Establishment Of Pig Fibroblasts And Neural Stem Cell Lines And The Study On Biological Characteristics For I Psc Induced From NSCs

Background: Stem cell research is an important part of regenerative medicine, and the ultimate goal of stem cell research is the application of stem cells to treat disease.i PSC are pluripotent cells induced by the somatic cells, which have the similar function to the embryonic stem cells(ESCs). So, they will not only solve the ethical barriers of the ESCs, but also provide a new approach for obtaining ESCs, and has important value in theoretical and practical. Neural stem cell transplantation for the treatment of Parkinson’s disease is widely recognized as the most promising treatment option. The neural stem cells which were transgenic cultured under the hypoxic condition can be induced to differentiate into dopaminergic neurons efficiently, so as to provide cell source transplanted for the treatment of Parkinson’s disease.Objectives:(1) To establish the fibroblasts and neural stem cell lines of pigs used a primary explants technique and cryogenic preservation technology, and proceeded to Biology and Genetics detection according to ATCC quality control procedures.(2) To establish experimental technology system for the first time to induce pig i PS cell lines using pig’s own reprogramming factors.Methods: First, we used the 22 newly born pigs as experimental animals in this experiment to establish the fibroblasts and neural stem cell lines. Then, we reconstructed the lentivirus-mediated four plasmid system of Si Dansai Biotechnology Ltd. in Shanghai using pig’s own reprogramming factors. Four reprogramming factors genes were transfected into 293 FT cells by lipofectamine-mediated method and harvested the viruses. After purification and concentration, the viruses were utilized to introduce four genes Oct4, Sox2, Klf4 and c-Myc into fibroblasts and neural stem cell to induce to IPS cells in vitro.Results:(1) The establishment of fibroblasts cell lines: we sucessfully created the fibroblasts cell lines, and examined its genetic characteristics and biological quality. In order to ensure the stability of their genetics, especially hereditary characteristics, all cells used to preserve a species should undergo a minimum number of passages(<4) in vitro. By the morphology, bacteria, fungi, mycoplasma, growth curve, karyotype analysis and many other testing of cell line quality, the results show that the established porcine fibroblasts are stable. Six fluorescent protein genes were expressed in the fibroblast cell lines favourably, it showed that cells were transfected efficiently. Every index of the fibroblast cell line meets all the standard quality controls of American type Culture Collection, which will serve as a valuable resource for genome, postgenome and somacloning of pig.(2) The establishment of neural stem cell lines: we successfully established the NSC cell lines sampled from a new pig’s cerebral cortex. We used a primary explants technique, in vitro culture techniques, and cryogenic preservation technology to establish the NSC cell line and proceeded to Biology and Genetics detection. Pig NSC showed the typical neurosphere, and expressed Nestin efficently which is the specific gene of the neural stem cells, comply with essential characteristic of NSC described in a large number of the academic paper. After the induction of 7d, cells have the ability to differentiate into neurons and glial cells. In this research, we explored the technique and method to separate and culture NSC of pig in vitro, biological characteristics of the cells and factors of affecting the separation and proliferation. All research results described above laid the foundation for the application of neural stem cells in the medical field.(3) Induction of the i PS cell lines: Using a lentivirus-based vector method to introduce four reprogramming factor genes(Oct4, Sox2, Klf4 and c-Myc) into293 FT cells, virus packaging and recombinant virus titers. Then, the recombinant viruses were used to infect pig neural stem cells in vitro to induce the generation of i PS cell lines. Moreover, the surface antigen and marker gene of the i PS cells were detected, respectively.Conclusions:(1) We used a primary explants technique and cryogenic preservation technology to establish a pig fibroblast cell line and proceeded to genetic characteristics and biological characteristics detection according to ATCC quality control procedures, which will provide effective experimental material for inducing pig i PS cells and further genetic studies on the pig as well.(2) Isolated cerebral cortex tissue from newly born pigs, and made the tissue into single-cell suspension to culture cells with mechanical and enzymatic digestion method together. Furthermore, we cultured identified neural spheres and proved that they had the biological characteristics of neural stem cells. After recovering, cryopreserved neural stem cells show the same characteristic of NSC.(3) We established a lentivirus-mediated infection system in vitro for pig fibroblasts and neural stem cells. Reconstruction of viral vectors with Oct4, Sox2,Klf4 and c-Myc genes are efficiently tranfected and expressed. The recombinant viruses infected sucessfully pig neural stem cells in vitro and obtained N-i PS cell lines.Meanwhile, this research will made foundation for pig i PS cell transplantation to treat disease in experimental animal model.