| Retinal degenerations, the leading cause of visual impairment, such as retinitis pigmentosa and age-related macular degeneration, are characterized by progressive dysfunction of the photoreceptors and associated with progressive cell lose. It is a most meaningful challenge for eye doctors and researchers about how to prevent so many patients with retinal degenerations from progressive retinal damage and restore their visual function. In recent years, cell-replacement therapy have shown promising therapeutic implications and many kinds of cell types have been used in retinal transplantation experiment, including differentiated cells, such as IPE, and stem cells such as ESC, MSC, NSC and adult retinal stem cell. Although the effect is still unclear, and the restore of retinal function is far away from success, it is proved that the retinal transplantation is feasible and stem cells/progenitors is a most promising implication. Stem cells that have the self renewal potential and multilineage differentiation capacity are the relevant cells in regenerative medicine and have attracted special attention. During the past ten years, much improvement has made on the retinal transplantation, but it is evident that the poor retinal determined differentiation, limited resource and differentiated cells contamination are the restrictions for applications of the available stem cell sources. In this regard, it is a powerful strategy to explore alternative sources with higher purity cell type and retinal determined differentiation capacity. In the retinogenesis, embryo optic cup extended from forebrain vesicles contains two layers, the inner one give rise to neuronal retina during visual development, the outer one to be retinal pigment epithelium. It is reasonable that the cells harbored in embryo optic cup are the common progenitors of all the retinal cells. Given avoiding differentiated cell contamination, the cells isolated from optic cup before cell differentiation should be ideal as an alternative promising source of retinal stem cells for transplantation implication and a model suitable to study eye development mechanisms and for stem cells differentiation research. However, the isolation and biological properties of stem cells from the optic cup are still unknown. In this paper, We isolated and identified clonal population of retinal stem cells from optic cup of tail bud embryo of rat, and performed a series of experiments to study their biological properties, even the promoting effects on retinal degeneration recovery in RCS rats. The main results and conclusions are summarized as follows: 1. The distribution and differentiation of retinal stem cells of optic cup at embryonic day 12 to 14 (E12 d-E14 d ) was revealed by immunohistochemistry in order to find the appropriate embryonic stage to isolate retinal stem cell with higher purity . We found that the optic cup retinal stem cells, CHX10-positive, distribute in the two layers of optic cup at E12.5 d, which were organized as stratified epithelium arrangement in the middle of optic cup or clusters at the edge. In the E13.5d embryonic optic cup, pigment appeared in the outer layer and the CHX10-positive cells just stayed in the inner layer. Furthermore, differentiation to ganglion cells was initiated because the Thy1.1 expression was detected. At the E14.5 d stage, the pigment and the expression of Thy1.1 were more evident. By these results, we hypothesize that the cells distributed in the optic cup at E12.5 d were all retinal stem cells, which suggests that retinal stem cells isolated from optic cup at E12.5 d could be enriched easier. 2. The cells from optic cup at E12.5 d in rat were isolated and cultured with serum-free media. Immunofluorescence technique was employed to detect CHX10 antigen and specific antigens of mature retinal cells before and after differentiation. The study showed that the isolated cells derived from optic cup cultured in serum-free medium could give rise to neural spheres in the presence of bFGF. These cells could proliferate successively and generate secondary neural spheres, thus displaying potential to self-renew. These neural spheres could be expanded for up to 8 passages and expressed constantly the neuroectodermal marker Nestin and Chx-10, also retinal stem cell markers, showing the undifferentiated properties in vitro. After addition of fetal bovine serum (FBS) in the culture medium, the cells expressing specific antigens of retinal ganglion cell, bipolar cell, Müller cell and photoreceptor such as Thy1.1, PKCα, GFAP and rhodopsin were found. These results indicate that the stem cells from optic cup at E12.5 d of rat are stem cells of retina, it is optic cup-derived retinal stem cells (OC-RSCs).3. To understand the properties of OC-RSCs of rat, following study was proceeded. Flow cytometry results showed that the percentage of Chx-10 positive cells in the third passage was 92%. It is about 91.5% cells in G0/G1, and 8.5% in S stage by cell cycle detection. The doubling time of OC-RSCs was about 36 h and there was no obvious difference of the growth pattern and differentiation capacity between cells of the third, fifth and eighth passage according to MTT detection and induced differentiation by FBS. Electron micrographs evaluated that the OC-RSCs had a high nucleus-to-cytoplasm ratio. These results tell us that OC-RSCs could be enriched by passage three times at least, and have the basic properties of stem cells. 4. In regard to their unique derivation compared to adult retinal stem cells and other retinal stem cells from E14, E18, PN1 (1 day postnatal) of rat, the differentiation potential of OC-RSCs was analyzed in vitro with series concentration of FBS in culture medium under the condition of withdrawal of bFGF. Retinal cell type-special marker expression following 21-days treatment with 0.5%, 2.5%, 5% and 10% FBS on OC-RSCs was detected by flow cytometric analysis and represented the percentage of differentiation of retinal cells. Under the concentration of 0.5%, the cells expressing CHX10, GFAP and rhodopsin were 16.2%, 71.2% and 3.0% respectively. With the increase of FBS concentration, the number of cells expressing CHX10 increased, while the total of GFAP-positive cells reduced. As for rhodopsin-positive cells, rod photoreceptors, the highest differentiation rate of 12.6% was under the 2.5% FBS treatment, although the rhodopsin expression ratio at 5% and 10% FBS were much higher than that at 0.5%. It is reported firstly that the OC-RSCs differentiation pattern is FBS concentration-dependent, and the lower concentration of FBS benefit the Müller cell differentiation, and 2.5% FBS induced OC-RSCs differentiate to rod photoreceptors at maximum among the four FBS concentration. 5. Given the damage and lose of photoreceptors during retinal degeneration, it is a key problem to evaluate the differentiation potential of OC-RSCs to be photoreceptors treated by RA(retinoic acid) and VEGF(vascular endothelial cell growth factor), two factors promoting photoreceptor differentiation in vitro, by flow cytometric analysis. After 21-days treatment with RA (0.5μM) and VEGF (10ng/ml) alone, elevation of the number of rhodopsin-positive cells was observed, about 2-fold in RA group and 133% increase in VEGF treatment. These results indicate that both RA and VEGF can stimulate OC-RSCsdifferentiate to be photoreceptors in vitro and OC-RSCs generate more photoreceptors in the presence of RA than the presence of VEGF. 6. As an alternative promising source of retinal stem cell to therapeutic implication and a model for stem cell biology research, the properties such as survival, migration, differentiation to photoreceptor of OC-RSCs in the normal and degeneration retinal environment were studied, and the relationship of the donor cells variation with the receptor retinal function analyzed by Multifocal electroretinogram (mfERG) detection in retinal degeneration model rats (RCS) were also discussed. After being labeled with green fluorescent by EGFP plasmid transfection, OC-RSCs were injected into the subretinal space of Long Evans rats and RCS rats. The labeled cells were found throughout 4 weeks post-transplantation. By morphological observations and rhodopsin immunoreaction, the transplanted cells in RCS rats were survival and differentiated into rod photoreceptors, but the survival GFP+ cells at 1-week post-transplantation had no obvious photoreceptor differentiation. The migration at 4-weeks after transplantation was limited in RCS rats. These results indicate that OC-RSCs could survive and differentiate to photoreceptors in vivo, and the subretinal environment of RCS rats is more beneficial for OC-RSCs migration, partly due to the fact that the RCS rat retina is genetic injured. MfERG were recorded pre-and post-transplantation. P1 wave amplitude in the eye under 4-weeks post-transplantation of RCS increased compared with age-matched control eyes, which suggests some improvement of retinal function after OC-RSCs transplantation was existed. In conclusion, the differentiation to photoreceptor after OC-RSCs transplantation may be the reason of improvement of retinal function. Our observations suggest that cultured OC-RSCs can be a viable source for therapeutic transplantation. |
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