Human Embryonic and Induced Pluripotent Stem Cell Based Toxicity Testing Models: Future Applications in New Drug Discovery.

New drug discovery (NDD) is a fascinating discipline encompassing different facets of medicine, pharmacology, biotechnology and chemistry. NDD is very often restricted by efficacy or safety problems of the new clinical candidate in human patients. Drug regulatory authorities have provided various guidelines for advancement of safe new chemical entities (NCEs) in clinical trials which must be strictly followed. In spite of this, various drugs have failed in clinical trials or withdrawn from market because of human safety issues related to cardiotoxicity, hepatotoxicity, neurotoxicity and teratogenicity. The failure of safety prediction was pointed to species specificity issues, lack of mechanistic toxicity data and inadequate clinical trials. These drugs not only affect human health but also cause loss of resources and time. The species specificity issues are partially addressed by use of primary human cells but their availability is very limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) offer sources for generation of an unlimited number of human somatic cells. The emergence of mechanistic models for toxicity testing with transcriptomics, proteomics along with toxicokinetics readouts based on hESCs and hiPSCs is paving the way to design new human relevant testing strategies. Introduction of these models at the timeframe of lead selection and optimization in parallel with in vitro pharmacokinetic studies will significantly reduce compound attrition rate by selection of safer lead molecules. We focused on upcoming hESCs and hiPSCs based toxicity testing models and their future role to address safety gaps of present drug discovery and development.

The Potential Application of Biomaterials in Cardiac Stem Cell Therapy.

Biomaterials play a vital role in the field of regenerative medicine and tissue engineering. To date, a large number of biomaterials have been used in cardiovascular research and application. Recently, biomaterials have held a lot of promise in cardiac stem cell therapy. They are used in cardiac tissue engineering to form scaffolds for cellular transplantation, promote angiogenesis, enhance transplanted cell engraftment or influence cell migration. The science of biomaterial designing has evolved to an extent where they can be designed to mimic the microenvironment of a cardiac tissue in vivo and contribute in deciding the fate of transplanted stem cells and induce cardiac lineage oriented stem cell differentiation. In this review, we focus on biomaterials used in cardiovascular stem cell research, tissue engineering and regenerative medicine and conclude with an outlook on future impacts of biomaterial in medical sciences.

Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters.

Transcriptomics of Hepatocytes Treated with Toxicants for Investigating Molecular Mechanisms Underlying Hepatotoxicity.

Transcriptomics is a powerful tool for high-throughput gene expression profiling. Transcriptome microarray experiments conducted with RNA isolated from hepatocytes after exposure to toxicants enable a deep insight into the molecular mechanisms of hepatotoxicity. This understanding, along with structure-activity relationships underlying hepatotoxicity, will provide a novel strategy to design cost-effective and safer therapeutics. Transcriptomics studies conducted with established hepatotoxic drugs in various in vitro and in vivo hepatotoxicity test systems have contributed to the elucidation of the mechanistic basis of liver insults, which were later on substantiated at the proteomics and metabolomics levels. The present chapter is focused on comprehensive transcriptomics of cultured primary hepatocytes treated with chemicals by applying Affymetrix microarray technology. It also describes the detailed protocol for culturing of hepatocytes, their exposure to toxicants as well as sample collection, including RNA isolation, RNA target preparation and finally the hybridization to gene chips for microarray expression analysis.

Signaling molecules, transcription growth factors and other regulators revealed from in-vivo and in-vitro models for the regulation of cardiac development.

Several in-vivo heart developmental models have been applied to decipher the cardiac developmental patterning encompassing early, dorsal, cardiac and visceral mesoderm as well as various transcription factors such as Gata, Hand, Tin, Dpp, Pnr. The expression of cardiac specific transcription factors, such as Gata4, Tbx5, Tbx20, Tbx2, Tbx3, Mef2c, Hey1 and Hand1 are of fundamental significance for the in-vivo cardiac development. Not only the transcription factors, but also the signaling molecules involved in cardiac development were conserved among various species. Enrichment of the bone morphogenic proteins (BMPs) in the anterior lateral plate mesoderm is essential for the initiation of myocardial differentiation and the cardiac developmental process. Moreover, the expression of a number of cardiac transcription factors and structural genes initiate cardiac differentiation in the medial mesoderm. Other signaling molecules such as TGF-beta, IGF-1/2 and the fibroblast growth factor (FGF) play a significant role in cardiac repair/regeneration, ventricular heart development and specification of early cardiac mesoderm, respectively. The role of the Wnt signaling in cardiac development is still controversial discussed, as in-vitro results differ dramatically in relation to the animal models. Embryonic stem cells (ESC) were utilized as an important in-vitro model for the elucidation of the cardiac developmental processes since they can be easily manipulated by numerous signaling molecules, growth factors, small molecules and genetic manipulation. Finally, in the present review the dynamic role of the long noncoding RNA and miRNAs in the regulation of cardiac development are summarized and discussed.

diXa: a data infrastructure for chemical safety assessment.

MOTIVATION: The field of toxicogenomics (the application of '-omics' technologies to risk assessment of compound toxicities) has expanded in the last decade, partly driven by new legislation, aimed at reducing animal testing in chemical risk assessment but mainly as a result of a paradigm change in toxicology towards the use and integration of genome wide data. Many research groups worldwide have generated large amounts of such toxicogenomics data. However, there is no centralized repository for archiving and making these data and associated tools for their analysis easily available. RESULTS: The Data Infrastructure for Chemical Safety Assessment (diXa) is a robust and sustainable infrastructure storing toxicogenomics data. A central data warehouse is connected to a portal with links to chemical information and molecular and phenotype data. diXa is publicly available through a user-friendly web interface. New data can be readily deposited into diXa using guidelines and templates available online. Analysis descriptions and tools for interrogating the data are available via the diXa portal. AVAILABILITY AND IMPLEMENTATION: http://www.dixa-fp7.eu CONTACT: d.hendrickx@maastrichtuniversity.nl; info@dixa-fp7.eu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Detection of Cryptosporidium and Giardia in agricultural and water environments in the Qinghai area of China by IFT and PCR.

Qinghai Province in northwest China is strongly influenced by agricultural activities and is an important source of food and drinking water. Here, we present findings regarding the occurrence and molecular epidemiology of Cryptosporidium and Giardia species based on a large-scale investigation of areas of Qinghai Province. The diagnosis and molecular detection of Cryptosporidium oocysts and Giardia cysts was carried out using immunofluorescence microscopy (IFT), whereas nested polymerase chain reaction (PCR) in fecal smears and water samples was used for the detection and molecular characterization of the species. In total, 561 samples (260 water samples and 301 fecal samples from animals) were collected and analyzed. Of the 260 water samples, 66 samples were Cryptosporidium-positive by IFT and 71 samples were positive by nested PCR; in addition, 39 samples were Giardia-positive by IFT and 40 samples were positive by nested PCR. Of the 301 fecal samples from animals, 98 samples were Cryptosporidium-positive by IFT and 61 samples were positive by nested PCR, whereas 52 samples were Giardia-positive by IFT and 31 samples were positive by nested PCR. We showed that the water supplies and animals investigated contained Cryptosporidium and Giardia (oo)cysts. Thus, we recommend that the Chinese Government and Chinese health authorities undertake control measures to protect the food and drinking water sources in Qinghai from these pathogenic protozoa.

Molecular identification of Giardia and Cryptosporidium from dogs and cats.

The aim of the present study was to diagnose the presence of Giardia cysts and Cryptosporidium oocysts in household animals using nested polymerase chain reaction (PCR) and sequence analysis. One hundred faecal samples obtained from 81 dogs and 19 cats were investigated. The Cryptosporidium genotypes were determined by sequencing a fragment of the small subunit (SSU) rRNA gene, while the Giardia Assemblages were determined through analysis of the glutamate dehydrogenase (GDH) locus. Isolates from five dogs and two cats were positive by PCR for the presence of Giardia, and their sequences matched the zoonotic Assemblage A of Giardia. Cryptosporidium spp. isolated from one dog and one cat were both found to be C. parvum. One dog isolate harboured a mixed infection of C. parvum and Giardia Assemblage A. These findings support the growing evidence that household animals are potential reservoirs of the zoonotic pathogens Giardia spp. and Cryptosporidium spp. for infections in humans.

Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP).

Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

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. The average number of cultivated cells per microsphere was optimised once the most suitable agitation conditions and the optimal timeframe of cultivation were identified. Our study shows that 72% of CultiSpher-S beads were colonised by cardiomyocytes under optimal conditions. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) showed that colonization of the beads was not limited to the surface, but that cells also invaded the inner surfaces of the microspheres. Electrophysiological experiments demonstrated that the action potentials (APs) of alpha-MHC(+) cardiomyocytes entrapped in microspheres were identical to action potentials of control cells. This attractive approach for cultivation and expansion of functional cardiomyocytes in biodegradable macroporous may offer a perspective for higher transplantation efficiencies of ES cell-derived cardiomyocytes.

Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT).

The development and evaluation of a 1-step single-tube accelerated loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Toxoplasma in water samples is described. The method has been evaluated based on the amplification of B1 and TgOWP Toxoplasma genes, and it demonstrated a sensitivity detection limit of 0.1 tachyzoites' DNA for both genes. LAMP detection was evaluated and compared with nested polymerase chain reaction (PCR) in 26 water sample pellets spiked with known numbers of Toxoplasma oocysts. After DNA extraction, the detection sensitivity in spiked pellets was 100% by LAMP and 53.8% by PCR. Subsequently, 52 natural water samples of different origin were directly investigated by 3 assays: LAMP, PCR, and immunofluorescence test (IFT). Twenty-five (48%) of 52 have been found positive for Toxoplasma DNA by LAMP, whereas nested PCR products were generated in 7 of 52 (13.5%) water samples. All 52 water samples were negative for Toxoplasma by IFT. These data clearly indicate LAMP as a rapid, specific, and sensitive tool for the detection of Toxoplasma contamination in water samples.

A chemical genetics approach for specific differentiation of stem cells to somatic cells: a new promising therapeutical approach.

Cell replacement therapy of severe degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease through transplantation of somatic cells generated from embryonic stem (ES) cells is currently receiving considerable attention for the therapeutic applications. ES cells harvested from the inner cell mass (ICM) of the early embryo, can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells thereby providing an unlimited renewable source of somatic cells. In this context, identifying soluble factors, in particular chemically synthesized small molecules, and signal cascades involved in specific differentiation processes toward a defined tissue specific cell type are crucial for optimizing the generation of somatic cells in vitro for therapeutic approaches. However, experimental models are required allowing rapid and "easy-to-handle" parallel screening of chemical libraries to achieve this goal. Recently, the forward chemical genetic screening strategy has been postulated to screen small molecules in cellular systems for a specific desired phenotypic effect. The current review is focused on the progress of ES cell research in the context of the chemical genetics to identify small molecules promoting specific differentiation of ES cells to desired cell phenotype. Chemical genetics in the context of the cell ES-based cell replacement therapy remains a challenge for the near future for several scientific fields including chemistry, molecular biology, medicinal physics and robotic technologies.

Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning.

BACKGROUND: Bone morphogenetic protein (BMP)2 is a late mesodermal marker expressed during vertebrate development and plays a crucial role in early embryonic development. The nature of the BMP2-expressing cells during the early stages of embryonic development, their transcriptome and cell phenotypes developed from these cells have not yet been characterized. RESULTS: We generated a transgenic BMP2 embryonic stem (ES) cell lineage expressing both puromycin acetyltransferase and enhanced green fluorescent protein (EGFP) driven by the BMP2 promoter. Puromycin resistant and EGFP positive BMP2+ cells with a purity of over 93% were isolated. Complete transcriptome analysis of BMP2+ cells in comparison to the undifferentiated ES cells and the control population from seven-day-old embryoid bodies (EBs; intersection of genes differentially expressed between undifferentiated ES cells and BMP2+ EBs as well as differentially expressed between seven-day-old control EBs and BMP2+ EBs by t-test, p < 0.01, fold change >2) by microarray analysis led to identification of 479 specifically upregulated and 193 downregulated transcripts. Transcription factors, apoptosis promoting factors and other signaling molecules involved in early embryonic development are mainly upregulated in BMP2+ cells. Long-term differentiation of the BMP2+ cells resulted in neural crest stem cells (NCSCs), smooth muscle cells, epithelial-like cells, neuronal-like cells, osteoblasts and monocytes. Interestingly, development of cardiomyocytes from the BMP2+ cells requires secondary EB formation. CONCLUSION: This is the first study to identify the complete transcriptome of BMP2+ cells and cell phenotypes from a mesodermal origin, thus offering an insight into the role of BMP2+ cells during embryonic developmental processes in vivo.

An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples.

The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 x 10(4) was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied. The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes.

BACKGROUND: Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes. RESULTS: A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway. CONCLUSION: The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.