Induction And Differentiation Of Microglia Derived From Human Induced Pluripotent Stem Cells And Their Application In The Research Of AD Pathogenesis And New Drugs

Alzheimer’s disease(AD)is a chronic neurodegenerative disease,mainly manifested by progressive memory loss and cognitive impairment.Pathogenesis of AD is complex and has not yet been elucidated,which seriously hinders the development of effective drugs for the prevention and treatment of AD.In recent years,more and more studies have shown that neuroinflammation plays an important role in the occurrence and development of AD.As the permanent immune cells of the central nervous system,microglia cells play a key role in neuroinflammation.An increasi ng number of evidences show that the mechanism of neuroinflammation mediated by microglia cells is closely related to the occurrence and development of AD.On the one hand,microglia cells play a key role in tissue maintenance,injury response and pathogen defense of the central nervous system;on the other hand,continuously activated microglia can release a good deal of cytotoxic factors and mediate the phagocytosis of neuronal synapses.Microglia are very sensitive to external stimuli.Under the stimulation of various pathological conditions of AD,such as the deposition of β-amyloid protein(Aβ)and oxidative stress,microglia cells will be continuously activated,which is closely related to the occurrence and development of AD.Microglia has become one of the hot spots in AD research.The central nervous system is a complex system composed of neurons and a variety of glial cells.AD is not a lesion of a si ngle system or a certain cell group.More and more studies have shown that there is an inseparable relationship between the activation of microglial cells and neuronal injury in AD.Aβ and hyperphosphorylated Tau protein produced by AD neurons can activate microglia cells,suggesti ng that the overactivation of microglia cells may be the result of pathological damage of neurons.However,in recent years,there have also been reports about the overactivation of microglia cells before the occurrence of neuroinflammation,suggesti ng that the overactivation of microglia cells may be the cause of pathological damage of neurons.Therefore,the interaction between neurons and microglia plays an important role in the regulation of neuroinflammation and neurodegeneration,and the study of the interaction between them is of great significance for the understandi ng of the pathogenesis of AD.At present,the most commonly used sources of AD cells are immortalized cells and primary cells.But both sources of cells have their limitations.Although immortalized cells can expand and freeze and thaw repeatedly,they lose their physiological or pathological relevance as cell models due to genetic modification.Primary cells have good physiological or pathological correlation,but their source is very limited,especially human primary cells.Human induced pluripotent stem cells(HIPSCs)are reprogrammed from human cells to carry the genetic material of the patient,and the differentiated nerve cells contain genetic modifications and mutations that are involved in the development of the disease.Microglia induced by hiPSCs derived from patients with AD provide a good cell model for researchers and have a wide application prospect in the study of the pathological mechanism of AD and the research and development of anti-AD drugs.First of all,we used hiPSCs microglia differentiation scheme that our team set up early,the source of you ng people,cognitively normal elderly people and AD patients hiPSCs differentiation respectively for the correspondi ng microglia,observe the three sources microglial phenotype and function of the difference,and compare a variety of AD related stimulati ng factors of its influence;Then the interaction between microglia and neurons from three sources was studied.Finally,the anti-inflammatory immune activity of anti-AD drugs was evaluated by microglia derived from hiPSCs.Chapter 1 The induced differentiation of microglia derived from hiPSCs and the effects of various exogenous stimuli on its phenotype and function.Firstly,hiPSCs were induced into the correspondi ng microglia cells in you ng people,cognitively normal elderly people and AD patients usi ng the previously established protocol of induci ng the differentiation of HIPSCs into microglia cells.The effect of lipopolysaccharide(LPS)and various AD-related stimulati ng factors,includi ng hydrogen peroxide(H2O2),cortisol(CORT),β-amyloid oligomer(oAβ),on the phenotype and function of microglia cells from the three hiPSCs were observed.And differences in the response of microglia derived from hiPSCs to exogenous stimuli in you ng adults,cognitively normal elderly and AD patients.1.hiPSCs induced differentiation into microgliaWe applied the experimental scheme of induci ng differentiation of hiPSCs into microglia cells previously established in our laboratory,and induced differentiation of hiPSCs into microglia cells from three sources:you ng people,cognitively normal elderly people and AD patients.Flow cytometry was used to detect the positive cell rates of hiPSCs-derived hematopoietic progenitor cells CD43 and CD34 duri ng the differentiation process.The results showed that the positive cell rates of CD43 and CD34 were both over 90%,and there was no significant difference in the positive cell rates amo ng all groups.Immunofluorescence assay was used to detect microglia specific surface proteins IBA1 and TMEM119,and the results showed that the positive rate of microglia IBA1 and TMEM119 from HIPSCs was more than 90%,and there was no significant difference in the positive rate of microglia from HIPSCs amo ng all groups.These results suggest that hiPSCs from the three sources can all induce differentiation into microglia cells,and there is no significant difference in the differentiation efficiency amo ng all groups.2.Effects of various exogenous factors on phenotype and function of microglia derived from hiPSCs.HipSCs from you ng people,cognitively normal elderly people and patients with AD were successfully induced to differentiate into microglia cells.The cells were treated with LPS and AD-related stimulants H2O2,CORT and oA-β to observe the effects of various exogenous stimuli on the phenotype and function of microglia from different HipSCs sources,and the differences in response of microglia from the three sources.Application CCK-8 testi ng hiPSCs source microglia cell vitality,the application of neutral red devouri ng experimental detection hiPSCs source microglia ability,application lu minex testi ng cells secreted cytokines level(IL-10,TNF-α,IL-1β,IL-6,CXCL10 and CCL7),the application of chemilu minescence detection hiPSCs source microglia reactive oxygen species(reactive oxygen species,ROS)level,the application of ELISA kits oA beta levels in the cell.2.1 Effects of LPS on phenotype and function of microglia derived from hiPSCs.There was no significant difference in the cell viability of microglia derived from hiPSCs between the you ng and the cognitively normal elderly without LPS stimulation.The cell activity of microglia derived from hiPSCs in AD patients was significantly higher than that in cognitively normal elderly people.1μg/ml LPS significantly increased the cell viability of microglia derived from the three hiPSCs.There was no significant difference in cell viability between you ng hiPSCs-derived microglia and cognitively normal elderly.Cell viability of microglia derived from hiPSCs in AD patients was still significantly higher than that in cognitively normal elderly people.Without LPS stimulation,there were no significant differences in the phagocytic ability,ROS levels and cytokine secretion levels of hiPSCs-derived microglia between you ng and cognitively normal elderly.Phagocytic ability and ROS levels of microglia derived from hiPSCs in AD patients were significantly higher than those in the cognitively normal elderly,and there was no significant difference in cytokine secretion levels.1μg/ml LPS significantly increased the phagocytic capacity,cytokine secretion and ROS levels of microglia derived from hiPSCs in you ng adults,cognitively normal elderly and AD patients.There were no significant differences in the phagocytic ability,ROS level and cytokine secretion levels of microglia derived from hiPSCs between the you ng and the cognitively normal elderly.The phagocytic capacity,cytokine secretion and ROS levels of hiPSCs-derived microglia in AD patients were still significantly higher than those in cognitively normal elderly people.2.2 Effects of H2O2 on the phenotype and function of microglia derived from hiPSCs.There was no significant difference in the cell viability of microglia derived from hiPSCs between the you ng and the cognitively normal elderly without H2O2 stimulation.Cell activity of hiPSCs-derived microglia in AD patients was significantly higher than that in cognitively normal elderly people.25 μM H2O2 significantly increased the cell viability of microglia derived from the three hiPSCs.There was no significant difference in cell viability between you ng hiPSCs-derived microglia and cognitively normal elderly.However,the cell viability of hiPSCs-derived microglia in AD patients was still significantly higher than that of you ng people and cognitively normal elderly people.Without H2O2 stimulation,there were no significant differences in the phagocytic ability,ROS levels and cytokine secretion levels of hiPSCs derived microglia between you ng and cognitively normal elderly.Phagocytic ability and ROS levels of microglia derived from HipSCs in AD patients were significantly higher than those of you ng people and cognitively normal elderly people,and there was no significant difference in cytokine secretion levels.25 μM H2O2 significantly increased the phagocytosis ability,cytokine secretion and ROS levels of microglia derived from hiPSCs in you ng adults,cognitively normal elderly and AD patients.There were no significant differences in the phagocytic ability,ROS level and cytokine secretion levels of microglia derived from hiPSCs between the you ng and the cognitively normal elderly.The phagocytic ability,cytokine secretion and ROS levels of hiPSCs-derived microglia in AD patients were still significantly higher than those in cognitively normal elderly people.2.3 Effects of CORT on the phenotype and function of microglia derived from hiPSCs.There was no significant difference in the cell viability of microglia derived from hiPSCs between the you ng and the cognitively normal elderly without CORT stimulation.Cell activity of hiPSCs-derived microglia in AD patients was significantly higher than that in cognitively normal elderly people.25 μM CORT significantly increased the cell viability of hiPSCs-derived microglia from the three sources,and the cell viability of hiPSCs-derived microglia from AD patients was still significantly higher than that of you ng people and cognitively normal elderly people.There were no significant differences in the phagocytosis,ROS levels and cytokine secretion levels of hiPSCs-derived microglia between you ng and cognitively normal elderly without CORT stimulation.The phagocytosis and levels of ROS from hiPSCs in AD patients were significantly higher than those from you ng people and cognitively normal elderly people,and there was no significant difference in cytokine secretion levels.25 μM CORT significantly increased the microglial uptake ability,cytokine secretion and ROS levels in you ng people,cognitively normal elderly people and AD patients.There were no significant differences in the phagocytic ability,ROS level and cytokine secretion levels of microglia derived from hiPSCs between the you ng and the cognitively normal elderly.The phagocytic capacity,cytokine secretion and ROS levels of hiPSCs-derived microglia in AD patients were still significantly higher than those in cognitively normal elderly people.2.4 Effects of Aβ oligomer(oAβ)on phenotype and function of microglia derived from hiPSCs.There was no significant difference in the cell viability of hiPSCS-derived microglia between the you ng and the cognitively normal elderly without oAβ stimulation.The cell activity of hiPSCs-derived microglia in AD patients was significantly higher than that in cognitively normal elderly people.1 μM oAβ significantly increased the cell viability of microglia derived from three hiPSCs.There was no significant difference in the cell viability of microglia derived from hiPSCs between you ng and cognitively normal elderly people.Cell activity of hiPSCs-derived microglia in AD patients was still significantly higher than that of you ng people and cognitively normal elderly people.Without oAβ stimulation,there were no significant differences in the phagocytic ability,ROS levels and cytokine secretion levels of hiPSCs derived microglia between you ng and cognitively normal elderly people.The phagocytosis and levels of ROS from hiPSCs in AD patients were significantly higher than those from you ng people and cognitively normal elderly people,and there was no significant difference in cytokine secretion levels.1 μM oAβ significantly increased the phagocytic capacity,cytokine secretion and ROS levels of microglia derived from hiPSCs in you ng adults,cognitively normal elderly and AD patients.There were no significant differences in the phagocytic ability,ROS level and cytokine secretion levels of microglia derived from hiPSCs between the you ng and the cognitively normal elderly.However,the phagocytic ability,cytokine secretion and ROS levels of hiPSCs-derived microglia in AD patients were still significantly higher than those in you ng people and cognitively normal elderly people.2.5 Ability to degrade Aβ oligomer(oAβ)The content of 1 μM oAβ in cells at 0,3,6 and 9h after incubation was deter mined by ELISA kit.The results showed that the intake of oAβ in hiPSCs-derived microglia of AD patients was higher than that of you ng and cognitively normal elderly microglia.Ter mination of 0～9h after incubation,oAβ content in you ng people and cognitively normal elderly hiPSCs source microglial cells fall faster,9h when you ng and cognitively normal elderly hiPSCs source microglia in the oA no difference between the residual content of oAβ AD hiPSCs source of microglia in the oA beta residual content is significantly higher than cognitively normal elderly hiPSCs source microglia,prompt oAβ removal ability is lower than you ng people and cognitively normal elderly hiPSCs source microglia.These results suggested that all the four exogenous stimuli could cause phenotypic and functional cha nges of microglia from the three sources of hiPSCs.The cell viability,phagocytosis,cytokine secretion and ROS levels of hiPSCs-derived microglia in AD patients were significantly higher than those in cognitively normal elderly people before and after stimulation,but the ability to degrade oAβ was lower than that of you ng and cognitively normal elderly people.These results suggested that hiPSCs-derived microglia from the three sources had different responses to exogenous stimuli.Before and after multiple exogenous stimuli,hiPSCs-derived microglia from AD patients showed higher inflammatory response and oxidative stress response,and weaker ability to clear oAβ.Chapter II Interactions between microglia and neurons derived from hiPSCs,in you ng adults,cognitive normal elderly and AD patients.hiPSCs derived from you ng people,cognitively normal elderly people and AD patients were successfully induced to differentiate into microglia cells,and the effect of exogenous stimulation on microglia derived from hiPSCs was observed.In this part,the interaction between hiPSCs derived neurons and microglia cells was further studied.1.Effects of hiPSCs-derived microglia on the phenotype of hiPSCs-derived neurons.Application of you ng people,cognitively normal elderly people and AD patients hiPSCs source microglia medium supernatant,intervention in you ng people,cognitively normal elderly people and AD patients respectively source hiPSCs source neurons,observation to stimulate and give LPS stimulation condition,the three sources of hiPSCs source microglia supernatant of hiPSCs source neurons phenotypic effects of three types of sources.Cell viability was detected by CCK-8,neurite le ngth was detected by InCuCyte ZOOM real-time dynamic imagi ng analysis system,apoptosis was detected by TUNEL,and ROS level was detected by chemilu minescence.The results showed that the supernatant of hiPSCs-derived microglia cells in AD patients without LPS stimulation had no significant effects on the cell viability,neurite le ngth,apoptosis level and ROS level of hiPSCs-derived neurons in you ng adults.The cell viability,neurite le ngth and apoptosis level of hiPSCs-derived neurons in cognitively normal elderly people were not significantly affected,but the ROS level was significantly increased.Can significantly reduce the cell viability and neurite le ngth of hiPSCs-derived neurons in patients with AD,and significantly increase the level of neuronal apoptosis and ROS.The supernatant of microglia derived from hiPSCs in you ng adults,cognitively normal elderly and AD patients treated with LPS stimulation could significantly reduce the cell viability of hiPSCs derived neurons in each group,reduce the neurite le ngth,and increase the level of neuronal apoptosis and ROS.Compared with you ng people and cognitive normal elderly people,the supernatant of hiPSCs-derived microglia in AD patients after LPS stimulation decreased the cell viability and neurite le ngth of hiPSCs-derived neurons from the three sources most significantly,and increased the level of neuronal apoptosis and ROS,and the effect on HiPSCS-derived neurons in AD patients was the most obvious.It is suggested that hiPSCs-derived microglia and three kinds of hiPSCs-derived microglia can damage neurons in AD patients after stimulation by LPS,and the damage effect of AD microglia on neurons from all sources is the most obvious,and the effect on hiPSCs-derived neurons in AD patients is the most significant.2.Effects of hiPSCs-derived neurons on the phenotype of hiPSCs-derived microglia.The cell culture medium supernatant of hiPSCs-derived neurons in you ng,cognitively normal and AD patients was used to intervene hiPSCs-derived microglia in you ng,cognitively normal and AD patients,respectively,to observe the effects of hiPSCs-derived neurons from three sources on the phenotype and function of hiPSCs-derived microglia from three sources.The results showed that the cell viability,phagocytosis ability and ROS level of hiPSCs-derived microglia were not affected by the supernatant of hiPSCs-derived neurons in you ng and cognitively normal elderly.In AD patients,the supernatant of hiPSCs-derived neurons significantly increased the cell viability and phagocytosis of microglia from the three hiPSCs-derived cells.The ROS level of hiPSCs-derived microglia in you ng people was not significantly affected,but the ROS level of hiPSCs-derived microglia in cognitively normal elderly people and AD patients was significantly increased,and the ROS level of hiPSCs-derived microglia in AD patients was the most significantly increased.It is suggested that hiPSCs-derived neurons can activate hiPSCs-derived microglia in patients with AD,and the effect on hiPSCs-derived microglia in patients with AD is the most obvious.Chapter III Evaluation of anti-inflammatory immunoactivity of anti-AD drugs based on microglia derived from hiPSCs.Our previous study found that LWD-b,one of the active components of Rehmanniae glutinosa(LW-AFC),can improve the cognitive function of AD model mice by regulati ng the balance of neuroendocrine and immunomodulatory network.The anti-inflammatory immune activity of LWD-B and its 7 active components was further studied by usi ng microglia cells derived from hiPSCs in you ng people,cognitively normal elderly people and AD patients.The concentration of LWD-b was 100μg/ml,and the concentration of paeoniflorin,mononoside,sweroside,ergosteroside,equinoside,equinoside and gallic acid was 100 μM,and the concentration of gallic acid was 50 μM.Solvent-control group and LPS stimulation group were set up in the experiment.CCK-8 assay was used to detect the viability of Himgls cells,neutral red phagocytosis assay was used to detect the phagocytosis ability of Himgls cells,Luminex assay was used to detect the level of cytokines secreted by cells,and chemiluminescence assay was used to detect hiPSCs-derived microglia ROS level.1.Effects of LWD-b and its active monomer on cell viability of LPS-induced hiPSCs-derived microglia.CCK-8 assay was used to detect the cell viability of microglia from hiPSCs.The results showed that LPS stimulation significantly increased the cell viability of microglia from three HiPSCs.LWD-b and its active components had no significant effect on the cell viability of microglia derived from hiPSCs in you ng adults,cognitively normal elderly people and AD patients.LPS stimulation significantly increased the cell viability of hiPSCs-derived microglia in you ng adults,cognitively normal elderly adults,and AD patients.Loganin could significantly inhibit the cell viability of microglia derived from you ng hiPSCs after LPS stimulation.Morroniside and loganin significantly inhibited the cell viability of microglia derived from hiPSCs in cognitively normal elderly after LPS stimulation.Loganin,morroniside,paeoniflorin,Loganic acid,gallic acid,Sweroside had significant inhibitory effects on the cell viability of microglia derived from hiPSCs in AD patients after LPS stimulation.2.Effects of LWD-b and its active monomers on phagocytosis of LPS-induced hiPSCs-derived microglia.Neuter red phagocytosis assay was used to observe the effects of LWD-b and its active monomer on the phagocytosis of YC hiPSCs-derived microglia,CNC hiPSCs-derived microglia and AD hiPSCs-derived microglia induced by LPS.The results showed that LPS stimulation could significantly enhance the viability of YC hiPSCs-derived microglia,CNC hiPSCs-derived microglia and AD hiPSCs-derived microglia.LWD-b and its active monomer had no significant effect on the phagocytosis capacity of YC hiPSCs-derived microglia,CNC hiPSCs-derived microglia and AD hiPSCs-derived microglia without LPS stimulation.Loganin had a significant inhibitory effect on phagocytosis of YC hiPSCs-derived microglia after LPS stimulation.Morroniside and loganin had significant inhibitory effects on phagocytosis of CNC hiPSCs-derived microglia after LPS stimulation.Paeoniflorin,morroniside,sweroside,loganin,loganin acid and gallic acid had obvious inhibitory effect on t phagocytosis of AD hiPSCs-derived microglia stimulated by LPS.3.Effects of LWD-b and its active monomers on cytokine secretion ability of LPS-induced hiPSCs derived microglia.Millipore kit was used to observe the effects of LWD-b and its active monomer on the secretion of anti-inflammatory factor IL-10 and pro-inflammatory factor IL-6,IL-1β TNF-αchemotactic factor CCL7 and CCL10 of YC hiPSCs-derived microglia,CNC hiPSCs-derived microglia and AD hiPSCs-derived microglia induced by LPS.After LPS stimulation,the secretion levels of YC hiPSCs-derived microglia,CNC hiPSCs-derived microglia and AD hiPSCs-derived microglia IL-10,IL-6,IL-1β TNF-α,CCL7 and CCL10 were significantly increased.LWD-b and its active monomer have tendency to inhibit the secretion levels of various cytokines of YC CNC and AD hiPSCs-derived microglia.Effects on the cytokine secretion capacity of YC hiPSCs-derived microglia loganin,loganin acid,gallic acid and LWD-b can significantly inhibit the secretion of TNF-α in YC hiPSCs-derived microglia after LPS stimulation.Loganin can significantly inhibit the secretion of IL-6 by YC hiPSCs-derived microglia after LPS stimulation.Effects on the cytokine secretion capacity of CNC hiPSCs-derived microglia:morroniside,loganin,loganin acid and gallic acid had significant inhibitory effects on the secretion of TNF-αlevel of CNC hiPSCs-derived microglia after LPS stimulation;Morroniside significantly inhibited the level of IL-10 secreted by CNC hiPSCs-derived microglia after LPS stimulation.Morroniside and gallic acid had significant inhibitory effects on CXCL10 secreted by CNC hiPSCs-derived microglia after LPS stimulation.Effects on the cytokine secretion capacity of AD hiPSCs-derived microglia:Loganin significantly inhibited the secretion of IL-6 and IL-1β by YC hiPSCs-derived microglia after LPS stimulation;morroniside,loganin,loganin acid,gallic acid and LWD-b can significantly inhibit the secretion of TNF-α and CXCL10 levels of AD hiPSCs-derived microglia after LPS stimulation.morroniside,loganin and gallic acid had significant inhibitory effects on CCL7 secreted by AD hiPSCs-derived microglia after LPS stimulation.Through the above research,this study mainly got the followi ng conclusions:(1)hiPSCs from you ng people,cognitively normal elderly people and AD patients were successfully induced to differentiate into microglia cells.(2)All the 4 kinds of exogenous stimuli can cause the inflammatory response and oxidative stress response of hiPSCs-derived microglia in you ng people,cognitively normal elderly people and AD patients.Before the addition of stimulus factors,microglia derived from hiPSCs in AD patients had shown stro ng inflammatory response and oxidative stress response.After the addition of stimulus factors,the response of microglia derived from hiPSCs in patients with AD was more obvious,which could better reflect the pathological cha nges related to AD.(3)hiPSCs-derived microglia and hiPSCs-derived neurons can interact with each other in you ng people,cognitively normal elderly people and AD patients.To LPS stimulation of source microglia can damage neurons in patients with AD,you ng people after LPS stimulation,cognitively normal elderly people and AD patients hiPSCs source of the role of microglia were damaged neurons,hiPSCs source microglia in patients with AD on three sources of neurons damage more pro minent,and the extent of the source of hiPSCs neuronal damage in patients with AD the worst;Neurons derived from hiPSCs in AD patients can activate microglia derived from hiPSCs in you ng people,cognitively normal elderly people and AD patients,and the effect on microglia derived from hiPSCs in AD patients is most significant.(4)LWD-b and its active components can reduce the inflammatory response and oxidative stress response of hiPSCs-derived microglia cells from three sources after LPS stimulation.Amo ng them,loganin,morroniside,loganic acid,gallic acid had the most significant effects,suggesti ng that the above drugs had good anti-inflammatory immune activity.It provides experimental basis for further study on the mechanism of action of the drug.