Efficient Differentiation Of Neurons Derived From Human Induced Pluripotent Stem Cells And Its Application In Drug Toxicology Analysis And Molecular Mechanism Research Of Nervous System Diseases

Objective:iPSC-derived(induced pluripotent stem cell)motor neurons(MNs)play an important role in disease modeling and therapy development for motor neuron diseases,such as amyotrophic lateral sclerosis(ALS).So far,traditional strategies of inducing iPSC differentiation into motor neurons are based on compounds,which are relatively inefficient.Therefore,highly efficient differentiation methods are urgently needed.Synthetic mRNA is an advanced method for somatic cell reprogramming and has been successfully used in inducing differentiation in recent years.Therefore,in this study,we aimed to establish an efficient method to induce iPSC differentiation into motor neurons by applying synthetic mRNA,and provide models and platforms for further investigations into motor neuron diseases.ALS is a neurodegenerative disease characterized by progressive degeneration of cerebrospinal motor neurons with poor prognosis.Currently,only two drugs,riluzole and edaravone,have been approved by Food and Drug Administration(FDA)for the treatment of ALS.However,the clinical efficacy of edaravone in ALS treatment remains to be improved,and relevant mechanisms have not been clarified.Therefore,we aimed to elucidate the cellular actions and molecular mechanism of edaravone in motor neurons,and provide basis for clinical treatment with edaravone in ALS and other neurodegenerative diseases.Methods:We applied synthetic mRNA to explore efficiency of a variety of strategies and schemes of inducing iPSC differentiation into motor neurons.Quantitative reverse transcription PCR(RT-qPCR)and immunofluorescence staining were used to analyze the expression of neuronal markers.The patch clamp and multielectrode array(MEA)were used to evaluate the electrophysiological activity.Then,mRNA-induced motor neurons(miMNs)were generated from iPSC from healthy people and ALS patients using the method and applied in high-throughput platforms of neurite tracing and MEA to evaluate effects of edaravone,and RNA sequencing was performed to analyze transcriptional signatures.Methods including RT-qPCR,Western blot and enzyme-linked immuno-sorbent assay(ELISA)were used to investigate molecular mechanism of edaravone,which was further validated in mice by Western blot and ELISA.Results:In this study,we established a protocol efficiently inducing iPSC differentiation into motor neurons in healthy and ALS patients based on synthetic mRNAs of Olig2 and Ngn2 with modification at phosphorylation sites(Duration:4 days,motor neuron purity:90%),showing strengths over traditional differentiation strategies based on compounds(Duration:10-14 days;motor neuron purity:50%-70%).Further studies showed edaravone could protect miMNs from hydrogen peroxide-induced neurite damage and impairment of electrical activity by neurite tracing and MEA analysis.Transcriptomes were analyzed and results showed that edaravone activated neurotrophic factor signaling pathway(GDNF/RET pathway),which was validated in miMNs and mouse models.Results also showed addition of edaravone in medium without neurotrophic factor(GDNF/BDNF)promoted miMNs survival and maturation.Neurotrophic factor(VGF)induced by edaravone may be used as a potential biomarker for the treatment of ALS.In addition,we found edaravone also regulated expression of antioxidant enzymes(CAT,GPX7),glutamate receptors(such as GRIA1,GRID2,and GRIK4),and pre-apoptotic proteins(BAX).Conclusion:Synthesized mRNAs of Olig2 and Ngn2 with modification at phosphorylation sites highly efficiently induced iPSC differentiation into motor neurons,and could be applied in high-throughput platforms including neurite tracing and MEA analysis,providing important basis for researches of ALS and other motor neuron diseases.Edaravone could activate GDNF/RET neurotrophic signaling pathway and protect miMNs,and combination therapy of edaravone and GDNF may improve the clinical efficacy,and VGF may be a potential biomarker of response to edaravone treatment,providing evidences for the treatment with edaravone in ALS and other neurodegenerative diseases.Objective:The advancement of induced pluripotent stem cell(iPSC)technology has revolutionized human disease research and provided ideal humanized models for elucidating pathogenic mechanism,as well as for analyzing environmental toxins.Autism Spectrum Disorder(ASD)is a highly heterogeneous neurodevelopmental disorder characterized by a specific combination of impairments in social communication and repetitive behaviors,highly restricted interests and/or sensory behaviours.ASD was once thought to be relatively rare(1 in 1,000 children),but recent epidemiologic studies have estimated the prevalence of ASD at 1 in 54 children.The pathogenic mechanism of ASD has not yet been elucidated,and substantial evidences showed environmental factors played a role in the development of ASD.Styrene is a chemical solvent widely used in industrial production.Styrene oxide(SO)is the main metabolite of Styrene.Recent epidemiological evidences suggested styrene was associated with increased ASD risk,but the precise mechanism has not been clarified.In this study,we used human iPSC-based models to study styrene oxide-induced neurotoxicity and its association with ASD.Methods:We generated cortical neurons from induced pluripotent stem cells through doxycycline-inducible Ngn2 expression,which served as models for studying styrene oxide.We evaluated neurotoxicity of styrene oxide by lactate dehydrogenase assay,Calcein AM staining and neurite tracing as well as MEA analysis.RNA sequencing and transcriptome profiling were performed to reveal molecular changes induced by styrene oxide,and the association between styrene oxide and ASD was further investigated by RT-qPCR,western blot and immunofluorescence staining.Results:In this study,human induced pluripotent stem cells were converted into cortical neurons by doxycycline-inducible Ngn2 expression,and neuron markers were detected by immunofluorescence staining.Results showed styrene oxide(2000 μM)could induce 50%neuron death by lactate dehydrogenase assay,and low concentration of styrene oxide(10 μM)could cause neurite damage by Calcein AM staining.Lower concentration of styrene oxide(1μM)could inhibit spontaneous electrical activity by microelectrode array analysis,including total spike counts,mean firing frequency and total burst counts.ASD-associated synaptic proteins(SHANK1,PCDH19,SCN1A)were inhibited by styrene oxide through transcriptome profiling,which was validated by real-time quantitative PCR,western blot and immunofluorescence staining.Finally,quantitative real-time PCR and western blot results showed styrene oxide could inhibit expression of ASD-related synaptic proteins(SHANK1,PCDH19,SCN1A)in cerebral cortex of mice.In addition,results showed styrene oxide could also inhibit expression of multiple membrane receptor proteins(CHRNA10,UTS2R,SSTR1).Conclusion:Cortical neurons derived from human induced pluripotent stem cells provide practical models for studying environmental toxins.Styrene oxide could inhibit the spontaneous electrical activity,induce neurite injury and neuron death.Styrene oxide could inhibit expression of autism-related synaptic proteins(SHANK1,PCDH19,SCN1A)in vitro and in vivo,revealing the association between styrene oxide and ASD.Styrene oxide could also inhibit expression of multiple membrane receptor proteins(CHRNA10,UTS2R,SSTR1),suggesting broader neurotoxicity.