The Rapeutic Effect Of HESC-derived OPC On Ischemic Stroke In Rats

Objective: To induce functional hOPCs from hESCs in vitro efficiently and to verify the therapeutic effect of OPC implantation on Rat ischemic stroke during in vivo animal experiments.Methods: 1)hESCs were induced to differentiate into 3D OPC and 2D OPC in vitro using traditional and optimized culture protocols.Real-time fluorescence quantitative PCR(qPCR)was used to detect the OPC’s transcripts,such as OCT4,OLIG2,NKX2.2,SOX10 and PDGFR?,compareing to the expression of these genes in undifferentiated hESC;2)The genomics experiments were divided into 3D OPC(n=4),2D OPC(n=4)and undifferentiated hESC(n=4)groups.We analyzed the gene expressions in 2D OPC and 3D OPC comparing to hESC;3)We established rat MCAO/R(middle cerebral artery occlusion reperfusion)model.Nine SD rats were randomly divided into three groups,Sham(n=3),MCAO/R 1d(n=3)and MCAO/R 21d(n=3).TTC staining was used to evaluate cerebral infarct volume;4)Thirty-eight SD rats were randomly divided into Sham(n=7),MCAO(n=8),MCAO+ Vehicle(n=7),MCAO+ 3D OPC(n=8)and MCAO+ 2D OPC(n=8).Rats in MCAO+ 3D OPC and MCAO+ 2D OPC groups were treated with microinjection of OPCs into the brain,and MCAO+ Vehicle was injected with HBSS instead.On the 56 th day after surgery,we took the brain and observed the brain damage and evaluated cerebral cortex expansion.5)The above five groups of rats were subjected to neurological behavioral scoring at 1d,7d,14 d,21d,28 d,35d,42 d,49d,and 56 d after MCAO surgery,including Garcia score,adhesion removal test,forelimb use asymmetry test,and Rotarod test.Results: 1)Compared with undifferentiated hESCs,3D OPC and 2D OPC transcripts can express high level of OLIG2,NKX2.2,SOX10,and PDGF Ra genes as specific markers of OPCs(P<0.0001),while the undifferentiated stem cell markers OCT4 was relatively lower expressed(P<0.0001).2)After 3D OPC group,2D OPC group and undifferentiated hESC group were subjected to gene sequencing,principal component analysis(PCA)and differential gene analysis showed that there was no significant difference between 3D OPC and 2D OPC,while the two groups were significantly different from undifferentiated hESC.3)The brain tissue infarction in MCAO / R 1d group and MCAO / R 21 d group was significa ntly higher than that in Sham group(P <0.05).4)Compared with MCAO and MCAO + Vehicle groups,MCAO + 3D OPC group and MCAO + 2D OPC group had significantly reduced brain injury,and the cortex width index recovered significantly(P <0.05).5)Compared with MCAO and MCAO + Vehicle groups,MCAO + 3D OPC group and MCAO + 2D OPC group significantly improved their sensory and motor coordination ability and function(p <0.05).Conclusion: 1)hESC can efficiently and rapidly induce the differentiation of OPC,and the optimized experimental protocol can shorten the time course of the entire induction process.2)High-quality 3D OPC and 2D OPC can be induced from hESC by using both differentiation schemes.3)Transplantation of 3D OPC and 2D OPC can reduce brain damage caused by cerebral ischemia-reperfusion in rats,and significantly restore their neurological damage.