Retinal degeneration mainly include age-related macular degeneration, retinitispigmentosa and Stargardt’s disease. Although its expression is slightly different, its pathogenesis is photoreceptor cells and/or retinal pigment epithelial (RPE) cel1 damage or degeneration. Because of the 1ack of self-repairing and renewal of retinal photoreceptor cells and RPE cells, cell replacement therapy is one of the most effective methods for treating such diseases.The stem cells currently used for the treatment of retinal degeneration include embryonicstem cells (ESC) and various adult stem cells, such as retinal stem cells (RSC), induced pluripotent stem cells (iPSC). and mesenchyma1 stem cells (MSC). Understanding the currentbasic and clinical application progress of ESC, iPSC, RSC, MSC can provide a new idea for the treatment of retinal degeneration.
Purpose To investigate the development of embryonic stem cells (ESC)in the subretinal space． Methods ESC were cultivated in suspension for 4 days till they developed into cell aggregates,i.e.embryonic body(EB).ESC as well as EB combined with or without RA were respectively transplanted into vitreous cavity and subretina1 space in SD rats,and the subretinal transplanted eyes,transient ischemia-reperfusion injuries were made by ligating the ophthalmic artery for 40 seconds before the transplantation .The experimental eyes were enucleated for histological and immunohistochemical assays after 14～28 d. Results The EB was found to develope into photoreceptors induced by RA in the subretinal space under an ischemia-reperfusion condition，and EB transplantation without RA induction induced multiple differentiations in the subretinal space.The single injection of RA without EB induced hyperplasia of the neural retinal cells.ESC transplanted into vitreous cavity rapidly proliferated and developed into atypical hyperplastic mass. Conclusion EB derived from ESC can differentiate into photoreceptors induced by RA in the host subretinal space under an ischemia-reperfusion condition. (Chin J Ocul Fundus Dis，2000，16：213-284)
The capacity of embryonic spinal cord tissue in the repair of injured structure of spinal cord has been noted for years. In order to investigate the embryonic spinal cord graft in the repair of motor function of injured spinal cord, the embryonic spinal cord tissue was transplanted to the hemisection cavity in spinal cord in adult rat. One hundred adult Wistar Rats were used to simulate the hemisectional injury of spinal cord by drilling 2-3 mm cavity in lumbar enlargement. Sixty rats were treated with rat embryonic spinal cord tissue grafting while the other forty were chosen as control. The outcome was evaluated according the combined behavioural score (CBS) and motor evoked potential (MEP) in the 1, 2, 4 and 12 weeks. The grafting group was superior to the control as assessed by CBS (P lt; 0.05), especially within 4 weeks. (P lt; 0.01). The restoration of the latent peak of early wave(P1, N1) was better in the grafting group, too. This suggested that embryonic spinal cord graft could improve the recovery of motor function of injured spinal cord in adult rat. The effect of the embryonic spinal cord tissue graft might be concerned with its secretion of several kinds of neurotrophic factors, nerve growth factor, nerve transmitted factor, or adjustment of hormone.
Purpose To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. Methods The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. Results Two to three days after transplantation,yellowish-white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. Conclusion The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina-like structure. (Chin J Ocul Fundus Dis，2000，16：213-284)
Diabetic retinopathy is a serious complication of diabetes and is the leading cause of blindness in people with diabetes. At present, there are many views on the pathogenesis of diabetic retinopathy, including the changes of retinal microenvironment caused by high glucose, the formation of advanced glycation end products, oxidative stress injury, inflammatory reaction and angiogenesis factor. These mechanisms produce a common pathway that leads to retinal degeneration and microvascular injury in the retina. In recent years, cell regeneration therapy plays an increasingly important role in the process of repairing diseases. Different types of stem cells have neurological and vascular protection for the retina, but the focus of the target is different. It has been reported that stem cells can regulate the retinal microenvironment and protect the retinal nerve cells by paracrine production, and can also reduce immune damage through potential immunoregulation, and can also differentiate into damaged cells by regenerative function. Combined with the above characteristics, stem cells show the potential for the repair of diabetic retinopathy, this stem cell-based regenerative therapy for clinical application provides a pre-based evident. However, in the process of stem cell transplantation, homogeneity of stem cells, cell delivery, effective homing and transplantation to damaged tissue is still a problem of cell therapy.
ObjectiveTo investigate the effects of over expression of Mash-1 gene on the differentiation of embryonic stem cells (ESC) into neural cells in vitro. MethodsThe ESC of rats (CE3 cells) were transfected with MSCVMash- 1 (MSCV-Mash-1-CE3 group) or MSCV (MSCV-CE3 group). The expression of Mash-1 gene was detected by RT-PCR. After transfection, hanging-drop culture was used to form embryonic bodies, and then embryonic bodies were cultured with neural induction medium. The cell morphology was observed under inverted phase contrast microscopy at 7 and 21 days; the positive rates of neural stem cells marker protein (nestin) and neuron marker protein (β-tubulin Ⅲ) were measured by immunofluorescence staining after cell attachment; and the gene expressions of α-fetal protein (AFP), Brachyury, fibroblast growth factor 5 (FGF-5), Oct3/4, nestin, and β-tubulin Ⅲ were detected by real-time fluorescence quantitative PCR at 0, 1, 7, 14, and 21 days after culture. The CE3 cells were used as control (CE3 group). ResultsCompared with MSCV-CE3 and CE3 groups, the expression of Mash-1 gene in MSCV-Mash-1-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-1-CE3 group had axons growth and showed neural stem cell-like and neuron cell-like morphology (unipolar, bipolar, and multipolar neurons), but few cells had axons growth in MSCV-CE3 and CE3 groups. The positive rates of nestin at 7 days and β-tubulin Ⅲ at 21 days in MSCV-Mash-1-CE3 group were significantly higher than those in MSCV-CE3 and CE3 groups (P<0.05). Real-time fluorescence quatitative PCR results showed that the gene expression of Brachyury was significantly decreased after 1 day (P<0.05), and the gene expressions of FGF-5 and nestin were significantly increased after 1 day (P<0.05) in MSCV-Mash- 1-CE3 group when compared with CE3 and MSCV-CE3 groups; the gene expression of β-tubulin Ⅲ was significantly increased after 7 days (P<0.05). There was no significant difference in above indexes between CE3 and MSCV-CE3 groups (P>0.05). The expressions of AFP and Oct3/4 showed no significant difference among groups at each time point (P>0.05). ConclusionOver expression of Mash-1 gene can promote differentiation of ESC into neural cells in vitro.
Objective To investigate the protective effect of nerve growth factor (NGF) on apoptosis of cultured human fetal retinal pigment epithelium (HFRPE) cells induced by indomethacin (IN) in vitro.Methods Subcultured HFRPE cells were treated with different concentrations of IN to establish apoptotic model. The protective effect of NGF on apoptosis of cultured HFRPE cells were assessed using an acridine orange (AO) staining method and transmission electron microscopy (TEM).Results HFRPE cells exposed by 200-600 μmol/L IN for 24 hours elicited typical apoptosis morphological changes, including condensed chromation, nuclear fragmentation and reduction of nuclear size and cell volume. There was a statistically difference in HFRPE cells with apoptosis between 200 μmol/L IN+500 μg/L NGF and 200 μmol/LIN groups ( q=3.9204，P=0.0320); there was a significant difference in HFRPE cells with apoptosis in 400 μmol/L IN+500 μg/L NGF and 400 μmol/ L IN as well (q=9.7915，P=0.0001). Conclusion NGF has an protective effect on IN-induced HFRPE cells apoptosis. (Chin J Ocul Fundus Dis，2003，19：38-41)
To investigate the processes of retinal vascular development of human fe- tus. METHODS:Eighty-six specimens of retinas of human fetus from 13th to 38th gestational week were studied by immunohistochemieal method(ABC). RESULTS:The spindle cells of mensenchymal origin was firstly found to migrate into the retina from optic disc at 12--13 weeks ,and spreaded subsequently toward the periphery of the retina. At the same time ,the consequential processes of differentiation,proliferation,canalilization and remodelling developed into vascular plexus in ganglion cell layer (GCL). Vascular buds formed in GCL in 26th gestational week extended toward nerve fiber layer (NFL)and inner nuclear layer(INL)and developed into capillary plexuses in NFL and inner and outer margin of INL respectively. CONCLUSIONS.. Four vascular layers could be distinguished in the ratina of full term fetuses,and these layers were formed through two principal developmental processes. （Chin J Ocul Fundus Dis,1996,12: 88- 90）
Objective To investigate the possibility of commitment differentiation of embryonic stem cells induced by the medium of cultured retinal neurons of SD rats. Methods The medium from cultured retinal neurons of SD rats were collected, sterilized and mixed with DMEM medium according to 2∶3 proportion, ES cells were cultured with these mixed medium and were observed under the phase contrast microscope daily, the induced cells were identified by NF immunohistochemistry methods. Results The ES cells cultured with these mixed medium can differentiate into neuron-like structure, and the induced cells were positive in NF immunofluorescence staining. Conclusion The medium from cultured retinal neurons of SD rats can induce ES cells commitment differentiation into neuron-like structure. (Chin J Ocul Fundus Dis, 2002, 18: 134-136)