Establishment Of Epilepsy-specific IPS Cells And Induced Them Into Neurons In Vitro

Epilepsy is one of the most common neurological diseases, characterized mainly by atypical neuronal network electric activity. The pathogenesis of epilepsy is still not very clear. In order to study the pathogenesis of epilepsy and mechanisms of resistance,animal models are widely used, but the animal models are limited and usually do not fully recapitulate the human neural phenotype.Like the ESCs, iPSCs have the ability to self-renew and their developmental potential to become all cell types in the body. The reprogrammed iPSC acquired from patients somatic cells retain the same genetic background of the donor individual,and there is no the shortcoming of ESCs for its shortage in the resource, so in the field of regenerative medicine and cell replacement therapy iPSc have broad application prospects. At present, the iPSC technology is applied to the reports of neurological diseases, but iPSC technology applied to epilepsy is still lack of relevant experience.Efficiency and security of different types of somatic cells induced to iPS cells is quite different. To date, the research of reprogramming neuroectodermal cells to iPSCs is still lacking.In our study, we propose the strategies for how iPSCs can potentially be used to gain insights into the biology of epilepsy by analyzing the electrophysiological characteristics of the candidate GABRG2gene.Parti Reprogramming studies of neural progenitor cells in patients with epilepsyObjective:To evaluate the feasibility of transferring four genes (Oct4, Sox2, c-Myc, and Klf4) into the neural progenitor cells of patients with epilepsy to acquire induced pluripotent stem cells in retroviral vector and compare the results with the fibroblast acquired iPSC.Method:1, The hippocampus tissue removed by surgery in patients with drug-resistant epilepsy were isolated and cultured to get neural progenitor cells. Cut part of the scalp tissue to obtain skin fibroblasts;2, Established retroviral transfection system;3, To construct the retroviral expression vector carrying the four factors preparation of neural progenitor cells and adult fibroblasts integrated GFP-Nanog promoter;4, Transfected and produce iPSCs;5, Take the selection of iPS clone and identification of the iPSC, including alkaline phosphatase staining (AP) and immunohistochemistry, teratoma generated experiments.Results:18days after the retroviral transfection, the iPS clone similar to hES cell colony morphology appeared.25to30days, about0.2%of transfected neural progenitor cells and0.1%of transfected fibroblasts showing Nanog promoter-GFP (+).12weeks after the formation, the iPSC clones showed positive alkaline phosphatase staining, inoculated into SCID mice can be formed teratomas.Conclusion:Transferred four kinds of genes into neural progenitor cells with the use of retroviral could obtain iPSCs, the iPSC established ESC morphology and characteristics of pluripotent differentiation. Under the same conditions, induced human neural progenitor cells generate iPSC efficient than fibroblasts. Part2IPSC into neurons in vitro directed differentiation of epilepsy patientsObjective:To study the conditions of iPSC neurons directed differentiation in vitro and to analyze if the iPSC aquired from the patients with epilepsy have the same characteristic of epilepsy neural electrophysiological performance.Methods:1, Culture the iPS cells derived from fibroblast and neural progenitor cell in the MEF feeder layer;2, Amplification for three generations or more, clone isolated from MEF on collagen;3, Using N2B27medium induced to differentiate into neurons;4, Immunofiuorescence staining neurons, analysis Tujl and MAP2expression;5, Patch clamp technique to detect any abnormal discharge in the neurons and any related receptor function change.Results:iPSC derived from fibroblasts and neural progenitor cells can be stabilized passaged20generations, and high expression of OCT4and SOX2, Tuj1positive cells in the induction of2-3days can be detected and10days MAP2positive cells can be detected. To normal neurons in contrast, the frequency of spontaneous discharge in the neurons increased significantly, part of them reduce the reaction to exogenous GABA.Conclusion:Through the N2B27medium iPSC can be induced to neurons in vitro. These neurons spontaneous discharge frequency increases, has the characteristics of neurons with epilepsy. Part of the neurons for external GABA reaction down tip mechanisms of drug-resistance epilepsy and GABA receptors can be relevant. Part3Drug-resistant epilepsy GABRG2gene mutation and polymorphismObjective:To explore the possible genetic mechanism of drug-resistant epilepsy through analysis of the mutation and polymorphism in GABRG2gene, which coding the y2subunit of the GABAA receptor.Methods:12drug-resistant epilepsy patients undergoing surgery, extracted peripheral blood for DNA extraction and determination of DNA concentration, PCR amplification of10exon sequence of the GABRG2gene, PCR products were gel purified using the sequencing box to its fluorescently labeled bi-directional sequencing, and analyzed results by ChromasPro and AlignX.Results:No.1,3,4,6,7,10,11,12patients were found exon3105Asn C315T AAC/AAT, C mutated to T; No.2,3,4,5,7,8,9,10,11patients were found exon5196Asn C588T AAC/AAT, C mutated of T. AAC and AAT are the encoding asparagine genetic code. The mutations detected were synonymous mutations. The exon3105Asn C315T AAC/AAT is polymorphism:C%is0.33, T%is0.67; exon5196Asn C588T AAC/AAT polymorphism:the same C%is0.25, T%is0.75.Conclusion:The study of drug-resistant epilepsy patients’ GABRG2gene, not found previously reported pathogenic mutations, but there exon3105Asn C315T AAC/AAT and C588T AAC/AAT polymorphisms, suggesting that the mechanism may be associated with drug-resistant epilepsy.