Fig 2: t-hCO neurons undergo advanced maturation.a, 3D reconstruction of biocytin-filled hCO and t-hCO neurons at 8 months of differentiation. b, Quantification of morphological features (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 and ***P < 0.0001). c, 3D-reconstructed dendritic branches of hCO and t-hCO at 8 months of differentiation. The red asterisks indicate putative dendritic spines. Quantification of dendritic spine density (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0092). d, Quantification of the resting membrane potential (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P < 0.0001). e, Repetitive action potential firing in hCO and t-hCO induced by increasing current injections, and quantification of the maximal firing rate (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P < 0.0001). f, Spontaneous EPSCs (sEPSCs) in hCO and t-hCO neurons at 8 months of differentiation, and quantification of the frequency of synaptic events (n = 25 hCO neurons, n = 17 t-hCO neurons; ***P < 0.0001). For b–f, hCO and t-hCO from line 1208-2 are taken from the same differentiation batch maintained in parallel. g, Gene set enrichment analysis (one-sided Fisher’s exact test) of genes significantly upregulated (adjusted P < 0.05, fold change > 2, expressed in at least 10% of nuclei) in t-hCO glutamatergic neurons compared with hCO glutamatergic neurons with gene sets of both early-response (ERG) and late-response (LRG) activity-dependent genes identified from an in vivo mouse study16 and human-specific LRGs from in vitro neurons17. The dashed line denotes Bonferroni-corrected P value of 0.05. h, GluN gene expression (pseudobulk and scaled for each gene) across snRNA-seq replicates of LRG genes significantly upregulated in t-hCO glutamatergic neurons. i, Immunostaining showing SCG2 expression in t-hCO (top) and hCO (bottom) neurons. White arrowheads indicate SCG2+ cells. Scale bar, 25 µm. Data are presented as mean ± s.e.m.

Fig 3: Correlation analysis between key genes and immune cells. (A) Correlation analysis for SCG2. (B) Correlation analysis for CCL19. Size of the dots represents the strength of the correlation between genes and immune cells, and color of the dots represents p-value. p < 0.05 was considered statistically significant. (C,D) Scatter plot showing the correlation between SCG2/CCL19 and immune cells. The X-axis represents the mRNA expression level of genes, the Y-axis represents the expression abundance of immune cells, and the R value represents the correlation coefficient.

Fig 4: Validation at single cell level and tissue level. (A) tSNE visualization of clustering revealed 12 cell populations. Cell population identities were determined based on marker gene expression. (B) Heatmap of top 5 marker genes for each cell cluster. (C) Heatmap shows the expression of SCG2 and CCL19 in 12 clusters of cells in CAVD group and normal control group, respectively. The transition from blue to orange represents the increased expression level. (D) The heatmap of GSEA analysis results shows the hallmark functional pathways enriched in each of the calcified or normal aortic valve cell clusters. The transition from blue to red indicates the increased enrichment of pathways. (E) H and E staining and immunohistochemical staining of human aortic valve tissue sections. Macroscopic and micrographs views of immunohistochemical staining of SCG2 and CCL19 from left to right. (F) Histograms of quantitative immunohistochemical staining results. The expression of SCG2 and CCL19 was significantly increased in CAVD group. AUC: Area under the ROC curve; 95% CI: 95% confidence interval; Norm: normal; CAVD: calcific aortic valve disease; VICs: valvular interstitial cells; VECs: valvular endothelial cell; TCs: T cells. ***p < 0.001.

Fig 5: Validation of key genes SCG2 and CCL19. (A,B) Validation of the expression and receiver operating characteristic (ROC) curve of SCG2 and CCL19 in the merged metadata cohort. (C,D) Validation of the expression of SCG2 and CCL19 and ROC curve for evaluating the discriminatory accuracy of SCG2 and CCL19 in the validation dataset GSE83453. (E,F) GSEA analysis result show the top ten enrichment terms (GO and KEGG respectively) of regrouped high SCG2 expression group. (G,H) GSEA analysis result show the top ten enrichment terms (GO and KEGG respectively) of regrouped high CCL19 expression group. **p < 0.01, ****p < 0.0001.