Assistant Professor Kyoto University, Kyoto, Japan
Abstract: Transcription factors and co-regulators play a crucial role in determining the functions and properties of cells. Since these factors form complex regulatory networks, it is essential to elucidate the dynamics of the entire regulatory network for understanding of cellular properties both in undifferentiated states and during differentiation processes. Using time-series perturb-seq data, we have previously elucidated the structure of a gene regulatory network involved in maintaining the undifferentiated state of human pluripotent stem cells (Ishikawa M et al., 2023, Commun Biol). In this study, we aim to control the dynamics of the regulatory network by manipulating its core factors, ultimately seeking to efficiently induce differentiation of human pluripotent stem cells into specific cell lineages. To achieve this, we improved conventional CRISPR-a/i transcriptional control systems and established human iPS cells capable of simultaneously perturbing (activating or repressing) multiple factors in various combinations within a single cell. By comparing the previously mentioned perturb-seq data with public scRNA-seq data from early human development, we theoretically predicted which factors should be considered as core of the regulatory network and how these factors should be regulated to induce differentiation of cells into specific lineages. These predictions were experimentally validated using CRISPR-a/i-modified human iPS cells. As a result, we were able to confirm the expression of marker genes for the predicted cell lineage through the simultaneous manipulation of seven factors. Currently, we are further refining the manipulation system and analyzing the molecular mechanisms underlying this differentiation induction.
