Experimental Study Of Embryonic Stem Cell Differentiated Into Retinal Progenitor Cells

Embryonic stem cells derived from the undifferented inner cells of the blastocyst, which have the ability of infinite proliferation. Under the specific condition, ESCs can different into the endoderm, mesoderm and ectoderm. The studies found that m ESCs can be directional induced into RPCs in vitro. The regeneration ability of cells of visual organ is limited. And they cannot repair themselves effectively after injuring. To investigate the method of inducing m ESCs differentiation into RPCs in vitro can provide the theory basis of seed cells for blinding retinal degeneration diseases treated cells.So far, the methods of inducing m ESCs differentiation into RPCs in vitro mainly include natural differentiation method, stromal cell-derived inducing activity, serum-free embryoidbody–like method and the modified serum-free embryonic body–like method, among them, the stromal cell-derived inducing activity is the classical induction method, but this method can only induce very few m ESCs into the cranial nerve; the serum-free embryonic body–like method can greatly improve the efficiency of differentiation, however, in order to induce the mature photoreceptor cells, m ESCs must be co-cultured With embryonic retinal cells. On the basis of the serum-free embryonic body–like method, Osakada and histeam used the modified serum-free embryoid body–like method, which greatly improved the differentiation efficiency and received the mature photoreceptor cells. This experiment use the modified serum-free embryonic body–like method inducing m ESCs into RPCs, then identified the specific markers(Pax6 、 Sox2 and Otx2)of RPCs by immunofluorescence staining and flow cytometry. Further research the trend of the three markers, which will provide the basis for the future study of the differentiation. Objective1.Cultured ESCs(V6.5) and identified the specific markers of ESCs SSEA1 and Ki67.2. To investigate the method of inducing mouse embryonic stem cells differentiation into retinal progenitor cells in vitro.3.Research the trend of the three markers Pax6、Sox2 and Otx2 during this differentiation. MethodsThe specific marker of mouse embryonic stem cell(V6.5) SSEA1、Ki67、cell cycle and alkaline phosphatase were identified by immunofluorescence stai ning and flow cytometry.Then embryonic stem cells were cultured under serum free suspension culture to form embryonic bodies. Retinal progenitor cells wer e induced by Dicckopf-1、Lefty-A and N-[N-(3,5-Difluorophenacetyl)-L- alanyl]-2- phenylglycine tert-butyl ester, their specific markers Pax6、Sox2 and Otx2、multiplication capacity and cell cycle were identified by immunofluorescence st aining and flow cytometry on D0、D10 and D20. Results1.The specific markers of mouse embryonic stem cells SSEA1 and alkaline phosphatase were positively ex RPEssed, Ki67 and cell cycle were positively ex RPEssed showed a potential of self-proliferation ability.2.Induced retinal progenitor cells were positive to Pax6、Sox2 and Otx2, The results identified by immunofluorescence staining were(50.87±2.97)%、(49.10±2.6)% and(32.01±3.87)% on 20 d, the positive ex RPEssion of Ki67 and cel l cycle showed a potential of self-proliferation ability.3.During the process of the differentiation, Pax6、Sox2 and Otx2 were identified by immunofluorescence staining and flow cytometry on 5d、10d、20d. The results show that Pax6、Sox2 and Otx2 were positively ex RPEssed, The ex RPEssion level of Pax6 and Sox2 increased during 0d to 10 d, and then decreased gradually, 10 d The ex RPEssion of Pax6 and Sox2 reached the highest level(77.30±2.50)% and(91.89±1.50)%. However, the ex RPEssion of Otx2 increased gradually, D10 The ex RPEssion of Otx2 reached(32.01±3.87)%. The positive ex RPEssion of Ki67 and cell cycle on 20 d showed that RPCs have a potential of self-proliferation ability. Conclusions1.Cultured ESCs(V6.5) can used foe the next directed differentiation exp eriment.2.Under the effect of specific cytokines, mouse embryonic stem cell can be induced into retinal progenitor cells.3.During the differentiation, Pax6、Sox2 both shows the tendency of first increasing and then decreasing, 10 d The ex RPEssion of Pax6 and Sox2 reached the highest level; the ex RPEssion of Otx2 increased gradually.