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Developmentally informed large-scale production and cryopreservation of human induced pluripotent stem cell-derived microglia

ABSTRACT: Purpose: mRNA expression profiles of iPSC-derived microglia (iPSdMiG) generated with a new cutting-edge protocol were compared to human primary microglia and cortex samples, iPSC-derived microglia-like cells (iMGL), THP1 macrophages, and parental iPSCs. Methods: Library preparation was performed using the Quantseq 3’mRNA-Seq kit (Lexogen, USA). Reads were generated on a HiSeq 2500 V4 sequencer (Illumina, USA) and quality as well as adapter trimming were performed using BBDuk from BBMap. Reads were aligned to hg38 with the ensemble gene annotation version 97 using STAR and read counts were generated using featureCounts ignoring multimapping reads. Unwanted variance due to differences in library preparation and sequencing were removed using RUVSeq and subsequent differential gene expression analysis was performed with DESeq2. Transcription factor enrichment analysis was performed with ChEA3 and co-expression analysis was performed using WGCNA. Results: Overall, our iPSdMiG highly resemble human primary microglia. They have a similar expression of lineage-specific genes and cluster in close proximity in PCA. Our iPSdMiG and human primary microglia share key transcription factors driving cellular identity and co-express genes belonging to a immunity-related gene module.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178846 | GEO | 2022/08/22

REPOSITORIES: GEO

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Project description:To determine IL-17-induced global transcriptome changes in midbrain neurons derived from induced pluripotent stem cells (iPSC) from three sporadic Parkinson's disease (PD) patients and three age- and sex-machted controls, deep RNA sequencing (RNA-Seq) of IL-17-treated and untreated PD and control iPSC-dderived midbrain neurons was performed. Total RNA was isolated from untreated and IL-17-treated cells with the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems) and cluster generation was performed on the cBot with the TruSeq SR Cluster Kit v3 (Illumina). The sequencing run was performed on a HiSeq 1000 instrument (Illumina) using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit (Illumina). Image analysis and base calling resulted in .bcl files that were then converted into .fastQ files by the CASAVA1.8.2 software. FastQ files were aligned to the human genome (hg19) using STAR.and annotated with gencode.v19. DESeq2 was used to determine differential expression. Criteria to determine significantly dysregulated genes were as follows: adjusted p-value below 0.05 and log2FC (fold change) of greater than one. Only genes with a mean expression value of greater than one RPKM (reads per kilobase per million mapped reads) throughout the dataset were considered. Control and PD samples were analyzed as two independent datasets. Upon IL-17 treatment only 17 genes were found to be dysregulated in controls but 125 genes were dysregulated in iPSC-derived midbrain neurons from PD patients. The 125 IL-17-dependent genes in PD iPSC-derived neurons separated the treated from untreated PD samples using an unsupervised, hierarchical clustering applying an Euclidean distance metric. More detailed study information can be found in Sommer A, Maxreiter F, Krach F, Fadler T, Grosch J, Maroni M, Graef D, Eberhardt E, Riemenschneider MJ, Yeo GW, Kohl Z, Xiang W, Gage FH, Winkler J, Prots I, Winner B. Th17 Lymphocytes Induce Neuronal Cell Death in a Human iPSC-Based Model of Parkinson's Disease. Cell Stem Cell. 2018 Jul 5;23(1):123-131.e6. doi: 10.1016/j.stem.2018.06.015. PMID: 29979986

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Developmentally informed large-scale production and cryopreservation of human induced pluripotent stem cell-derived microglia

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