| ObjectiveIn order to obtain a sufficient amount of human neural stem cells(h NSCs),the monolayer differentiation cell culture method was used to differentiate human embryonic stem cells(h ESCs)into h NSCs that can be used for transplantation by using small chemical molecules.MethodsUsing the monolayer differentiation cell culture method,h ESCs were inoculated onto radiative mouse embryonic fibroblasts(r MEFs)feeder cells and the small chemical molecules DMH1 and SB431542,which are SMAD signaling pathway inhibitors,were added,and the solution was changed every other day.After 6 days of adherent culture,the cells were digested with dispase and inoculated at the ratio of 1:2.Continuing to culture for another 6 days,that is,after a total of 12 days of adherent culture,the cells were suspended in culture.After 18-20 days of differentiation,the neurospheres were digested into single cells.A part of the cells were adhered to a polyguanylate/laminin-coated(PLO/laminin)coverslip and cultured.At different stages of cell differentiation,the expression of related functional markers Foxg1,Otx2,Sox1,Nestin,Ki67,Pax6,Tuj1,Map2,Synapsin,etc.were detected by immunofluorescence staining.Results 1.Under the action of SMAD signaling pathway inhibitors DMH1 and SB431542,when h ESCs were continuously induced for 10 days,the typical rosette-like neuroepithelial cells were evidently appearing,which could express forebrain markers Foxg1,Otx2 and neuroepithelial cells markers Sox1,Pax6.2.In the neural precursor stage,immunofluorescence staining results showed that the cells were differentiated toward the direction of dorsal forebrain and expressed the markers of forebrain neural stem cells Ki67,Nestin,Foxg1,and Pax6,a marker of dorsal forebrain.3.The cells were adherently cultured in vitro for 1 month,that is,after 50 days of differentiation,the cells could differentiate into mature neurons,which were glutamatergic neurons,and emitted nerve filaments and could express synapsin marker Synapsin.ConclusionsUsing a monolayer differentiation cell culture method,h ESCs could be induced to differentiate into the dorsal forebrain h NSCs when the SMAD signaling pathway inhibitors DMH1 and SB431542 were added at a specific stage,and the h NSCs could eventually differentiate into mature glutamatergic neurons and had the ability to form synaptic connections between cells.ObjectiveTo observe the effects of repetitive transcranial magnetic stimulation(r TMS)combined with h NSCs transplantation on the improvement of motor function in rats with cerebral ischemia,and to explore the possible mechanism of neurological function improvement in rats after cerebral ischemia.MethodsMale Sprague-Dawley(SD)rats,weighing 230-250 g,were selected.The model of cerebral ischemia was generated by middle cerebral artery occlusion(MCAO),then the rats were divided into four groups:(1)the control group(vehicle): rats were transplanted with cell culture medium;(2)h NSCs transplantation group(h NSCs): 2.5 × 105 h NSCs were transplanted into the rats' striatum;(3)r TMS treatment group(r TMS): rats were injected with an equivalent cell culture medium and were given r TMS treatment;(4)h NSCs + r TMS combined treatment group(h NSCs + r TMS): 2.5 × 105 h NSCs were transplanted into rats,and r TMS treatment was also given at the same time.HNSCs transplantation was performed 4 days after the surgery of ischemia stroke and r TMS stimulation was given 24 hours later,which was continuously administered for 28 days.At the same time,each group was injected with the immunosuppressant cyclosporine A(10 mg/kg)daily.At different time points,that was,before modeling,before cell transplantation,and after cell transplantation,the behavioral changes of rats were detected by grabmeter,rope grabbing test,and tail lifting test.After all animals completed the behavioral tests,some rats were deeply anesthetized with 10% chloral hydrate and decapitated to get fresh material.Western blot was applied to detect the expression of related protein,such as BDNF,Trk B,and p-Trk B.Another part of rats were perfused with physiological saline and 4% paraformaldehyde.Then tissues were sectioned to detect the the size of rat cerebral infarction and the proliferation of NSCs in the affected SVZ area by nissl staining and immunofluorescence staining,respectively.Results1.MCAO model of SD rats ischemia-reperfusion were successfully established,and h NSCs transplantation and r TMS treatment were also performed.The analysis results of the survival rate of the rats in each group showed that the survival rate of rats was not statistically significant between groups(P>0.05).2.The infarct volume test results showed that compared with the vehicle group,the cortical width index of the h NSCs + r TMS group increased significantly(P<0.05).The results of nissl staining showed that compared with the vehicle group,the infarct volume of the h NSCs + r TMS group was significantly reduced,and it was statistically significant(P<0.05).3.The results of animal behavior studies showed that the combined treatment of h NSCs and r TMS significantly improved the grip on the hemiplegia side of the rats,compared with the vehicle group,h NSCs group,and r TMS group(P<0.001,h NSCs + r TMS vs vehice;P<0.001,h NSCs + r TMS vs h NSCs;P<0.01,h NSCs + r TMS vs r TMS);compared with the vehicle group,the r TMS group had a statistically significant recovery in the grip function of the hemiplegia side(P<0.01).In the rope grabbing experiment,compared with the vehicle group and the h NSCs group,the combined treatment of h NSCs and r TMS had a significant effect on the recovery of the forelimb grasping function in rats(P<0.001,h NSCs + r TMS vs vehice;P<0.01 h NSCs + r TMS vs h NSCs);compared with the vehicle group,the r TMS group had a statistically significant recovery in grip function(P<0.05);in the grip test and the rope grabbing experiment,the combination of h NSCs and r TMS accelerated the neurological recovery in rats compared with the vehicle group,h NSCs group,and r TMS group.In the tail lifting experiment,the functional improvement in combined treatment of h NSCs and r TMS was statistically promoted compared with the vehicle group(P<0.05),and there was no statistical significance between the other groups(P>0.05).4.Using Ki67+/Nestin+ to detect the proliferation of NSCs in the affected SVZ area,the results showed that compared with the vehicle group,the number of NSCs in the h NSCs group,r TMS group,h NSCs and r TMS combination treatment group was more and there were significant differences(P<0.05,h NSCs vs vehicle;P<0.01,r TMS vs vehicle;P<0.001,h NSCs + r TMS vs vehicle);compared with h NSCs group,the number of NSCs in the h NSCs and r TMS combined treatment group had a statistically significant difference(P<0.01).5.The results of western blot study showed that compared with the vehicle group,the expression of BDNF and p-Trk B in the r TMS group were increased significantly and there were significant differences(P<0.01);compared with the vehicle group,the h NSCs and r TMS combined treatment group had a statistically significant expression of BDNF(P<0.01),and p-Trk B expression was also increased significantly(P<0.001);compared with the h NSCs group,the h NSCs and r TMS combined treatment increased the expression of BDNF with statistical differences(P<0.05),p-Trk B expression was also increased significantly(P<0.01);there was no significant difference in Trk B expression between groups(P>0.05);compared with the vehicle group,p-Trk B/Trk B in the r TMS group and h NSCs and r TMS combination treatment group were significantly increased and significantly different(P<0.01,r TMS vs vehicle;P<0.001,h NSCs + r TMS vs vehicle);compared with h NSCs group,p-Trk B/Trk B was increased significantly with significant differences(P<0.01).ConclusionsThe recovery of motor function in rats treated with r TMS combined with h NSCs transplantation was better than the treatment with h NSCs or r TMS,but the combination treatment couldn't improve the survival rate of rats with cerebral infarction.The therapeutic effects may be related to reducing the infarct volume of brain tissue,promoting the proliferation of NSCs in SVZ,and activating BDNF-Trk B signaling pathway.ObjectiveThe exogenous h NSCs were transplanted into the striatum of MCAO rats.Under the intervention of r TMS,the effects of r TMS on the survival,proliferation and differentiation of h NSCs were studied,and the maturation of h NSCs and the formation of synaptic connections were also observed.MethodsMale Sprague-Dawley(SD)rats,weighing 230-250 g,were selected.The model of cerebral ischemia was generated by middle cerebral artery occlusion(MCAO),then the rats were divided into two groups:(1)h NSCs transplantation group(h NSCs): 2.5 × 105 h NSCs were transplanted into the rats' striatum;(2)h NSCs + r TMS combined treatment group(h NSCs + r TMS): 2.5 × 105 h NSCs were transplanted into rats,and r TMS treatment was given at the same time.HNSCs transplantation was performed 4 days after the surgery of ischemia stroke and r TMS stimulation was given 24 hours later,which was continuously administered for 28 days.At the same time,each group was injected with the immunosuppressant cyclosporine A(10 mg/kg)daily.After 28 days of r TMS treatment intervention,some rats were deeply anesthetized with 10% chloral hydrate,and then perfused with physiological saline and 4% paraformaldehyde.Tissue sections were examined by immunofluorescence for the survival,proliferation and differentiation of transplanted h NSCs.Another part of rats continued to be observed and were sacrificed by anesthesia at 3 months,and tissue sections were stained by immunofluorescence staining.Results1.Compared with the h NSCs group,with the intervention of r TMS,the survival number of transplanted cells in the h NSCs + r TMS group was significantly increased and statistically significant(P<0.05).2.The results of immunofluorescence staining showed that compared with the h NSCs group,the percentage of Ki67+/Hu Nu+ in the h NSCs and r TMS combination treatment group was significantly reduced(P<0.05),the percentage of Tuj1+/Hu Nu+ in the h NSCs and r TMS combination treatment group was significantly higher than that in the h NSCs group(P<0.05).3.After 3 months of cell transplantation,the transplanted cells could still survive in MCAO rats;morever,mature neurons expressing Neu N were significantly increased,while proliferating cells were significantly decreased;3 months later,the results of immunofluorescence staining showed that the transplanted cells could differentiate into glutamatergic neurons and express synaptic-associated proteins Synaptophsin.Conclusionsr TMS could promote the survival of transplanted h NSCs,reduce the proliferation of h NSCs,and promote their differentiation into neurons;Three months after cell transplantation,h NSCs could differentiate into mature glutamatergic neurons and had the ability to form synaptic connections and rebuild neural circuits. |
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