Correlation Between Genetic Abnormalities in Induced Pluripotent Stem Cell-Derivatives and Abnormal Tissue Formation in Tumorigenicity Tests Supplementary figures

Fig. S1. A: Morphology of iPSC clones with genomic mutations in the Census database and Shibata’s list. B: HE staining of transplanted H9-non RPEs, 16E84-non RPEs, 16H12-non RPEs, and 15M38-non RPEs. Genes listed in the Census database and Shibata's List from hg19/ver88 with VAF (%) values above the detection limit (24%) are highlighted in pink, and those below the detection limit are indicated in gray. VAFs of autosomal dominant genes reaching approximately 50% are shown in blue, indicating the clonality of the cells in the transplant. C: HE staining of transplants of H9, 16E84, 16E85, 16H12, and 15M38 in the cardiomyocyte lineage. Genes listed in the Census database and Shibata's List from hg19/ver88 are highlighted in pink. VAFs below the detection limit are highlighted in gray. VAFs of autosomal genes reaching approximately 50% are shown in blue, indicating the clonality of the cells in the transplant. D: Genomic-PCR and qRT-PCR detection of plasmid expression and POU5F1, POU5F1B, and NANOG expression. E: HE staining of transplants of Ff-I01-RPEs or Ff-I01-NSCs and VAFs of relevant transplants, as determined by WGS. Genes listed in the Census database and Shibata's List from hg19/ver88 are highlighted in pink. VAFs below the detection limit are highlighted in gray. VAFs of autosomal genes reaching around 50% or those of sex chromosomal genes reaching 100% are displayed in blue to show the clonality of cells in the transplant. F: Transition of the genetic instability of Ff-I01-RPEs or Ff-I01-NSCs and VAFs of relevant transplants by WGS. Karyotyping of Ff-I01-NSCs at p10 in culture (without transplantation). Genes listed in the Census database and Shibata's List from hg19/ver88 are highlighted in pink. VAFs below the limit of detection (24%) are shown in gray. VAFs of autosomal genes reaching approximately 50% are displayed in blue, indicating the clonality of the cells in the transplant. Fig. S2. The detection limit (LOD) and decision limit for VAFs, as determined by WGS and WES. WGS and WES were conducted in parallel. To ensure consistency between the results of the WGS and WES, the LOD (LD) and decision limit (critical value, LC) (IUPAC Commission on Analytical Nomenclature, Pure & Appl Chem. 1995;67:1699-1723) for VAFs were examined, based on the relative standard deviations (RSDs) of VAFs obtained by WGS and WES for common SNVs/del in common samples of iPSCs and iPSC derivatives from cell lines 16E84, 16E85, 16H12, and 15M38, according to the method reported by Miura et al. (Miura T et al., BMC Genom Data. 2021;22:8). A and B show the relationships between the mean VAFs of SNVs/del measured by WGS and WES and their RSDs. WES was conducted in parallel with WGS, as shown in Table 1. The purple, orange, and dark blue lines represent moving averages of 13, 17, and 21 data points, respectively. In C and D, the RSD of the mean VAF of an SNVs/del measured by WGS and WES was plotted against the VAF of the SNVs/del measured by WGS. In E and F, the RSD of the average VAF of an SNVs/del measured by WGS and WES was plotted against the VAF of the SNVs/del measured by WES. Based on these results, when the average VAF of an SNVs/del measured by WGS and WES was less than or equal to 30% (i.e., its LOD), which gave an RSD of 0.30, the SNVs/del were detected with sufficient certainty, and if the average VAF was greater than or equal to 9% (i.e., its decision limit), which gave an RSD of 0.61, the SNVs/del were detected above the LOD (A and B). When the VAF of an SNV/del was greater than its LOD and decision limit, the VAF of the SNV/del measured by WGS was greater than 24% and 12% in most cases, respectively (C and D). In the current study, these values are referred to as the LOD and decision limit for VAF in WGS, respectively. Fig. S3. A: Profile of CNVs from 16E84-iPSCs and their derivatives. B: Profile of CNVs from 16E85-iPSCs and their derivatives. C: Profile of CNVs from 16H12-iPSCs and their derivatives. D: Profile of CNVs from 15M38-iPSCs and their derivatives. E: Profile of CNVs from H9-iPSCs and their derivatives. Fig. S4. A: CNVs detected in Ff-WJ14s01-iPSCs and their derivatives. B: CNVs detected in 1210B2-iPSCs and their derivatives. C: CNVs detected in Ff-I01-iPSCs and their derivatives. D: CNVs detected in H9-ESCs and their derivatives. E: CNVs detected in 16E84-iPSCs and their derivatives. F: CNVs detected in 16E85-iPSCs and their derivatives. G: CNVs detected in 16H12-iPSCs and their derivatives. H: CNVs detected in 15M38-iPSCs and their derivatives. Fig. S5. CNs detected at 14q32.33 or 17q12 by ddPCR. CN: copy number. The ribonuclease P RNA component H1 gene (RPPH1) on Chr. 14q11.2 was used as a stable control for diploid copies (two copy control, CN = 2). The target locus in 14q32.33 was Chr.14:106260714, and that in 17q12 was Chr.17:36120285 on GRCh38. The amplicon lengths were approximately 100 bp. Samples with a copy number greater than 3 are highlighted in yellow.

图S1。A：携带癌症基因普查（Cancer Census）数据库及柴田列表（Shibata's List）中基因组突变的诱导多能干细胞（induced pluripotent stem cell, iPSC）克隆形态。B：移植后H9-non RPE、16E84-non RPE、16H12-non RPE及15M38-non RPE的苏木精-伊红（HE）染色结果。将人类基因组参考序列hg19/版本88（hg19/ver88）中收录于癌症基因普查数据库及柴田列表，且变异等位基因频率（Variant Allele Frequency, VAF）高于检测限（24%）的基因标记为粉色；VAF低于检测限的基因标记为灰色。常染色体显性基因的VAF接近50%时标记为蓝色，以体现移植细胞的克隆性。C：心肌细胞谱系来源的H9、16E84、16E85、16H12及15M38移植体的HE染色结果。将hg19/ver88中收录于癌症基因普查数据库及柴田列表的基因标记为粉色，VAF低于检测限的基因标记为灰色；常染色体基因的VAF接近50%时标记为蓝色，以体现移植细胞的克隆性。D：通过基因组PCR及实时定量逆转录PCR（qRT-PCR）检测质粒表达情况，以及POU5F1、POU5F1B、NANOG的表达水平。E：Ff-I01-视网膜色素上皮细胞（retinal pigment epithelium, RPE）或Ff-I01-神经干细胞（neural stem cell, NSCs）移植体的HE染色结果，以及通过全基因组测序（Whole Genome Sequencing, WGS）测定的相关移植体VAF。将hg19/ver88中收录于癌症基因普查数据库及柴田列表的基因标记为粉色，VAF低于检测限的基因标记为灰色；常染色体基因的VAF接近50%，或性染色体基因的VAF达到100%时标记为蓝色，以体现移植细胞的克隆性。F：Ff-I01-RPE或Ff-I01-NSCs的遗传不稳定性动态变化，以及通过WGS测定的相关移植体VAF；同时展示了未进行移植、体外培养至第10代的Ff-I01-NSCs的核型分析结果。将hg19/ver88中收录于癌症基因普查数据库及柴田列表的基因标记为粉色，VAF低于检测限（24%）的基因标记为灰色；常染色体基因的VAF接近50%时标记为蓝色，以体现移植细胞的克隆性。

图S2。基于全基因组测序（WGS）与全外显子组测序（Whole Exome Sequencing, WES）测定的变异等位基因频率（VAF）检测限与判定限。本研究同步开展WGS与WES实验。为保证两种测序结果的一致性，参考三浦等（Miura T et al., BMC Genom Data. 2021;22:8）报道的方法，基于诱导多能干细胞（iPSC）及iPSC衍生细胞系16E84、16E85、16H12、15M38的共通样本中，WGS与WES检测到的共通单核苷酸变异（single nucleotide variants, SNVs）/缺失（deletion, del）的VAF相对标准偏差（relative standard deviation, RSD），对VAF的检测限（LD）与判定限（临界值，LC，国际纯粹与应用化学联合会IUPAC分析化学命名委员会，Pure & Appl Chem. 1995;67:1699-1723）进行了验证。A与B展示了WGS与WES检测到的SNVs/del的平均VAF与其RSD的相关性。本研究同步开展WGS与WES实验，具体信息见表1。紫色、橙色及深蓝色曲线分别代表13、17、21个数据点的移动平均线。C与D中，将WGS与WES检测到的单SNV/del的平均VAF的RSD，相对于WGS检测到的该SNV/del的VAF进行绘图。E与F中，则将该平均VAF的RSD相对于WES检测到的该SNV/del的VAF进行绘图。基于上述结果，当WGS与WES检测到的单SNV/del的平均VAF≤30%（对应RSD=0.30）时，可认为该SNV/del的检测具有足够置信度；当平均VAF≥9%（对应RSD=0.61）时，该SNV/del的VAF高于检测限（A、B）。若单SNV/del的VAF同时高于检测限与判定限，则其WGS检测得到的VAF通常分别大于24%与12%（C、D）。本研究将上述数值分别定义为WGS中VAF的检测限与判定限。

图S3。A：16E84-iPSC及其衍生细胞的拷贝数变异（copy number variation, CNV）谱。B：16E85-iPSC及其衍生细胞的CNV谱。C：16H12-iPSC及其衍生细胞的CNV谱。D：15M38-iPSC及其衍生细胞的CNV谱。E：H9-iPSC及其衍生细胞的CNV谱。

图S4。A：Ff-WJ14s01-iPSC及其衍生细胞中检测到的CNV。B：1210B2-iPSC及其衍生细胞中检测到的CNV。C：Ff-I01-iPSC及其衍生细胞中检测到的CNV。D：H9-胚胎干细胞（embryonic stem cell, ESC）及其衍生细胞中检测到的CNV。E：16E84-iPSC及其衍生细胞中检测到的CNV。F：16E85-iPSC及其衍生细胞中检测到的CNV。G：16H12-iPSC及其衍生细胞中检测到的CNV。H：15M38-iPSC及其衍生细胞中检测到的CNV。

图S5。通过数字液滴PCR（digital droplet PCR, ddPCR）检测到的14q32.33或17q12位点的拷贝数（CN，copy number）。以14号染色体14q11.2区域的核糖核酸酶P RNA组分H1基因（RPPH1）作为二倍体稳定对照（拷贝数为2，CN=2）。人类基因组参考序列GRCh38中，14q32.33的靶位点为Chr.14:106260714，17q12的靶位点为Chr.17:36120285。扩增子长度约为100 bp。拷贝数大于3的样本标记为黄色。