Inflammatory Cytokines Regulate The Migration,Proliferation And Differentiation Of Oligodendrocyte Progenitor Cells

Background:Hypoxia-ischemia activates the innate immune cells in the central nervous system and peripheral immune cells,producing the tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)that impede the development of oligodendrocyte progenitor cells(OPCs)and disrupt the normal myelination process.Preterm infants are more sensitive to hypoxic-ischemic injury,resulting in preterm white matter injury(PWMI).OPCs have the potential abilities of migration,proliferation and differentiation and differentiated into mature OLs,wrapping neuronal axons to form myelination.Even though they promote remyelination in vivo,exogenous OPCs transplantation has not yet to be a completely effective therapy.However,inflammatory microenvironment in the brain after PWMI may influence the function of transplanted OPCs.Therefore,in order to improve the success of transplantation,it is necessary to comprehend the effect of inflammatory cytokines on the function of transplanted OPCs.Objective:Based on the established cultivation system of human OPCs(hOPCs)derived from human adult neural stem cells(NSCs),to explore OPCs differentiation into mature oligodendrocytes(OLs)in vitro;to explore the effect of the addiction or withdrawal of TNF-α and IL-1β on the hOPCs migration,proliferation and differentiation.Methods:After 7 days in vitro,the immunocytochemistry and flow cytometry were performed to identify the specific marker of hOPCs,including PDGFR-α,A2B5,Olig2 and Sox1O.After differentiation of 16 days,the immunocytochemistry identified the myelination protein of mature OLs including specific markers Galc,PLP1 and MBP.With the addiction of inflammatory cytokines,Cell Counting Kit-8(CCK-8),Transwell assay,immunocytochemistry of proliferation marker Ki-67 or cell proliferation fold change,immunocytochemistry of PLP1 or RNA-sequencing(RNA-seq)are respectively performed to study the effect of inflammatory cytokines on OPCs cytotoxicity,migration,proliferation and differentiation.After incubating with OPCs for 7 days,the inflammatory cytokines were withdrawn to explore human OPCs migration,proliferation and differentiation.Results:The immunocytochemistry and flow cytometry identified the specific marker of human OPCs derived from NSCs including PDGFR-α,A2B5,Olig2 and Sox10.After 16 days of differentiation,OPCs differentiated into mature OLs with web-like processes or a membrane-like structure.Immunocytochemistry confirmed that OLs are positive of Galc,PLP1 and MBP,while negative of PDGFR-α.Without the cytotoxicity on OPCs,compared with the control group(without inflammatory cytokines),human OPCs migration cell number decreased in 10 ng/mL TNF-α,30 ng/mL and 60 ng/mL IL-1β treated group.10 ng/mL TNF-αand 30 ng/mL IL-1β inhibited the positive rate of proliferation marker Ki-67 and decreased cell proliferation fold change.Besides,they decreased the positive rate of mature oligodendrocyte marker PLP1 and activated signaling pathways that keep cells in the pluripotent state.Withdrawing 10 ng/mL TNF-α and 30 ng/mL IL-1β,human OPCs relative migration cell number increased but Ki-67 positive rate decreased.Withdrawing 30 ng/mL IL-1β,after OPCs differentiation,PLP1 positive rate was normal,while withdrawing 10 ng/mL TNF-α,PLP1 positive rate decreased.Conclusion:A large number of high purity human OPCs are induced from human adult NSCs,and have the potential of rapid differentiation in vitro.Both TNF-α and IL-1β inhibited the migration,proliferation and differentiation.Withdrawing inflammatory cytokines,human OPCs migration was improved while proliferation and differentiation were more sensitive to the stimulation of cytokines and failed to restore the normal level.