Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation

Cell differentiation is controlled by transcription factors (TFs). However, we still do not fully understand which TFs are needed for even one mammalian differentiation pathway. To address this, we developed a detailed and integrated method. This method combines pluripotent stem cell differentiation in the lab, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen. Using this approach, we identified all the TFs required for spinal motor neuron (MN) differentiation in mice and their requirement in human MN differentiation. Our transcriptomic analysis revealed that while over 1,300 TFs are transcribed during ventral spinal cord differentiation, only 55 have annotated functions in the literature. The screen identified 232 TFs and other transcription-regulating genes essential for the progression of ventral spinal cord differentiation, including 116 TFs specifically required for MN differentiation. This approach led to three critical observations. First, the motor neuron progenitor (pMN) stage is a regulatory hub that correlates with a reduction in cell potential. Second, a secondary screen in human ventral spinal cord differentiation revealed a greater degree of conservation j between mouse pMNs and the human-specific ventral MN progenitors (vpMNs). This finding was unexpected, as the conservation was anticipated to be with the canonical human pMN stage. Third, the screen underscored the importance of the numerous and yet understudied ZF TFs controlling mammalian cell differentiation. In summary, we present a universal, unbiased strategy for studying TFs in mammalian differentiation, comparable to methodologies used in simpler organisms. This approach enhances our understanding of MN differentiation and provides a practical framework to identify all TFs required for any differentiation trajectory that can be recapitulated in vitro, offering many applications in various fields of developmental biology. Overall design: Mouse ES cell line E14 Tg2a was differentiated with retinoid acid and smoothened agonist as embryoid bodies into ventral spinal cord. Cells were sampled at day 0, 2, 3, 4, 5, and 6 of differentiation and cryopreserved until proceeded with single-cell sequencing.