Experimental Research Of Differentiation Of Human Embryonic Stem Cells Into Retinal Photoreceptor Cell

Objective1. Different combinations of small molecules were used to induce the differentiation of human embryonic stem cells into retinal photoreceptor cells, and the morphological changes of the cells were observed at different stages of differentiation.2. The expression levels of Ki67, Sox2, Otx2, and Rhodopsin were detected from the cell level, protein level and gene level during the differentiation of human embryonic stem cells into retinal photoreceptor cells. The expressions of Ki67, Sox2, Otx2, and Rhodopsin in different groups of small molecule compounds were compared and the differentiation and efficiency of each group were explored.Methods1. Human embryonic stem cells were cultured by adherent culture. ABSRT, ABSRTA,which was used in the course of culture, were used to act on human embryonic stem cells and to observed cells morphological in each period in the process of differentiation in zero days, tenth days, twentieth days, thirtieth days. After collecting specimens, using immunofluorescence staining to detect the expression patterns of Ki67, Sox2, Otx2 and Rhodopsin in different stages of differentiation.2. In the differentiation of zero days, tenth days, twentieth days, thirtieth days,collecting specimens, using Western Blot, qRT-PCR to detect the expression level of Ki67, Sox2, Otx2, and Rhodopsin cells in each period, from the protein level and gene level, analyzed expression characteristics of Ki67, Sox2, Otx2, Rhodopsin and compared differentiation efficiency in each group.Results1. Adherent human embryonic stem cells morphology were observed under microscope,cells gradually being stretched from round to fusiform, cell body translucent and strong refractive, large nucleus, obvious nucleolus, more common mitotic figures, strong cell proliferation and division, and the cell clones are integrated into the net. In the 60 hours after cell resuscitation, the cell fusion rate was about 80%.2. Human embryonic stem cell small colony group were growing in six holes plate with 1%Matrigel, on the first day, we can observe cell small clonogenic group adherent growth, intermediate density bigger, and cells gradually climb out, turning from round to long spindle shape. Covering cells for two days with 2% Matrigel, we can observe cell colony group to formulate columnar epithelial nerve, and on tenth days can observe the rosette like structure formation, in addition of small molecules, with the prolongation of time, the cells axons gradually increasing and the elongating with gradually mature, morphology close to nerve cells.3. Small molecule compounds ABSRT group and ABSRTA group could induce differentiation of human embryonic stem cells into retinal photoreceptor cells. ABSRT group and ABSRTA group showed that the expression level of Ki67 was decreased gradually with the induction time, and the levels of Sox2 were positive in two groups,and the expression levels of Sox2 in two groups were up-regulated, and the up-regulation degree of different stages was different. Before the D30 Higher than the ABSRT group, D30 when the ABSRT group higher than the ABSRTA group. The expression of Otx2 and Rhodopsin were up-regulated and the expression levels were up-regulated in different stages. The up-regulation degree of ABSRTA group was higher than that of ABSRT group. The efficiency of induction of human embryonic stem cells into retinal photoreceptor cells induced by different combinations of small molecule compounds was different. The induction efficiency of ABSRTA group was higher than that of ABSRT group, and ABSRTA group was 24.94% higher than that of ABSRT group.Conclusions1. The ABSRTA group and ABSRT group can induce the differentiation of human embryonic stem cells into retinal photoreceptor cells.2. The small molecule compound ABSRT group and ABSRTA group, in the induction differentiation of human embryonic stem cells into retinal photoreceptor cells, stem cell marker gene expression level of Ki67 decreased gradually, relating to the development of regulated gene, Sox2 and Otx2 positive expression, increasing, and the expression of retinal photoreceptor cell Rhodopsin specific gene expression were all positive, increasing. The efficiency of induction of human embryonic stem cells into retinal photoreceptor cells induced by different combinations of small molecule compounds was different. The induction efficiency of ABSRTA group was higher than that of ABSRT group, and ABSRTA group was higher than ABSRT group in inducing efficiency of 24.94%.