Fig. 2: Characterization of chromosomal instability and DNA damage in TSCs.

A Representative FISH images showing aneuploid cells (white arrows) in nTSCs, nbTSCs, ccTSCs, and pdTSCs, detected using probes for chromosomes 7, 12, and 18. Scale bar, 20 μm. B Fold change of aneuploid cell with chromosomes 7, 12, and 18, normalized to the baseline control primed hPSCs (≥ 100 cells counted per line, n = 3 independent biological cell populations). Data are presented as mean ± SD. Significance was analyzed by two-sided Student’s t-test. P < 0.05: a, compared to primed hPSCs; b, compared to primary CT. For chromosome 7, compared to primed hPSCs, nTSC vs. hPSC: P = 0.01; nbTSC vs. hPSC: P = 0.02; ccTSC vs. hPSC: P = 0.01; pdTSC vs. hPSC: P = 0.3. For chromosome 12, compared to primed hPSCs, nTSC vs. hPSC: P = 0.04; nbTSC vs. hPSC: P = 0.04; ccTSC vs. hPSC: P = 0.2; pdTSC vs. hPSC: P = 0.2. For chromosome 18, compared to primed hPSCs, nTSC vs. hPSC: P = 0.0003; nbTSC vs. hPSC: P = 0.01; ccTSC vs. hPSC: P = 0.049; pdTSC vs. hPSC: P = 0.07. Compared to primary CT, significant differences were only found in chromosome 7: pdTSC vs. primary CT: P = 0.03; hPSCs vs. primary CT: P = 0.03. The TSC passage number is from 12 to 16. Source data are provided as a Source Data file. C Flow cytometry analysis of DNA content in TSCs and primed hPSCs. N, haploid number. Cell cycle phase: G1, S, G2/M. The percentages of different phases were measured. The percentage of aneuploidy with DNA content of <2 N and >4 N was plotted. D Percentage of aneuploid cells, identified by DNA content outside the 2 N and 4 N ranges. TSCs exhibit elevated aneuploidy levels compared to parental hPSCs. E Representative image of micronuclear DNA (white arrow) in TSCs. DAPI for nuclear staining. Scale bar, 20 μm. F Percentage of cells with micronuclei in TSCs, primed hPSCs, and primary CT. TSCs were analyzed at early passages (passages 3–5) and late passages (≥ 12). Mean ± SD (≥ 300 cells counted per line, n = 3 independent biological cell populations). Significance was analyzed by two-sided Student’s t-test. P < 0.05: a, early-passage TSCs, late-passage TSCs, and primary CT compared to primed hPSCs, respectively; b, early-passage TSCs compared to late-passage TSCs. For late-passage TSCs, compared to primed hPSCs, nTSC vs. hPSC: P = 0.02; nbTSC vs. hPSC: P = 0.00003; ccTSC vs. hPSC: P = 0.03; pdTSC vs. hPSC: P = 0.0007. For early-passage TSCs, compared to primed hPSCs, nTSC vs. hPSC: P = 0.02; nbTSC vs. hPSC: P = 0.005; ccTSC vs. hPSC: P = 0.0001; pdTSC vs. hPSC: P = 0.01. Significant differences were observed between early- and late-passage nbTSC (P = 0.001) and pdTSC (P = 0.004). hPSCs vs. primary CT: P = 0.045. No significant difference was observed between TSCs and primary CT. Source data are provided as a Source Data file. G Immunofluorescence staining for γ-H2AX, indicating double-strand DNA breaks, in nTSCs, nbTSCs, ccTSCs, pdTSCs, and hPSCs. Red arrow indicates apoptosis with diffuse expression of γ-H2AX; orange arrow indicates cells with double-strand DNA breaks, dotted expression of γ-H2AX. Scale bar, 20 μm. H Quantification of γ-H2AX foci per nucleus in TSCs and primed hPSCs. Mean ± SD of >50 cells per line (n = 3 independent biological cell populations). Significance was analyzed by two-sided Student’s t-test. *P < 0.05 compared to primed hPSCs. nTSC vs. hPSC: P = 0.01; nbTSC vs. hPSC: P = 0.03; ccTSC vs. hPSC: P = 0.003; pdTSC vs. hPSC: P = 0.01. Source data are provided as a Source Data file.