Selective GSK3a Inhibition Promotes Self-Renewal Across Different Stem Cell States [scRNA-seq]

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent the naïve and primed pluripotent states, respectively, each requiring distinct culture conditions. In this study, we show that BRD0705, a selective GSK3a inhibitor, significantly enhances the self-renewal of both ESCs and EpiSCs. When combined with IWR1, BRD0705 sustains long-term maintenance of ESCs in a naïve state and EpiSCs in a primed state, even preserving their unique identities under long-term co-culture. Single-cell RNA sequencing and histone mark profiling confirm that this regimen maintains distinct gene expression and epigenetic signatures corresponding to each pluripotent state. Importantly, unlike pan-GSK3 inhibitors CHIR-99021 (CHIR), BRD0705 acts independently of ß-catenin signaling, unveiling a novel mechanism for supporting pluripotency. Finally, BRD0705/IWR1 also supports the maintenance of the formative pluripotent stem cells and neural stem cells. Collectively, we propose that GSK3a inhibition may preserve diverse stem cell states by insulating stem cells from differentiation cues and promoting their intrinsic self-renewal capacity. We believe our findings lay the foundation for universal stem cell culture methods with significant regenerative medicine applications. Overall design: Co-cultured cells were trypsinized and dissociated into the single-cell suspension in DMEM supplemented with 10% FBS. According to the manufacturer's instructions, single-cell RNA sequencing was performed with the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1. ESC- and EpiSC-derived cells were separated based on GFP and RFP fluorescence by FACS sorting. Sequencing was carried out on Illumina sequencers.