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Howden, SE, Gore, A, Li, Z, Fung, HL, Nisler, BS, Nie, J et al.. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci USA 108: 6537-6542

Morgridge Institute for Research, Madison, WI 53715, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2011; 108(16):6537-42. DOI: 10.1073/pnas.1103388108
Source: PubMed

ABSTRACT

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.

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Available from: Brian Edward McIntosh
Genetic correction and analysis of induced pluripotent
stem cells from a patient with gyrate atrophy
Sara E. Howden
a,b,c
, Athurva Gore
d
, Zhe Li
d
, Ho-Lim Fung
d
, Benjamin S. Nisler
e
, Jeff Nie
a
, Goukai Chen
a,b,c
,
Brian E. McIntosh
a,b,c
, Daniel R. Gulbranson
a,b,c
, Nicole R. Diol
a,b,c
, Seth M. Taapken
e
, David T. Vereide
a,b,c
,
Karen Dyer Montgomery
e
, Kun Zhang
d
, David M. Gamm
f
, and James A. Thomson
a,b,c,g,1
a
Morgridge Institute for Research, Madison, WI 53715;
b
Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public
Health, Madison, WI 53707-7365;
c
Genome Center of Wisconsin, University of Wisconsin, Madison, WI 53706;
d
Department of Bioengineering, University of
California at San Diego, La Jolla, CA 92093;
e
Wicell Research Institute, Madison, WI 53707;
f
Department of Ophthalmology and Visual Sciences, Waisman
Center Stem Cell Research Program, Eye Research Institute, University of Wisconsin, Madison, WI 53705; and
g
Department of Molecular, Cellular, and
Developmental Biology, University of California, Santa Barbara, CA 93106
Contributed by James A. Thomson, March 1, 2011 (sent for review January 21, 2011)
Gene-corrected patient-specic induced pluripotent stem (iPS) cells
offer a unique approach to gene therapy. Here, we begin to assess
whether the mutational load acquired during gene correction of
iPS cells is compatible with use in the treatment of genetic causes
of retinal degenerative disease. We isolated iPS cells free of trans-
gene sequences from a patient with gyrate atrophy caused by a
point mutation in the gene encoding ornithine-δ-aminotransferase
(OAT) and used homologous recombination to correct the genetic
defect. Cytogenetic analysis, array comparative genomic hybridiza-
tion (aCGH), and exome sequencing were performed to assess the
genomic integrity of an iPS cell line after three sequential clonal
events: initial reprogramming, gene targeting, and subsequent
removal of a selection cassette. No abnormalities were detected
after standard G-band metaphase analysis. However, aCGH and
exome sequencing identied two deletions, one amplication,
and nine mutations in protein coding regions in the initial iPS cell
clone. Except for the targeted correction of the single nucleotide in
the OAT locus and a single synonymous base-pair change, no ad-
ditional mutations or copy number variation were identied in iPS
cells after the two subsequent clonal events. These ndings con-
rm that iPS cells themselves may carry a signicant mutational
load at initial isolation, but that the clonal events and prolonged
cultured required for correction of a genetic defect can be accom-
plished without a substantial increase in mutational burden.
A
utologous therapies based on induced pluripotent stem
(iPS) cells (13) have the potential to treat a wide range of
acquired and inherited diseases, but the extensive culture period
required could introduce a mutational burden incompatible with
specic clinical applications. For example, diabetes is a disease
that continues to have a serious impact on quality and length
of life despite its clinical management with insulin and is an
attractive target for iPS cell-based transplantation therapies.
However, pancreatic cancer is generally fatal within a few months
of diagnosis. Thus, it will be essential to determine the accu-
mulated mutational load of cells before transplantation and to
determine whether these mutations may lead to a signicantly
increased risk of oncogenesis. A recent study revealed that a
signicant number of point mutations are generally present in
iPS cell clones relative to the average sequence of the parental
somatic cells (4). Exome sequencing of 22 independent human
iPS cell lines reprogrammed by using four different methods
revealed a range of 114 point mutations in each of the lines
analyzed, with a projected average of six protein-coding muta-
tions per genome.
Because of the proliferative potential of iPS cells, homologous
recombination can be used to correct specic genetic defects
before transplantation. However, gene targeting typically requires
prolonged culture, drug selection, and additional clonal genetic
bottlenecks beyond initial iPS cell generation and may, therefore,
be expected to introduce high mutational loads. Here, we assess
the mutational load accumulated in iPS cells after three clonal
events that led to the targeted correction of a single base-pair
mutation in the OAT locus.
Homozygous mutations in the OAT locus result in Gyrate at-
rophy (GA) of the choroid and retina, a disease characterized by
progressive loss of visual acuity and night vision with eventual loss
of central vision typically occurring in the fourth to fth decades
of life (5). Although the exact pathophysiological mechanism of
GA remains unknown, the retinal pigmented epithelium (RPE) is
thought to be the initial site of degeneration (6). RPE dysfunction
is also involved in macular degeneration, the leading cause of
incurable blindness in developed countries, affecting >14 million
people worldwide (7). Diseases involving the RPE are attractive
targets for iPS cell-based therapies, because there are already
protocols for deriving RPE from human iPS cells (8, 9) and the
site can be easily monitored with noninvasive techniques. Fur-
thermore, the physiological function of the RPE depends on fairly
simple anatomical relationships so it is reasonable to expect that
transplanted cells could sustain retinal function if the structural
damage is not yet too severe. Indeed, a recent study has dem-
onstrated protective effects of iPS-derived RPE after trans-
plantation into RCS dystrophic rats (10), and the two most recent
human embryonic stem (ES) cell clinical trials approved in the
United States involves transplantation of RPE cells (11).
In this study, we used an episomal reprogramming method
(12) to generate iPS cells free of transgene sequences from a GA
patient homozygous for a deleterious mutation in the OAT gene.
We then used homologous recombination with a BAC-based
vector to correct the disease-causing mutation. Vector-free,
correctly targeted iPS cell clones were identied by custom high-
density array comparative genomic hybridization (aCGH) and
conrmed by standard techniques. We also performed sequential
aCGH and exome sequencing of the parental broblast line, the
iPS cell line before targeting, the iPS cell line after gene tar-
geting, and the iPS cell line after removal of the selection cas-
sette to assess the accumulation of genomic changes during these
procedures. We found that although there was a fairly substantial
mutational load in the iPS cell line at the time of derivation
before targeting, homologous recombination and cassette re-
moval could be carried out with minimal additional changes.
These results suggest that the accumulation of mutations may
Author contributions: S.E.H., K.Z., D.M.G., and J.A.T. designed research; S.E.H., A.G., Z.L.,
H.-L.F., B.S.N., J.N., G.C., B.E.M., D.R.G., N.R.D., S.M.T., and K.D.M. performed research;
A.G., Z.L., H.-L.F., B.S.N., J.N., S.M.T., and K.D.M. contributed new reagents/analytic tools;
S.E.H., A.G., Z.L., H.-L.F., B.S.N., J.N., S.M.T., D.T.V., K.D.M., and K.Z. analyzed data; and
S.E.H. and J.A.T. wrote the paper.
The authors declare no conict of interest.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: jthomson@morgridgeinstitute.
org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1103388108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1103388108 PNAS
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not limit the clinical utility of gene targeting approaches, but that
extensive genetic characterization of the initial iPS cell clones
will be critical.
Results
We obtained primary dermal broblasts from a GA patient from
the Coriell Institute for Medical Research. This donor is homo-
zygous for a C > T transition at nucleotide 677 (677C > T) in exon
7 of the OAT gene that converts alanine 226 (GCG) to valine
(GTG). To generate patient-specic iPS cells free of transgene
sequences, we used an episomal reprogramming strategy based
on the transfection of oriP-containing plasmid vectors encoding
seven reprogramming factors (OCT4, SOX2, NANOG, LIN28,
c-Myc, KLF4, and SV40 large T-antigen) (12). These EBV-based
episomal vectors are replicated in synchrony with the host cells
and are relatively stable, but in the absence of selection, are
nonetheless lost from dividing cells at a rate of 26% per cell
generation (13, 14), allowing the vectors to persist long enough
to induce reprogramming before being ultimately lost, resulting
in iPS cells that are free of transgene sequences. After a sin-
gle transfection, iPS cell clones were selected based on basic
morphological criteria (e.g., compact colonies, high nucleus-to-
cytoplasm ratios, and prominent nucleoli) and six clones were
expanded for further analysis. Total DNA was extracted and PCR
analysis was performed to examine whether episomal DNA used
for reprogramming remained. In four iPS cell clones, episomal
sequences were not detected (Fig. S1).
Two iPS cell lines (5 and 12) were selected for subsequent
gene targeting experiments. We designed a gene targeting con-
struct carrying a 37.4-kb fragment from a human BAC clone that
encodes the entire OAT coding region. A puromycin resistance
cassette anked by loxP sites was inserted just downstream of the
OAT coding region, resulting in two homologous arms 27.7 kb
and 8.8 kb in size (Fig. 1). We successfully isolated and expanded
15 and 5 puromycin-resistant colonies after the electroporation
of the linearized targeting vector into iPS cell clones 5 and 12,
respectively. Genomic DNA was extracted from each clone, and
PCR analysis was performed to test for the presence of vector
backbone sequences because clones that had undergone the
desired homologous recombination event should not contain
these sequences (Fig. S2). To assess expression of wild-type (w/t)
OAT mRNA, total RNA was extracted and RT-PCR was per-
formed to amplify exon 7 of the OAT transcript, which was then
sequenced. Exon 7 of the OAT gene was also amplied from the
genomic DNA of each iPS cell clone and sequenced. After
scoring each line for the presence or absence of vector backbone
sequences and a w/t copy of the OAT gene and/or transcript
(Table S1), we identied two potential gene-targeted iPS cell
clones (5.12 and 12.4). These two clones also displayed an almost
1:1 ratio of mutant to w/t transcript as evidenced by sequencing
analysis of the OAT RT-PCR products (Fig. 2A). Fluorescent in
situ hybridization (FISH) analysis revealed only two signals
corresponding to the endogenous OAT loci on chromosome
10q26 and failed to detect any additional random integration
events in these clones (Fig. 2B). FISH analysis performed on an
iPS cell clone in which backbone targeting vector sequences were
detected by PCR revealed the two endogenous loci and a third
signal corresponding to a random integration event (Fig. 2C).
Although we conrmed a gene targeting event in 2 of 20 (10%)
drug-resistant clones, we could not detect a w/t copy of the OAT
gene or transcript or any vector backbone sequences in 10 (50%)
clones. Therefore, we cannot exclude that these clones did un-
dergo homologous recombination but with a crossing over event
occurring between exon 7 and the puromycin cassette rather than
upstream of exon 7 (Fig. 1).
We next wished to examine whether aCGH could also be used
as a method to distinguish gene-targeted iPS cell clones from
drug-resistant clones arising from random integration of the
gene targeting vector. Genomic DNA from a gene-targeted iPS
line (clone 12.4) and a random integrant (clone 5.15) conrmed
by FISH were mixed with sex-mismatched reference DNA and
then hybridized to a custom-designed array containing probes
spanning our OAT gene targeting vector. No copy number var-
iation (CNV) involving the OAT coding sequence could be
detected for the gene-targeted line (clone 12.4) or its parental
iPS cell line, whereas increased hybridization corresponding to
the integrated puromycin cassette was observed as expected (Fig.
3A). Furthermore, CNV was not detected across the endogenous
OAT locus, providing further evidence that homologous re-
combination of the gene targeting vector had occurred in iPS cell
line 12.4 (Fig. 3B). In contrast, in the random integrant (5.15),
we detected more than two copies of the OAT gene along with
the puromycin cassette and all of the gene targeting vector
backbone sequences (Fig. 3 A and B).
Our custom array was also designed to include probe sequences
spanning the three episomal vectors used for reprogramming. We
detected no vector sequences for the gene-targeted line (clone
12.4) and its parental iPS cell line (clone 12), but an 1,850-bp
region of the reprogramming vectors corresponding to the 5 end
of oriP and C-terminal portion of the EBNA1 gene was detected
in both the random integrant (clone 5.15) and its parental iPS cell
line (clone 5) (Fig. 2C). PCR analysis using primers specic to this
sequence conrmed its presence in iPS cell lines 5, 5.12, and 5.15
but not 12 or 12.4 (Fig. S3). These results indicate that, in addition
to conrming homologous recombination, aCGH is useful in
excluding any iPS cell clones with integrated vector sequences.
Although all clones had normal karyotypes after G-banding
analysis, aCGH analysis revealed a number of subkaryotypic
alterations in both the parental and gene-corrected iPS cells that
were not detected after aCGH analysis of the original patient
broblast line. These genomic alterations included two small
deletions in the random integrant (clone 5.15) and its parental
line (clone 5), and two separate small deletions and a single
amplication in the gene-targeted line (clone 12.4) and its pa-
rental line (clone 12) (Table 1). No additional genomic aberra-
tions were detected by aCGH in the gene-targeted iPS cell line
(clone 12.4) compared with its parental iPS cell line (clone 12).
Analysis of iPS cells derived from the same patient line but using
lentiviral vectors also revealed several other amplications and de-
letions, indicating that reprogramming with episomal vectors per
se is unlikely to be the cause of large-scale genomic aberrations.
To remove the puromycin cassette, we transfected in vitro
transcribed mRNA-encoding Cre recombinase into the gene-
targeted iPS cell line (clone 12.4). Loss of the selection cassette
was observed in 8/8 independent colonies as evidenced by sen-
sitivity to puromycin in the culture medium and by PCR analysis
of the extracted genomic DNA (Fig. S4A). One clone (12.4.2)
was selected for further analysis. Southern blot conrmed the
loss of the puromycin cassette and G-band analysis conrmed
a normal karyotype (Fig. S4 B and C). Furthermore, when cells
from this line were injected into immunocompromised mice, we
loxP
loxP
8.8 kb
27.7 kb
PGK-Puro
vector backbone
PCR1 PCR2
OAT
E7*
probe
Fig. 1. Schematic diagram of the BAC-based gene targeting vector used in
the study. A loxP-anked selection cassette (PGK-P uro) was inserted 2kb
downstream of the OAT coding region by recombineering. The sizes of the
homologous arms are shown. The point mutation in exon 7 (E7*), the
regions amplied by PCR to identify random integrants, and the probe used
in Southern blot experiments are also indicated.
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observed teratomas comprising all three primary germ layers
(Fig. S5). This line was also analyzed by aCGH, which also
revealed loss of the puromycin cassette, lack of CNV at the OAT
locus, absence of any reprogramming vector sequences, and lack
of additional changes in genome integrity in comparison with its
parental iPS cell lines (Fig. S6).
To investigate the accumulation of mutations through reprog-
ramming, prolonged iPS cell culture, and the serial clonal events
needed for gene targeting, we performed exome sequencing on
the parental patient broblast cell line, an uncorrected iPS cell
line (clone 12), the gene-targeted iPS cell line (clone 12.4), and
the cassette-free iPS cell line (clone 12.4.2). We searched for
novel mutations between each successive clonal line relative to
its direct progenitor cell line sequence and discovered nine
mutations in the original iPS cell clone (Table 2). Somewhat
surprisingly, the genomes were remarkably stable through the
subsequent clonal events. Exome sequencing successfully detec-
ted the correct 677T > C substitution introduced by homologous
recombination in the gene-corrected iPS cell lines (clones 12.4
and 12.4.2). Although all nine original mutations were conrmed,
no additional mutations were detected in the targeted iPS cell line
(clone 12.4), and only one additional synonymous mutation was
detected in the cassette-free iPS cell line (clone 12.4.2).
Discussion
A central challenge to the therapeutic use of either human iPS
or ES cells is understanding what specic types and number of
mutations and genomic aberrations are clinically acceptable for
a given application, for such changes will always accumulate with
continued cell division. Indeed, recent analyses of large sets of
iPS cell lines identied a substantial number of cell lines carrying
full and partial chromosomal aberrations that arose either from
culture adaptation or from clonal selection of the parental cell
line (15, 16). Although all of the iPS cell lines in this study had
normal karyotypes by standard G-band analysis, aCGH and
exome sequencing revealed at least three regions of CNV and
nine mutations in the original iPS cell line before targeting.
These ndings are comparable to the results of other recent
studies, suggesting that iPS cells themselves may carry a signi-
cant number of genomic aberrations and protein-coding muta-
tions arising either in the donor, during somatic cell expansion,
or during reprogramming itself (4). Importantly, we found that
our current culture conditions can allow extensive expansion and
genetic manipulations of human iPS without a dramatic increase
in genomic instability and mutational burden. Clearly additional
studies are required to better estimate the mutational load ac-
quired during iPS cell culture, yet our results nonetheless allow
an initial crude estimate of this load. The observation that only
a single point mutation was acquired after gene correction and
cassette removal is consistent with the expected load based on
the average mutation rate in human somatic cells, while also
being above the expected load based on average mutation rate in
human germ-line cells (17).
Although BAC vectors have widely been used for the genetic
modication of murine ES cells, only a single study had reported
the use of BAC-based vectors for gene targeting in human plu-
ripotent stem cells (18). Here, we demonstrate the use of a BAC-
based homologous recombination strategy to successfully repair
a single base-pair mutation in a patient-specic iPS cell line that
is also free of reprogramming transgenes. We also found that
transfection of mRNA-encoding Cre recombinase is a highly
efcient method for removal of a loxP-anked selection cassette
in gene-corrected iPS cells. Although the residual 35-bp loxP site
that remains in the genome of the cassette-free iPS cells is un-
likely to have any detrimental effect, it would nonetheless be
desirable to have the correction be entirely seamless. A seamless
correction could be achieved by performing two sequential ho-
mologous recombinations, using both positive and negative se-
lection, or by anking the selection cassette with terminal repeats
A
BC
BAC control
Patient iPSCs Gene corrected iPSCs:
Random integrant
Gene corrected iPSCs:
Targeted
Fig. 2. Gene correction of patient iPS cells. (A) Chromatograms resulting from Sanger sequencing of OAT exon 7 PCR products amplied from the gene
targeting vector or cDNA from an uncorrected iPS cell line and gene-corrected iPS cell lines arising from a random integration or gene targeting event. Arrows
indicate the location of the point mutation in the patient line. The antisense strand is shown. FISH analysis of a gene-targeted iPS cell line (B) and a random
integrant (C ) using a uorescently labeled probe specictotheOAT locus. Yellow arrows indicate endogenous signals on chromosome 10q; red arrow
indicates a third signal caused by random integration of the gene-targeting construct.
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GENETICS
Page 3
from the piggyBac transposon, which can be seamlessly excised
after transient expression of the piggyBac transposase (19).
In this study, we used an episomal approach to produce
integration-free iPS cells, which has the advantage of requiring
just a single transfection. Although there are now several other
approaches to produce vector-free iPS cells, including seamless
removal of piggyBac vectors (20), protein transduction (21), and
RNA transfection (22), previous exome sequencing studies failed
to nd differences in mutational load between iPS cells derived
by integration-free episomal or RNA methods, or between
methods that included SV40 large T-antigen and those that did
not (4). Although episomal sequences have the potential to in-
tegrate into the genome, as evidenced in this study, the detailed
characterization required to assess genomic integrity of iPS cell
clones could simultaneously be used to identify and exclude any
iPS cell clones carrying integrated transgenes with no additional
cost to the analysis. We demonstrate the feasibility of using
customized aCGH for this purpose.
The mutational load observed in our iPS cell line is within the
range of mutations detected in the recent exome sequencing
study of 22 different iPS cell lines (4). Interestingly, analyses in
that report revealed that a majority, but not all, of these muta-
tions were present in subpopulations of the parental cell lines.
Therefore, simply changing iPS cell generation methods or cul-
ture conditions cannot eliminate such mutations. Because iPS
cell lines from different patients will also have unique sets of
mutations, it will be extremely challenging to assign relative risks
to different combinations of mutations. Clearly an ongoing di-
iPS 12
reprog. vector 1
iPS 12.4
iPS 5
iPS 5.15
+2
-2
0
pEFx
OCT4
IRES
SOX2
pEFx
SV40pA
IRES
SV40 LT KLF4
SV40pA
OriP
EBNA1 Amp/pUC
SV40pA SV40pA
0
0
0
0
+2.5
-2.5
0
0
0
CA
T
ParC
ParB
ParA
RepE
OriP
OAT
PGKpuro
SV40pA
OriP/EBNA1
iPS 12.4 (targeted)
iPS 5.15 (random)
iPS 12
OAT
chr 10
iPS 12
iPS 12.4
iPS 5
iPS 5.15
genes
vector backbone
iPS 5
PGKpuro
LHPP
NKX1
125950000
126000000
126050000
126100000
126150000
126200000
0
+1.5
-1.5
0
0
0
A
B
C
Fig. 3. High-density aCGH analysis of gene-corrected iPS cells. aCGH data for the OAT gene targeting vector (A), endogenous OAT locus (B), and one of the
three reprogramming vectors (C ) are shown for gene-corrected iPS cells resulting from a gene targeting (clone 12.4) or random integration (clone 5.15) event.
Data for the two parental iPS cells (clones 12 and 5) are also shown.
Table 1. Genomic aberrations identied in patient iPS cell lines by aCGH
iPS line Chromsome Region Size Type Genes
12, 12.4 1p21.3 9685653997322458 466 Kb Del 1
3p12.1 8521168385484482 273 Kb Del 2
5q23.3 128528748128683706 155 Kb Amp 0
5, 5.15 10p11.22 3283730333179113 341 Kb Del 2
13q31.332.1 9374504193803505 58 Kb Del 1
5.15 10q26.13 126073879126101050 27 Kb Amp 1 (OAT )
20q11.21 2929706230479976 1.2 Mb Amp >20
V* 1p21.3 9783256597927692 95 Kb Amp 1
3q11.2 9923999999546452 306 Kb Amp 6
6q25.1 151460582151594818 134 Kb Amp 2
6q27 168184657168243139 58 Kb Del 2
7p13 4422205144300697 79 Kb Del 1
*Patient iPS line generated with lentiviral reprogramming vectors.
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alogue between stem cell and cancer biologists will be needed as
the rapid advances in genomic sequencing yield greater insights
into the genesis of cancer. Further work is also needed to de-
termine whether there is a consistently different mutational load
in different starting somatic cell types inuencing the choice of
cellular source for iPS cell generation, and whether the muta-
tional load increases dramatically with aging, which might justify
the banking of neonatal cells. With the advent of high-density
arrays and modern sequencing methods, even minor changes in
genome integrity can be detected, allowing an extensive evalu-
ation of genome integrity and mutational load. The mutational
burden observed in iPS cells and its impact on clinical utility are
critical issues that must be addressed before the broad thera-
peutic application of gene-corrected iPS cells in transplantation
medicine.
Materials and Methods
iPS Cell Derivation and Culture. Patient broblasts were obtained from Coriell
Laboratories (ID no. GM06330) and cultured in DMEM (Invitrogen) supple-
mented with 10% FBS (HyClone) at 37 °C, 5% CO
2
,5%O
2
. The episomal
plasmids and protocols used for reprogramming were described (12). After
isolation, iPS cells were maintained and expanded in TeSR and passaged
routinely with EDTA as described (23).
Transfection. Gene targeting experiments were performed by using con-
ditions optimized for human ES cells (24). Cells were harvested with TrpLE
(Invitrogen) and resuspended at a concentration of 5 × 10
6
cells per mL;
0.5 mL of the cell suspension was mixed with 50 μg of linearized gene
targeting vector in 300 μL of PBS and electroporated in a 0.4-cm cuvettete.
Cells were then plated on a 10-cm matrigel-coated dish in TeSR medium
with 10 μM HA100 (Sigma). Puromycin was added to the culture medium
(1 ng/mL) after 3 d. To remove the loxP-anked selection, cassette cells were
harvested and electroporated as described with 15 μg in vitro transcribed
mRNA-encoding Cre recombinase.
FISH. Metaphase spreads were prepared according to a standard procedure.
The OAT gene targeting vector, labeled with Spectrum Green-dUTP (Vysis),
was used as the probe.
Genomic PCR and RT-PCR. Total genomic DNA was extracted by using the
Nucleon BACC3 kit (GE Healthcare). Total RNA was extracted by using the
RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized by using SuperScript III
(Invitrogen), according to the manufacturers protocol. PCR was performed
by using the Taq PCR Core Kit (Qiagen).
aCGH. A custom array was designed that consisted of the NimbleGen human
CGH 2.1M Whole-Genome-Tiling v2.0D array in addition to 4,633 custom
designed probes that cover the gene targeting construct and the reprog-
ramming plasmid vectors used in this study. The original whole-genome-
tilling design has the resolution of 5 kb over the entire human genome,
whereas our custom ne-tilling design could detect 200-bp CNV regions. CGH
experiments were performed at Roche NimbleGen and the WiCel l Cytoge-
netics Laboratory (WiCell Research Institute). Experiments performed at
WiCell involved a multistep labeling procedure as described (25). Data were
analyzed using SignalMap software (Roche NimbleGen). aCHG data can be
found at http://www.ncbi.nlm.nih.gov/geo/ under accession no. GSE26773.
Whole-Genome Library Construction. Whole-genome libraries for each cell line
were constructed as described (4). Briey, genomic DNA for each of the four
samples was sheared by using a Covaris AFA and enzymatically end-repaired.
Sheared DNA was then enzymatically ligated to common sequencing pri-
mers and amplied by using PCR to generate a who le-genome library.
In-Solution Hybridization Capture with DNA Baits. Whole-genome libraries
were enriched for exomic regions by using the NimbleGen SeqCap EZ Exome
kit. Libraries were denatured and hybridized to the SeqCap EZ Exome library
and then captured using streptavidin beads by following the manufacturers
protocol. Enriched libraries were sequenced on an Illumina Genome
Analyzer IIx.
Consensus Sequence Generation and Variant Calling. Novel reprogramming-
associated mutations were identied as described (26). Briey, Illumina GA IIx
reads were ltered by using GERALD and mapped to the genome using
Bowtie or BWA. Consensus sequence generation was performed by using
SAMtools or GATK. Each iPS consensus sequence was directly compared with
its progenitor consensus sequence, and candidate sites indicating a gain of
a new allele in the iPS line were chosen.
Sanger Validation of Candidate Mutations. Each candidate mutation was
validated via amplication with specially designed primers and capillary
Sanger sequencing performed by GeneWiz.
ACKNOWLEDGMENTS. We thank Dr. David Frisch and Dr. Fred Blattner for
providing the hES-2Ainducible BAC vector. This study is funded by a Wynn-
Gund Translational Award from the Foundation Fighting Blindness and par-
tially funded by the Charlotte Geyer Foundation and National Institutes of
Health Grant R01 HL094963. S.E.H. is supported by a National Health and
Medical Research Council Overseas Biomedical Fellowship. A.G. is supported
by the Focht-Powell Fellowship and a California Institute for Regenerative
Medicine predoctoral Fellowship.
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Table 2. Mutations in protein coding regions identied via exome sequencing
iPS cell line
Mutation (chromosome,
position, bases) Gene Codons AA Sub. SNP type
SIFT functional
prediction
Gene mutated in cancer?
(COSMIC)
12, 12.4, 12.4.2 15,40166820,G/A PLA2G4D GCC-GCt A75A Synonymous N/A No
14,21172795,T/C OR10G2 GAT-GgT D15G Nonsynonymous Tolerated No
14,63713924,GG/AA SYNE2 GAG-GAa E2227E Synonymous N/A Yes
GAC-aAC D2228N Nonsynonymous Damaging
18,18783610,AAA/GAC RBBP8 AAA-gAc K62E Nonsynonymous Tolerated Yes
19,58264996,C/A ZNF160 ATG-ATt M201I Nonsynonymous Tolerated Yes
13,42464175,C/G EPSTI1 GGT-cGT G43R Nonsynonymous Tolerated No
19,6263204,T/C ACER1 ATA-ATg I102M Nonsynonymous Tolerated No
19,826291,C/A MED16 AGC-AtC S575I Nonsynonymous Damaging No
17,44364015,G/A SNF8 CGG-tGG R210W Nonsynonymous Damaging No
12.4, 12.4.2 10,126082451,G/A OAT GTG-GcG V226A Nonsynonymous N/A (Corrected) No
12.4.2 11,62157118,C/A GANAB CTG-CTt L250L Synonymous N/A Yes
N/A (not applicable) is listed for the three mutations that are synonymous or the gene-corrected base-pair change that was brought about by homologous
recombination. Synonymous changes do not alter amino acid sequence and there fore should not be damaging to the cell.
Howden et al. PNAS
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    • "Multiple studies have proved the utility of this approach (reviewed in Hanna et al., 2010; Han et al., 2011; Bellin et al., 2012; Imaizumi and Okano, 2014). Moreover, recent studies showed that mutations in patient-derived iPSCs can be corrected by genomic manipulations (Raya et al., 2009; Zou et al., 2009; Howden et al., 2011). However, many ASD-and schizophrenia-associated mutations exhibit incomplete penetrance, suggesting that genetic background effects significantly influence the clinical presentation and could contribute to the observed phenotypes. "
    [Show abstract] [Hide abstract] ABSTRACT: Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Aug 2015 · Cell Stem Cell
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    • "RPE differentiated from the uncorrected GA hiPSCs showed very low levels of OAT enzymatic activity, whereas RPE from gene-corrected hiPSCs had OAT activity comparable to human prenatal RPE and RPE differentiated from hESC and control hiPSC lines (Meyer et al., 2011). In addition, it was shown that the process of OAT gene repair did not add to the mutational load, nor did it increase genetic instability (Howden et al., 2011). However, the cost and effort that would be required to produce and test clinical grade, gene-corrected cells makes this option unwieldy with currently available technology. "
    [Show abstract] [Hide abstract] ABSTRACT: Human pluripotent stem cells have made a remarkable impact on science, technology and medicine by providing a potentially unlimited source of human cells for basic research and clinical applications. In recent years, knowledge gained from the study of human embryonic stem cells and mammalian somatic cell reprogramming has led to the routine production of human induced pluripotent stem cells (hiPSCs) in laboratories worldwide. hiPSCs show promise for use in transplantation, high throughput drug screening, "disease-in-a-dish" modeling, disease gene discovery, and gene therapy testing. This review will focus on the first application, beginning with a discussion of methods for producing retinal lineage cells that are lost in inherited and acquired forms of retinal degenerative disease. The selection of appropriate hiPSC-derived donor cell type(s) for transplantation will be discussed, as will the caveats and prerequisite steps to formulating a clinical Good Manufacturing Practice (cGMP) product for clinical trials.
    Full-text · Article · Jun 2014 · Experimental Eye Research
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    • "In the context of clinic development, the high targeting efficiency to generate CP hESC lines supports the feasibility to genetically modify any clinic-grade hESCs under the GMP conditions. While the genetic modification of hESCs is a known safety concern in human therapy associated with random integration of the exogenous DNA into the human genome, the knockin approach used here should minimize such risk because homologous recombination can be achieved without any apparent random integration of the exogenous DNA (Howden et al., 2011; Song et al., 2010). To further address this concern before clinic application of the genetically modified hESCs, the "
    [Show abstract] [Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knockin hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T cell costimulatory pathways, and PD-L1, which activates T cell inhibitory pathway, is required to confer immune protection, as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression.
    Full-text · Article · Jan 2014 · Cell stem cell
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