Supplementary Figure 1: Pluripotency of Y-iPSC, A-iPSC-ZSCAN10, and A-iPSC.

(a) Teratoma formation assay for ESC, Y-iPSC, A-iPSC-ZSCAN10, and A-iPSC. H/E staining was carried out with harvested teratoma to demonstrate the potential for tissue differentiation to three germ layers. Scale bar = 400 μm. (b) AP staining. (c) OCT4 expression levels in embryonic fibroblasts (MEF), ESC, Y-iPSC, A-iPSC-ZSCAN10, and A-iPSC by qPCR. Three representative independent clones of each cell line are shown. (d) Immunostaining for SSEA-1 and NANOG in ESC, Y-iPSC, A-iPSC-ZSCAN10, and A-iPSC. Nuclei were stained with DAPI. No NANOG expression was detected in fibroblasts, whereas iPSC from B6CBA expressed NANOG at low levels¹. (e) Generation of chimeric mice by injection of Y-iPSC, A-iPSC, and A-iPSC-ZSCAN10 with GFP+ marker in E12.5 embryos. Two clones of each cell type were used to test the chimerism. Placentas/placental fetal membranes (red arrows) were included in the images as negative controls. Images on the TRITC filter were included to verify minimal auto-fluorescence signals. (f) Confirmation of transgene silencing in Y-iPSC, A-iPSC-ZSCAN10, and A-iPSC. Silencing of transgene expression (OCT4, SOX2, KLF4, and MYC) was confirmed by qPCR and RT-PCR using 1 μg of RNA and the primers specified in Methods. Shown here are three representative independent clones of each cell line. Uninfected fibroblasts were used as a negative control and day 3 fibroblasts transfected with Yamanaka factors were used as a positive control. No detectable expression of the four transgenes was observed after 40 cycles of amplification. We detected the signal in positive controls of OCT4 in PCR cycle 22.4, SOX2 in PCR cycle 18.5, KLF4 in PCR cycle 16, MYC in PCR cycle 17, and ACTIN in PCR cycle 13.5. (g) An example of normal karyotype in cytogenetic analysis of ESC, ntESC, Y-iPSC, A-iPSC-ZSCAN10, and A-iPSC. All three clones of ntESC showed a normal karyotype. (h) An example of a detailed analysis of chromosome instability in A-iPSC. (i, j) DNA damage response of A-iPSC (n = 10 independent clones in each cell type) generated from lung and bone marrow with pATM after phleomycin treatment (2 h, 30 μg ml⁻¹). qPCR of ZSCAN10 mRNA levels (showing poor activation of ZSCAN10 expression) and GSS mRNA levels (indicating excessive expression) in A-iPSC generated from lung (n = 12) and bone marrow (n = 9). Error bars indicate standard error of the mean of the number of clones indicated above. Statistical significance was determined by two-sided t-test. Detailed information provided in Supplementary Table 5.