Isogenic hiPSC models of Turner syndrome development reveal shared roles of inactive X and Y in the human cranial neural crest network

Abstract

Viable human aneuploidy can be challenging to model in rodents due to syntenic boundaries or primate-specific biology. Human monosomy-X (45,X) causes Turner syndrome (TS), altering craniofacial, skeletal, endocrine, and cardiovascular development, which in contrast remain unaffected in X-monosomic mice. To learn how monosomy-X may impact embryonic development, we turned to 45,X and isogenic euploid human induced pluripotent stem cells (hiPSCs) from male and female mosaic donors. Because the neural crest (NC) is hypothesized to give rise to craniofacial and cardiovascular changes in TS, we assessed differential expression of hiPSC-derived anterior NC cells (NCCs). Across three independent isogenic panels, 45,X NCCs show impaired acquisition of PAX7⁺SOX10⁺ markers and disrupted expression of other NCC-specific genes relative to isogenic euploid controls. Additionally, 45,X NCCs increase cholesterol biosynthesis genes while reducing transcripts with 5' terminal oligopyrimidine (TOP) motifs, including those of ribosomal and nuclear-encoded mitochondrial proteins. Such metabolic pathways are also over-represented in weighted co-expression modules that are preserved in monogenic neurocristopathy and reflect 28% of all TS-associated terms of the human phenotype ontology. We demonstrate that 45,X NCCs reduce protein synthesis despite activation of mammalian target of rapamycin (mTOR) but are partially rescued by mild mTOR suppression. Our analysis identifies specific sex-linked genes that are expressed from two copies in euploid males and females alike and qualify as candidate haploinsufficient drivers of TS phenotypes in NC-derived lineages. This study demonstrates that isogenic hiPSC-derived NCC panels representing monosomy-X can serve as powerful models of early NC development in TS and inform new hypotheses toward its etiology.

Keywords: Turner syndrome; X chromosome inactivation; Y chromosome; gene dosage; haploinsufficiency; human iPSC; neural crest; protein synthesis; ribosome biogenesis; sex chromosome evolution.

Figure 1

Monosomy-X impact on neural crest differentiation relative to isogenic euploid controls (A) Representative immunofluorescence (IF) of PAX7 (green), SOX10 (red), and nuclei (Hoechst 33342) in hiPSC-derived pairs of 45,X and euploid control NCCs from Male1, Male2, and Female donors. Scale bars, 100 μm. (B) CellProfiler quantification of 4–7 rounds of differentiation. Brackets and p value (two-tailed Mann-Whitney U test) indicate grouped cell lines compared within and across isogenic panels (colored by donor, symbols denoting karyotype).

Figure 2

Reduction of neural crest marker expression in monosomy-X relative isogenic euploid controls (A) Principal-component analysis (PCA) segregates samples by “condition” (donor and karyotype), with percent of variance of PC1/PC2 as indicated. (B) Variance-stabilized counts (vst) of early and late anterior NC (eANC/lANC), retinoic acid-treated NC (raANC), and neuromesodermal progenitor (NMP) markers from Frith et al. (vertical panels) were median normalized and averaged over euploid (EU) and 45,X (XO) NCCs of each (horizontal) donor panel (significant differences denoted by two-tailed Mann-Whitney p value). (C and D) As in (B) for, respectively, p75−/p75+ associated markers and chick NC marker sets. (E) Pearson correlation of the matching PAX7⁺SOX10⁺ percentage with averaged marker sets (Lee, 2007, Frith, 2018, Simões-Costa, 2015⁴¹) from (B)–(D), alongside cycling markers (Hsiao, 2020⁴⁴), NC markers from a 3D folding human neural tube model (Karzbrun, 2021⁴³), and averaged pseudo-autosomal, X/Y pair, and other escapee (PAR/PAIR/other ESC) expression, as annotated in Tukiainen et al., Wainer Katsir and Linial,^,, and Sauteraud et al.^,, Only significant pairwise correlation is shown (p ≤ 0.05, Fisher-transformed Pearson R).

Figure 3

Concordant impact of monosomy-X on NCC transcriptomes across isogenic panels (A) Left: Venn diagram of differentially expressed genes (DEGs) in female (fXO), male1 and male2 (mXO1, mXO2) 45,X NCCs. Significance of pairwise overlapping DEGs shown by boldface p values (hypergeometric distribution). Right: differential vst heatmaps for overlapping DEG sets from the Venn diagram (left) as denoted by arrows. Ratio and p value (sign-test) denote the number of DEGs with concordant direction (“Dir.”) in the triple and all pairwise overlapping DEG sets, also shown in divergent (fXO, mXO1/2) annotation panels showing the Wald statistic (DESeq2) for each gene. Dendrogram segregates samples by karyotype, irrespective of donor. (B) Wikipathway gene set enrichment analysis (GSEA), ordered by the quantile-normalized mean Wald statistic across 45,X conditions (ave.XO). The x axis denotes log-scaled GSEA adjusted p value. Colors indicate normalized enrichment score of upregulated (red) and downregulated (blue) gene sets (sized by number of genes). Results for all isogenic monosomy-X comparisons (fXO, mXO1/2), averaged (ave.XO/ave.mXO), and control comparisons (fmE1/2: female-male euploids; m1m2O: male1/2 45,X samples). (C) Human Phenotype Ontology (HPO) GSEA results for significantly (p.adjust ≤ 0.1) enriched terms associated with Turner syndrome (Orphanet ID: 881), ordered by the quantile-normalized mean (mXO1/2) Wald statistic (ave.mXO, otherwise as in B).

Figure 4

Monosomy-X-sensitive gene modules correlate in human development and hiPSC-derived NCC models of monogenic neurocristopathy (A) Color labels and genes per WGCNA module (1–29) with corresponding headings. (1) Module-trait correlation matrix (Pearson R) of module eigengene to karyotype status (euploid, 45,X) and %PAX7⁺SOX10⁺, sex-linked gene expression (PAR, X/Y-pair, other escapees, and “All” classes combined), and averaged expression of published marker gene sets (labeled as in Figures 2 and S2). Only significant correlations (p ≤ 0.05, Fisher transformation) are colored, with log-scaled adjusted p values displayed to the nearest integer. (2) Significantly enriched (p.adjust ≤ 0.1, hypergeometric distribution) gene terms from the Hallmark and canonical pathways collections (MSigDB) representing metabolism and development. Shading and integer correspond to the number of enriched MSigDB terms falling into a given category (column). (3) Preservation statistics (shading and integer Z score for Z ≥ 5) in transcriptomes of the developing human face^, (Carnegie stages CS13–17 and CS22) and heart (CS13–CS23), alongside hiPSC-derived NCC models of monogenic neurocristopathy syndromes (Pierre-Robin/SOX9, Waardenburg/SOX10, familial dysautonomia/IKBKAP, Bohring-Opitz/ASXL1, floating-harbor/SRCAP, and branchio-oculofacial/TFAP2A³³). Monosomy-X human iPSC-derived trophoblast-like cells (TBL¹²) and pancreatic tumor stroma (Stroma³⁴) as respective positive and negative controls. (4) DESeq2-derived Wald statistic averaged by module. (B) Module eigengene correlations (kME²) for each sex-linked gene over its correlation with the PAX7⁺SOX10⁺ rate (colored by assigned module, sized by %pHI rank). (C) Dotplot of significantly enriched (p.adjust ≤ 0.1, hypergeometric distribution) TS-HPO terms (x axis, numeric module labels, above gene total). Dot color and size denote log-scaled adjusted p value and fraction of term-associated genes per module. (D) Modules with significant correlation (R, with regression line and gray 95% confidence interval [CI]) between genes’ TOPscores (y axis) and their Pearson coefficient with the PAX7⁺SOX10⁺ rate (x axis), indicated by contour plots. (E) Median TOPscores over median Wald statistic by gene module (colors, size relative to gene total) with standard error bars per module. Pearson R correlation (black line, 95% CI) and p value indicated for each comparison (fXO, mXO1, mXO2), alongside TOPscores by DEG category (right).

Figure 5

Monosomy-X impairs translation, but mild mTOR suppression improves 45,X NCC survival (A) OPP incorporation levels normalized to isogenic XY SOX10⁺ cells (XY+ group) by (n) differentiation rounds (bottom, in parentheses), in both male donor-derived iPSC panels segregated by group (karyotype in SOX10⁺ and SOX10^– cells). Median of averaged OPP incorporation by image (data points) are indicated below boxplots, with symbols above indicating significance of median difference relative to isogenic XY+ cells (two-tailed Mann-Whitney U test: ns, p > 0.05; ^∗∗∗∗p ≤ 10⁻⁴). (B and C) (B) Percentage of SOX10⁺ cells and (C) cells per image in both male donor-derived iPSC panels segregated by karyotype, differentiated without (0 nM) or with increasing concentrations of rapamycin (5, 10, 20 nM), across (n) rounds of differentiation (bottom, in parentheses). Median of image-averaged %SOX10 (B) and cells/image (C) are indicated below each boxplot, with significance of median difference relative to isogenic XY cells (two-tailed Mann-Whitney U p value) indicated above each boxplot.