Role Of Synapsin-Ⅰ During Neural Differentiation And Transplantation For Cerebral Ischemia Of Embryonic Stem Cells

Background and ObjectiveCell replacement therapy has given a charming prospect in treatment for cerebral ischemia recent years. Embryonic stem (ES) cells can proliferate indefinitely without senescence and are able to differentiate into neural cells in large amount. These properties make ES cells an attractive source for the cell replacement therapy. Implantation of Undifferentiated ES cells leads to formation of benign teratoma in the recipients. ES cells are usually induced into neural precursor cells or neural cells in vitro before transplanting into the ischemia brain. That is, ES cells treatment for cerebral ischemia includes two parts: First, neural differentiation of ES cells in vitro. Second, migration, expansion and interaction of ES cells with the brain cells after their implantation.Cell differentiation is the result of appropriate spatial and temporal expression of specific genes. It means that exact gene is stimulated at the exact time and site. Complex regular mechanisms are required for the neural differentiation as well as the migration, expansion and integration of ES cells. As a result, finding a controllable point of the regular mechanisms is valuable for ES cells treatment for cerebral ischemia.Synapsin-I is a representative phosphoprotein associated with the membranes of synaptic vesicles. It plays a broad role during neural development and in the regulation of neurotransmitter release. As we found before in our study, the expression of synapsin-I had its rule after transient ischemia followed by reperfusion. Since synapsin-I is associated with not only neural differentiation but also cerebral ischemia, we single it out as the candidate gene for the controllable point of the ES cells treatment for cerebral ischemia.In this study, we attempt to study the role of synapsin-I during neural differentiation and transplantation for cerebral ischemia of ES cells. We try to verify the possibility that synapsin-I be used as a controllable point during this process and offer some valuable information for the regular mechanisms research.Methods1. Neural differentiation of ES cells was induced by two approaches: the five step approach and the retinoic acid (RA) approach. A tumor cell line called PC 12 cell which can also differentiate into neural cells was compared with at the same time. Synapsin-I expression was accessed by immunohistochemistry, semi-quantified RT-PCR and Western-blot analysis respectively at different time point to find the common rule of synapsin-I expression during neural differentiation in different cell lines and different inducing approaches.2. Synapsin-I expression was inhibited by antisense oligonucleotides at different stage during the neural differentiation. At different time point, morphology, differentiated efficiency and neural specific marker were compared between the normal differentiation and the intervened differentiation to observe the effect of the suppression of synapsin-I on the neural differentiation of ES cells and PC 12 cells.3. Rats were subjected to 10 minutes of middle cerebral artery occlusion (MCAO) and PC 12 cells were subjetted to 20 minutes of oxygen and serum deprivation. At the time point of 1, 3, 6, 12, 24 and 72 hours after reperfusion and giving oxygen again, expression of synapsin I was observed by immunohistochemistry. 4. ES cells and differentiated PC 12 cells were cocultured in transwell plate to establish a model of ES cells migration to ischemia cells in vitro. Migration of ES cells to PC 12 cells was relatively accessed by Hoechst33342 staining and fluorescence-activated cells sorter (FACS). Migration of ES cells was compared between Undifferentiated and differentiated ES cells, PC 12 cells subjected to oxygen and serum deprivation and normal PC 12 cells, before and after synapsin-I antisense oligonucleotides transfection of ES cells.Results1. Immunohistochemistry, semi-quantified RT-PCR and Western-blot analysis combined with morphology and other neural specific marker showed that synapsin-I expression during neural differentiation in different cell lines and different approaches had a common rule. Synapsin-I appeared at the early stage, then increased progressively, and reached a plateau at the full differentiation stage, after that declined slowly at the late stage.2. Suppression of synapsin-I at different stage during the neural differentiation of ES cells resulted in a delay of differentiation course and a reduction of neural differentiation efficiency, especially at the early stage. Considerable decreases of neural precursor cell (nestin positive cell) ratio and neurite growth rate were observed when synapsin-I antisense oligonucleotides were applied at the third and forth stages. At the end of the third stage, the nestin positive cell ratio decreased from 76.2±5.1% to 68.5±4.2% (P<0.05). At the end of the forth stage, the nestin positive cell ratio decreased from 90.2±4.3% to 75.1±4.7% (P<0.05). MAP2 positive cell ratio and the interaction among cells reduced at the fifth stage when synapsin-I ntisense oligonucleotides were applied. The MAP2 positive cell rate deceased from 41.2±2.7% to 30.7±3.2% (P<0.05) at the end of the fifth stage. On the other hand, suppression of synapsin-I on PC 12 cells also led to a delay of differentiation course. Neurites of intervened cells were significantly shorter than the normal differentiated cells at the same time point. Proportion of differentiated cell after intervention at 1, 4, 7 and 10 days post-induction obviously decreased from 1.81±0.40% 45.13±4.17%, 90.26±4.68% and 84.66±4.81% to 0.33±0.46% 9.78±3.47% 45.3±7.98% and 34.2±5.89% (P<0.01).3. In the MCAO models, no obvious changes in synapsin-I immunoreactivity were detected within 12 hours of reperfusion after transient ischemia. At the time point of 24 hours after reperfusion, a considerable reduction in synapsin-I immunoreactivity of the caudate putamen and frontparietal cortex was observed (t=6.59 and 7.28, p<0.01). After that the synapsin-I immunoreactivity recovered to control level at the 72nd hour after reperfusion. In the oxygen and serum deprived cell model, synapsin-I immunoreactivity showed a similar trend of the MCAO model.4. PC 12 cells subjected to oxygen and serum deprivation attracted more ES cells migration than those without oxygen and serum deprivation by 68% to several times.A near half decline of ES cells migration was observed after antisense oligonucleotides's application.ES cells implanted at the time point of 24 hours after transient oxygen and serum deprivation of PC 12 cells migrated much more than those implanted immediately. On Undifferentiated ES cells, the ratio of ES cells migration between the time point of 24 hour and 0 hour was 1.97. On differentiated ES cells, the ratio of ES cells migration between the time point of 24 hour and 0 hour was 2.04.Conclusions1. Synapsin-I plays an important role during neural differentiation. Different roles of synapsin I function at different stages. It is likely that synapsin I plays a more important role at the early stage of neural differentiation. 2. ES cells migration maybe affected by the host internal environment and associated with the synapsin-I expression level of ES cells and the host cells in the excised model of ES cells transplantation for cerebral ischemia.3. Synapsin I may be implicated in the regular mechanism of neural differentiation and transplantation for cerebral ischemia of ES cells. It is hopeful that synapsin-I be used as a controllable point during this process to have a better prospect of ES cells treatment.4. The transwell coculture system may be used as a practical model for ES cells differentiation and migration research in vitro.