The Synergy Between IFNβ And BMP Signaling In Regulating The Proliferation And Quiescence Of Human Neural Stem Cells

Introduction:Adult neural stem cells promote damage repair,maintain organ plasticity and tissue homeostasis,and delay aging.Stem cell pools maintain self-renewal by quiescence and activation,whose imbalance may lead to developmental abnormalities,neurodegenerative disease and tumors.After injury these stem cells are induced to proliferate to quickly reestablish homeostasis.Conversely,excessive proliferation often results in the depletion of stem cell pools,leading to neurogenesis dsfunction.Now more and more research focus on how stem cells maintain the balance between quiescence/activation,to ensure neurogenesis and promote tissue damage repair after brain injury.Acute interferon(IFN)treatment of mice promotes the proliferation of dormant haematopoietic stem cells in vivo.The balance between quiescence and activation was orchestrated by texternal niche factors and intrinsic signaling pathways,including BMP,FGF,Notch and Wnt.BMP signaling has been reported to promote the quiescence of a variety of stem cells,including adult neural stem cells Disruption of BMP signaling induces excessive proliferation of stem cells and blocking IFN-I signaling in the brain of aged mice partially restored hippocampal neurogenesis Furthermore,BMP and IFN activity is positively correlated during tissue injury.Based on the dynamic expression and important function of BMP4 and IFNβ in the development of central nervous system,we focus on the role of IFNβ and BMP4 on human neural stem cells Our study will help to understand how neural stem cells can response to the niche factors and integrate the extrinsic and intrinsic signaling factors,which will shed light on the diagnosis and treatment of neurodegenerative diseases and brain tumors.Objective:The purposes of this study are as follows:1.Establish the culture system of quiescence and activation of human neural stem cells(hNSCs)in vitro.The establishment of the quiescence and activation system breaks the ethical barriers to human research and solves the bottleneck problem of limited neural stem cells isolated from mice available for genome-wide studies.2.Determine the synergy regulation of IFNβ/BMP4 in the proliferation-quiescence system of hNSC,which has a good technical and theoretical basis for resolving molecular network systems that control the proliferation-quiescence banlance of adult neural stem cells.3.Explore the synergy between IFNβ and BMP signalings in regulating the quiescence and proliferation of hNSCs.This study will provide sciencetific basis for the therapy of aging and glioma with BMP4 and IFNβ combination.Methods:1.Establishment of proliferation-quiescence system of human neural stem cells Human neural stem cells were derived from human ES.hNSCs were treated with different concentration of BMP4 and FGF.The proliferation ability and cell cycle changes were evaluated by ki67 staining and flow cytometry.2.Effect of IFNβ on the proliferation of human neural stem cells hNSCs were treated with different concentration of IFNβ and FGF.The proliferation ability and cell cycle change were detected by Ki67 immunofluorescence staining and flow cytometry3.Effect of IFNβ and BMP4 on the proliferation of human neural stem cells To determine the combined effect of IFNβ and BMP4 on the proliferation of human neural stem cells,hNSCs were treated with different concentration of IFNβ and BMP4.Cell growth and cell cycle were evaluated.4.Analysis of transcriptome level on the synergy action of BMP and IFNβ signaling in hNSCs Time series of transcriptome study was performed to explore the role of IFNβ and BMP signaling pathway in regulating quiescence and activation of human neural stem cells.Morpheus was applied to compare each experimental condition to the corresponding control and find the differential genes.The differential gene were import into the KEGG database in DAVID,the transcriptome data cluster analysis,the up-regulated or down-regulated gene signal pathway,the pathways were considered as differentially expressed between two conditions if they had a P-value<0.05 signal pathway.In a variety of processing from the regulatory role of the signal pathway.Analyze and compare the signal pathways that were up or down in different treatment groups.5.Analysis of mRNA and protein level on the synergy action of BMP and IFNβsignaling in hNSCs Changes of key regulators of IFNβ and BMP signaling during different treatment were examined by real-time PCR and Western blot.6.The synergy mechanism of IFNβ and BMP4 in regulating quiescence of hNSCs KEGG database,Gene Ontology(GO)database were used to manually annotate the functions of this group of genes and find out the pathways involved in these genes for preliminarily investigating the synergy mechanism of between BMP pathway and IFNβ signaling pathway.Results:1.The ESC derived human neural stem cells were positive for stem cell marks SOX2 and Nestin.The hNSCs are capable to differentiate into neurons and astrocytes.Furthermore,hNSC showed decreased proliferation rate and paused in G0/G1 phase upon BMP4 treatment.After BMP4 exposure,hNSCs are able to re-enter cell cycle with mitogen stimulation and differetionate into neuronas and astrocytes.2.IFNβ alone promoted the proliferation of hNSCs.3.IFNβ and BMP4 in incombination furthermore decreased the proportion of G0/G1 in hNSC compared with BMP4 alone.Thus,the treatment of IFNβ and BMP4 in combination enhanced the quiescence effect of BMP4,while inhibited the proliferation role of IFNβ.4.Upon the addition of BMP antagonist Noggin,the cell proliferation was almost recovered,in addition to cell morphology and cell numbers.The number of Ki67 positive cells were increased when Noggin was added to BMP4 and IFNβ treated hNSCs.The number of cells in G2-S phase were significantly increased.Antibody IFNAR1 failed to block the inhibitory effect from BMP4 and IFNβ cotreatment.5.Cluster analysis showed that differentially expressed genes of BMP4 and IFNβcotreatment were similar with these of BMP4 alone.6.Time series analysis of transcriptome data revealed that most IFNβ signaling pathway genes were strongly responsive to IFNβ and BMP4 treatment,compared to IFNβ alone.The BMP signal downstream genes,compared to BMP4 alone,were not sensitive to the treatment with combined BMP4 and IFNβ.7.Real-time PCR results showed key genes of IFNβ pathway respond quickly at 1.5 hour,reach the peak at 24h.We observed that four genes STAT1,IFIH1,ISG15,IRF7 and IFIT1 were significance increased when BMP4 and IFNβ treated together These four genes also increased when only IFNβ existence but the amplification range was lower than BMP4 and IFNβ treated together,at the same time,these four genes were almost no response to BMP4 only.Consistent with the result,the increased STAT1 protein was verified under BMP4 and IFNβ together by western blot.8.Bioinformatics analysis revealed that these genes were involved in IFN immune pathway,defense pathway,cell proliferation/differentiation/adhesion/migration/cell cycle pathway,apoptotic pathway,DNA remoding/DNA helicase activity,mRNA transport\RNA pol II promoter binding pathway.Among them,the anti-viral transcription factors induced by IFN immune response maybe involved the synergistical role of IFNβ/BMP4.Conclusion:1.We established the reversible proliferation-quiescence system of human neural stem cells in vitro.2.BMP4 inhibited hNSCs proliferation by inducing cells quiescence.IFNβdramatically stimulated the proliferation of hNSCs.3.BMP4 and IFNβ in combination dramatically reduced proliferation.The synergy regulation of BMP4 and IFNβ exist in hNSC.4.BMP4 promoted the response of IFNβ related genes and changed the effects of IFNβ on hNSCs.5.IFN immune pathway,defense pathway,cell proliferation/differentiation/adhesion/migration/cell cycle pathway,apoptotic pathway,DNA remoding/DNA helicase activity,mRNA transport/RNA pol Ⅱ promoter binding pathway maybe involved in the synergy regulation by IFNβ and BMP4.