Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC - Second round of pooled screening identified an initial set of TFs for inducing microglia gene expression.

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor (TF) screens have enabled efficient production of a few cell types; however, engineering cell types that require complex TF combinations remains challenging. Here, we report an iterative, high-throughput single-cell TF screening method that enables identification of TF combinations for specialized cell differentiation, which we validated by differentiating human microglia-like cells. We found that the expression of six TFs, SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8, is sufficient to differentiate human iPSC into cells with transcriptional and functional similarity to primary human microglia within 4 days. Overall design: scRNA-seq of second pooled TF screening. Following the dual transfection of 42 TF vectors and SFC (SPI1-T2A-FLI1-P2A-CEBPA) casette into hiPSCs, cells were purified and treated with dox for 4 days. Tra-1-60 negative cells were sorted for scRNA-seq. For the second iteration, 5% stem cells and 5% MG3.1-SFC were spiked in as undifferentiated and initial differentiation control.