The Establishment Of IPSCs Model And Neural Cell Model For Schizophrenia

BackgroundSchizophrenia(SZ)is a highly heritable and serious mental disease,occurring in adolescence frequently,caused by the interaction of multiple factors.Due to the lack of disease model,its pathogenesis is still unclear.Various types of neurons either derived from iPSCs or induced from somatic cells of SZ patients provide a ideal cell model for studying the molecular events of pathogenesis and the neural cells differentiation in SZ disease in vitro.ObjectivesThis study aims to establish iPSCs and neural cell model for SZ patients.One model is induced pluripotent stem cells model,which was acquired from the skin fibroblasts of a SZ patient by reprogramming factors delivered by lentiviral vectors,and the other was induced neural cell model,which was directly induced from skin fibroblasts through the combined expression of three neural development key transcription factors.Methods1.Lentiviral plasmid of reprogramming factors construction: two fragments of OCT4-IRES2-SOX2 and C-MYC-IRES2-KLF4 were amplified from the plasmid of pEP4EO2SEM2 K by PCR and were connected to the lentiviral vector pLVX-IRESmCherry.Recombinant plasmid p LVX-OCT4-IRES2-SOX2 and pLVX-C-MYC-IRES2-KLF4 were obtained by enzyme digestion,linkage and transformation to E.coli.2.Lentiviral plasmids construction for neuron induction: ASCL1、BRN2 and MTT1 L were amplified from the human blood cDNA by PCR and were connected to the lentiviral vector pLVX-IRES-mCherry.Recombinant plasmid pLVX-ASCL1,pLVX-BRN2 and pLVX-MYT1 L were obtained by enzyme digestion,linkage and transformation to E.coli.3.Recombinant lentiviruses preparation and their infection activities evaluation: Recombinant lentiviruses were packaged in 293 T cells by co-transfection of the recombinant plasmids and packaging plasmids of pMD2.G and pSPAX2,and then the viruses were collected by concentration and ultrafiltration after 36 hours after transfection.The infection activity was evaluated by infecting 293 T cells,then the amount of viruses for infecting target cells were determined.4.Induction and identification of iPSCs derived from HDF cells of SZ patients.Skin fibroblasts from a patient were reprogrammed into iPSCs by recombinant lentiviruses containing OCT4-IRES2-SOX2 and C-MYC-IRES2-KLF4,and the pluripotency of iPSCs were identified by reverse-transcription PCR and Western blotting.5.Induction and identification of neuron from patients HDF cells with SZ.Skin fibroblasts derived from a patient were directely induced into neuron cells by recombinant lentiviruses containing ASCL1,BRN2 and MYT1 L.The induced neuron cells were indentified by reverse-transcription PCR and immunofluorescence.Results1.The sequencing results showed that the recombinant lentiviral vectors pLVX-OCT4-IRES2-SOX2(OCT4/SOX2)、pLVX-C-MYC-IRES2-KLF4(C-MYC/KLF4)for iPSCs were constructed successfully and the recombinant lentiviral vectors pLVX-ASCL1,pLVX-BRN2 and pLVX-MYT1 L for neuron induction were constructed successfully.Each recombinant lentilvirus was packaged in 293 T cells effectively.2.HDFs derived from a schizophrenia patient were successfully induced to iPSCs,and the clones were identified.3.HDFs derived from a schizophrenia patient were successfully derectly induced to neuron cells,and the induced neurons were identified.ConclusionsSkin fibroblast cells from a schizophrenia patient were successfully induced into iPSCs by co-expression of reprogramming factors,and into neurons by combined expression of three neural development related factors.The study provides approachs for making the cell models of schizophrenia,and thus facilitates the model the mechanism study and drug development of the disease in the future.