ziPS cells for Biomedicine: zebrafish induced pluripotent stem cells for disease modeling

Zebrafish is an attractive organism for disease modeling because it is easy and cheap to grow and maintain. Stem cells are a useful cell type for disease modeling. At the time the project started, no stem cells from zebrafish were available. We have generated cells from zebrafish embryos at very early stages of development. We have designed methods for isolation and culture of cells from embryos at various developmental stages and have generated many cell cultures. We have tested factors that promote expansion of these cells in culture, as well as the maintenance of gene expression profiles that resemble those of embryos the cells came from originally. Gene expression was measured by RNA-sequencing (at the whole genome level) and for specific genes. We have also mapped at the whole genome level changes in several biochemical modifications of DNA and associated proteins (called histones, which together with DNA make up the chromosomes) during early zebrafish development. The results show that much of the so-called epigenetic patterning of developmental gene expression in embryos is set up before the embryo turns on its own gene expression program. This finding is important because it shows an epigenetic pre-programming of gene expression and development by chemical ?marks? of DNA and chromosomes. This work has been highly profiled worldwide through high-level publications and in many seminars. We further show how exposure of embryos or cells to drugs affects epigenetic marks, gene expression, development and health of the cells. In the course of this work, we have also designed new bioinformatics (computing) methods to more precisely analyze gene expression patterns, epigenetic modifications of DNA and chromosomes, and the 3-dimensional structure (in space) of chromosomes in cells or embryos. These tools are versatile and can be used for similar analyses in other animal species. In addition, injection of embryo-derived cells into host zebrafish embryos shows that the cells integrate into various tissues during development but unfortunately cannot be detected in the germline (that is, as eggs or sperm). This means that cells derived from zebrafish embryos in this study show some level of pluripotency (competence to develop into several cell types), but are not able to give rise to gametes or to whole fish. Still, the embryo-derived cells, which we term ?stem-like cells?, can be cultured and used in the testing of drugs for biomedical applications.

Direct reprogramming of fibroblasts to a pluripotency through generation of induced pluripotent stem cells (iPS cells) has sparked studies on how to create patient-specific pluripotent cells to study disease and develop drug screens. However, despite the pre-eminence of mouse in modeling human disease, several aspects of murine and human biology limit its use in high-throughput and in vivo genetic and therapeutic screening. Zebrafish has become an attractive organism for embryologically and genetically tr actable disease modeling, but cell types suitable for such investigations have been lacking. The goal of this proposal (ziPS CELL) is to produce pluripotent zebrafish ziPS cells for the study of disease and drug screening in vivo. Advantages of using ziPS cells over human cells in biomedicine are enablement of high-throughput and relatively cheap disease study and drug screens at several stages of development and in several generations. The approach will consist of 1) inducing pluripotency in embryo-deriv ed and somatic cells by expressing pluripotency-associated factors which we already have identified by RNA deep-sequencing, 2) enhancing pluripotency of the cells with specific inhibitors, 3) generating chimeric fish from ziPS cells and 4) providing proof -of-concept of using ziPS cells in a biomedical context. Scientific competence lays in the combined expertise of the PIs and collaborators in all aspects of the project. The project is led by the two PIs and will be undertaken at the Norwegian School of V eterinary Sciences and at the University of Oslo Medical School. Stays abroad (GIS and NUS, Singapore) are planned but not finalized. This is a 3-year project for two motivated postdocs (tbn) already versed in pluripotency and/or zebrafish embryology.