Comparison Of Gene Expression Profiling Between Retinal Ganglion-like Cells With Bone Mesenchymal Stem Cells By Suppression Subtractive Hybridization Analysis

Although pathogenesy of retinal diseases are different,neuronal degeneration is the cause of debilitating visual impairment associated with prevalent ocular diseases such as retinal ischemia,retinal hypoxia,retinitis pigmentosa (RP),age-related macular degeneration(AMD),diabetic retinopathy, and glaucoma.Recent identification and characterization of bone mesenchymal stem cells properties have opened new avenues of treating irreversible functional impairments caused by the death of retinal ganglion cell populations.Recently, it has been reported that BMSCs differentiate into various cells, including osteoblast, chonseoblast, tendon cells, hepatocytes, endothelial cellse and other mesoblastema cells in certain conditions. It has also been reported that BMSCs differentiate into ectoblast cells,such as neural cells and astrocytes in vitro and also into astrocytes in vivo .Moreover, it has been improved that bone mesenchymal stem cells could incorporat and differentiatinto retinal neural cells in the injured retina. The incorporated cells expressed neuron-specific marker, such as calbindin and rhodopsin. This finding suggest that BMSCs can differentiate into retinal neural cells under the appropriate conditions.In 2003 Kicic A reported that BMSCs induced by activin A and EGF into cells could espress photoreceptor specific markers rhodopsin, opsin, and recoverin in vitro. Then the cells were injected into the subretinal space of adult rats. BMSCs integrated into the host retina, forming structures similar to the photoreceptor layer.Following these initial studies we cultured neonatal retinas with BMSCs that closely mimics retinal development in vivo. To identify differentially expressed genes governing bone mesenchymal stem cells transdifferentiation into retinal ganglion-like cells, we attempt to characterize gene expression pattern before and after neural differentiation by SSH analysis. The present study was designed to identify possible genes which may play important roles in inducing bone marrow mesenchymal stem cells into retinal ganglion-like cells.PartⅠ: Cultivation, purification and identification of Adult Bone Marrow Mesenchymal Stem Cells in vitroWe have successfully extracted BMSCs from the femurs of adult rats and propagated in vitro using density gradient and adherence to plastic dishes method. BMSCs maintained highly proliferative capacity before 7th passage.The BMSCs were identified using fluorescent-activated cell sorting (FACS). In brief, harvested cells were washed in PBS, and incubated for 30 min on ice with fluorescentconjugated monoclonal antibodies (1:100 dilution) directed against the following cell surface markers: CD29,CD71,CD90,CD105 and CD106. Cells were washed for two more times before analysis. The results show that population profiles of third-passage BMSCs comprised a higher purity population by flow cytometric analysis of expressed surface antigens .The cells were negative for CD34,CD45, while positive for CD29,CD71,CD90,CD105,CD106.For purification, the cells were sorted using a FACSAria cell sorter (Becton Dickinson) after the fluorescence-labeling procedure mentioned above, BMSCs expressing CD90+/CD71+ were then collected, recultured and used in all subsequent experiments. As the cells approached confluency, they assumed a more spindleshaped, fibroblastic morphology.In our methods, higher purity of BMSCs can be isolated and cultured by combining density gradient centrifugation with FACS sorting. BMSCs could be cultured stably. It would provide target cells for experiments in vitro.PartⅡ: The Study of induction of the Adult Bone Mesenchymal Stem Cells Differentiate into retinal ganglion-like Cells in vitro Third-Passage BMSCs were induced by incubation with retinal cells, and the differentiated retinal ganglion -like cells were identified with inverted mircroscopy .After the BMSCs were co-cultured with retinal cells, the BMSCs displayed some external features of neurons. The cells were immunoreactive for neuronal-lineage specific marker nestin on day 3, which was normally restricted to neuronal marker. They also expressed specific genes such as neurofilaments,thy1.1 andβ-tubulin III as detected by RT-PCR and immunohistochemistry analysis on day 10.In this study the BMSCs were co-cultured with neonatal retinas, and the results confirmed the expression of neuron and retinal's markers. Neonatal retinas produce some extrinsic factors that affect proliferation and differentiation of BMSCs. The results showed that extrinsic factors play an essential role in the differentiation of BMSCs. Neonatal retinal cells therefore seemed to provide the most suitable microenvironment for Bone marrow mesenchymal stem cells to differentiate into mature retinal cells.Previous studies have reported that cell–cell interactions determine neuronal phenotypes when retinal progenitor cells are cultured with rodent retinal cultures, and this culture system has the advantage of keeping the cell–cell interactions intact which may play an important role in cell survival and differentiation.In summary, we successful induced BMSCs into retinal ganglion-like cells by BMSCs/retinal cells co-culture system, however, there are still a number of fundamental problems about BMSCs need to be resolved before they can be used for safe and effective clinical cell and gene therapy.PartⅢ: Comparison of Gene Expression Profiling between Retinal ganglion-like cells with Bone mesenchymal stem cells by Suppression Subtractive Hybridization (SSH) AnalysisTo construct a subtractive library of Retinal ganglion-like cells,suppression subtractive hybridization(SSH) was performed. The SSH library contained 120 clones. Analysis of 23 clones with enzyme restriction showed that 6 clones contained cDNA fragments which were distributed between 200-800bp.Based on the function of the 6 known genes, many of these genes function in signal transduction, ca2+ transportation the evasion of apoptosis and neurite generation.The construction of subtractive library of retinal ganglion-like cells will provide valuable information for a better understanting of the molecular mechanism of induction.