Fetus Rat RSCs To Ganglion Cells Differentiated Research

Retinal stem cells(RSCs) may potentially serve as an unlimited source of self duplication and multi-directional differentiation cells and replace degenerated retinal and RPE cells, For example, age-related macular degeneration, pigmentary degeneration of retina, glaucoma optic atrophy et al. In vitro RSCs induced differentiation, we may transplant differentiated functional cells into retina and replace dysfunctional or degenerated retina, then reconstructing retinal structural. So, it provides a new method to cure these optic nerve and retinal diseases ultimately for us. amongIn 1980, with stem cells discovered, RSCs were isolated and cultured from mammalian and Human being in vitro. It may maintain self-renewal or differentiate into all types of retinal cells. Research discovered, fetal rat RSCs show time and space specificity, when it differentiates into retinal cells, and, at first, express preferentially retinal ganglion cells( RGCs) in vivo. This suggested there are some cytokines and extracellular matrix(ECM) to promote stem cells differentiation in retinal microenvironment. However, we do not understand whether RSCs of different period in culture in vitro display the same time specificity. At present, direction differentiation regulation of RSCs is hot spot topics of stem cells research. There are close correlation between proliferation and differentiation of RSCs and neurotrophic factors, growth factors and hormones. Many factors show important effect when stem cells are induced differentiation. But, it is not impossible that stem cells are induced direction differentiation to functioning cells to depend certain a factors.According to study conditions at present, this topic compared differentiation outcome of different time's RSCs to RGCs in culture in vitro. Then, taking RSCs of certain period when can differentiate to relative higher proportional RGCs, on the basis of serum inducing differentiation, admoveatured singly bFGF, BDNF and insulin inducing factor by turn, and induced differentiation. Observing differentiation proportion that RSCs differentiated to RGCs, we expected to attain much higher proportional RGCs again in order to establish foundation for cell transplantation. This research includes three parts:Part 1: to isolate, culture, identify fetal rat RSCs and study cell characteristic.To isolate, culture, identify RSCs of E18D SD rat and study cell characteristic. RSCs were isolated from days 18 SD fetal rat retina by using improving culture method,and the cells were cultured and passaged in vitro.Immunocytochemistry were used to identify RSCs and their potentiality of differentiation. The primary cultured retinal cells could form neurospheres and showed ability of proliferation.Neurospheres expressed the neuroectodermal marker Nestin.Part 2: to compare proportion that RSCs of different fetal age SD rats differentiated to RGCs.On the same condition, culturing RSCs of E16D, E18D, E20D fetal rat by turn, When RSCs are induced differentiation by 5%fetal bovine serum, which withdrew simultaneously bFGF in midium, we observed differentiation proportion of RGCs of different period.Result showed:1,When fetal rat RSCs were induced 7 days later, the parts of cells expressed markers of RGCs(Thy1.1), rod(opsin) and neuroglial cells(GFAP).2,When E16D, E18D, E20D fetal rat RSCs were induced 7 days later by turn, positive rate of cells expressed Thy1.1 was13.8±3.1%, 8.0±3.2% and 7.5±2.5%. Positive rate of E16D was the highest. Difference have statistical significance, which compared with other periods(P<0.01).Part 3: to compare proportion that RSCs of the same fetal age which were induced by different inducing factors differentiated to RGCs.Culturing RSCs of E16D fetal rat, on the basis of serum inducing differentiation, admoveatured singly different bFGF, BDNF and insulin inducing factors in medium or not. So, they included four teams: serum, bFGF, BDNF and insulin. We observed that proportion RSCs differentiated to RGCs. Result showed:When each team was induced 7 days later, positive rate of cells of serum team, bFGF team, BDNF team and insulin team expressed Thy1.1 was 14.2±1.8%,3.5±2.0%,18.1±2.5% and 6.0±2.2%. Positive rate of BDNF team was the highest. Difference of between teams all have statistical significance(P<0.01).Conclusion:1,The isolated cells from SD fetal rats retinal have the ability of proliferation and differentiation in vitro, and differentiated cells express characteristics of retinal neural cells and glial cells.2,E16D fetal rat RSCs in culture in vitro differentiated to proportion of RGCs was relative higher. It suggested RSCs in culture in vitro had time specificity when it differentiated.3,E16D fetal rat RSCs in culture in vitro differentiated to proportion of RGCs was relative higher after it was induced by BDNF. It suggested BDNF raised differentiation ratio of RGCs.