(A) Spectral karyotype of Control, POF and Turner syndrome (TSC1, TSC2, TSN and TSF) fibroblasts. White box indicates two X chromosomes in Control and POF fibroblasts and only one X chromosome in all Turner syndrome fibroblasts. (B) X chromosome deletion map from genome-wide human SNP array for fibroblast samples. Red shaded box indicates region of monoallelic SNPs, indicating deletion of second X chromosome in Turner syndrome fibroblasts. Arrowhead marks centric portion of TSC1 with biallelic SNPs. (C) Phase contrast image of one iPSC subclone (C = subclone) colony, grown on MEFS or matrigel, derived from Control, POF and Turner syndrome fibroblasts along with their spectral karyotype. White box indicates two X chromosomes in Control and POF iPSC subclones and only one X chromosome in all Turner syndrome iPSC subclones. (D) Immunofluorescence for cell surface markers TRA-1-60, TRA-1-80 and SSEA4 (green) along with nuclear marker OCT4 (red) marking pluripotent iPSC subclones. Cell nuclei were costained with DAPI (blue). Scale bar, 150 μm. (E) Immunofluorescence for markers of the three germ layers after spontaneous differentiation of iPSC subclones, demonstrating cells expressing α Feto Protein (endoderm), Smooth Muscle Actin (mesoderm) and βIII Tubulin (ectoderm) (green). Cell nuclei were costained with DAPI (blue). Scale bar, 150 μm. (F) In vitro (teratoma) differentiation of subclones from all iPSC lines with evidence of all three germ layers, gut epithelium (endoderm), cartilage and smooth muscle (mesoderm) and neural rosettes and pigmented epithelium (ectoderm).

(A) Schematic of sorting method used to identify and isolate single pluripotent cells for gene expression analysis. (B–E) Violin plot representation of Log₂Ex values (fold change above background) of single cells for (B) pluripotency genes in fibroblasts, hESC or iPSC subclones (C = subclone), (C) XIST, (D) genes on PAR1 and PAR2 and (E) genes that escape XCI genes on the X chromosome in hESC and iPSC subclones. Asterisk indicates a significant difference in expression (p-value < 0.01) when compared to H9 hESCs. Color-coded subclone information is provided.

(A) Schematic of in vitro differentiation of VASA:GFP transfected pluripotent stem cells across genotypes for seven days with BMP 4/8 and RA. GFP+ cells were sorted and then purity sorted each day for single cell qRT-PCR analysis. (B) Heatmap of single GFP+ cells from h9 hESC, Control iPSCs, TSC1 iPSCs and Triple X iPSCs throughout the seven day directed differentiation. Global Log₂Ex values (fold change above background) of germ cell genes is shown with grey indicating no detectable expression.

(A) Multi-dimensional scaling (MDS) plot of RNA-seq samples. Similarities of gene expression patterns were calculated and mapped for H9 hESCs, Control iPSCs (subclones 7 and 8), TSC1 iPSCs (subclones 1 and 2) and Triple X iPSCs as well as day 7 differentiated H9 cells sorted for GFP (H9 GFP- and H9 GFP+), day 7 Control subclone 8 cells sorted for GFP (Control GFP- and Control GFP+) day 7 differentiated TSC1 subclone 2 cells sorted for GFP (TSC1 GFP- and TSC1 GFP+) and day 7 differentiated GFP+ Triple X cells (Triple X GFP+). (B). Hierarchical clustering of X aneuploid samples, pairwise scatterplots of gene expression levels, and the similarity matrix. The similarity matrix is colored from yellow to red, from the most similar to the most different sample comparisons. C) X chromosome scanning of pluripotent cells. The length of X chromosome was scanned in 10,000 bp windows. The reads mapping to each bin were normalized for the number of loci it maps to and the fraction of the mapped length that overlaps the bin. The reads from clones of control and TSC1 iPSCs were averaged. D) X chromosome scanning of differentiated cells. The reads mapping across the X chromosome was calculated as above. The GFP+ and GFP- reads were averaged for H9, Control and TSC1 differentiated cells.

(A) Schematic of xenotransplantation procedure. Control and X chromosome aneuploid pluripotent stem cells were injected into the seminiferous tubules, via the efferent ducts of busulfan treated, immune deficient mice. (B) Histological cross sections from Human fetal testis (20 weeks), uninjected mouse testis, male control iPSCs, H9 hESCs, Control iPSCs, TSC1 iPSCs and Triple X iPSC-injected murine testis analyzed by immunohistochemistry using human specific antibody, NUMA and germ cell marker VASA antibody. All images include: i) single channel for NUMA (green); ii) VASA (red) co-stained with nuclear DAPI (blue); iii) merge of NUMA/VASA/DAPI. Scale bar, 40 um. White dashed line indicates border of seminiferous tubule. White box within merge demonstrates magnified region (Scale bar, 10 um) showing injected samples with human germ cells with colocalization of NUMA and VASA in murine seminiferous tubules (iv).

(A–G) Immunofluorescence images of cross-sectioned Human fetal testis (20 week), mouse uninjected testis and xenografts from mouse testis injected with H9 hESC, Control iPSC, Triple X iPSC and TSC1 iPSCs. Positive NUMA (green) or STELLA (red) staining indicated human derived cells. Cross-sections were analyzed for (A) OCT4 (red), (B) DAZL (red), (C) STELLA (red), (D) CKIT (green) and (E) H3K27me3 (green). White arrow indicates human derived cells, marked with either NUMA or STELLA, that are positive for additional markers. Scale bar, 25 um.