Effect of chemopreventive agents on differentiation of mouse embryonic stem cells.

Chemopreventive agents are derived from edible plants and from ancient time is a part of daily intake for many humans and animals. There are several lines of compelling evidence from epidemiological, clinical and laboratory studies that these dietary constituents are associated in reducing cancer risks. However, developmental toxicity of these natural compounds cannot be excluded.

Specific gene signatures and pathways in mesodermal cells and their derivatives derived from embryonic stem cells.

The vertebrate early stage embryo is consisting of the three primary germ layers ectoderm, mesoderm and endoderm, from which all organ tissues are developed. During early embryonic development, mesodermal cells become sequentially determined to more precisely defined cell types including muscle, heart, vasculature, blood, kidney, gonads, dermis and cartilage. How the prospective mesodermal cells integrate the various signals they receive and how they resolve this information to regulate their morphogenetic behavior and cell fate decisions is largely unknown.

Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing.

Human embryonic stem cells (hESCs) can be propagated indefinitely in vitro in an undifferentiated pluripotent state, can differentiate into derivatives of all three germ layers and are of considerable interest for applications in regenerative medicine. Clinical application of hESCs, however, requires reliable protocols for cryopreservation. Current protocols for cryopreservation of hESCs suffer from low recovery rates of hESCs and loss of pluripotency after thawing.

Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.

Background And Purpose: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals.

Isolation and functional characterization of alpha-smooth muscle actin expressing cardiomyocytes from embryonic stem cells.

Early mammalian heart development is characterized by transient expression of alpha-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter.

Initial colony morphology-based selection for iPS cells derived from adult fibroblasts is substantially improved by temporary UTF1-based selection.

Background: Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells. Recently, selection of fully reprogrammed cells was achieved based on colony morphology reminiscent of embryonic stem (ES) cells. The maintenance of pluripotency was analysed.

Global transcriptomic analysis of murine embryonic stem cell-derived brachyury(+) (T) cells.

Brachyury(+) mesodermal cell population with purity over 79% was obtained from differentiating brachyury embryonic stem cells (ESC) generated with brachyury promoter driven enhanced green fluorescent protein and puromycin-N-acetyltransferase. A comprehensive transcriptomic analysis of brachyury(+) cells enriched with puromycin application from 6-day-old embryoid bodies (EBs), 6-day-old control EBs and undifferentiated ESCs led to identification of 1573 uniquely up-regulated and 1549 uniquely down-regulated transcripts in brachyury(+) cells. Furthermore, transcripts up-regulated in brachyury(+) cells have overrepresented the Gene Ontology annotations (cell differentiation, blood vessel morphogenesis, striated muscle development, placenta development and cell motility) and Kyoto Encyclopedia of Genes and Genomes pathway annotations (mitogen-activated protein kinase signaling and transforming growth factor beta signaling).

Induced pluripotent stem cells as a model for accelerated patient- and disease-specific drug discovery.

Human induced pluripotent stem (iPS) cells hold great promise for therapy of a number of degenerative diseases such as ischemic heart failure, Parkinson's disease, Alzheimer's disease, diabetes mellitus, sickle cell anemia and Huntington disease. They also have the potential to accelerate drug discovery in 3 ways. The first involves the delineation of chemical components for efficient reprogramming of patient's blood cells or cells from biopsies, obviating the need for cellular delivery of reprogramming exogenous transgenes, thereby converting hope into reality for patients suffering from degenerative diseases.

The potential of embryonic stem cells combined with -omics technologies as model systems for toxicology.

The derivation of pluripotent embryonic stem (ES) cell lines has opened up new areas of research in basic and applied science, most significantly in developmental biology and regenerative medicine. While application-oriented research has for the most part focussed on obtaining differentiated, organotypic cells from ES cells for future cell grafting therapies, ES cells have more immediate potential for use in toxicological in vitro assays used during drug development. ES cells are derived from blastocyst-stage embryos and offer an in vitro model for early development, thus enabling tests for teratogenicity testing in a human cell culture system and avoiding the pitfalls of inter-species differences.

Somitovasculin, a novel endothelial-specific transcript involved in the vasculature development.

Objective: We recently isolated and characterized endothelial-like CD31(+) cells derived from mouse embryonic stem (mES) cells and identified their transcriptome. The main objective of this study was to determine the functional relevance of the transcripts of unknown function (TUF) for vasculature development.

Methods And Results: We selected 2 TUFs of more than 27 to study their role for blood vessel development in zebrafish.

Chemoprotective mechanism of the natural compounds, epigallocatechin-3-O-gallate, quercetin and curcumin against cancer and cardiovascular diseases.

Cancer and cardiovascular disease (CVD) chemoprevention can be achieved by the use of natural, synthetic, or biologic compounds to reverse, suppress, or prevent the development of diseases. Chemoprevention is a potential anti-cancer approach, which has reduced secondary effects in comparison to classical prophylaxis. Natural compounds such as flavonoids reduce oxidative stress, which is the most likely mechanism in the protective effects of these compounds.

Micro-arrayed wheat seed and grass pollen allergens for component-resolved diagnosis.

Background: Wheat is a potent allergen source and can cause baker's asthma, food and pollen allergy. The aim of the study was to develop an allergen micro-array for differential diagnosis of baker's asthma, wheat-induced food allergy and grass pollen allergy.

Methods: We analysed the immunoglobulin-E reactivity profiles of patients suffering from baker's asthma, wheat-induced food allergy and grass pollen allergy to micro-arrayed recombinant wheat flour allergens and grass pollen allergens and compared these results with clinical results and diagnostic tests based on crude wheat flour, wheat pollen and grass pollen allergen extracts.

Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells.

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells.

Human ES cell derived cardiomyocytes for cell replacement therapy: a current update.

Cardiovascular diseases are the leading cause of death globally. The pluripotency and indefinite proliferative capacity of embryonic stem (ES) cells make them a promising candidate for the cell replacement therapy where the damaged cells are replaced by the functional cells derived from stem cells in vitro. Emerging results with human ES cells for the myocardial repair are encouraging, but this approach is still in its infancy and is under extensive investigation.

Entrapment of embryonic stem cells-derived cardiomyocytes in macroporous biodegradable microspheres: preparation and characterization.

Embryonic Stem (ES) cells-derived cardiomyocytes can possibly be applied for cell therapy of diseases such as heart failure. Biodegradable scaffolds will significantly improve the expansion of sufficient functional ES cell-derived cardiomyocytes and may also increase the survival rate of cardiomyocytes after their transplantation. In the present study, we cultivated cardiomyocytes isolated from a transgenic a-myosin heavy chain (alpha-MHC) ES cell lineage expressing both puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter in macroporous gelatine microspheres using small-scale bioreactors and proved that cardiomyocytes function after their cultivation in micropsperes.

Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated.

Scalable selection of hepatocyte- and hepatocyte precursor-like cells from culture of differentiating transgenically modified murine embryonic stem cells.

Potential therapeutic applications of embryonic stem cell (ESC)-derived hepatocytes are limited by their relatively low output in differentiating ESC cultures, as well as by the danger of contamination with tumorigenic undifferentiated ESCs. To address these problems, we developed transgenic murine ESC clones possessing bicistronic expression vector that contains the alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live" fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under established culture conditions these clones allowed for both monitoring of differentiation and for puromycin selection of hepatocyte-committed cells in a suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]).

Tolerization of a type I allergic immune response through transplantation of genetically modified hematopoietic stem cells.

Allergy represents a hypersensitivity disease that affects >25% of the population in industrialized countries. The underlying type I allergic immune reaction occurs in predisposed atopic individuals in response to otherwise harmless Ags (i.e.

Intracellular and surface RANKL are differentially regulated in patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is characterized by ankylosis of axial joints but osteoporosis is also a well-reported feature. T cells have been implicated as a source of receptor activator of NFkappaB ligand (RANKL) in inflammatory bone diseases. Hence, we assessed whether T cells in patients with AS act as a source of RANKL too.

Bone structure and metabolism in a rodent model of male senile osteoporosis.

Osteoporosis is a common and severe condition in elderly men, which is poorly characterized. In order to identify the hallmarks of age-related bone loss in the male mammalian skeleton, we studied several aspects of bone structure and metabolism in 23-month-old male Sprague-Dawley rats and compared them to 5-month-old animals. Cancellous bone mineral density, bone volume and trabecular number were markedly reduced in the proximal tibia of aged rats when compared to the young rats.

Identification of the scavenger receptors SREC-I, Cla-1 (SR-BI), and SR-AI as cellular receptors for Tamm-Horsfall protein.

Tamm-Horsfall protein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in urine. An important role for THP in antibacterial host defense but also in inflammatory disorders of the urogenital tract has been suggested. In line with this, THP has been shown recently to potently activate macrophages and dendritic cells (DC) via the toll-like receptor 4 (TLR4) pathway.

Prevalence of IgE-binding to Art v 1, Art v 4 and Amb a 1 in mugwort-allergic patients.

Background: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract.

Methods: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA.

Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor.

The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels.