Supplementary Figure 5: Pluripotency of human Y-hiPSC, A-hiPSC-ZSCAN10, and A-hiPSC.
From: ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors

(a) Teratoma formation assay for hESC (H9), Y-hiPSC, A-hiPSC-ZSCAN10, and A-hiPSC. Hematoxylin and eosin staining of teratomas derived from immunodeficient mice injected with hESC (H9), Y-hiPSC, A-hiPSC, or A-hiPSC-ZSCAN10 reveals differentiation to all three embryonic germ layers, including cartilage (mesoderm), muscle (mesoderm), neuroepithelial rosettes (ectoderm), brain (ectoderm), intestinal epithelium (endoderm), and respiratory epithelium (endoderm). Scale, 100 μm. (b) Human pluripotent gene expression in hESC (H9), Y-hiPSC, A-hiPSC, and A-hiPSC-ZSCAN10 were confirmed by flow cytometry analysis (TRA181, SSEA3, and SSEA4) and qPCR (OCT4). Error bars on qPCR indicate standard error of the mean of two replicates with three independent clones in each sample group (n = 6). Fibroblasts were used as a negative control. (c) Quantification by image-analysis of apoptotic response by DNA fragmentation assay after phleomycin treatment. Error bars indicate standard error of the mean of n = 10 biological replicates. Statistical significance was determined by two-sided t-test. (d,e) DNA damage response with ATM after phleomycin treatment (2 h, 30 μg ml−1) in A-hiPSC-AG4 clones that were reprogrammed with lentivirus expressing hOCT4, hSOX2, and hKLF4 without hMYC expression (d) and with integration-free episomal vectors (e). (f) Immunoblot showing impaired ATM DNA damage response in Y-hiPSC (H1OGN) with ZSCAN10 shRNA expression in three independent clones after phleomycin treatment (2 h, 30 μg ml−1). (g) Confirmation of the ZSCAN10 binding activity to the consensus binding motif on the GSS promoter that reported by Yu et al. J. Biol. Chem., 2009 using ChIP-qPCR14. Error bars indicate standard error of the mean of three replicates for AiPSC and YiPSC (n = 3) and three replicates for positive/negative controls (n = 3) Statistical significance was determined by two-sided t-test followed by post-hoc Holm–Bonferroni correction for a significance level of 0.05, ∗ indicates significant and NS: not significant. Oct4 promoter region was used as a positive control and an 94 bp region on chromosome 14 was used as a negative control. Detailed information provided in Supplementary Table 5. (h) Copy number profiling analysis of various hiPSC, including examples of non-rearranged A-hiPSC, rearranged A-hiPSC, non-rearranged A-hiPSC-ZSCAN10, and non-rearranged Y-hiPSC. (i) Representative high-resolution G-banded karyotyping analysis of human iPSC clones. Y-hiPSC, A-hiPSC, and A-hiPSC ZSCAN10 showed normal karyotypes. However, A-hiPSC showed a higher frequency of cytogenetic abnormalities (Fig. 5e). A-hiPSC-JA also had an abnormal karyotype, with partial deletion of the p-arm of chromosome 20 and a lost Y chromosome. (j) Genes mutated in the coding region of A-hiPSC and A-hiPSC-ZCAN10. Number of variants found with a sequencing depth of at least 20 and a consensus quality score of at least 20.