Differentiation Of Human Induced Pluripotent Stem Cells To Neural Stem Cells

Objective:Induced pluripotent stem cell (iPS cell) is a kind of pluripotent stem cell which can be generated from adult cells through reprogramming technology. It has the same characteristics with embryonic stem cells and can differentiate to all types of cells derived from three germ layers. The use of iPS cells can decrease the ruin of embryo and avoid ethical problems. Additionally, transplantation with iPS cells derived specific cells can reduce the immunological rejection. So iPS cell is the most promising seed cell and can provide new therapy in the treatment of some intractable diseases in clinic. Repair of nerve injury has been one of the hotspots for many years, neural stem cells which can replace the injury tissues were considered the most effective treatment. Neural stem cells derived from iPS cells which were generated from patients themselves are optimal seed cells because it can avoid immunological rejection and can get enough amount for the treatment. Here, we establish a stable culture system of human iPS cells and find an effective neural differentiation inducing method. Finally, a stable culture system for iPS derived neural stem cells is also set up. This will be helpful for basic research as well as clinic use of neural stem cell-based transplantation.Methods:1. Subculture of human iPS cells. Human iPS cells were maintained on feeder layers, collagenaseⅣwas used for the passage.2. Preparation of embryoid bodies. iPS clones detached from feeder layers were collected and the remaining feeder cells were removed, then clones were cultured in suspension for 4 days with EB medium.3. Inducing process of iPS cells to NSCs. There were three groups: natural differentiation group (EBs were cultured in EB medium), retinoic acid (RA) inducing group (EBs were cultured in EB medium supplied with RA for 4 days and then transferred to EB medium), RA and serum free medium inducing group: EBs were cultured in EB medium supplied with RA for 4 days and then transferred to serum free medium).4. Growth of iPS cells and the change of cell appearance in 3 groups were observed under invert microscope; RT-PCR was used to detect the expression of pluripotent-associate genes. Fold changes of pluripotent-associate genes in EB cells were also measured by real-time PCR. We also compared Nestin expression level of cells on the 7th day after RA inducing process. Additionally, immunostaining was used to detect the expression of Nestin,β-tubulinⅢand GFAP in protein level.5. iPS derived NSCs (termed iPSd-NSCs) cells were collected and cultured in serum free medium. Neuospheres were observed everyday, photos were taken and diameters were measured. Potencies of terminal differentiation and clone formation were also tested. Moreover, cells before and after differentiation were all subjected to immunostaining for Nestin,β-tubulinⅢand GFAP.Results:1. Human iPS cells maintained on feeder layers tended to form packed clones, cells inside clones had small volume and a high nucleus/ cytoplasm ratio. iPS cells expressed Oct4 and Sox2. When cultured in suspension condition, iPS cells subjected to natural differentiation and form EBs, the expression of Oct4 and Sox2 in EBs decreased significantly.2. On the third day after RA inducing process, rosette constructs were observed in RA inducing group as well as RA and serum free medium inducing group, number of rosette constructs increased until the 7th day. In RA inducing group, most cells inside the rosette constructs expressed Nestin and could differentiate to neurons which expressedβ-tubulinⅢbut rarely GFAP positive astrocytes. Rosette cells in RA and serum free medium inducing group tended to detach from the bottom and form neurosphere-like clone, all clones had clear and bright borders, and some newly proliferated cells on the surface. Immunostaining results showed that all these clones expressed Nestin and can differentiate toβ-tubulinⅢand GFAP positive cells in serum-containing medium. EBs in natural differentiation group adhered to the bottom and then some fibroblast-like cells grew out around, small parts of these cells expressed Nestin,β-tubulinⅢor GFAP. On the 7th days after RA inducing process, real-time PCR indicated that the expression level of Nestin gene in RA inducing group as well as RA and serum free medium inducing group were significantly higher than natural differentiation group (p<0.05), the fold changes were 3.71±0.03 and 7.14±1.96 respectively; Moreover, compare to RA inducing group, Nestin expression level in RA and serum free medium inducing group also much higher (p<0.05), the fold change was 1.93±0.52. Additionally, the immunostaining results showed that, Nestin positive cell rates were 23.77±2.96%, 53.25±4.52% and 87.54±3.67% in natural differentiation group, RA inducing group and RA and serum free medium inducing group. There were significant diversity between groups (p<0.05).3. iPSd-NSCs proliferated fast in serum free medium, passage were carried out every 6-8 days; Single iPSd-NSC cell can form neurospere monoclone; when transferred to serum containing medium, iPSd-NSCs clones can differentiate toβ-tubulinⅢand GFAP positive terminal neural cells.Conclusions:1. Human iPS cells can proliferate stably, keep self-renewal and differentiation potency in our culture system, so our culture system is suitable for the in vitro propagation of human iPS cells.2. RA can promote neural differentiation of human iPS cells, however, RA and serum free medium screening method can promote neural differentiation of human iPS cells as well as enhance the survival and proliferation ability of iPSd-NSCs, so it is an effective neural inducing method and can get enough amount of neural stem cells.3. Human iPSd-NSCs propagate stably in serum free medium, and keep undifferentiation as well as neural differentiation potency, so we establish a stable induction and culture system of iPSd-NSCs, it will be helpful for the mechanism research of neural proliferation and differentiation.