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LTR EF1α OCT4 AA KLF4 AA SOX2 IRES cMYC WPRE LoxP LTR
OKSIM
cMYC
OCT4
SOX2
KLF4
individual vector
0.8
0.6
OCT4
0.4
Primary Antibody
KLF4
0.2
SOX2 0
OCT4 KLF4 SOX2 cMYC
cMYC
Individual vectors 1 1 1 1
GFP OKSIM 1.027 1.052 0.596 0.293
β-Actin
SUPPLEMENTARY FIG. 1. OKSIM polycistronic viral vector. (A) Diagram of vector design. EF1α promoter drives the
expression of a polycistronic gene that contains the human cDNA sequences of OCT4, KLF4, and SOX2 bound by self-
cleaving 2A elements and the human cDNA sequence of cMYC separated by internal ribosomal entry site (IRES) sequence.
Abbreviations: LTR, long terminal repeat; EF1-α, elongation factor 1α; IRES, internal ribosome entry site; WPRE, Woodchuck
hepatitis virus post-transcriptional regulatory element. (B) Western blot analysis of HEK293 cells transfected with plasmids
coding all factors together (OKSIM) or individual ones as indicated. A plasmid coding for GFP was co-transfected with the
single-factor vectors to control for potential differences in transfection efficiency. (C) Densitometry analysis of western blot
results. The expression level achieved with either the polycistronic vector or single-factor vectors is shown. The densitom-
etry measure for each gene was normalized to β-actin and then expressed as a proportion to the level achieved by individual
factor vectors.
DNA SSEA4 NANOG
0 0.5 1
SUPPLEMENTARY FIG. 3. Genome-wide methylation of xeno-free induced pluripotent stem cell (iPSC). (A) Heat map
and cluster analysis of xeno-free iPSCs, input fibroblasts, and a human embryonic stem cell (ESC) indicating that the overall
pattern of methylation of iPSCs closely matches ESC while it differs from fibroblasts (only 1,936 CpGs that were significantly
different between ESCs and fibroblasts were used for this graph). Methylation levels were determined using Illumina’s
Infinium Human Methylation27 BeadChip (in the Color Key 0 indicates hypomethylation and 1 hypermethylation). (B)
Scatter plots comparing the methylation level at 27,570 different CpGs between different cell lines as indicated. A high cor-
relation is observed between xeno-free iPSC and H7 hESC (r = 0.97), while the correlation is lower when ESC or iPSC are
compared to fibroblasts (r = 0.79 and 0.80, respectively).
1.2
Relative Transgene
Abundance 1
0.8
0.6
0.4
0.2
0
0 0.2 μL 1 μL 5 μL Skin
Fibroblasts
iPSC-Derived Fibroblasts Infected
With Different Levels of Ad-Cre
Locus Buccal Swab Xeno-free iPS cells IMR90-iPS cells MSU-iPS cells H9 hESC
Amelogenin XY XY XX XY XX
vWA 17, 17 17, 17 16, 19 15, 17 17,17
TPOX 10, 11 10, 11 8, 9 8, 8 10, 11
THO1 6, 8 6, 8 8, 10 9.3, 9.3 9.3, 9.3
D5S818 11, 12 11, 12 12, 13 11, 13 11, 12
D13S317 11, 11 11, 11 11, 13 11, 12 9, 9
D7S820 10, 12 10, 12 9, 12 10, 12 9, 11
D16S539 10, 10 10, 10 10, 13 11, 12 12, 13
CSF1PO 10, 10 10, 10 11, 13 12, 13 11, 11
D8S1179 15, 16 15, 16 13, 15
FGA 19, 21 19, 21 19, 25
D3S1358 15, 19 15, 19 14, 18
D21S11 28, 29 28, 29 28, 32
D18S51 17, 17 17, 17 15, 18
Penta E 13, 16 13, 16
Penta D 11, 13 11, 13
hESC, human embryonic stem cell.