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

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. Overall design: Examination of mRNA profiles of iPSC-derived microglia (iPSdMiG), parental iPSC lines and THP1 macrophages. Third-party-reanalysis: In silico comparison with human primary microglia and cortex samples from GSE99074 and iPSC-derived microglia-like cells (iMGL) from GSE89189.