Fig. 1: Development of D-OSKM tumors that resemble human germ cell tumors.

a Schematic illustration of the genomic construct of ESCs containing single- or double-OSKM polycistronic cassettes (S-/D-OSKM). b qRT-PCR analyses for expression of reprogramming factors in S-/D-OSKM MEFs at Day 3 after Dox treatment. Data are presented as means ± SD of biological triplicates. The mean expression level of S-OSKM MEFs treated with 2.0 μg/mL Dox was defined as 1 (two-way ANOVA and Tukey’s multiple-comparison test, two-sided). c Western blot analysis for each reprogramming factor in S-/D-OSKM MEFs after Dox treatment for three days. Fold increase in normalized intensity for each reprogramming factor in densitometric analysis is OCT4: 4.61, SOX2: 2.58, KLF4: 3.04, and c-MYC: 3.36. d Quantification of NANOG-positive iPSC colonies in S-/D-OSKM MEFs. Data are presented as means ± SD of three independent experiments (two-way ANOVA and Sidak’s multiple-comparison test, two-sided). e Principal component analysis (PCA) plot of gene expression profiles of mCherry-positive cells at the indicated time points in S-/D-OSKM MEFs, as determined by RNA-seq (S-/D-OSKM MEFs Day 0, n = 1; mCherry + MEFs Day 3, n = 3; Day 7, n = 3; Day 14, n = 3). f Schematic illustration of the genomic construct of D-OSKM ESCs for tissue-specific expression of OSKM in the kidney or pancreas (upper panel). The lower panel shows the protocol for Dox treatment of chimeric mice. g Representative macroscopic images of S-/D-OSKM kidney tumors. Scale bars: 5 mm. h Representative histological images and immunostaining for OCT4 and NANOG in S-/D-OSKM pancreatic tumors. Scale bars: 500 μm. i Quantification of the OCT4-positive cell area in S-/D-OSKM kidney tumors (normal kidney, n = 5; S-OSKM kidney tumor, n = 18; D-OSKM kidney tumor, n = 20). Data are presented as means ± SD of biologically independent samples (Kruskal–Wallis test and Dunn’s multiple-comparison test, two-sided). j Representative histological images and OCT4 immunostaining of splenic invasion of D-OSKM pancreatic tumor cells. Scale bars: 1000 μm. k qRT-PCR analyses for expression of pluripotency-related genes in S-/D-OSKM kidney tumors. Data are presented as means ± SD of biological triplicates. Relative expression levels to ESCs are shown (one-way ANOVA and Dunnett’s multiple-comparison test, two-sided). l Representative histological images of S-/D-OSKM tumors in the kidney and pancreas. Scale bars: 500 μm (upper) and 100 μm (lower). m Heatmap showing relative expression of uniquely upregulated genes in human testicular germ cell tumors (TGCTs) (n = 158). For identification of the uniquely upregulated genes, RNA-seq data from 23 types of human cancer were obtained from the Cancer Genome Atlas (TCGA) in the NIH GDC Data portal (https://portal.gdc.cancer.gov/) (see Fig. 7a). Uniquely upregulated genes in TGCTs were defined as genes that are expressed higher (>2 folds) in TCGTs than any other cancer type. Color range is shown using a log₂ scale. n Quantification of the OCT4-positive cell area in D-OSKM kidney tumors after Dox treatment at various doses (2.0 mg/mL, n = 20; 0.4 mg/mL, n = 4; 0.25 mg/mL, n = 7; 0.13 mg/mL, n = 4). Data are presented as means ± SD of biologically independent samples (Mann–Whitney test, two-sided).