Selective GSK3α Inhibition Promotes Self-Renewal Across Different Stem Cell States

Abstract

Pan-GSK3α/β inhibition promotes stem cell self-renewal through activation of WNT/β-catenin signaling, but its broad effects complicate the precise control of stem cell states. Here, we show that selective inhibition of GSK3α with BRD0705 supports the long-term self-renewal of mouse embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), and neural stem cells (NSCs), independent of β-catenin signaling. When combined with the tankyrase inhibitor IWR1, BRD0705 broadly supports the maintenance of diverse pluripotent stem cell states, including ESCs, EpiSCs, and formative pluripotent stem cells. This BRD0705/IWR1 cocktail enables stable co-culture of naive ESCs and primed EpiSCs while preserving their distinct molecular and functional identities. Single-cell transcriptomics, epigenomic profiling, and functional assays confirm sustained lineage-specific features across stem cell types. These findings demonstrate that selective GSK3α inhibition enhances stemness by buffering against differentiation cues and promoting intrinsic self-renewal capacity. This work identifies GSK3α as a key regulator of self-renewal across distinct stem cell states and establishes a versatile culture system with broad applications.

Figure 1.. BRD0705 Effectively Supports the Self-renewal of Both ESCs and EpiSCs

(A) Alkaline phosphatase (AP) staining of ESC colonies formed under treatment with various compounds from the chemical inhibitor library. Cells were cultured in DMEM/FBS medium for 4 days. Scale bar, 100 μm. NT, no treatment control. (B) Quantification of AP+ colonies. Data represent mean±SEM. from triplicate experiments. *, p<0.05, indicating a statistically significant difference in the number of AP+ clones compared to the control under different inhibitor treatments. (C) Left panel: a representative phase-contrast image showing the morphology of ESCs at passage 25 cultured in DMEM/10%FBS under BRD0705 treatment. Right panel: A representative phase-contrast image showing the morphology of ESCs cultured in 2iL conditions for three passages after being maintained in BRD0705 for 25 passages. Scale bar: 100 μm. (D) qRT-PCR analysis of pluripotency and germ layer-specific gene expression in ESCs maintained in 2iL, BRD0705, or in cells transitioned from BRD0705 to 2iL, normalized to GAPDH. Error bars are SEM from technical triplicates. *, p<0.05. (E) Representative phase-contrast and fluorescence images of Oct4-GFP ESCs cultured in LIF alone or with BRD0705.The basal cell culture medium was DMEM/10%FBS. Scale bar, 100 μm. (F) Quantification of Oct4-GFP+ colony ratio from the experiments shown in (E). Data are presented as mean ± SEM from three biological replicates. *, p<0.05. **, p<0.01. ***, p<0.001. (G) Phase-contrast images of mouse embryo blastocysts (E3.5), passage 18 ESCs derived from blastocysts under BRD0705+PD03 conditions, and ESCs cultured under CHIR+PD03 conditions. Scale bar: 200 μm. (H) Bright-field and fluorescent images of E10.5 mouse embryos generated after blastocyst injection of BRD0705+PD03 cultured ESCs with ubiquitous GFP. Scale bars, 350 μm. (I) Representative phase-contrast images of EpiSCs treated with BRD0705 or IWR1 from the compound library on day 3. Scale bar 100μm.

Figure 2.. Characterization of ESCs and EpiSCs Cultured under BRD0705/IWR1

(A) Top panel: Phase-contrast images showing ESCs cultured in BRD0705/IWR1 for 25 passages, then transitioned to 2iL or ActivinA/bFGF/XAV condition for 3 passages (scale bar: 100 μm). Bottom panel: phase-contrast images showing EpiSCs cultured 15 passages in BRD0705/IWR1, then transitioned to 2iL or ActivinA/bFGF/XAV conditions for 3 passages (scale bar: 200 μm). (B) qRT-PCR analysis of marker gene expression in ESCs cultured in BRD0705/IWR1 for 15 passages, 2iL, and 2iL after transition from BRD0705/IWR1-cultured ESCs, normalized to GAPDH. Data represent mean ± SEM, n=3.qRT-PCR analysis of marker gene expression in EpiSCs treated with BRD0705/IWR1, ActivinA, bFGF, and IWR, with normalization to GAPDH. Error bars indicate the SEM from technical triplicates. n=3. (C) qRT-PCR analysis of marker gene expression in EpiSCs maintained in BRD0705/IWR1 and ActivinA/bFGF/XAV939, normalized to GAPDH. Error bars indicate the SEM from technical triplicates, with N=3. (D) Schematic representation of the experimental workflow showing the injection of P10 BRD0705/IWR1-expanded ESCs or EpiSCs into E3.5 blastocysts, followed by embryo development to E10.5 (Left). Representative phase-contrast and fluorescence images of E10.5 mouse embryos derived from blastocyst injection of GFP-expressing ESCs or EpiSCs cultured with BRD0705/IWR1 (right). Scale bars, 500 μm. (E) Shown are phase-contrast and fluorescence images of gonads originating from E15.5 mouse embryos obtained from blastocyst injection of ESCs-GFP cultured with BRD0705/IWR1. Scale bars, 350 μm. (F) Phase-contrast and fluorescence images show #1 embryonic gonad cells (EGCs) derived from the gonads of E15.5 chimeric embryos formed by ESCs-GFP cultured in BRD0705/IWR1. EGCs cultured under 2iL conditions. Scale bars, 250 μm. (G) IF analysis verifies the expression of GFP and OCT4 in EGCs cultured in 2iL. Scale bar 50μm. (H)The schematic illustrates the co-culture of RFP-Zeocin-resistant ESCs and GFP-Puromycin-resistant EpiSCs under BRD0705/IWR1 conditions for 12 passages, followed by selection with Zeocin or Puromycin to isolate ESCs or EpiSCs, respectively. (I) Bright-field and fluorescent images show RFP/GFP expression in co-cultured ESCs (RFP-Zeocin) and EpiSCs (GFP-Puromycin) under BRD0705/IWR1, AFX, and 2iL conditions, following the transition of passage 12 ESCs/EpiSCs maintained in BRD0705/IWR1. Scale bar: 100 μm. (J) The bar graph represents the ratio of ESCs (RFP-Zeocin) and EpiSCs (GFP-Puromycin) shown in Figure I, under BRD0705/IWR1, AFX, and 2iL conditions. (K) Bright-field and fluorescent images show RFP and GFP expression in ESCs (RFPZeocin)/EpiSCs (GFP-Puromycin) co-cultured cells after zeocin selection, followed by 3 passages in BRD0705/IWR1, AFX, and 2iL culture conditions. Scale bar: 100 μm. The bar graph represents the proportion of GFP+ or RFP+ cells under different conditions in the panel above. Scale bar: 100 μm. (L) Bright-field and fluorescent images illustrate the RFP and GFP expression in ESCs (RFPZeocin)/EpiSCs (GFP-Puromycin) co-cultured cells after puromycin selection, subsequently cultured for 2 passages in BRD0705/IWR1, AFX, and 2iL conditions. Scale bar: 500 μm. The bar graph represents the proportion of GFP+ or RFP+ positive cells under different conditions in the panel above. Scale bar: 100 μm. (M) The schematic shows the generation of chimeric embryos by injecting ESCs (RFPZeocin)/EpiSCs (GFP-Puromycin) mixed cells, co-cultured for 14 passages with BRD0705/IWR1, into WT E3.5 embryos, resulting in their contribution to E10.5 embryonic development. (N) Representative morphology of P14 co-cultured ESCs (RFP+) and EpiSCs (GFP+) under BRD0705/IWR1 conditions used for injection into WT E3.5 blastocysts, along with the respective proportions of these two cell types. Scale bar: 100 μm. (O) Images show E10.5 embryos derived from E3.5 blastocyst injection of ESCs (RFPZeocin)/EpiSCs (GFP-Puromycin) mixed cells co-cultured in BRD0705/IWR1 for 14 passages, illustrating the distribution and integration of the injected cells in the embryos. Scale bar: 500 μm. (P) Summary of chimera experiments at E10.5.

Figure 3.. scRNA seq and Chromatin Landscape Analysis

(A) Schematic illustration of the experimental design. Naive ESCs (RFP+) and EpiSCs (GFP+) were co-cultured under BRD0705/IWR1 conditions for 16 passages, followed by scRNA-seq and Cut&tag analyses. (B) UMAP plots displaying the expression of Vimentin, GFP, and RFP confirm the presence of ESCs and EpiSCs in co-culture under BRD0705/IWR1 conditions. (C) UMAP visualization of key pluripotency-related genes, including the PSC marker Nanog; ESC markers (Esrrb, Zfp42, Nr0b1, Tfcp2l1, Tbx3, Tcl1, and Prdm14); and the EpiSCs marker Fgf5 and Pitx2, highlights the transcriptional differences between ESCs and EpiSCs. (D) UMAP clustering of single-cell transcriptomes reveals distinct cell populations within the co-culture cells, labeled as clusters 0–5. (E) Clusters 1–5 from (D) were projected onto a UMAP plot of early embryonic developmental stages. (F) Heatmap showing the transcriptomic correlation between co-cultured cells under BRD0705/IWR1 conditions and reference ESCs, EpiSCs, and formative cells cultured under different conditions, indicating their resemblance to naive and primed pluripotent states. scNaive: single-cell sequencing results of naive ESCs in BI co-cultured cells. scPrime: single-cell sequencing results of primed PSCs in BI co-cultured cells. (G) Bar graph quantifying the number of naive ESCs-specific and primed EpiSCs-specific chromatin accessibility peaks for H3K4me3, H3K27ac, and H3K27me3 modifications. (H) Heatmaps displaying Cut&Tag profiles for H3K4me3, H3K27ac, and H3K27me3 modifications in naive ESCs (2iL), primed EpiSCs (AFX), and BRD0705/IWR1 co-cultured ESCs (RFP+) and EpiSCs (GFP+). Naive ESCs exhibit distinct chromatin accessibility patterns compared to primed EpiSCs, and co-cultured ESCs and EpiSCs maintain their respective epigenetic signatures, confirming the long-term maintenance of their identities under BRD0705/IWR1 conditions.

Figure 4.. BRD0705 enhances stemness maintenance in other types of stem cells

(A) Morphological comparison of #1 Gsk3αβ DKO ESC line under different treatment conditions. Cells were cultured in non-treated control conditions, PD03, BRD0705, and CHIR. Cell cultured in DMEM/FBS medium for Passage 3. Scale bars represent 100 μm. (B) Quantification of clone numbers corresponding to the images in Figure A. PD03 treatment significantly increases Gsk3α/β DKO ESCs clone numbers compared to control and other conditions (BRD0705 and CHIR), which show minimal clone formation. Error bars indicate mean ± SEM. (C) Morphology of WT, Gsk3α^−/−, and Gsk3β^−/− ESCs treated with PD03, PD03+CHIR, or PD03+BRD0705. PD03+CHIR induces ‘dome-like’ colonies in WT, Gsk3α^−/− and Gsk3β^−/−. PD03+BRD0705 induces ‘dome-like’ colony formation only in Gsk3β^−/− cells, while other genotypes show a spread-out morphology. Cell cultured in N2B27 for day 4. Scale bars, 200 μm. (D) The top row shows Gsk3α^−/−-Gsk3αKD ESCs under NT, BRD0705 condition, and WT ESCs treated with BRD0705 after 10 passages (P10). The bottom row displays the morphology of the corresponding P10 cells after being transitioned to 2iL conditions for 3 passages. Scale bars represent 100 μm. (E) Survival of ESCs lines under different conditions. Representative images of WT, Gsk3α^−/−+Gsk3α-L195G, and Gsk3β^−/−+Gsk3β-L132G ESCs cultured with or without 3MB-PP1. #1 and #2 represent two different cell lines. WT ESCs displayed complete cell death by passage 3 (P3) under both conditions, while Gsk3-modified ESCs maintained viability at passage 5 (P5) with visible cell colonies. Scale bars represent 200 μm. (F) Morphology of Gsk3α^−/−+Gsk3α-L195G ESCs under different culture conditions. Representative images of cells at passage 11 (P11) cultured in +3MB-PP1 (left) and following a switch to 2iL (right). Scale bars, 200 μm. (G) Morphology of WT, Gsk3α^−/−+Gsk3α-L195G, and Gsk3β^−/−+Gsk3β-L132G EpiSCs under different treatments. Representative images show cells cultured in DMEM/FBS medium for 3 passages in NT, +3MB-PP1, and +BRD0705 conditions. Scale bars, 200 μm. (H) Morphological analysis of β-Catenin^−/− ESCs cultured in LIF/N2B27 on feeder under different treatment conditions. Representative images display cells cultured in NT, IWR1, BRD0705, IWR+BRD0705, and CHIR conditions. Scale bars, 100 μm. (I) Quantification of Figure I. Clone numbers of β-Catenin^−/− ESCs under different treatments: Ctr, IWR1, BRD0705, IWR1/BRD0705, and CHIR. Data are presented as mean ± SEM, *, p < 0.05, **, p < 0.01. (J) Western blot analysis of β-Catenin in WT and β-Catenin^−/− ESCs. β-Catenin is detected in WT but absent in β-Catenin^−/− cells. GAPDH serves as the control. (K) Western blot analysis of phosphorylated β-catenin (p-β-catenin), total β-catenin, and GAPDH in response to different concentrations (0, 4, 8, 20 μM) of BRD0705 and CHIR.