SOX2+ sustentacular cells are stem cells of the postnatal adrenal medulla

Abstract

Renewal of the catecholamine-secreting chromaffin cell population of the adrenal medulla is necessary for physiological homeostasis throughout life. Definitive evidence for the presence or absence of an adrenomedullary stem cell has been enigmatic. In this work, we demonstrate that a subset of sustentacular cells endowed with a support role, are in fact adrenomedullary stem cells. Through genetic tracing and comprehensive transcriptomic data of the mouse adrenal medulla, we show that cells expressing Sox2/SOX2 specialise as a unique postnatal population from embryonic Schwann Cell Precursors and are also present in the normal adult human adrenal medulla. Postnatal SOX2⁺ cells give rise to chromaffin cells of both the adrenaline and noradrenaline lineages in vivo and in vitro. We reveal that SOX2⁺ stem cells have a second, paracrine role in maintaining adrenal chromaffin cell homeostasis, where they promote proliferation through paracrine secretion of WNT6. This work identifies SOX2⁺ cells as a true stem cell for catecholamine-secreting chromaffin cells.

Fig. 1. Single-cell RNA sequencing of the mouse adrenal medulla.

A Experimental workflow. B UMAP of adrenomedullary cell types (2708 cells). C Heatmap showing the transcriptional signatures of 8 clusters (top 5 differentially expressed genes). D Violin plots indicating expression of different markers to identify each cluster. E UMAP showing the distribution of cell cycle states over the dataset. Computationally identified percentages of medullary cells in different cell cycle phases: 44.61% (1208 cells) are in G1, 31.75% (859 cells) are in S, 23.67% (641 cells) in G2/M; F Featureplots of known sustentacular cell and SCP markers S100b, Plp1, Gfap, Sox10, and of newly identified marker Sox2. Colour scale represents Log-normalised expression level, with 0 (grey) representing no expression, and 4 (red) representing highest expression.

Fig. 2. SOX2⁺ cells are present in the adrenal medulla and are derived from Schwann Cell Precursors.

A Immunohistochemistry with antibodies against SOX2 (brown) at P15 and P365 in wild type adrenal medullae. Nuclei counterstained with Hematoxylin, scale bar 20 μm. B Quantification of SOX2⁺ cells over the total nuclei of adrenal medulla. n = 3 females, 3 males for each timepoint, except for 365-day lineage tracing n = 2 females, 2 males, 3 technical replicates for each timepoint, plotted mean and SEM. One-way ANOVA multiple comparisons test: P15 vs. P17 (P-value 0.0008); P15 vs. P21 (P-value 0.9925); P15 vs. P28 (P-value 0.0386); P15 vs. P42 (P-value 0.0006); P15 vs. P84 (P-value 0.0003); P15 vs. P178 (P-value 0.0006); P15 vs. P365 (P-value 0.0013). Source data are provided as a Source Data file. C Immunofluorescence staining of P15 Sox2^eGFP/+ adrenal medulla using antibodies against SOX10 (magenta) or S100β (magenta) and GFP (green), shows double-positive cells in both (arrowheads). Immunofluorescence staining of a P15 wild type (WT) sample using antibodies against GFAP (magenta) and SOX2 (green) shows double-positive cells (arrowheads). Nuclei counterstained with Hoechst, scale bars 10 μm. n = 2 females, 2 males, 3 technical replicates. D RNAscope mRNA in situ hybridisation on wild type P15 samples shows double-positive cells for S100b (red) and Sox2 (blue), Gfap (red) and Sox2 (blue), Sox10 (red) and Sox2 (blue), Plp1 (red) and Sox2 (blue) respectively - (black arrowheads) or single positive (white arrowheads); all nuclei counterstained with Hematoxylin, scale bar 10 μm. n = 2 females, 2 males, 2 technical replicates. E UMAP of the neural crest and SCP lineages between 9.5dpc and 12.5dpc from ref. : BCC, boundary cap cells; ChC, chromaffin cells; enteric neu., enteric neurons; SC, Schwann cells; sat. glia, satellite glia; sens. neu., sensory neurons; symp. neu., sympathetic neurons, endo. fib., endoneurial fibroblasts; tSC, terminal Schwann cells; mSC, myelinating Schwann cells; nmSC, non-myelinating Schwann cells. Trajectory of chromaffin cell (ChC) transitioning from the Hub cells (range from blue (uncommitted hub cell) to yellow (chromaffin cell)). Featureplots showing expression of Sox10, Sox2, Chga (scale Log-normalised expression with k-nearest neighbour (kNN) smoothing, ranging from blue (low expression) to red (high expression)). Graph of single features for Sox10 (blue), Sox2 (orange) and Chga (green) along pseudotime. Sox10 expression precedes that of Sox2 in this trajectory, which is maintained until expression of Chga marking chromaffin cells. F Immunofluorescence on P15 mouse adrenals from Wnt1^Cre/+;R26^mTmG/+ genotypes. Immunostaining with antibodies against SOX2 (magenta) and GFP (green) shows double-positive cells (arrowheads). Nuclei are counterstained with Hoechst, scale bars 10μm. n = 1 male, 1 female, 2 technical replicates. G Immunofluorescence on P15 mouse adrenals from a Sox10^CreERT2/+;R26^mTmG/+ line induced with tamoxifen (TMX) at 11.5dpc – immunostaining with antibodies against SOX2 (magenta) and GFP (green) shows double-positive cells (arrowheads). Nuclei counterstained with Hoechst, scale bars 10 μm.

Fig. 3. Adrenomedullary SOX2⁺ cells have stem cell properties in vitro and in ovo.

A Experimental workflow. B Crystal violet staining of fixed cell colonies following 14-day culture of GFP⁺ (SOX2⁺) and GFP^- (SOX2^-) Sox2^eGFP/+ cells under clonogenic conditions. C Immunofluorescence staining of GFP⁺ primary cells from Sox2^eGFP/+ medulla cultured for 14 days: GFP (green), SOX2 (red), nuclei stained with Hoechst, scale bar 50μm. n = minimum 8 adrenals pooled from minimum 4 mixed-sex mice, 4 technical replicates. D Quantification of GFP⁺ cells via flow cytometry after 14 days of culture, bar graph n = 3 independent biological replicates. E Experimental design of chick chorioallantoic membrane (CAM) assays for ex vivo 3D xenograft culture. F Representative images of resulting xenograft before removal from CAM (left) and after isolation (right) following 18 days of incubation. Representative images of wholemount native EGFP expression in Sox2^eGFP/+-derived xenograft (bottom). Scale bars 200 μm. n = minimum 8 adrenals pooled from minimum 4 mixed-sex mice, 4 technical replicates. G Immunofluorescence staining using antibodies against TH (red) or PNMT (red) on xenografts at day 18 (n = 2). Nuclei counterstained with Hoechst. Box plot showing quantification of TH and PNMT positive cells after immunofluorescence staining, as a proportion of total nuclei. Whiskers min to max, the box extends from the 25th to the 75th percentile, line is median. Source data are provided as a Source Data file. RNAscope mRNA in situ hybridisation using mouse-specific probes against Sox2 (red) and Th (blue) on xenografts. Nuclei counterstained with hematoxylin. Scale bars 10 μm.

Fig. 4. Adrenomedullary SOX2⁺ cells are stem cells in vivo.

A Experimental design indicating tamoxifen (TMX) induction at P14 and timepoints of collection and analysis. B Immunofluorescence using antibodies against GFP, on Sox2^CreERT2/+; R26^mTmG/+ adrenals induced with tamoxifen at P14 and collected after 72 h or 365 days. GFP in green, nuclei counterstained with Hoechst. Inserts magnified boxed regions. Scale bar 100 μm. C Quantification of GFP+ cells/total nuclei of adrenal medulla at different timepoints. n = 3 females, 3 males, for each timepoint, except for 365-day lineage tracing n = 2 females, 2 males, 3 technical replicates for each timepoint, plotted mean and SEM. One-way ANOVA multiple comparisons test: 72 h vs. 7 days (P-value > 0.9999); 72 h vs. 14 days (P-value = 0.2698); 72 h vs. 28 days (P-value = 0.2035); 72 h vs. 70 days (P-value = 0.0285); 72 h vs. 178 days (P-value = 0.0005), 72h vs. 365 days (P-value < 0.0001). Source data are provided as a Source Data file. D Double immunofluorescence on Sox2^CreERT2/+;R26^mTmG/+ adrenals induced at P14 and collected after 365 days, using antibodies against GFP (green) and SOX2 (magenta). Arrowhead indicates a double-labelled cell. Nuclei counterstained with Hoechst (blue). Scale bar 10 μm. n = 2 females, 2 males, 3 technical replicates. E Double immunofluorescence on Sox2^CreERT2/+;R26^mTmG/+ adrenals induced at P14 and collected after 178 days, using antibodies against GFP (green) and specific cell markers (magenta) TH (all chromaffin cells), PNMT (adrenaline chromaffin cells) or PENK (noradrenaline chromaffin cells). Note the presence of double-labelled cells (arrowheads). Nuclei counterstained with Hoechst (blue), scale bar 10μm. n = 3 females, 3 males, 3 technical replicates. F Double immunofluorescence on Sox2^CreERT2/+;R26^mTmG/+ mice induced at P189 (6 months) and collected after 28 days, using antibodies against GFP (green) and TH (magenta). Note the presence of double-labelled cells (arrowheads). Nuclei counterstained with Hoechst (blue), scale bar 10 μm. n = 3 females, 2 males, 6 technical replicates.

Fig. 5. SOX2⁺ adrenomedullary stem cells promote proliferation of chromaffin cells through secretion of paracrine WNT ligands.

A Featureplots for expression of WNT targets Lef1, Axin2 and Lgr5 in the mouse postnatal adrenal medulla dataset, Key of clusters, grouping by lineage. Colour scale represents Log-normalised expression level, with 0 (grey) representing no expression, and 2.5 (red) for Lef1 and Axin2, or 3 (red) for Lgr5 representing highest expression. B Immunofluorescence using antibodies against TH (chromaffin cells) and GFP (cells that have responded to WNT) on mouse TCF/Lef:H2B-EGFP adrenal medulla at P21. Nuclei are counterstained with Hoechst, scale bars 50 μm. n = 1 female, 1 male, 1 technical replicate. C Dot plots of all Wnt genes expressed in the mouse adrenal medulla dataset (left) and in the isolated SOX2-EGFP⁺ cell dataset (right), subdivided by cell clusters. Colour scale represents average expression level, as a sliding scale of expression magnitude from blue (lowest expression) to red (highest expression). Dot size reflects percentage of cells with gene expression. D RNAscope mRNA in situ hybridisation using probes against Sox2 (red) and Wnt6 (blue), showing co-expression. Image in right is magnified region (i). Nuclei counterstained with hematoxylin. Scale bars 20 μm. n = 2 females, 2 males, 2 technical replicates. E Featureplots for Wls in the mouse adrenal medulla dataset (left) and in the isolated SOX2-EGFP⁺ cell dataset. Colour scale represents Log-normalised expression level, with 0 (grey) representing no expression, and 3 (red) in ‘Adrenal Medulla’ or 1.6 (red) in ‘SOX2-EGFP⁺’ representing highest expression. F Representative immunofluorescence using antibodies against Ki-67 marking cycling cells in Sox2^+/+; Wls^fl/fl (control, top) and Sox2^CreERT2/+;Wls^fl/fl (mutant, bottom) samples following tamoxifen induction at P13/13/15 and analysis at P21 (n = 8 controls, 6 mutants). Nuclei counterstained with Hoechst, scale bars 50 μm. Graph showing percentage of Ki-67 positive cells across replicates, revealing a statistically significant reduction in cycling cells in the mutant. Two-sided unpaired t-test, P-value = 0.0083. Whiskers min to max, the box extends from the 25th to the 75th percentile, line is median. Source data are provided as a Source Data file. G Immunofluorescence staining using antibodies against TH (chromaffin cells, green) and Ki-67 (cycling cells, magenta) in Sox2^+/+;Wls^fl/fl (control) and Sox2^CreERT2/+;Wls^fl/fl (mutant) samples following tamoxifen induction at P13/14/15 and analysis at P21 (n = 3 controls, 3 mutants). Scale bars 50μm. Bar graph showing percentage of Ki-67 positive cells, split into TH positive cells and non-TH positive cells. Two-sided unpaired t-test, P-value = 0.0320. Source data are provided as a Source Data file.