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.