| BACKGROUNDSensorineural hearing loss (SNHL) is a kind of hearing impaired mainly in consequence of degeneration of hair cells (HCs) and spiral ganglion neurons (SGNs). However there’s always no chance for regeneration for postnatal mammals, under severe trauma such as ototoxic drugs, infections, and noises, inner ear cells probably cannot survive and may become degeneration, necrosis or apoptosis, which will result in permanent hearing loss. As an option for inner ear disease, stem cell transplantation into cochlea is expected to replace damaged HCs and SGNs, and reconstruct sense of hearing. Induced pluripotent stem cells (iPSCs) is a reprogramming technique of inducing somatic cells into pluripotent cells of which characteristics are similar to that of embryonic stem cells’(ESCs), and it offer an ideal resource for regenerative medicine which has been proved excellent results in many areas. Nevertheless, cell therapy in the inner ear was stuck in two main problems, which were the harmful micro-environment and the low survival rate of cell transplantation. Whether there is a way that combines cell therapy and gene therapy to solve the two issues simultaneously? Base on situation above, we carried out the following research.OBJECTIVETo set up urine-derived induced pluripotent stem cells as candidates for treatment of inner ear disorders. To set up anti-apoptosis Bcl-2 overexpression iPSCs for paving the way of exploring the possibilities combined cell therapy and gene therapy in the area of inner ear disease.METHODSFirstly, iPSCs were obtained via four classic key factors (Oct4, Sox2, c-myc and Klf4) induction from normal urine, and verified the pluripotent characteristics. Secondly, iPSCs were infectious by letivirus with anti-apoptosis Bcl-2, and verified if Bcl-2 was overexpression in and whether the gene manipulation would influence the abilities of its survival, differentiation, and pluripotency. Finally, the neurotrophic factors level of neurons derived from Bcl-2 overexpression iPSCs were tested.RESULTS1. Obtain normal urine derived-iPSCs1.1 Obtain a normal urine derived epithelial cell line which presented an appearance of fibre and epithelium with rapid proliferation and good condition.1.2 Obtain iPSCs induced by 4 key factors, and clones of which were round, with clear and keen boundary, and cells in the clones appeared uniform, compact arrangement, large nuclear with high nucleo-cytoplasmic ratio.1.3 The activity of alkaline phosphatase was strong positive. The surface antigens of SSEA4, TRA-1-60, TRA-1-81, OCT4, NANOG were positive as well. The endogenous of ESCs symbolic genes of OCT4, SOX2, KLF4, NANOG were positive. Meanwhile the exogenous genes of Oct4, Sox2, c-myc, and Klf4 were integrated into iPSCs genome without heterogenous expression. Embryoid bodies and teratoma formation were identified positive. It was confirmed that the iPSCs we obtained with pluripotency and without expression of c-myc oncogene.2. Obtain Bcl-2 overexpression iPSCs2.1 Obtain recombinant lentivirus carryng target gene Bcl-2.2.2 Obtain Bcl-2 overexpression iPSCs, and verify the abilities of survival, embryoid bodies formation and differentiation into neurons were not affected.2.3 BDNF and NT3 of neurons derived from Bcl-2 overexpression iPSCs increased significantly, while NGF did not.CONCLUSION1.Epithelial cell lines with good proliferation can been isolated from human urine.2. Urine-derived iPSCs can been proved with pluripotency.3. Recombinant lentivirus carrying Bcl-2 can been used to infect iPSCs.4. The abilities of survival, differentiation, and pluripotency of Bcl-2 overexpression iPSCs appear not significantly different from iPSCs.5. The neurotrophic factor level of BDNF and NT3 of neurons derived from Bcl-2 overexpression iPSCs significantly increased, while NGF did not. |
