Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in... Show moreTechnological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to __screening on a run-away train.__ Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research. Show less
Human embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on... Show moreHuman embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on comprehensive knowledge of their biology and control of their signaling mechanisms and fate choices. To begin to address this, we developed a standardized feeder-free hESC culture protocol. This system is optimized and tested for 12 independently derived stem cell lines, and optimal for clonal growth and efficient gene transfer without loss of pluripotency (Chapter 2,3). Using these protocols we created stem cells ubiquitously expressing EGFP, showed efficient SOX2 knockdown and created a fluorescent reporter stem cell line for the stem cell regulator OCT4. Next we used mass spectroscopy to investigate the plasma membrane of hESC (Chapter 4). We were able to show that these cells express a uniform epithelial plasma membrane profile and that VIMENTIN, normally associated with mesenchymal cells is also expressed. This expression turned out to be related to stress and associated with hardness of the tissue culture plastic substrate rather than differentiation. We continued to investigate the plasma membrane of hESC and decided to focus on integrins, the cell surface receptors that bind extracellular matrix proteins. Functional analyses of their function showed human embryonic stem cells have the capacity to bind to a wide range of extracellular matrix proteins via specific integrin receptors. We were able to show that recombinant vitronectin robustly supports the maintenance of hESC in an undifferentiated state in completely defined culture medium. Having validated a completely defined culture protocol we began to investigate differentiation mechanisms under defined conditions (Chapter 5). We used Stable Isotope Labeling in Cell Culture (SILAC) and quantitative phopspho-proteomics to investigate how human embryonic stem cells exit the pluripotent state. BMP4 was used to trigger differentiation and protein samples were analyzed at 30 min, 60 min and 240 min. We showed that approximately 50% of the 3067 identified phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships we reconstructed the hESC kinome, with CDK1/2 emerging as a key kinase in controlling self-renewal and lineage specification. Next we used gene targeting to create a fluorescent cardiac reporter cell line. EGFP was targeted into one allele of the NKX2-5 gene. EGFP fluorescence driven by the endogenous Nkx2-5 promoter faithfully reported cardiovascular lineage commitment of differentiating hESC under defined culture conditions. Using fluorescence activated cell sorting we showed that the early NKX2-5 positive cell population contained multipotent progenitor cells capable of directed differentiation to cardiomyocytes, endothelial and vascular smooth muscle cells (Chapter 7). Finally, we used the cardiomcyocytes from hESC to develop a system for cardiac safety pharmacology (Chapter 8). Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. In particular, blocking of the human Ether-a-go go Related Gene ion channel is associated with life-threatening arrhythmias, such as Torsade de Pointes. We demonstrated that, as predicted, patient serum levels of drugs and known responses on QT interval overlapped with field potential duration values derived from hESC-CM,. On this basis, we propose field potential duration prolongation as a directly applicable safety criterion for pre-clinical evaluation of new drugs in development. In conclusion, the availability of human cardiomyocytes from stem cell sources is now expected to accelerate cardiac drug discovery and safety pharmacology by offering more clinically relevant human culture models than presently available (Chapter 9,10). Show less
