Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC -Single cell transcriptome timecourse analysis of TFiMGLs after SCF-MCI (SPI1, FLI1, CEBPA, CEBPB, IRF8, and Mef2C) induction.

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. We conducted a time course study to assess transcriptional profiles of hiPSC derived TFiMGLs under two different cell culture conditions. In the first condition, TFiMGLs were induced following the conditions of the transcription factor screen – in short, cells were continuously cultured in mTeSR Plus (+MT) media supplemented with Dox for 6 days. Whereas in the second condition, mTeSR Plus media was switched on day 2 to commercially available microglia-specific media (+MG) supplemented with Dox until day 6. Cells grown in both conditions were collected at days 2, 4, and 6 post-induction for scRNA-seq.