reciprocally communicate. While this evidence has been obtained in experimental animal models, whether this bidirectional signaling between astrocytes and neurons occurs in human brain remains unknown. We have investigated the existence of astrocyte-neuron communication in human brain tissue, using electrophysiological and Ca(2+) imaging techniques in slices of the cortex and hippocampus obtained from biopsies from epileptic patients. Cortical and hippocampal human astrocytes displayed spontaneous Ca(2+) elevations that were independent of neuronal activity. Local application of transmitter receptor agonists or nerve electrical stimulation transiently elevated Ca(2+) in astrocytes, indicating that human astrocytes detect synaptic activity and respond to synaptically released neurotransmitters, suggesting the existence of neuron-to-astrocyte communication in human brain tissue. Electrophysiological recordings in neurons revealed the presence of slow inward currents (SICs) mediated by NMDA receptor activation. The frequency of SICs increased after local application of ATP that elevated astrocyte Ca(2+). Therefore, human astrocytes are able to release the gliotransmitter glutamate, which affect neuronal excitability through activation of NMDA receptors in neurons. These results reveal the existence of reciprocal signaling between neurons and astrocytes in human brain tissue, indicating that astrocytes are relevant in human neurophysiology and are involved in human brain function.

The case for applying tissue engineering methodologies to instruct human organoid morphogenesis.