Valproic Acid Exposure Inhibits The Development Of Outer Radial Glial Cells In Human Brain Organoids And The Underlying Mechanism

BackgroundAutistic Spectrum Disorder（ASD）is a severe neuropsychiatric disorder characterized by emotional expression difficulties,social interaction impairment,stereotyped and repetitive movements and behaviors,and inability to carry out normal language expression and social activities.In recent decades,growing evidence has led to the concept that neurogenesis defects,especially the abnormal formation of cerebral cortex structures during critical periods of development,may be a common pathophysiological mechanism of ASD.Taking the antiepileptic drug valproic acid（VPA）during pregnancy significantly increases the risk of ASD in offspring.The administration of high-dose VPA on gestational day 12.5,a critical stage of embryonic neurogenesis,can cause autism-like behavior in offspring rodents,which has been widely used as an animal model for autism research.However,the key pathways and molecular mechanisms of VPA causing autism are still unclear.The cortical developmental disorder is regarded as an important structural basis for the development of autism-like behaviors.Recently,single-cell sequencing results show that upper-layer cortical neurons are the earliest to show abnormalities and are most correlated with the clinical severity of ASD.In POGZ^WT/Q1038R mouse model of ASD,the number of excitatory pyramidal neurons in the upper cortical layer were decreased.Therefore,the generation and migration of upper pyramidal neurons are key to the construction of the normal cortical structure,and abnormalities in the number and morphology can lead to the occurrence of ASD.During embryonic development,the upper-layer pyramidal neurons are mainly generated by outer radial glia cells（o RGCs）,which locate in the outer subventricular zone（o SVZ）.Importantly,the upper-layer pyramidal neurons migrating along with the radial processes of o RGCs to the designated position in the cortical plate（CP）.o RGCs are extremely conserved in humans,and are largely absent in the developing cortex of lissencephalic rodents.Ethical issues pose a unique challenge to investigating the different developmental stages of fetal brain,and hinder our comprehensive understanding of the role of o RGCs in pathogenesis of ASD.Recent studies have shown that using three-dimensional（3D）culture combined with the sequential addition of low-concentration growth factors in suspension culture,human embryonic stem cells（h ESCs）or induced pluripotent stem cells（i PSCs）can spontaneously form brain organoids with typical cortical-like structures in vitro.More importantly,the cortical-like structures formed in vitro can recapitulate the dynamic developmental process of the human cerebral cortex,and can detect the o SVZ containing the o RGCs.Thus,the brain organoids are a good model for studying the VPA involve in ASD through influencing the development of o RGCs.Methods1.The dorsal cerebral organoids were obtained by 3D culture of h ESCs combined with cyclopamine A（Cyc A）to enhance dorsal forebrain identity,and defined as―dorsal^{Cyc A}‖,while cerebral organoids without Cyc A were defined as―dorsal^Unt‖.Identification of the dorsal specificity of dorsal^{Cyc A} organoids using immunofluorescence staining and quantitative real-time PCR（q RT-PCR）on days 20（D20）.Investigation of the specificity and continuity of dorsa^{l Cyc A} organoids using immunofluorescence staining.Dorsal^{Cyc A} organoids at different developmental stages（D28,D56,and D84）were sequenced by RNA-Seq,pearson correlation analysis was performed with human organ tissues to verify the reliability of dorsal^{Cyc A}organoids mimicry of the early development of the human embryonic cortex.The VPA-exposed dorsal^{Cyc A} organoid model was well established,the differential expression genes（DEGs）were obtained by RNA-Seq,and compared with the ASD risk-related gene set to clarify the changes of autism-related genes in the VPA-exposed organoids.2.To examine the effect of VPA exposure on the morphology and size of organoids on D28,D42 and D56,we performed immunohistochemical staining to detect the expression characteristics of radial glia cells（RGCs）markers（SOX2）and neuronal differentiation markers DCX in the dorsal^{Cyc A} organoids,then quantified the thickness of SOX2⁺ventricular zone-like（VZ）layer and DCX⁺CP layer.The proliferation and apoptosis of SOX2 in the VZ region were detected by ki67,Ed U,and TUNEL staining.Finally,the effect of VPA exposure on early cortical neurogenesis was clarified.3.Immunofluorescence staining was used to detect the expression and distribution of SOX2,intermediate progenitor cell marker（TBR2）,and deep neuron marker（CTIP2）in dorsal^{Cyc A} organoids at D56.The expression and distribution of outer radial glia cells（o RGCs）markers（HOPX）in the dorsal^{Cyc A} organoids on D56,D70 and D84 were detected.The expression and distribution of deep cortical neuron marker（TBR1）and upper-layer neuron marker（SATB2）were detected to clarify the effect of VPA exposure on the distribution and migration of dorsal^{Cyc A} organoids.RNA-Seq analysis to detect the gene expression in dorsal^{Cyc A} organoids on D56 and D84.The DEGs were screened for Gene ontology（GO）and pathway enrichment.4.RNA-Seq analysis to detect the gene expression in dorsal^{Cyc A} organoids at D28,and q RT-PCR technology was used to verify the m RNA expression of four selected genes to clarify the reliability of sequencing results.According to the criteria,DEGs were screened out,and GO functional enrichment,pathway enrichment,and Protein-Protein Interaction（PPI）Networks analysis were performed on DEGs.The effect of VPA on early neurogenesis was determined to be mediated through the Wnt/β-catenin pathway.The expression and distribution of acetyl-H3 were detected by immunofluorescence technique to determine the protein acetylation in VPA-exposed forebrain organoids.VPA analog VPD andβ-catenin inhibitor IWR-1 combined with VPA intervened in the dorsal^{Cyc A} organoids,using morphological methods,immunofluorescence,q RT-PCR,and western blotting techniques to detect whether VPD can reproduce the function of VPA and whether IWR-1 can rescue the effect of abnormal neurogenesis in the VPA-exposed dorsal^{Cyc A} organoids.Results1.The successful establishment of h ESCs derived 3D dorsal forebrain organoids model can well simulate the early and middle development of the human embryonic cerebral cortex.（1）Immunohistochemistry detected the expression of dorsal forebrain specific marker PAX6 and forebrain specific marker FOXG1 in dorsal^{Cyc A} organoids in the D20.q RT-PCR confirmed that the expression of PAX6 was increased,while the ventral forebrain-specific marker NKX2.1 was decreased in the dorsal^{Cyc A} organoids compared with the dorsal^Untorganoids,demonstrating the successful establishment of a 3D dorsal forebrain organoids model.（2）The immunostaining results of organoids at different developmental times（D20,D28,D42,D56,D70 and D84）show that the dorsal^{Cyc A} organoids displayed developmental specificity and continuity,and it can be used to detect key cytological events of cortical development in a strict temporal and spatial order,including cell proliferation,differentiation,and maturation,a large number of astrocytes are formed at D84 and present a typical lamellar structure.（3）Pearson’s correlation analysis indicated that the dorsal^{Cyc A} organoids from three time points（D28,D56,D84）were strongly correlated with fetal brains and spinal cord,but had little or no correlation with other fetal somatic tissues.Further comparison with the transcriptomes of human dorsolateral prefrontal cortex samples from six life stages,ranging from fetal development to aged human tissue,showed the highest correlation with fetal brain tissues,with the best correlation for D56 organoids.Comparison with transcriptomes from 16different human brain regions at 8 developmental stages,showed D28 organoid profiles were closely related to several subregions of the prefrontal cortex at 8 post-conception week（PCW）,and profiles at D56 and D84 organoids were more closely related to 12-24 PCW in multiple cortical areas.The comparisons of organoid profiles with transcriptomes of the human dorsolateral prefrontal cortex,orbital frontal cortex,and ventrolateral prefrontal cortex showed a temporal correlation between our preparation and the development of the dorsolateral prefrontal cortex.Thus,the dorsal^{Cyc A} organoid can simulate the development process of the human fetal cortex,and it can be used as a good model to study the early development of cortex in VPA-induced autism.（4）We compared the three different concentrations（100μM,250μM and 500μM）of VPA-induced DEGs transcriptional profiles with three sets of ASD risk-associated genes.The results showed 14,50,and 80 genes overlapping with 100μM,250μM,and 500μM from the SFARI database.Moreover,these three different concentrations of VPA-induced DEGs significantly overlapped with genes dysregulated in Psych ENCODE ASD brains（Overlapped genes=16,65,123）and ASD patient-derived organoids（Overlapped genes=83,219,271）.Notably,4 DEGs such as ABRB2,SCN1A,GRID2,and SLC24A2,shared in all the three ASD gene sets with 250μM and 500μM VPA.Thus,the dorsal^{Cyc A} organoids can be used to study the functional impact of dynamic expression of ASD risk genes during human brain development.2.Effects of VPA exposure on the development of RGCs and lamellar structure of dorsal^{Cyc A} organoids.（1）At D28,both surface area and diameter were indistinguishable in the organoids with VPA treatment at 100μM and 250μM compared with control organoids.However,the organoids with 500μM VPA treatment displayed markedly smaller surface area and diameter than control organoids.On D42 and D56,both 250μM and 500μM VPA treated organoids were markedly decreased in the surface area and diameter compared with control organoids,whereas alterations were not detected in the organoids with 100μM VPA treatment.These results suggest that VPA treatment induced a reduction in the organoid size in time-and dose-dependent.（2）The statistical results of VZ thickness in dorsal^{Cyc A} organoids at different developmental stages（D28,D42 and D56）showed that 250μM and 500μM VPA treatment caused a substantial reduction in the VZ thickness on D28,D42,and D56 organoids,whereas100μM VPA treated organoids were not significantly altered in the VZ thickness although a slight decrease trend was observed at D56 organoids.The statistical results of CP thickness show that 100μM,250μM and 500μM VPA treatment did not alter CP thickness at D28,and100μM and 250μM VPA treatment did not alter CP thickness at D42 and D56,whereas500μM VPA treatment caused a significant reduction.It is supposed that higher doses of VPA treatment delayed neural differentiation related to a decrease in the NPCs pool.（3）The proliferation of NPCs was detected with Ed U and Ki67 immunostaining.Ed U pulse experiments and immunostaining of Ki67 revealed that 250μM and 500μM VPA treatment displayed a decreased percentage of Ed U⁺and Ki67⁺cells in the VZ-like regions.While,100μM VPA treatment did not affect the ratios of Ed U⁺and Ki67⁺cells in the VZ significantly.We further found markedly increased TUNEL⁺cells in the VZ-like regions of250μM and 500μM VPA treated organoids on D28,D42,and D56.The organoids treated with 100μM VPA showed no statistical difference in the percentage of TUNEL⁺cells in the VZ on D28,D42,and D56 compared to control organoids.Therefore,these results suggested that a higher dosage of VPA treatment decreased the NPCs pool due to inhibition of the proliferation of NPCs and promotion of its apoptosis.（4）At D56,VPA treatment induced a notable increase in IPCs distribution in the VZ-like region whereas a reduction in the SVZ-like region.Both 100μM and 250μM VPA treatment increased the distribution of CTIP2 in the VZ-like region,but 500μM VPA treatment caused a decreased trend of CTIP2 distribution in the SVZ-like region.In addition,analysis of the relative cell proportion revealed that VPA treatment caused a significant decrease in the SOX2⁺cells in the VZ-like regions and IPCs in the SVZ-like regions.We also noticed that 500μM VPA treatment reduced the CTIP2⁺neurons in the CP-like regions.These results suggested that VPA exposure dampened the migration of IPCs and early born neurons.RNA-Seq results showed that VPA exposure led to changes in biological processes related to neurogenesis,generation of neurons,regulation of cell differentiation,embryo development,and positive regulation of cell population proliferation.（5）We can detect SOX2⁺/HOPX⁺double-stained o RGCs in the VZ as early as D56,and VPA treatment reduced the SOX2⁺/HOPX⁺cells.On D70 and D84,the organoids contained lots of o RGCs located in the o SVZ-like layer,and VPA treatment reduced the ratios of SOX2⁺/DAPI⁺and HOPX⁺/DAPI⁺.we performed RNA-seq in whole organoids of 0μM,100μM,250μM,and 500μM VPA treatment on day 84.Several genes associated with RGCs and o RGCs markers were down-regulated in VPA treated organoids,especially the o RGCs markers,RT-q PCR further verified the RNA-Seq results.GO and pathway enrichment on the DEGs revealed several biological processes related to reproduction and embryo development and NPCs proliferation（SOX2,OCT4,NANOG）pathways that were significantly altered.On D70,VPA treated organoids at different concentrations showed comparable TBR1 expression in the cortical bins compared with control organoids.In contrast,250μM and 500μM VPA treated organoids showed decreased SATB2 expression across the entire CP,although 100μM VPA treatment did not alter the SATB2 expression in cortical bins.These results revealed that250μM and 500μM VPA treatment impaired neuronal fate specification of upper layers in the CP of cultured organoids.3.The mechanism of abnormal development of RGCs in VPA treated dorsal^{Cyc A}organoids.（1）The RNA-seq on D28 organoids showed that there were 528,670,and 794differentially expressed genes（DEGs）between the 100μM and 0μM,250μM and 0μM,and500μM and 0μM,respectively,with an overlap of 31 genes,280 genes co-regulated by250μM and 500μM.（2）Gene Ontology（GO）and pathway enrichment were performed on these 31 DEGs.GO enrichment revealed significant enrichment for genes involved in several biological processes such as anatomical structure formation involved in morphogenesis,tissue morphogenesis and stem cell differentiation.In accordance,significant enrichment in pathway analysis showed to be associated with Wnt signaling pathway and pluripotency,signaling pathways regulating pluripotency of stem cells,and transcriptional regulation of pluripotent stem cells.（3）GO enrichment was performed on the co-regulated 280 DEGs and revealed significant enrichment for genes involved in the―regulation of cell population proliferation‖,suggesting a potential involvement of higher doses of VPA in the inhibition of NPCs proliferation.PPI network showed that the Wnt signaling pathway community contains the most pathway-associated genes,supposed to contribute to maintaining the NPCs pool within the early organoid development.（4）VPA treatment elevated the level of Acetyl-H3 in cortical organoids significantly compared to controls.Western blotting（WB）showed that 500μM VPA sharply increased theβ-catenin level,which can be inhibited by IWR-1.IWR-1 efficiently rescue VPA-induced reduction in the surface area and VZ-like thickness,while exposure to VPD did not affect the VZ-like thickness.These results suggest that Wnt/β-catenin pathway and inhibition of HDAC play a critical role in VPA induced abnormal neurogenesis.ConclusionThe present study has successfully established of h ESCs derived 3D dorsal forebrain organoids model and the possible mechanism that VPA exposure leads to abnormal neurogenesis and reduction of o RGCs production,resulting in the disorder of lamellar structure development,which may be the possible mechanism underlying VPA exposure involved in the development of ASD.