Catalpol promotes the generation of cerebral organoids with oRGs through activation of STAT3 signaling

Abstract

The generation of human cortical organoids containing outer radial glia (oRG) cells is crucial for modeling neocortical development. Here we show that Catalpol, an iridoid glucoside derived from Rehmannia glutinosa, significantly enhances the generation of cerebral organoids with expanded oRG populations and increased neurogenic potential. Catalpol-treated organoids exhibited thicker ventricular zone/subventricular zone (VZ/SVZ) and outer subventricular zone (oSVZ) regions, with increased numbers of SOX2 + HOPX+ and SOX2 + TNC+ oRG cells and elevated expression of oRG markers HOPX and FAM107A. We found that Catalpol promoted oRG generation through non-vertical divisions of ventricular radial glia (vRG) cells, indicating enhanced oRG generation via asymmetrical divisions. Furthermore, we demonstrated that Catalpol augmented oRG cell numbers through activation of the STAT3 signaling pathway. These findings highlight Catalpol's potential in promoting the generation of cerebral organoids with expanded oRG populations and increased neurogenic potential through STAT3 activation, offering new insights into neocortical development modeling.

Keywords: Catalpol; STAT3 signaling; cerebral organoids; outer radial glia cells.

FIGURE 1

Catalpol increases the development of VZ/SVZ regions and the number of outer radial glia (oRG) in cerebral organoids. (a) Chemical structure of Catalpol. (b) Schematic outline of cerebral organoids differentiation protocol with or without Catalpol (10 μM) treatment. (c) Immunofluorescence staining for TBR1 (red), SOX2 (green), and DAPI (blue) in day 30 control and Catalpol‐treated organoids. Scale bar, 50 μm. (d) Immunofluorescence staining for MAP2 (red), TBR2 (green), and SOX2 (blue) in day 30 control and Catalpol‐treated organoids. Scale bar, 20 μm. (e) Thickness of VZ/SVZ region and oSVZ region in day 30 control and Catalpol‐treated organoids. Data represent mean ± SEM. two‐tailed Student's t‐test, ***p < 0.001; n = 5 per group. (f) Quantification of SOX2 + HOPX+ and SOX2 + TNC+ oRG cells in oSVZ region. Data represent mean ± SEM. two‐tailed Student's t‐test, **p < 0.01; n = 3 per group (g) Immunofluorescence staining for SOX2 (red), HOPX (green), and DAPI (blue) in day 30 control and Catalpol‐treated organoids. Scale bar, 50 μm. (h) Immunofluorescence staining of SOX2 (red), TNC (green), and DAPI (blue) in day 30 control and Catalpol‐treated organoids. Scale bar, 50 μm. Arrows represent SOX2 + HOPX+ and SOX2 + TNC+ oRG cells. The dotted lines highlight the regions of the VZ/SVZ, oSVZ, and cortical plate (CP). Scale bar, 50 μm.

FIGURE 2

Enhanced generation of outer radial glia (oRG) by non‐vertical division of mitotic ventricular radial glia (vRG) in Catalpol‐treated organoids. (a) Schematic of vRG cells undergoing mitosis at the ventricular surface (solid orange line) with vertical (60–90°), oblique (30–60°) and horizontal (0–30°) cleavage angles. Mitotic spindle orientation was analyzed with respect to the ventricular surface. (b) Immunofluorescence staining for p‐Vimentin (pVIM; green), pericentrin (red), and DAPI (blue) in day 20 control and Catalpol‐treated organoids. The dotted square lines highlight the vRG division. Arrows represent centrosomes. Scale bar, 20 μm. (c) Quantification of mitotic spindle orientation in day 20 control and Catalpol‐treated organoids. Data represent mean. Two‐tailed Student's t‐test, *p < 0.05, *p < 0.01, and ***p < 0.001; n = 3 per group. (d) Immunofluorescence staining for pVIM (green), phospho‐histone H3 (pH 3; red), and DAPI (blue) in day 20 control and Catalpol‐treated organoids with or without TMZ. Scale bar, 20 μm. (e) The number of pVIM+pH 3+ cells in ventricular surface. Data represent mean ± SEM. two‐way ANOVA, **p < 0.01; n = 3 per group. (f) Immunofluorescence staining for SOX2 (red), HOPX (green), and DAPI (blue) in day 30 control and Catalpol‐treated organoids with or without TMZ. Arrows represent SOX2 + HOPX+ oRG cells. Scale bar, 20 μm (g) Quantification of SOX2 + HOPX+ oRG cells in oSVZ region on day 20 and 30 control and Catalpol‐treated organoids with or without TMZ. Data represent mean ± SEM. two‐way ANOVA, *p < 0.05, ****p < 0.0001; n = 3 per group. ns, not significant.

FIGURE 3

STAT3 activation by Catalpol promotes the generation of outer radial glia (oRG) in cerebral organoids. (a,b) Immunofluorescence staining for p‐Y705‐STAT3 (red) and SOX2 (green) in day 20 control and Catalpol‐treated organoids with or without STAT3 inhibitor III (Inhi. III). Scale bar, 20 μm. (c) Quantification of SOX2 + pSTAT3+ cells in oSVZ region. Data represent mean ± SEM. two‐way ANOVA, **p < 0.01, ****p < 0.0001; n = 3 per group. (d) Relative gene expression level of STAT3‐related genes (cyclinD1, BclXL, c‐Myc). Data represent mean ± SEM. two‐way ANOVA, *p < 0.05, ***p < 0.001; n = 3 per group. (e) Immunofluorescence staining for SOX2 (red), HOPX (green), and DAPI (blue) in day 30 control and Catalpol‐treated organoids with or without STAT3 inhibitor III (Inhi. III). Arrows represent SOX2 + HOPX+ oRG cells. Scale bar, 50 μm. (f) Quantification of SOX2 + HOPX+ oRG cells. Data represent mean ± SEM. Two‐way ANOVA, ***p < 0.001, ****p < 0.0001; n = 3 per group. ns, not significant.

FIGURE 4

Catalpol enhances the neurogenic capacity of cerebral organoids during cortex development. (a) Relative gene expression level of early deeper layer (SOX5, CTIP2) and early layer (ZBTB20, BRN2) in sorted outer radial glia (oRG) cells from day 30 control and Catalpol‐treated organoids. Data represent mean ± SEM. Two‐tailed Student's t‐test, *p < 0.05, **p < 0.01; n = 3 per group. (b) Immunofluorescence staining for TBR1 (green), SATB2 (green), SOX2 (red), and DAPI (blue) in day 60 control and Catalpol‐treated organoids. The dotted lines highlight the regions of the VZ/SVZ, and layers (V–VI, II–IV) of cortex regions. Scale bar, 50 μm. (c) Quantification of TBR1+ cells (V–VI layer) and SATB2+ cells (II–IV layer) in day 60 control and Catalpol‐treated organoids. Data represent mean ± SEM. two‐tailed Student's t‐test, *p < 0.05; n = 4 per group. (d) Volcano plot for Catalpol‐treated versus control differentially expressed genes (DEGs) in human cerebral organoids (log2FC >1, log2FC < −1, p value <0.05). (e) Bar graph showing GO categories from upregulated genes in Catalpol treated organoid. (f) The GSEA plot indicates a positive correlation between STAT3 signaling pathway target genes and differentially expressed genes in Catalpol‐treated condition. (NES = 0.48, FDR‐q value = 0.007). (g) The GSEA plot indicates a positive correlation between JAK2 signaling pathway target genes and differentially expressed genes in Catalpol‐treated condition. (NES = 0.56, FDR‐q value = 0.034).