Induced Pluripotent Stem Cell (iPSC)-derived Microglia And Their Application In The Study Of Neurodevelopmental Disorder

Microglia,or MG,is a crucial cell type for maintaining homeostasis in the central nervous system and play a significant role in neurodevelopmental disorders.The occurrence of autism has been closely linked to dysregulated inflammatory activation of MG,and short-chain fatty acids(SCFAs),which are metabolic products of the gut microbiota,can cross the blood-brain barrier(BBB)and regulate the inflammatory activation status of MG.However,the mechanisms underlying SCFA regulation of MG inflammatory responses and their regulatory effects on autism-related MG remain is unclear.To address this issue,we developed an efficient system for preparing induced pluripotent stem cell(iPSC)-derived MG without using feeder cell or xenogenic serum.This system mimics the metabolic pathway of MG development,and optimal concentrations and combinations of BMP4 and CHIR99021 were determined for mesoderm differentiation on the basis of HAND 1 expression from day1 to day4 induction.Optimization of the concentrations and ratios of bFGF,VEGF,and other cytokines from day 4 to 6 of induction achieved a high efficiency of hematopoietic progenitor cell transformation.Flow cytometry data showed that approximately 90%of cells were doublepositive for the mesodermal cell marker KDR and the hematopoietic stem cell marker CD34.MG progenitor cells with a purity of more than 90%were obtained after a day 640 cytokines induction,including SCF,IL-3,TPO,Flt3-Ligand,M-CSF,and GM-CSF.Mature MG cells with a purity of more than 95%(96.64%±1.33%)were obtained on day 12 after treatment with IL-34 and GM-CSF,with a cell number 35 times(35±4.04)that of the initial iPSCs.Differentiated MG demonstrated M1 polarization potential and phagocytosis of E.coli after stimulated with IFN-y and LPS.Transcriptome data analysis from cells on day 0,4,6,14,and 40 of differentiation showed a clear iPSC-mesodermhematopoietic progenitor cell-MG differentiation pathway,and cluster analysis of the transcriptome data of MG differentiated from this system and human primary MG showed high similarity.The study also established iPSC lines from autism patients with CHD8 gene doublesite mutations and prepared MGs from them.The effects of SCFAs on their inflammatory response were observed,and it was found that SCFAs treatment significantly reduced the inflammatory activation and phagocytic activity of MG induced by LPS and IFN-γ,and reduced the damage of M1-activated neurons.Inhibiting the expression of monocarboxylate transporter 1/4(MCT1/MCT4)reversed the protective effect of SCFAs on neurons,indicating that MCT4 plays a role in the protective effect of SCFAs on MGmediated neural damage.Further studies have found that inhibition of the expression of monocarboxylate transporter 1/4(MCT1/MCT4)can reverse the protective effect of shortchain fatty acid(SCFAs)treatment on neurons,indicating that MCT4 plays an important role in the regulation of the function of M1 microglia by SCFAs.Transcriptome data analysis from different sources has shown that the expression level of the MCT4 gene is significantly increased in autism patients,suggesting that MCT4 may be one of the reasons for the difference in the regulation of SCFAs to the microglia between autism patient and healthy people.In summary,this study established a preparation method for human induced pluripotent stem cell-derived microglia without using xenogenic serum or feeder cells,with high homogeneity,yield and transcriptome similarity with primary microglia.The prepared MG could survive inside the corresponding cerebral organoids.This provides an experimental model for further research on the interaction between microglia and neurons.At the same time,this study explores the effects of SCFAs on the polarization and immune regulation abilities of microglia with different genetic backgrounds,and for the first time to propose the key role of the MCT4 gene in regulating the biological function of microglia and protecting neurons through the metabolites of intestinal flora.This provides an effective model for the etiological research,objective disease indicator finding and medical intervention method development for neurodevelopmental disorders.