Chemicals orchestrate reprogramming with hierarchical activation of master transcription factors primed by endogenous Sox17 activation

Abstract

Mouse somatic cells can be chemically reprogrammed into pluripotent stem cells (CiPSCs) through an intermediate extraembryonic endoderm (XEN)-like state. However, it is elusive how the chemicals orchestrate the cell fate alteration. In this study, we analyze molecular dynamics in chemical reprogramming from fibroblasts to a XEN-like state. We find that Sox17 is initially activated by the chemical cocktails, and XEN cell fate specialization is subsequently mediated by Sox17 activated expression of other XEN master genes, such as Sall4 and Gata4. Furthermore, this stepwise process is differentially regulated. The core reprogramming chemicals CHIR99021, 616452 and Forskolin are all necessary for Sox17 activation, while differently required for Gata4 and Sall4 expression. The addition of chemical boosters in different phases further improves the generation efficiency of XEN-like cells. Taken together, our work demonstrates that chemical reprogramming is regulated in 3 distinct “prime–specify–transit” phases initiated with endogenous Sox17 activation, providing a new framework to understand cell fate determination.

Chemical Pretreatment Activated a Plastic State Amenable to Direct Lineage Reprogramming

“…To investigate how chemical compounds alter cell fate, we previously studied the chemical reprogramming process from mouse embryonic fibroblasts (MEFs) to XEN-like cells. The study revealed a hierarchical activation of XEN cell master TFs primed by Sox17 and the different roles of essential chemicals during the process (Yang et al, 2020). In parallel to that study, we measured the global gene expression profiles by RNA sequencing during reprogramming.…”

“…The data used in the RNA-Seq analysis was combined with our previous data (Yang et al, 2020), and we additionally performed RNA-Seq on the C6F5UE treatment group. Gene expression levels were normalized as log 2 (FPKM+1) in all bulk RNA-Seq data.…”

“…Interestingly, these upregulated lineage-associated TFs were highly overlapping in different chemical cocktails (Figure 1G). Thus, we refer to the induction phase with the activation of multi-lineage TFs, including Sox17 for XEN-like cell induction (Yang et al, 2020), as chemically activated multi-lineage priming (CaMP).…”

“…To clarify these two scenarios, gene expression in individual cells needs to be investigated. Thus, we re-analyzed our single-cell RNAsequencing data obtained with SMART-seq2 (Yang et al, 2020).…”

“…Inspired by the molecular frameworks during cell fate specification in another study of us (Yang et al, 2020), the endogenously activated TFs of multiple lineages in the CaMP induction might be beneficial to induce cell types of other lineages. We found that the TFs of neurons, including Ascl1, a master gene for neuronal reprogramming, were also activated in the CaMP phase.…”