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A

LTR EF1α OCT4 AA KLF4 AA SOX2 IRES cMYC WPRE LoxP LTR

B C Individual vectors OKSIM


1.2
Transfected Vector

Expression level relative to


1

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

DNA OCT4 SOX2

SUPPLEMENTARY FIG. 2. Expression of embryonic stem


cell (ESC) markers in another xeno-free induced pluripotent
stem cell (iPSC) line. Immunofluorescence staining indicat-
ing expression of SSEA4, NANOG, OCT4, and SOX2 by the
xeno-free iPSCs. DNA was counterstained with HOECHST.
A B

0.0 0.2 0.4 0.6 0.8 1.0


XF-IPSC
Color Key

0 0.5 1

0.0 0.2 0.4 0.6 0.8 1.0


H7 hESC

0.0 0.2 0.4 0.6 0.8 1.0


Fibroblasts
0.0 0.2 0.4 0.6 0.8 1.0
XF-IPSC

0.0 0.2 0.4 0.6 0.8 1.0


Fibroblasts
Fibroblasts XF-IPSC H7 hESC

0.0 0.2 0.4 0.6 0.8 1.0


H7 hESC

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

SUPPLEMENTARY FIG. 4. Transient expression of Cre-


recombinase excises the viral transgene from the cell genome.
Fibroblasts explanted from the xeno-free induced pluripotent
stem cell (iPSC) teratoma were infected with different levels
of an adenovirus that codes for Cre-recombinase. Seven days
after infection, genomic DNA was harvested from the cells
and primers for the viral transgene (KS) and an endogenous
gene (REX1) was used in a real-time PCR to measure the rel-
ative transgene abundance in the infected and uninfected
cells. Also, nontransgenic cells were included as controls. As
shown, with increasing levels of adenovirus, a decrease in
the abundance of the transgene was observed, which indi-
cates that transient Cre-recombinase expression can remove
the polycistronic transgene from the cell genome.
Supplementary Table 1. Antibodies Used for Immunostaining

Antigen Catalog # Isotype Manufacturer

OCT4 sc-8628 Goat IgG Santa Cruz Biotechnologies


SOX2 5603 Rabbit IgG Chemicon
NANOG sc-33759 Rabbit IgG Santa Cruz Biotechnologies
LIN28 sc-67266 Rabbit IgG Santa Cruz Biotechnologies
SSEA4 MC-813-70 Mouse IgG Developmental Studies
Hybridoma Bank
NESTIN Ab5968 Rabbit IgG Abcam
MYF-5 sc-302 Rabbit IgG Santa Cruz Biotechnologies
P4H AF0910-1 Mouse IgG Acris GmbH
Alexa 488 Donkey anti- A21202 NA Invitrogen
mouse IgG
Alexa 594 Donkey anti- A21207 NA Invitrogen
rabbit IgG
Alexa 488 Donkey anti- A11055 NA Invitrogen
goat IgG
Supplementary Table 2. Primers Used for RT-PCR

Gene Forward primer (5′–3′) Reverse primer (5′–3′)

OCT4 endogenous AAACCCTGGCACAAACTCC GACCAGTGTCCTTTCCTCTG


SOX2 endogenous CACATGTCCCAGCACTACC CCATGCTGTTTCTTACTCTCCTC
NANOG ACTCTCCAACATCCTGAACCTC CTTCTGCGTCACACCATTGC
REX1 GTGGGAAAGCGTTCGTTGAG CGCTTTCCGCACCCTTC
OKSIM—OK TCTCCCATGCATTCAAACG GTGGAGAAAGATGGGAGCAG
OKSIM—KS GACCACCTCGCCTTACACAT TTCAGCTCCGTCTCCATCAT
LEFTY TTAGCAAGTTACTCCATCCCAATTTA GGACGGGAAAGACAGGAAATG
AFP GAGGGAGCGGCTGACATTATT TGGCCAACACCAGGGTTTA
PITX2 CGTCGGGCCAAATGGA CGAAGCCATTCTTGCATAGCT
NESTIN AGCCCTGACCACTCCAGTTTAG CCCTCTATGGCTGTTTCTTTCTCT
NEFL GAGGACAAGCAGAACGCCGA GTTGAGGAGGTCTTGGTATTCTT
NODAL TGAGATTTTCCACCAGCCAAA TCCATCTGAAACCGCTCTAAGC
GAPDH ACAGTCAGCCGCATCTTC CTCCGACCTTCACCTTCC
Supplementary Table 3. Fingerprinting Analysis (STR Profile) of Tissue Donor, Xeno-Free Induced Pluripotent
Stem Cell Line, and Pluripotent Cells Grown in Our Laboratory

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.

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