Cryopreservation of gametes and embryos is extremely important for maintenance of genetic variability in small wild populations, however these materials are difficult to obtain. Studies with human and mouse induced pluripotent stem cells (iPS) have showed that cells are capable to differentiate into germ cells, sperm and oocytes. Studies with iPS in cervids are novel, so we have as focus on this project to establish protocols for obtaining stable iPS lines in Blastocerus dichotomus species to produce viable gametes in the future. Reprogramming of somatic cells into iPS cells is facilitated by the use of adult stem cells and, because little is known about the multipotent stem cells in cervids, the first phase of the project aims to obtain biopsies from various tissues (adipose tissue, dental pulp, antler button), that will be compared with unipotentes cell lines derived from skin biopsies (negative control). The viability of the cells will be assessed with trypan blue, and markers of mesenchymal cells will be assessed by RT-qPCR. Subsequently, these tissues will be cryopreserved and your viability after thawing will be tested. The second phase will focus in the derivation of iPS cells from multipotent lines of the preceding phase, which is conducted by means of gene transduction using the PiggyBac tool. The cultures for derivation of iPS will be made in the presence of mouse embryonic fibroblast (MEF) blocked by mitomycin, and colonies selected will be subcultured to maintain lines for further characterization by immunocytochemistry, RT-qPCR, alkaline phosphatase activity, lacZ dye, karyotype, ability to form embryoid bodies, cells differentiate into germline of females, evaluation of markers of tissues derived from the three germ layers, and in vitro differentiation in immunodeficient mice to assess the formation of the three germ layers.
News published in Agência FAPESP Newsletter about the scholarship: