Human Embryonic Stem Cells and Gene Therapy

review
The American Society of Gene Therapy
Human Embryonic Stem Cells and Gene Therapy

Yael Strulovici1, Philip L Leopold1, Timothy P OConnor1, Robert G Pergolizzi1 and Ronald G Crystal1
Department of Genetic Medicine, Weill Medical College of Cornell University, New York, New York, USA
Human embryonic stem cells (hESCs) theoretically represent an unlimited supply of normal differentiated
cells to engineer diseased tissues to regain normal function. However, before hESCs can be useful as human
therapeutics, technologies must be developed to provide them with the specific signals required to differentiate in a controlled fashion, to regulate and/or shut down the growth of hESCs and their progeny once they
have been transferred to the recipient, and to circumvent the recognition of non-autologous hESC-derived
cells as foreign. In the context that gene therapy technologies represent strategies to deliver biological signals to address all of these challenges, this review sets out a framework for combined gene transfer/hESC
therapies. We discuss how hESCs are derived, characterized, and differentiated into specific cell lineages, and
we summarize the characteristics of the 500 hESC lines reported to date. The successes and failures of gene
transfer to hESCs are reviewed for both non-viral and viral vectors, as are the challenges to successful use of
gene transfer in developing hESC therapy. We also consider gene transfer as a means of facilitating growth
and isolation of genetically modified hESCs and as a mechanism for mitigating adverse effects associated with
administration of hESCs or their derivatives. Finally, we evaluate the challenges that are likely to be encountered in translating the promise of hESCs to the clinic.
Received 3 November 2006; accepted 23 January 2007; published online 20 March 2007. doi:10.1038/mt.sj.6300125
Introduction
Embryonic stem cells are pluripotent cells derived from the early
embryo that are characterized by the ability to proliferate over pro
longed periods of culture while remaining undifferentiated and
maintaining a stable karyotype, but with the potential to differen
tiateinto derivatives of all three germ layers.18 Human embryonic
stem cells (hESCs) were first derived from the inner cell mass (ICM)
of the blastocyst stage (100200 cells) of embryos generated by
in vitro fertilization,9 but methods have been developed to derive
hESCs from the late morula stage (3040 cells)10 and, recently,
from arrested embryos (1624 cells incapable of further develop
ment)11 and single blastomeres isolated from 8-cell embryos.12 The
ability to culture hESCs and the potential of hESCs to differentiate
into derivatives of all three germ layers provide valuable tools for
studying early human embryonic development and cell differenti
ation and for developing in vitro culture models of human genetic
disorders.13,59,13 Because hESCs have the potential to differentiate
into normal tissues of all types, the ability to derive and maintain
hESCs in culture has captured the imagination of scientists and
the lay public in terms of the possibility of having an unlimited
supply of normal differentiated cells to engineer diseased tissues
to regain normal function.13,59
Although this is exciting in theory, there are significant hur
dles to translating the ability to culture and differentiate hESCs
in vitro into the generation in a reproducible fashion of normal,
functional human tissue that could be safely used to treat human
disease. Independent of the ethical and political controversies
surrounding the generation and use of hESCs,1417 there is only a

rudimentary knowledge of the complex biological signals required
to differentiate hESCs into the specific cell types required for nor
mal organ function.13,59 At present, most studies demonstrating
hESC differentiation into specific cell lineages use feeder layers of
heterologous (often xenogenic) cells to maintain hESCs in culture
and specific lineage-relevant protein mediators to maintain hESCs
in culture and to signal the hESCs to differentiate into specific
cell types.13,59,18 Little attention has been paid to ensuring that,
after transplantation into the recipient, the hESCs and their prog
eny could be exogenously controlled if they differentiated into
malignant cells or if they otherwise grew and/or functioned in an
unwanted fashion. Finally, if hESCs are to be useful in generating
normal tissues for the treatment of human disease, the tissues to
be transplanted must be compatible with the host such that the
cells derived from the hESCs will not be recognized as foreign
and rejected as would any transplanted tissue from an unrelated
donor.
For hESCs to be useful for therapy, technologies must be
developed to provide them with the specific signals required to
differentiate in a controlled fashion, to regulate and/or shut down
the growth of hESCs and their progeny once they have been
transferred to the recipient, and to circumvent the host rejection
of transplanted, non-autologous hESC-derived cells. Although
gene transfer is not a solution to all of the hurdles of moving
hESCs to the clinic, the technology of gene therapy represents a
delivery system for biological signals that addresses many of these
Correspondence: Ronald G. Crystal, Department of Genetic Medicine, Weill Medical College of Cornell University, 515 East 71st Street, S-1000,
New York, New York 10021, USA. E-mail: geneticmedicine@med.cornell.edu
850
www.moleculartherapy.org vol. 15 no. 5, 850866 may 2007
Genetic Modification of Human Embryonic Stem Cells
challenges.19 In this context, the focus of this review is on provid

ing an overview of the synergy of the technologies of gene therapy
and hESCs and describing how therapies for a variety of human
disorders can be developed through the combination of these
technologies.
Overview of hescs
Since the first derivation of hESCs by Thomson and colleagues in
1998,9 as of October 2006, 500 hESC lines had been reported in
the literature, including lines reported in peer-reviewed articles,
reviews, online lists, and posters and abstracts from meetings. Of
these, 455 (91%) are normal, non-disease-related cell lines and
45 are disease related. The normal cell lines include both hESC
lines eligible for funding by the United States government through
the National Institutes of Health (NIH) and hESC lines that are
not eligible for NIH funding; none of the disease-related hESC
lines is eligible for NIH funding. These 500 hESC lines have been
derived from a variety of early human embryonic stages, includ
ing the inner cell mass of the blastocyst, late-arrested embryos
that did not develop into a blastocyst, blastomeres from latemorula-stage embryos, and single blastomeres from embryos
at the 8-cell stage (Figure 1). New efforts are aimed at deriving
hESCs using alternative strategies such as nuclear transfer and
re-programming and parthenogenesis.2027 As part of this review,
we have compiled information regarding the criteria used to
characterize the stemness of hESCs (Table 1), markers used
to define the in vitro differentiation lineage derived from hESCs
(Table 2), and an overview of the reported hESC lines (Table 3). As
Figure 1 Derivation of human embryonic stem cells (hESCs).

(a)Sources of hESCs. Shown are the early stages of human embryogenesis. The most common source of hESCs is the inner cell mass of the
blastocyst, but techniques have been developed to derive hESCs from a
single blastomere from the eight-cell morula stage, multiple blastomeres
of the late morula stage (3040 cells), the eight-cell early-morula stage,
and arrested 1624-cell embryos. (b) Initiating new hESC lines. Independent of the source of the cells, the general strategy is to plate the
source cells on a feeder layer or on an acellular matrix. As an example,
shown are hESCs cultured on a feeder layer. As the hESCs attach and
grow, colonies are isolated and passaged, and continue to proliferate.
Single-cell clones can be isolated from the colonies; groups of hESCs or
individual hESCs are then characterized.
Molecular Therapy vol. 15 no. 5 may 2007
supplementary material, we provide a description of how hESCs

are derived, maintained, and characterized, including informa
tion on culture substrates, media, methods for propagating cells,
pluripotency, and expression of differentiation markers, detailing
how hESCs are characterized based on morphology, karyotype,
cell markers and gene expression, and the potential to differentiate
in vitro and in vivo (Supplementary Materials and Methods and
Supplementary Figure S1). These characteristics are described for
each cell line in tabular form in three tables: hESC lines eligible for
NIH funding (Supplementary Table S1), the non-disease-related
hESCs that are not eligible for NIH funding (Supplementary
Table S2), and the disease-related hESCs that are not eligible for
NIH funding (Supplementary Table S3).
Reported hESC Lines

The following summary of the 500 hESCs reported to date
(Table3; detailed in Supplementary Tables S1S3) includes data
from the publication describing the methods and conditions used
for the derivation and initial culture of the hESC line.
hESC lines eligible for NIH funding

In response to concerns regarding the ethics of deriving hESCs
from early-stage embryos, the United States federal government
initiated a moratorium on using federal funds to derive and/or
work on hESCs derived after 9 August 200128. A total of 71 hESC
lines were identified as being acceptable for study using United
States government funds (Table 3, Supplementary Table S1). The
hESC lines that are eligible for NIH funding were derived using
a variety of methods, all using the ICM as a starting material. All
of these lines were derived on xenogenic fibroblast feeder layers
and, therefore, might be contaminated with animal proteins or
with murine viruses, raising the possibility that these lines will
not be suitable for human therapeutics owing to a perceived risk
of cross-transfer of animal pathogens. Only 15 of the 71 hESC
lines that are eligible for NIH funding (21%) are fully character
ized and available for research to scientists around the world.
hESC lines not eligible for NIH fundingnot disease
related
All hESC lines derived after 9 August 2001 are considered by the
United States government not to be eligible for NIH funding. As
of October 2006, 384 of these lines were derived from normal
embryos (hESC lines not eligible for NIH fundingnot disease
related in Table 3; see details in Supplementary Table S2). Of
these 384, 73 (19%) have been fully characterized, karyotyped,
shown to express hESC stemness markers, demonstrated to
differentiate into derivatives of all three germ layers both in vitro
and in vivo, and fully described with respect to derivation and
propagation methods (Supplementary Table S2). Only a small
number of these cell lines have been assessed for HLA expression
or other genetic differences. Most of these lines were derived under
conditions that exposed the cells to xenogenic components. There
is no apparent difference in the derivation success rate between
using feeder layers and using media with human versus animal
supplements. No apparent difference has been observed in the
success rate of derivation using feeder layers mitotically arrested
by either irradiation or mitomycin C. Propagation of hESCs using
851
Table 1 Characterization of stemness markers of human embryonic stem cells

Category
Parameter
Remarks
Gene expression atmRNA levela
Oct-4
POU-domain transcription factor expressed only

in pluripotent cells; expression down-regulated with
differentiation
120
Nanog
Homeodomain protein, obligatory for maintaining

pluripotency in hESCs
102
REX-1
Acidic zinc-finger transcription factor; specific regulatory

target of Oct-4 in hESCs
102
Sox-2
Transcription factor, essential for self-renewal and

maintenance in undifferentiated hESCs
29
Gene expression assessed by

immunohistochemistry
Protein function
Gene expression pattern
References
GCTM-2
Epitope on the surface of hESCs
120
FGF-4
Fibroblast growth factor-4 is expressed in the ICM;

transcriptional regulator of Oct-4 and a target gene for
Sox-2
29
SSEA-3, SSEA-4
Stage-specific embryonic antigens; glycolipid proteins,

expressed uniquely in hESCs; expression significantly
down-regulated with differentiation
TRA-1-60, TRA-1-81
High-molecular-weight glycoprotein antigens expressed

on the surface of hESCs, embryonic carcinoma cells, and
embryonic germ cells
Alkaline phosphatase
High levels in undifferentiated hESCs
Telomerase
High levels correlate with the ability of hESCs to divide

indefinitely
Microarray assessment of
relative expression of the
entire genome
General patterns described, but definitive criteria not

establishedb
121127
Abbreviations: hESC, human embryonic stem cell; RT-PCR, reverse-transcriptase polymerase chain reaction; ICM, inner cell mass. aTypically assessed by qualitative
RT-PCR. bAnalysis of gene expression patterns of different hESCs has identified 11 genes with elevated expression in all lines compared with differentiated cells (see
text).
mechanical techniques is growing in preference to enzymatic pas

saging, with advantages in terms of reducing exposure to xeno
genic compounds and maintaining karyotype stability. About half
of the cell lines were derived from morula-stage embryos and half
from blastocyst-stage embryos using isolated ICM or the whole
embryo in direct culture. Two of these hESC lines were derived
from a single blastomere of an eight-cell-stage embryo and one
from a late-arrested embryo that failed to develop into a blasto
cyst. Some of the normal hESC cell lines that are not eligible for
NIH funding have been reported to exhibit an abnormal karyo
type at early passages, even though they were derived from what
was considered a normally developed embryo, and some of these
hESC lines have changed karyotype during prolonged periods of
culture. As new hESC lines have been derived, the success rate has
increased. New feeder layers, media, and derivation methods are
being tested to increase the efficiency of hESC line derivation and
maintenance and to reduce dependence on xenogenic products.
New methods also include further characterizations with more
embryonic markers.
hESC lines not eligible for NIH fundingdisease

related
In addition to the normal hESC lines that are not eligible for
NIH funding, 45 hESC lines derived since 9 August 2001 origi
nated from embryos found by pre-implantation genetic diagnosis
to be either homozygous or heterozygous for 20 different genetic
852
disorders, and these are denoted as disease-related (Table 3,

Supplementary Table S3). These hESC lines may be valuable for
in vitro models of human disease. Unfortunately, not all of these
hESC lines have been confirmed actually to have the genetic
abnormality attributed to them.29,30 A number of these diseaserelated lines are carriers for the genetic disease, thus limiting
their utility as disease models. Disease-related hESC lines may be
especially relevant for studying dominant genetic diseases where
there are no live experimental models. Furthermore, some genetic
diseases cannot be studied in mice owing to the differences in
expression of the disease in humans and mice or lack of expression
of the disease in mice, such as the Lesch-Nyhan syndrome.31,32 The
ability to differentiate hESCs into different cell types should enable
the study of the disease effects on the different tissues and the tox
icity and efficacy of new drugs or therapies in different cell types.
Genetic Modification of hescs

There has been considerable interest in genetically modifying
hESCs using both non-viral (Table 4) and viral (Table 5) gene
transfer vectors. The ability to deliver genes to and express them
in hESCs provides a valuable set of tools for a variety of applica
tions, including studies of developmental pathways, positive or
negative selection, directing differentiation, reducing or eliminat
ing immunogenicity, and using hESCs to deliver gene products.
Not surprisingly at this early stage of development of the field,
the vast majority of work on strategies for gene transfer to hESCs
www.moleculartherapy.org vol. 15 no. 5 may 2007
Table 2 Markers to define in vitro differentiation lineages derived from human embryonic stem cells
Germ layera
Markersb
Cell typesc
Detection methodsd
Ectoderm
Microtubule-associated protein2 (MAP2),

neurofilament (NF-H), -tubulin III (Tuj1), nestin,
neural cell adhesion molecule (NCAM), synaptophysin,
keratin, vimentin, sex-determining region Y (SRY)-box 1
(Sox-1), paired box gene 6 (Pax-6), neurogenin, neuronspecific enolase, brain glutamic acid decarboxylase
Neuron
PCR, immunohistochemistry
Network of cells with elongated processes
Neuron
Morphology
-Cardiac actin, smooth muscle actin (SMA), bone

morphogenic protein 4 (BMP4), cardiac specific
troponin, cardio-atrial naturietic factor (ANF),
enolase, desmin, myoglobin
Cardiomyocyte, skeletal muscle
Sarcomeres, contraction
Skeletal and cardiac muscle
Morphology, function
Albumin, -fetoprotein, pancreatic amylase, insulin,

glucagon, somatostatin, Nk1 transcription factor related
locus 1 (Nkx 6.1), paired box gene 4 (Pax 4), -1
anti-trypsin
Hepatocytes, pancreatic acinar cell,

pancreatic islet cells
Small, single cells with morphologic characteristics

of hematologic cell types
Blood cells
Morphology
Platelet-endothelial cell adhesion molecule

epithelial-like cells
Endothelial cells
Secreted human chorionic gonadotropin
Trophoblast
Enzyme-linked immunosorbent
assay
Caudal type homeobox transcription factor 2 (Cdx2)
Trophoblast
Immunocytochemistry
MSH homeobox homolog-2 (MSX-2)
Trophoblast
PCR
Mesoderm
Endoderm
Trophectoderm
Abbreviation: PCR, polymerase chain reaction. aSome germ layers contain cells with common biochemical markers or similar morphologies; e.g., both mesoderm
and endoderm express GATA binding protein 4 transcription factor (GATA-4), Nk2 transcription factor related locus 5 (Nkx 2.5), and markers of cardiomyocyte
differentiation. bMarkers may include gene expression, protein expression, cell morphology, or cell function; markers in bold text represent the most commonly
used markers for the lineage. cCell types listed are commonly observed progeny from germ layers; the list is representative but not exhaustive. dMethods listed are
commonly used techniques for detecting markers; the list is representative but not exhaustive.
has focused on comparing various methodologies to transfer and

express reporter genes such as enhanced green fluorescent protein
(EGFP) or -galactosidase in hESC lines that are eligible for NIH
funding.
Non-viral strategies
Non-viral strategies that have been assessed for gene delivery
to hESCs include chemical transfection with lipid or cationic
polymer transfection reagents and physical transfection using
electroporation or nucleofection (a combined electroporation/
chemical method that facilitates trafficking of DNA to the nucleus;
Table 5). For chemical transfection, most methods yield low trans
fection efficiencies regardless of the transfection reagent used,
although there is considerable variability that could be due to dif
ferences among hESC lines, promoters, transfection reagents, and
laboratories. Most of these strategies yield transient expression of
the transgene, but when selective methods using antibiotic resis
tance or other selection genes have been used, stable hESC lines
have been obtained that persistently expressed the transferred
gene.
Eiges et al.33 compared transfection efficiencies in the H9 hESC
line using the lipid-based transfection reagents lipofectamine
(Invitrogen, Carlsbad, CA) and FuGENE (Roche, Indianapolis,
IN), the cationic polymerbased reagent ExGen 500 (Fermentas,
Hanover, MD) and electroporation using as a reporter construct
the human elongation factor 1- (EF1) promoter driving EGFP.
None of the methods yielded transfection efficiencies higher than

10%. Other experiments in the same report showed that transfec
tion efficiencies using a luciferase reporter gene driven by a thy
midine kinase promoter were also low, although the ExGen 500
reagent showed tenfold greater efficiency than the other meth
ods. Results from other studies with similar transfection reagents
show contrasting results. For example, Lebkowski et al.34 reported
transient transfection efficiencies of 3080% of H9 hESCs using
FuGENE and an expression cassette including a cytomegalovirus
(CMV) promoter and EGFP reporter gene. In a study of transient
transfection of H9 hESCs with a plasmid expressing EGFP driven
by a CMV promoter, Siemen et al.35 reported moderate transfec
tion efficiencies using FuGENE or lipofectamine (28% for each),
whereas efficiency was below 5% with ExGen 500. Vallier et al.36
also found transfection efficiencies of H9 hESCs to be four times
greater using lipofectamine compared with ExGen 500. Over
all, the current literature suggests that with protocols optimized
toward hESC applications, lipid-based reagents compare favor
ably with cationic polymerbased reagents for non-viral transfec
tion efficiency.
Initial work with hESCs, consistent with results from murine
ESCs, suggests that physical methods of transient transfection
such as electroporation are highly inefficient, in part because
of the resulting low cell viability.32,33 However, transfection effi
ciencies of up to 40% have been achieved in H9 hESCs using
electroporation to transfer the EGFP gene driven by a CMV
853
Table 3Overview of reported human embryonic stem cell linesa

Cell lines known to be derived on feeder layers
[N (%)] from:
Matrix
Cell lines
known to
have a normal
karyotype
[N (%)]
Cell lines
with complete
characterization
[n (%)]b
0 (0%)
0 (0%)d
25 (35%)e
18 (25%)
62 (16%)f
3 (1%)f
210 (55%)g
73 (19%)
0 (0%)i
0 (0%)i
26 (58%)j
3 (7%)
62 (12%)l
3 (1%)l
261 (52%)m
94 (19%)
Number of
institutions
reporting
cell lines
Number of
cell lines
Xenogenic
Human
hESC lines eligible for

NIH fundingc
14
71
38 (54%)
hESC lines not eligible

for NIH fundingnot
disease related
47
384
153 (40%)f
hESC lines not eligible

for NIH funding
disease relatedh
45
7 (16%)i
54k
500
198 (40%)l
hESC line
classification
Total
Abbreviations: hESC, human embryonic stem cell; NIH, National Institutes of Health. Details regarding the production and characterization of these 500 cell lines can be
found in Supplementary Tables 13. bFor this table, an hESC line is considered to be fully characterized if the following are described: karyotype, feeder layer and serum
source for derivation, splitting method, and markers used for characterization. cFive of these cell lines were registered twice although they resulted from a single derivation;
therefore, 71 independent derivations were approved for funding by the NIH, of which 25 have been shown to maintain a normal karyotype. dDerivation information
available for 38 of the published lines; percentage calculated based on a total of 71 hESC lines eligible for NIH funding. eKaryotype is provided for only 26 of the published
hESC lines (one cell line has an abnormal karyotype); percentage calculated based on a total of 71 hESC lines eligible for NIH funding. fDerivation information available
for 218 of the published lines; percentage calculated based on a total of 384 hESC non-disease-related lines not eligible for NIH funding. gKaryotype is provided for only
234 of the published hESC lines (24 cell lines have abnormal karyotypes); percentage calculated based on a total of 384 hESC non-disease-related lines not eligible for NIH
funding. hCell lines representing 20 genetic diseases have been established, including adrenoleukodystrophy (n = 1); Becker muscular dystrophy (n = 2, not characterized,
1 contaminated before characterization); -globin mutation IVS 110 (n = 2, both carriers); -thalassemia (n = 2, 1 not characterized); cystic fibrosis (n = 3, 1 homozygote
for F508 deletion, 1 heterozygote for F508 deletion and 5T variant, 1 not characterized); Duchenne muscular dystrophy (n = 3, 1 carrier, 1 carrier not characterized,
1 not characterized); Emery Dreifuss muscular dystrophy (n = 2, 1 line, carrier, 1 line affected); Fanconi Anemia-A (n = 1); Fragile-X syndrome (n = 2, 1 line, carrier, 1 line
affected); Huntingtons disease (n = 5, 1 not characterized); Lowe syndrome (n = 1, not characterized); Marfan syndrome (n = 1); metachromatic leukodystrophy (n = 1);
myotonic dystrophy (n = 7, 3 not characterized); neurofibromatosis type I (n = 6); ocular albinism (n = 1, not characterized); sickle cell disease (n = 1, not characterized);
spinal muscular atrophy (n = 2, 1 not characterized, 1 line lost before characterization); torsion dystonia (n = 1, not characterized); Van Waardenburg syndrome (n = 1).
i
Derivation information available for 7 of the published lines; percentage calculated based on a total of 45 non-NIH-approved, disease-related hESC lines. jKaryotype
is provided for only 27 of the published hESC lines (1 cell line has an abnormal karyotype); percentage calculated based on a total of 45 non-NIH-approved, diseaserelated hESC lines. kSome institutions have derived both NIH-approved and non-NIH-approved normal and disease-related hESCs. lDerivation information is provided
for only 270 of the published hESC lines; percentage calculated based on a total of 500 hESC lines. mKaryotype is provided for 287 of the published lines (24 cell lines
have abnormal karyotypes); percentage calculated based on a total of 500 hESC lines.
a
promoter.35 A modification to classic electroporation methodol

ogy is nucleofection, a strategy based on electroporation but using
a proprietary Nucleofector solution (Amaxa, Gaithersburg, MD)
and specific electric parameters to achieve enhanced delivery of
plasmid DNA to the cell nucleus. Nucleofection methodologies
have been reported to achieve transfection efficiencies of 65% in
H9 hESCs and 20% in H1 hESCs.35 In summary, whatever the
strategy, to be useful, transient transfection by physical methods
requires optimization for the transfection reagent and promoter,
and possibly also the transgene and specific cell line, but dam
age to the target cell population, with consequent low viability,
remains an issue.
One characteristic of non-viral transfection strategies is that
almost all the transgenes are not integrated into the genome, and
thus expression is transient, because the genome becomes diluted
with each cell replication. Applications that require persistent
expression of a transgene (including the assessment of whether
gene transfer affects the characteristics that define hESCs) and
maintenance of gene expression during self-renewal in an undif
ferentiated state as well as following differentiation necessitate the
generation of stable genetically modified hESCs. The rare hESCs
that integrate transgenes mediated by non-viral strategies can be
selected using antibiotic resistance genes. Several examples of the
generation of stable genetically modified hESC lines using trans
fection and selection for antibiotic resistance have been reported
(Table 5). For example, Eiges et al.33 used selection for neomycin
resistance to derive a stable line of H9 hESCs that expressed EGFP
854
under the control of the REX-1 promoter, a promoter active in

undifferentiated hESCs. Two weeks after chemical transfection,
neomycin-resistant hESC colonies were isolated and then further
propagated for up to 13 passages. As expected, the efficiency of
obtaining stable cells lines using this approach was very low, esti
mated to be 1 in 105. Vallier et al.36 generated stable H9 hESC lines
expressing fluorescent reporter genes under the control of a CAG
promoter (a hybrid of the CMV enhancer, chicken -actin pro
moter, and rabbit -globin intron) that stably expressed in undif
ferentiated hESCs as well as in cells differentiated into all three
germs layers. Stable H1 hESC lines expressing EGFP have also
been generated under the control of the EF1 promoter37 and the
Oct-4 promoter.38 In the case of the EF1 promoter lines, EGFP
expression was consistent over time in undifferentiated cells and
did not affect Oct-4 expression levels or the pluripotency of the
modified cells as determined by their ability to differentiate into
all three germ layers. With the Oct-4 promoter, EGFP expression
declined with directed differentiation, as expected.38
Zwaka and Thomson32 reported homologous recombination
in hESCs using electroporation in the presence of a protein-rich
isotonic solution at room temperature, a strategy that yielded
greater transfection efficiency than chemical transfection. As a
proof of principle using H1 hESCs, the gene encoding hypoxan
thine phosphoribosyltransferase was knocked out, Oct-4 GFP
was knocked in, and antibiotic resistanceselected recombinant
cells maintained the ability to differentiate, with GFP expression
decreasing upon differentiation.

H1
Lipofectamine or
FuGENE
FuGENE
Transfection with
chemical reagents
plus selection
H9
Lipofectamine or
FuGENE
H1
H9
FuGENE;
neomycin
selection
Lipofectamine or
ExGen 500,
puromycin
selection
SNUhES3
H9
ExGen 500
FuGENE
H9
FuGENE,
lipofactamine or
effectene
H9
HES-2d
Effectene
and silica
microspheres
HES-2d
HES-2d
Lipofectamine or
FuGENE
ExGen 500
H9
FuGENE
Transfection
with chemical
reagents, no selection
ExGen 500
H9
Reagenta
Category
hESC
line
CAG
hOct-4
CMV,
CBA or
EF1
CMV
EF1
Hb9
enhancer
TK
EF1
TK
CMV
CMV
CMV
CMV
Promoter
DsRed2,
hrGFP
EGFP
EGFP
EGFP
EGFP
EGFP
Luciferase
EGFP
Luciferase
EGFP
EGFP
EGFP
EGFP
Transgene
Four times greater

with lipofectamine
than with ExGen
Moderate (70%)
NA
Very low (<1%)
Low (<10%)
Low (<7%)
Low
Low (<10%)
High (relative to
Lipofectamine or
FuGENE)
Low (5%)
Moderate (1020%)
Moderate (28%)
Moderate (3080%)
Efficiencyb
Table 4 Genetic modification of human embryonic stem cells using non-viral vectors
Persistent
(12 passages) for
both transgenes
Persistent
(50 passages)
Transient
NA
Transient
NA
Transient
Transient
Transient
NA
NA
NA
Transient
Persistence of
expression in
undifferentiated
hESCs
Yes
Yes
NA
NA
No
Yes
No
No
No
NA
NA
No
No
Directed
differentiation
assessed
No
No
NA
NA
NA
No
NA
NA
NA
NA
NA
NA
NA
Effect of
transfection
on hESCs
36
38
130
128
33
129
33
33
33
128
128
35
34
References
Table 4 continued on next page
Yes
No
NA
NA
NA
Yes
NA
NA
NA
NA
NA
NA
NA
Expression in
differentiated
cells
855
856
H9
H1
FuGENE,
neomycin
selection
FuGENE,
neomycin
selection
H9
H1
Nucleofection,
neomycin
selection
H9
H9
Nucleofection
H1
Nucleofection
Electroporation
Electroporation
H9
H9
ExGen 500,
neomycin
selection
Electroporation
hESC line
Reagenta
CMV
CMV
CMV
TK
CMV
CMV
EF1
PGK
CMV,
PGK
Promoter
EGFP
EGFP
EGFP
Luciferase
EGFP
EGFP
EGFP
EpsteinBarr
nuclear
antigen 1
EGFP
Transgene
NA
Moderate (20%)
Moderate (65%)
Low
Low (5%)
Moderate (40%)
NA
NA
Low (<10%)
Efficiencyb
Persistent (25
passages)
NA
NA
Transient
NA
NA
Persistent (4 mo)
Persistent
(12 wk)
Persistent
(13 passages)
Persistence of
expression in
undifferentiated
hESCs
No
No
No
No
No
No
Yes
NA
Yes
Directed
differentiation
assessed
NA
NA
No
NA
NA
NA
No
No
No
Effect of
transfection
on hESCs
NA
NA
NA
NA
NA
NA
Yes
Yes (teratomas)
Yes
Expression in
differentiated
cells
35
132
35
33
132
35
37
131
33
References
Abbreviations: CAG, hybrid cytomegalovirus enhancer, chicken -actin promoter, and rabbit -globin intron; CBA, chicken -actin promoter; CMV, cytomegalovirus early promoter-enhancer; DsRed2, DsRed2 fluorescent
protein; EF1, elongation factor 1- promoter; EGFP, enhanced green fluorescent protein; H69, homeobox 9 promoter; hrGFP, humanized Renilla green fluorescent protein; PGK, phosphoglycerate kinase promoter;
TK, thymidine kinase promoter; NA, not assessed. aTransfection reagents indicated are available from commercial sources. FuGENE (Roche, Indianapolis, IN); lipofectamine (Invitrogen, Carlsbad, CA); effectene plus silica
microspheres (Polysciences, Warrington, PA); ExGen 500 (Fermentas, Hanover, MD); Nucleofection (Amaxa, Gaithersburg, MD). bHigh, >80%; moderate, 1080%; low, 110%; very low, <1%. cNucleofection uses a
proprietary chemical agent (Amaxa, Gaithersburg, MD) and specific, unspecified, electric parameters to deliver plasmid DNA to the nucleus. dHES-2 also known as hES2.
Transfection by
electroporation plus
chemical agentsc
Transfection by
electroporation alone
Category
Table 4 Genetic modification of human embryonic stem cells using non-viral vectors (continued)


EF1
EF1
EF1
H9
H9
H9
W, C, S
LVW, C, I
U3
H1
W, C, S, G
Very low (0.01%)

Very low (<0.01%)
Very low (<0.01%)
-galactosidase
-galactosidase
-galactosidase
EGFP
EGFP
EGFP
EGFP
EGFP
EGFP
EGFP
EGFP
EGFP
rhGFP +
puromycin
resistance gene
EGFP
EGFP
EGFP
EGFP
Moderate (2040%)
Low (~5%)
Moderate (~10%)
NA
NA
NA
NA
NA
High (87%)
Moderate (70%)
without selection; high
(90%) with puromycin
selection
Moderate (70%)
Moderate (3048%)
Low (7%)
High (~100%)
Moderate (5065%)
Low (11%)
-galactosidase
EGFP
Efficiency
Transgene
b
Persistent (6 wk)
Persistent (6 wk)
Persistent (6 wk)
NA
Persistent (2 y)
Persistent (60 d)
Persistent (60 d)
Persistent (60 d)
Persistent (60 d)
Persistent
(40 passages)
Persistent
(40 passages)
Persistent (38 wk)
NA
Yes
Yes
NA
NA
NA
NA
Persistence of
expression in
undifferentiated
hESCs
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
Yes: mixture of
cell types
Yes: mixture of
cell types
Directed
differentiation
assessed
No
No
No
No
No
No
No
No
No
No
No
No
NA
NA
No
NA
NA
No
No
Effect of
transfection
on hESCs
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
NA
NA
Yes
NA
NA
Low
Low
Expression in
differentiated
cells
46
46
46
41
43
40
40
40
40
42
42
45
45
44
34
39
39
39
39
References
Abbreviations: CAG, hybrid cytomegalovirus enhancer, chicken -actin promoter, and rabbit -globin intron; E1F-, elongation factor 1- promoter; EGFP, enhanced green fluorescent protein; HIV-1, human
immunodeficiency virus-1; MSCV-LTR, murine stem cell virus long terminal repeat; PGK, phosphoglycerate kinase; rhGFP, recombinant human gene fluorescent protein; RSV-LTR, Rous sarcoma virus long terminal
repeat; U3, U3 region of the myeloproliferative sarcoma virus; NA, not assessed. aAll lentivirus vectors listed are based on the HIV-1 virus backbone, including the central termination signal (CTS) sequence from HIV-1,
modifications to the 3 long terminal repeat (LTR) sequence to render the vector self-inactivating (SIN), and a vesicular stomatitis virus G protein (VSV-G)pseudotyped capsid. This basic lentivirus vector is indicated as LV;
additional modifications are indicated by superscripts, including W = inclusion of a post-transcriptional regulatory element from the woodchuck hepatitis virus; C = inclusion of the HIV-1 central polypurine tract sequence;
S = addition of a scaffold attachment region of the interferon- gene; I = addition of the chicken -globin 5 HS4 insulator gene into the 3 LTR; R = capsid pseudotyped with the RD114 feline endogenous virus instead of
VSV-G; G = capsid pseudotyped with the gibbon ape leukemia virus instead of VSV-G bHigh, >90%; moderate, 2090%; low, 120%; very low, <1%. cMSCV, murine stem cell virus is a hybrid recombinant virus derived
from a mutant variant of murine myeloproliferative sarcoma virus (see text).
LV
U3
H1
LVW, C, S, R
U3
CAG
HES-2
H1
CAG
H1
LVW, C, S
LV
LVC
W, C, S, I
LV
LV
EF1
H9
LVW, C
EF1 +
IRES
H1
Bicistronic
LVC
EF1
HES-1
EF1
PGK
H1
CAG
H1
HES-1
MSCVLTRc
RSV-LTR
RSV-LTR
RSV-LTR
RSV-LTR
Promoter
LVC
W, C
LV
LVW, C
Lentivirus
H9
Serotype 5
H7
H9
Serotype 4
VSV-G
pseudotyped
H9
Serotype 2
Adenoassociated
Retrovirus
H9
Serotype 5
Adenovirus
hESC line
Details
Vector
Table 5 Genetic modification of human embryonic stem cells using viral vectors
857
Viral strategies
The viral vectors most commonly assessed for gene transfer to
hESCs are based on DNA-based adenoviruses (Ads) and adenoassociated viruses (AAVs) and RNA-based retroviruses and
lentiviruses (Table 5). Lentivirus vectors have been most com
monly used to achieve chromosomal integration.
Smith-Arica et al.39 assessed the efficiency of delivering genes
to H9 hESCs using Ad serotype 5 and AAV serotypes 2, 4, and
5. When they used the -galactosidase reporter gene under the
control of the Rous sarcoma virus long-terminal repeat promoter,
the transduction efficiency of the Ad5 vector in undifferentiated
hESCs was 11%. Of AAV serotypes 2, 4, and 5, only AAV2 showed
evidence of an ability to transduce hESCs, but the efficiency
(~0.01%) was very low. Gene transfer using Ads may provide
high-level, transient gene expression that is useful for delivering
temporary differentiation signals to hESCs. Similar to non-viral
vectors, Ad vectors do not integrate, and thus the transferred gene
would be diluted with cell division.
One study assessed gene transfer to hESCs using a murine
stem cell virus (a hybrid recombinant virus derived from a
mutant variant of murine myeloproliferative sarcoma virus, with
the 5 untranslated regions replaced by functionally equivalent
sequences obtained from the dl587rev virus) with a vesicular
stomatitis virus G proteinpseudotyped capsid to deliver the
EGFP reporter gene under the control of the murine stem cell
virus long-terminal repeat.34 Transduction efficiency of H7 hESCs
with this vector was 5065%, and EGFP-positive cells continued
to express stage-specific embryonic antigens-4, indicating that
they could be maintained in an undifferentiated state, and were
capable of differentiating into all three germ layers with persistent
EGFP expression.
The most common, and generally most successful, strategy for
using viral vectors to deliver genes to hESCs is the use of vesicu
lar stomatitis virus G proteinpseudotyped, self-inactivating len
tivirus vectors (for an example, see Supplementary Figure S2).
The focus on lentivirus vectors for hESC applications relates to
their ability to infect both dividing and non-dividing cells, pro
vide persistent expression secondary to chromosomal integration
of the transgene, and accommodate larger transgenes than Ad or
AAV vectors. Depending on the promoter used, lentiviral vector
mediated gene transfer can result in persistent expression in cells
maintained in an undifferentiated state as well as in differentiated
cells derived from hESCs.4043 The first report of gene transfer into
hESCs, in which EGFP under the control of a CAG promoter was
delivered to the H1 hESC line, utilized a lentivirus vector with a
reported efficiency of nearly 100%.44 Several groups have assessed
alternative strategies using lentivirus vectors, including EF1 and
CAG promoters to ensure high expression levels, and modifica
tions to the vector, including the addition of a post-transcriptional
regulatory element from the woodchuck hepatitis virus and the
central polypurine tract from human immunodeficiency virus1.4043,45 Whereas lentiviral vectors pseudotyped with the vesicular
stomatitis virus G protein envelope transduce both hESCs and
mouse embryonic fibroblast feeder layer cells, lentivirus vectors
pseudotyped with envelopes from the gibbon ape leukemia virus
or the RD114 feline endogenous virus transduce hESCs but not
feeder layer cells.46
858
Applications of Gene Therapy to hescs

Most of what is currently known about the derivation, differentia
tion, genetic modification, and behavior following transplantation
of ESCs has been learned from studies of murine ESCs. Although it
is believed that this information can be extrapolated to the behavior
of hESCs, it remains to be seen whether this is an accurate assump
tion. In this section, we briefly review the major challenges to the
successful deployment of gene-modified hESCs as a tool to treat
human disorders.
Selection
The ability to produce genetically modified hESCs expands the
potential of hESCs to correct hereditary disorders and deliver
gene products for specific therapies. Integrating gene transfer vec
tors (retrovirus and lentivirus vectors) are able to deliver genes to
hESCs and, by virtue of becoming an integral part of the hESC
genome, if they are designed appropriately, can continue to exert
an influence in all cells and tissues derived from the transduced
cell as it divides. However, in most studies, transduction effi
ciency with these vectors has not been 100%, and an unselected
population containing both genetically modified and unmodified
hESCs results (Table 5). The unmodified cells have the potential
to diminish the impact of an hESC-derived transplant by compet
ing for engraftment within a niche with the transduced cells but
supplying no benefit to the recipient. Thus, the ability to select
genetically modified hESCs from a mixed population would
permit the transplant of cells likely to be more effective.
Despite numerous attempts to direct the differentiation of
hESCs toward particular cell types, all studies to date have resulted
in a mixed population of cell types that may still include some undif
ferentiated hESCs. As undifferentiated hESCs have the potential to
form teratomas, it is critical to develop gene transfer strategies to
select from the mixed population only those cells with the desired
characteristics. A thorough understanding of markers of differen
tiation is also important, so that the desired cells can be identified
and/or selected by antibodies specific to the appropriate marker.
A variety of drug selection systems could be used to eliminate
cells that do not express the cognate resistance genes within the
expression cassette, but selection of transduced cells using drug
resistance can be unpredictable and/or toxic.32,4750 Reporter genes,
such as GFP, have been used to identify transduced cells within a
mixed population and to allow the segregation of the transduced
population by flow cytometry.40 However, these techniques are
difficult to scale up and may not be suitable for use in a clinical
setting.40,51 Another approach, which is scalable and has already
been used in a humans (albeit not with hESCs), is the use of genet
ically encoded surface marker protein delivered by an integrat
ing vector, allowing the genetically modified cells to be purified
from a mixed population by magnetic-bead purification.52,53 One
example is the truncated human low-affinity nerve growth fac
tor (LNGFR), a non-toxic, non-tumorigenic, and non-functional
protein that can be expressed on the surface of transduced cells.53
The LNGFR complementary DNA is small (1.2 kilobases), leaving
room in expression cassettes for most therapeutic genes. T cells
marked with LNGFR and purified using magnetic beads tagged
with an anti-LNGFR antibody have been used clinically with no
adverse effects.54 The use of a similar strategy to identify and purify
transduced hESCs could permit the transplant of essentially 100%

transduced cells, assuming it could be demonstrated that LNGFR
expression would not alter the characteristics of the undifferenti
ated hESCs and would not interfere with the response of hESCs to
differentiation signals.
Control or elimination of hESCs and their progeny

Given their capacity for self-renewal and pluripotency, hESCs
havegreat potential as regenerative medicines, but experimen
tal models using differentiated ESCs have shown that even small
numbers of undifferentiated ESCs within a transplant can lead
to the development of teratomas.55,56 Another potential concern
with the use of integrating vectors (retrovirus or lentivirus; see
below) is insertional mutagenesis leading to dysregulation of cell
growth. Within the limitations of current technology, it has not
been possible to ensure that a population of differentiated ESCs
is entirely free of undifferentiated ESCs or that all gene insertion
sites are safe.
Strategies are being developed to enhance the safety profile
of hESC transplants by incorporating genetic elements into the
expression cassettes of gene transfer vectors to allow the use of
external stimuli to suppress or eliminate uncontrolled cell growth
(Figure 2). One example is to deliver the herpes simplex thymi
dine kinase gene with a therapeutic gene.5759 The herpes simplex
thymidine kinase gene is small (1.3 kilobases), and the use of an
internal ribosome entry site (or other strategy for multiple genes;
see below) avoids the need for a separate promoter, leaving suf
ficient space in the expression cassette for most transgenes. The
herpes simplex thymidine kinase gene product has no conse
quences for the transduced cell until the prodrug (ganciclovir) is
administered. Human thymidine kinase is unable to metabolize
ganciclovir, so untransduced cells are not affected by the presence
of the drug, whereas cells carrying the herpes simplex thymidine
kinase gene convert ganciclovir to a toxic product that kills the
expressing cell. This strategy has been shown to eliminate terato
mas resulting from subcutaneous murine stem cell transplants.60
Other suicide geneprodrug strategies (e.g., cytosine deaminase
with 5 fluorocytosine) also can be used.50
Modulation of differentiation
One important potential application of gene transfer to hESCs
is to provide to the hESCs and their progeny signals relevant to
differentiation. Gene transfer of growth factors or transcription
factors that modulate the differentiation pathway of ESCs may
be helpful in improving the outcome of differentiation protocols.
Given that undifferentiated hESCs represent a significant hazard
in the clinical use of hESCs, the ability to induce differentiation
in vitro along a particular developmental path is central to the
development of hESCs as a renewable, implantable tissue source
for clinical applications. There are reports of successful in vitro
differentiation of hESCs into neural precursors that, when trans
planted into rodent brains, developed into neurons, astrocytes,
and oligodendrocytes with no evidence of teratoma formation.61,62
Attempts to differentiate hESCs into differentiated cells of other
germ layers have been less successful, with the overall percentage
of cells staining positive after stimulation for specific differenti
ated cell markers being very low (often <1%).63
Figure 2Use of gene transfer to control differentiation and growth

of human embryonic stem cells (hESCs). To be useful in clinical applications, genetic modification of hESCs may be carried out using multifunctional gene transfer vectors that include a selectable marker, a therapeutic
gene, and a suicide gene. After transduction of hESCs, a population of
genetically modified hESCs may require selection to remove unmodified
hESCs, ensuring that all hESCs in the population carry the therapeutic gene
(red nuclei). The resulting genetically modified hESCs can be propagated
in vitro, expanded, and stored for future use, or they can be differentiated toward a desired cell type. As differentiation protocols typically
result in the production of multiple cell types, a second round of selection using lineage-specific markers is likely to be required to generate a
pure population of genetically modified differentiated cells that can be
delivered to the patient. If the genetically modified cells exhibit uncontrolled growth and/or inappropriate differentiation that could have
deleterious effects on the patient, it should be possible to ablate all of
the genetically modified cells by administration of a drug that activates
the suicide gene transferred along with the selectable marker gene and
therapeutic gene. If the failure of therapy is deemed to be correctable,
the original genetically modified hESCs can be cultured and the process
repeated. Nuclei are shown in blue; genetically modified nuclei shown
as a red dot in blue nuclei.
Delivery of gene products

It is likely that most gene transfer to hESCs will be performed
ex vivo, with subsequent delivery of the modified, differentiated cells
to a receptive niche in the recipient. In most cases, long-term expres
sion of the therapeutic gene will be required, but there are applica
tions where transient expression would be preferable. For example,
for wound healing, bone regeneration, repair of neural injury, or
growth of new blood vessels, the gene-based stimuli may be required
for only a short duration. To correct genetic diseases, however, per
sistent expression of the transferred genes will be required.
Independent of the strategy for gene delivery, the vector used
to transduce the hESCs must contain the gene or complemen
tary DNA and all regulatory signals within the maximum genetic
payload capacity of the vector. The genetic cargo must be expressed
in the differentiated tissue to produce the therapeutic product,
usually a protein but possibly a regulatory RNA. This will require
the appropriate promoters, enhancers, insulators, and other reg
ulatory elements to ensure that a physiologic level of expression
occurs to influence the disorder being treated. Additional space
in the payload could be devoted to conditional suicide genes as
described above, which would allow the removal of the genetically
modified cell or its progeny if it subsequently displayed unwanted
behavior.
859
Correction of genetic diseases

Among the important potential applications of gene therapy to
hESCs is the correction of genetic diseases. Although many hered
itary disorders can be targeted by gene therapy vectors alone, the
combination of gene therapy and stem cell therapy may have
added utility, where cells differentiated from hESCs would act as
factories to produce therapeutic proteins or where a high propor
tion of corrected cells could be developed.
The hESCs containing the genetic payload must be deliv
ered to an appropriate location to have the desired impact. In
the caseofcirculating proteins (e.g., factor IX, factor VIII, von
Willebrand factor, 1-antitrypsin), it may be possible to estab
lish tissuereservoirs distant from the normal site of the secreted
product. Such applicationswould permit the cells differenti
ated from hESCs to be delivered to a site that is accessible and
receptive to engraftment, even if that tissue is not the normal site
of production of that particular protein. In cases where the prod
uct is not secreted, the hESCs with their regulated genetic cargo
must be differentiated to the correct cell type for, and engrafted
to, a site associated with natural expression of the transgene (e.g.,
the cystic fibrosis transmembrane conductance regulator forcys
tic fibrosis or dystrophin for muscular dystrophy). Such pro
teins can exert their influence only at the appropriate site, and
there is no known mechanism by which cells expressing the
protein remote from the affected tissue could have a therapeutic
effect.
Additional hurdles to the successful therapeutic application of
gene-modified hESCs include whether the hESC themselves, or the
expressed product, will be toxic or immunogenic in the recipient.
A recipient of hESCs who has never been exposed to the protein
product before, as in deletion or nonsense mutants, may mount an
immune reaction against the product, limiting the effectiveness of
the therapy. A more likely immunologic problem will be that the
cells derived from the hESCs, unless they are derived from the
recipient, could arouse an immune response leading to elimination
of the therapeutic cells. The technology of nuclear transfer, in
which the created hESCs would be genetically indistinguishable
from the recipient (see below), is the most obvious strategy to
circumvent this problem.
Modification of feeder layers
Although gene transfer to stem cells per se is the primary focus at
the interface of the two technologies, another strategy where gene
transfer may be helpful is the genetic modification of the feeder
layers used to maintain hESCs and/or differentiate hESCs to the
desired cell phenotype. Genetic modification of feeder cell layers
could lead to constitutive production of key signaling molecules
in a cost-effective manner.
An integrating vector would have the advantage that a perma
nent genetic modification of feeder layer cells would enable clon
ing and characterization of a cell line that had a predictable level
of protein expression. However, given the relatively short useful
lifetimes of murine and human fibroblasts, transient modification
strategies such as Ad or non-viral vectors might also be useful.
The use of transient vectors would not pose a problem, because
the feeder layer cells are always rendered incapable of replication
by radiation or chemical treatment. In the absence of cell division,
860
expression from transient vectors should be stable for the lifetime

of the culture.
Feeder layer cells include immature fibroblasts (murine
embryonic fibroblasts, STO (Sandes inbred mice strain resistant
to thioguanine and ouabain) cells, human foreskin fibroblasts),
which are used to passage undifferentiated hESCs, and stromal
fibroblasts (OP9 cells), which are used to differentiate hESCs (see
Supplementary Data). Murine embryonic fibroblasts can be
genetically modified by non-viral or viral vectors, including ret
rovirus, lentivirus, Ad, Epstein-Barr virus, and SV40 virus.46,6470
Human foreskin fibroblasts can be genetically modified using
non-viral vectors, Ad, retrovirus, and lentivirus vectors.7175 OP9
cells can be transduced by transfection or through the use of Ad
and retroviral vectors.76,77 Despite this experience with genetic
modification of the cell types that form feeder layers, the use of
this technology for enhancing hESC differentiation has been lim
ited. Xu et al.74 delivered a telomerase gene to hESCs using a ret
rovirus and then developed a fibroblast-like cell line differentiated
from the hESCs that was capable of acting as a feeder layer for
maintenance of naive hESCs.
Challenges to Linking the Technologies of

hESCs and Gene Therapy
The combination of hESC and gene therapy has the potential to
be much more powerful than either technology alone, but cer
tain obstacles must be overcome before the combination can be
effectively applied. Considerable work needs to be done on the
efficiency of gene transfer, the ability to deliver multiple genes,
insertional mutagenesis, and control over gene expression.
Efficiency
With existing technology, integrating gene transfer vectors are
routinely able to transduce part of a population of hESCs in a
single round of infection, and transduced cells can be selected
using genetic markers, leading to a population of which more than
95% carries the transgene (Table 5).40,42,44 Consequently, the effi
ciency of transduction is already good (assuming the cells can be
selected), and it is probable it will get better as modifications are
made to vectors and/or the means by which they are delivered to
the hESC. However, a serious limitation to the efficiency of genemodified hESCs as a therapeutic modality is engraftment of the
cells in the recipient. In most cases, the transplanted cells lack a
selective advantage and must compete with normal endogenous
tissue to populate a niche in the host. Consequently, one major
area of interest is the development of genetic elements that would
be delivered along with the therapeutic transgene to provide a
selective advantage to the modified cells, allowing more efficient
engraftment and expansion. Although selective advantage is
achieved in bone marrow transplants using ablative chemo
therapyand/or radiation, immunosuppression will not likely be
appropriate for most applications, particularly if it is required
on a chronic basis. Furthermore, whereas bone marrow cells are
proliferative, many potential applications of hESCs are for organs
suchas the nervous system, heart, and liver, where cell prolif
eration is minimal. Together, these issues make engraftment/
selective advantage a major challenge for developing efficient hESC
therapies.
Delivery of multiple genes

For many applications, it is likely that more than one gene will need
to be transferred to hESCs. One approach for in vivo delivery of
dual cassettes is to express both proteins in a bicistronic vector that
uses a single promoter. The conventional method for bicistronic
expression cassettes uses internal ribosomal entry sites. How
ever, internal ribosomal entry sites often yield substantially lower
expression of the second gene than the first gene in the expression
cassette.78 Another strategy is the use of dual promoters, with each
promoter driving expression of a separate gene. The expression
cassettes can be arranged in tandem, separated by transcriptional
and translational termination signals.79,80 However, the use of iden
tical promoters in tandem increases the likelihood of homologous
recombination, leading to deletion of an intervening open read
ing frame. To avoid this problem, dual promoters from different
origins can be placed in reverse orientation in a back-to-back
configuration.81 Another strategy to express two proteins from a
single open reading frame is based on the foot-and-mouth-disease
virusderived 2A self-processing sequence.82 The 2A sequences are
specific oligopeptides that undergo self-cleavage to generate the
mature foot-and-mouth-disease viral proteins. Foot-and-mouthdisease virus 2A is short (13 amino acids) and is able to cleave at
its own C terminus between the last two amino acids. Using a footand-mouth-disease virus 2A sequence adjacent to a furin cleavage
site to link the genetic sequences, it should be possible to engineer
expression cassettes that result in high levels of both full-length
peptides in vivo.
Insertional mutagenesis
Retroviral and lentiviral gene transfer vectors randomly integrate
into the genome of cells that they infect, along with the genetic
cargo they carry.8386 Integration into the genome is a power
ful feature of these vectors, especially for rapidly and regularly
dividing cell types such as hESCs, as each daughter cell receives
a copy of the genetic payload at cell division. However, the pro
cess of random integration itself is inherently risky and can result
in insertional mutagenesis, where gene expression in the region
in which the new vector is inserted becomes dysregulated. If any
of the genes affected in this way are involved with growth con
trol (e.g., oncogenes) the cell can, in the worst case, develop into
a tumor. The issue of insertional mutagenesis takes on additional
importance in the context of ESCs developed by parthenogenesis
from an unfertilized ovum. In the haploid genome of these cells,
insertions that knock out single copies of tumor suppressor genes
may predispose cells to neoplasia. In contrast, in diploid cells, both
copies of a tumor suppressor gene would have to be knocked out
to lose the tumor suppressor function, a far less likely scenario. As
described above, the incorporation of conditional suicide genes
into the genetic payload can eliminate cells that have lost growth
control, although at the cost of losing the therapeutic benefit of
the transgene. Strategies are needed that can eliminate only those
cells that have escaped regulatory control, while leaving cells with
benign integrations unharmed.
Another approach to the problem of insertional mutagen
esis takes advantage of the fact that hESCs can be cloned and
expanded.8789 A clonal transduced hESC line can be analyzed for
the number and location of vector insertions, and transplanted
only when it is clear that the insertion points are not likely to
produce dysregulation of cell growth.
Control over gene expression

For gene transfer to be effective and safe, the expression of trans
genes must be controlled so that neither too little nor too much of
the gene product is generated. In the vast majority of gene transfer
studies, the vectors include strong constitutive viral promoters to
drive high expression levels in essentially all tissues. Examples of
promoters of this type are the CMV immediate-early promoter
enhancer, the simian virus 40 large T-antigen promoter, the Rous
sarcoma virus long terminal repeat promoter, and other retroviral
long terminal repeat promoters.90 However, among the impor
tant challenges to successful gene therapy is persistent transgene
expression, and the CMV promoter and other viral promoters
are known to undergo transcriptional inactivation in several tis
sues.90 One strategy for circumventing transcriptional silencing of
constitutive viral promoters is the use of eukaryotic housekeeping
gene promoters, such as hEF1, phosphoglycerate kinase, and the
hybrid CAG promoter.90 To date, the promoter most commonly
used in applications of lentiviral vector gene transfer to hESCs has
been the EF1 promoter (Table 5).
Expression systems have been developed that use tissuespecific regulation of transcriptional or regulatory elements that
can be turned on or off as required via the addition of an exoge
nous drug. These are either naturally occurring inducible promot
ers that exhibit tissue specificity or engineered systems, such as the
tetracycline-regulatable system, hormone-responsive promoters,
chemically induced dimerization of bipartite transcription factors,
and zinc-finger protein transcription factors, that can be activated
or inactivated as required. Most of these regulated systems are not
able to achieve the same level of transcription possible from the
constitutive viral promoters.9092 Whether such systems will be
useful in gene therapy/hESC applications remains to be assessed.
Moving hESC-based Therapy to the Clinic

At present, research into hESC therapeutics is focusing on the
generation and differentiation of pluripotent stem cells, with a
goal of creating cells that could replace the structure and function
of diseased or damaged cells. As detailed above, investigators pur
suing these experimental goals have already met with significant
challenges with in vitro studies, but even higher hurdles will be
encountered in the context of integrating regenerated tissues into
the physiology of an organism. In addition to challenges relating to
the nature of stem cell technology per se, a major challenge relates
to the implantation of non-autologous tissue as a therapeutic.
Immune rejection
Among the significant hurdles to considering hESC technology for
treating human disease is that, except in rare circumstances, the
source of the hESCs will be unrelated to the recipient. For bone
marrow and organ transplantation, the most successful outcome
can be predicted when there exists compatibility in HLA molecules
of donor and recipient, thus limiting immune rejection of the nonautologous tissue.93,94 For bone marrow transplantation, enhanc
ing the chances of survival of the transplanted tissue requires
large registries of potential donors (http://www.marrow.org).93,95
861
In this context, the generation of a large bank of stem cell lines

is an essential component of any realistic strategy for transform
ing stem cell therapy into a main-line therapeutic option. Thus, it
is unrealistic to consider the use of only hESC lines that are eli
gible for NIH funding as potential therapeutics for widespread use.
Independent of the issue of potential contamination of these lines
by animal proteins and/or pathogens, NIH funding for stem cell
research is limited to the use of 71 cell lines (of which only 15 have
been completely characterized and are generally available; Table 3,
Supplementary Table S1). Among other issues, in the context of
stem cell therapeutics, this is equivalent to limiting potential trans
plant donors to 15 individuals to supply all of the tissue that may
be needed in the future.
An alternative to creating a comprehensive bank of hESCs
would be to create stem cell lines specific to the patient. Several
strategies can be envisioned to achieve this. First, the technology
of nuclear transplantation opens the door, at least theoretically, to
uses that employ hESCs that contain a transferred nucleus.23,24,26
Our understanding of the biological processes governing the
de-differentiation and re-differentiation of a somatic nucleus is at
only a very early stage, and this technology has not been efficient
or easily transferable among species.21,22,27 However, it is important
to discuss the potential use of nuclear transfer because it could
solve the problem of immune rejection by incorporating into
hESCs the genome of the patient to be treated. Coupled with gene
transfer technology, nuclear transfer has the attractive potential to
provide a targeted gene correction in the context of the patients
own HLA molecules to generate cells and/or tissues that have
been damaged by disease.
Although a workable strategy combining gene transfer
and nuclear transfer for the development of autologous hESCs
remains to be determined, the basic concepts can be outlined
(Figure 3). The process would begin with isolation and culture
of a line of somatic cells easily cultured from the patient, such
as dermal fibroblasts obtained from a skin biopsy. The cultured
cells would be transduced with a gene transfer vector that deliv
ered persistent gene expression of the therapeutic gene. Retrovi
ral or lentiviral vectors would be suitable choices owing to their
ability to insertin the host cell genome. One or more cell lines
would be cloned from the transduced cells to confirm expression
of the therapeutic gene, permit characterization of the insertion
sites, and evaluate any effects on overall gene expression using
gene expression arrays. Once a cell line had been characterized,
it would be used as nuclear donor for the nuclear transfer pro
cess. Non-autologous eggs would be physically enucleated, and a
nucleus from the genetically modified patient-derived fibroblast
line would be delivered by microinjection into the egg. Physical
and/or chemical stimuli would be used to activate the egg cell
cycle, and the resulting embryo would be used as a source of
cells for the development of a new hESC line (Figure 1). Once an
hESC line was established, differentiation of the hESC line into
an appropriate cell or tissue would be required before returning
the genetically corrected cells to the patient.
Self ESCs might also be derived using other technologies
(Figure 3). Parthenogenesis (stimulation of cell division in an
unfertilized egg) can lead to the production of pluripotent stem
cells and thus, for female patients, may provide a ready source of
862
Figure 3 Strategies for combining gene transfer and nuclear transfer to derive autologous, pluripotent, genetically modified human
embryonic stem cells (hESCs) to treat a genetic abnormality. A major
limitation to the utility of hESC therapy may be the lack of cell lines
that match the immunologic self identity of most patients. As a result,
hESC-derived tissues may be subject to host rejection. To avoid this
problem, three strategies may be used to create autologous, pluripotent, genetically modified hESCs. (a) An adult somatic cell is isolated
from the patient and cultured. Since these cells would be derived from
the patient, they would also contain any genetic abnormalities that may
have contributed to the patients underlying condition. To compensate
for this, a gene transfer vector could be used to deliver a therapeutic
gene to the somatic cells. A non-autologous oocyte would be enucleated, and the nucleus from the genetically modified autologous cells
transferred to the enucleated egg. These cells would be used to derive
the genetically modified autologous hESCs that would then be propagated for maintenance and storage or differentiated into therapeutic
cells and delivered to the patient. Unmodified nuclei are shown in blue;
genetically modified nuclei are shown in red. (b) As an alternative to
nuclear transfer, a genetically modified somatic cell could be fused with
a pluripotent cell to expose the nucleus of the somatic cell to the reprogramming enzymes in the pluripotent cell. Eventually, it may be
possible to express the enzymes in somatic cells to achieve the same
result. (c) A third potential source of autologous stem cells might be
available for female patients through parthenogenic activation of autologous oocytes. Gene transfer to the resulting haploid cells could result in
autologous, pluripotent, genetically modified cells. Unmodified nuclei
are shown in blue; genetically modified nuclei are shown in red.
stem cells for therapy.20,25 Alternatively, the enzymes that account

for the re-programming of a somatic cell nucleus after nuclear
transfer, including de-methylases and de-acetylases, might be
expressed exogenously in a somatic cell or introduced by fusion
with a cytoplasm in which the enzymes are expressed, leading to
de-differentiation of the somatic cell nucleus in situ.23,9699 These
technologies require further characterization to determine whether
any additional risks might be introduced from haploid genomes or
de-differentiated genomes.
Xenogenic contamination
The United States Food and Drug Administration regulations do not
generally allow approval of biological therapeutics that have a his
torical connection to xenogenic substances.100 The history of hESC
lines is problematic in this regard, as among the hESC cell lines that
are eligible for NIH funding, all lines were established by growth on
murine embryonic fibroblasts (Table 3, Supplementary Table S1).
Although a number of non-eligible hESC lines have been isolated
by growth on human feeder cell layers, in many of these cases the
human feeder cells were cultured and propagated using xenogenic
serum (Supplementary Table 2). To get around this issue, the most
recently isolated hESC lines have been isolated in human serum
or in completely defined media containing specific mediators.101,102
However, even in these strategies, attention will have to be paid to
the source of enzymes used to dissociate the colonies during passag
ing, as most hESC lines are routinely passaged using combinations
of non-human-derived trypsin, collagenase, or dispase. Manually
splitting the cells avoids this problem and is also useful in the subculturing of only those cells with an undifferentiated morphology
than can be picked out of mixed populations of differentiated and
undifferentiated cells. However, manual sub-culturing of hESCs is
time consuming and labor intensive and cannot be used for bulk
hESC propagation. At present, only a minority of the hESC lines are
manually split or are propagated manually in combination with an
enzymatic reagent (Supplementary Tables 13).
Cell and tissue physiology

Even if we assume access to HLA-matched hESC lines for every
patient and that these hESC lines could be differentiated into any
specific cell type in the body, there remain additional hurdles to
therapeutic application of hESCs and their progeny relating to the
fundamental differences between propagation of hESCs in vitro
and differentiation of hESCs in vivo.
The first and most obvious difference between lab propagation
and in vivo differentiation is the number of times hESCs replicate
before differentiation. ESCs represent a transient stage of normal
development and give rise to adult tissue stem cells that maintain
the ability to re-populate differentiated cells in vivo. In the present
state of the art, hESCs maintained as colonies on either feeder cell
layers or extracellular matrix material divide every 2472 hours.2,103
Perpetual maintenance of hESC cell lines in vitro contrasts with
the natural physiology of hESCs. Under normal conditions after
fertilization, hESCs proliferate rapidly and then differentiate into
pluripotent progenitor cells that give rise to terminally differenti
ated cells. Continuous culture in an undifferentiated state repre
sents an artificial energetic and logistic stress on the cells, possibly
accounting for the spontaneous alterations in karyotype that have
been observed in many of the hESC lines.3,104,105
The second difference between propagation of hESCs in
culture and in vivo lies in the microenvironment of differentia
tion. Although the focus of the clinical use of hESCs is to gener
ate defined cell types, even in the best cases mixed populations
of cells are produced, requiring isolation of the desired cell type.
In comparison, in vivo differentiation involves the parallel, inter
dependent development of multiple cell types. Other physiologic
variables such as extracellular matrix interactions, intercellular
signaling, blood flow, and an airliquid interface are likely among
the many factors that can play a role in cell differentiation in vari
ous organs.106110
The specific conditions under which cell differentiation
occurs raise two important problems for the use of cells and
tissues derived from hESCs. If cells that have been differentiated

in vitro are to be transplanted, how will those cells integrate with
the existing tissue structures so that a physiologic function can be
performed? And at what stage of differentiation will transplanta
tion be most successful? At least part of this answer relates to the
idea of gaining access to stem cell niches. If an hESC is differen
tiated to become a committed, tissue-specific adult stem cell or
progenitor cell, one therapeutic strategy will be to engraft these
organ-specific precursors in organ-specific stem cell niches.111 In
the case of bone marrow transplants, an intravenous infusion of
bone marrow containing adult hematologic stem cells and pro
genitors is able to pass through the circulation, with some of the
populations taking up residence in the bone marrow.112 However,
the relative ease with which bone marrow stem cell niches are
accessed may be difficult to replicate in solid organs.
It has been possible to re-populate the liver in a murine
model of tyrosinemia with a normal population of hepatocytes
(presumably containing adult stem cells or progenitor cells with
the capacity to expand and differentiate) via the spleen (proximal
to the hepatic portal circulation).113 In this specific, rare applica
tion, normal, non-disease-carrying hepatocytes have a growth
advantage over endogenous hepatocytes carrying a mutation in
fumarylacetoacetate hydrolase, resulting in the gradual exchange
of corrected cells for disease cells within the context of the organ.
Re-population of keratinocytes in skin has been accomplished after
implantation of keratinocyte stem cells into the epidermis.114,115 As
with bone marrow transplantation, the success of such strategies is
likely to be organ and disease specific. It will be a major challenge
to direct the delivery of stem cells to their specific niches in most
applications; e.g., it is unlikely, given the current state of knowl
edge, that delivery of neuronal precursors to the sub-arachnoid
space, ventricles, or parenchyma of the brain will establish correct
functional neuronal circuits.
Similar, if not more challenging, logistical problems can be
envisioned if in vitro differentiation is allowed to proceed further.
Given the general lack of plasticity of fully differentiated cells,
the ability to integrate these cells functionally within a tissue is
a daunting hurdle. It is conceivable, however, that if a function
ing unit of cells could be formed in vitro, one could integrate an
autonomously functioning organ within the physiology of the
organism. The most relevant comparison for stem cell therapy
might be the recent developments in pancreatic islet transplan
tation. Isolation and delivery of small, avascular groups of dif
ferentiated cells to sites including the liver, kidney capsule, and
subcutaneous space have resulted in perfusion and survival of the
cells for long periods.116118 In this context, the differentiation of
hESC-derived pancreatic islets in vitro with subsequent surgical
implantation is feasible with existing technologies.
Selected larger organs might also be functionally established
in vitro with the possibility of surgical placement in vivo. In one
example, a kidney developed from bovine embryonic stem cells
was demonstrated to produce urine.119 However, unlike in the case
of transplanted organs, which arrive with a pre-existing vasculature
of their own, it is difficult to envision how development of an intact,
functional circulatory structure will be accomplished in vitro.
If the hurdles to in vitro expansion and differentiation can be
overcome, and if the physical and functional integration of the
863
cells with existing tissues can be accomplished, one still must

consider the possibility that the progeny of genetically modified
hESCs might pose additional risks for which safeguards should be
developed before therapy. The most basic risk would be the deliv
ery of undifferentiated hESCs capable of uncontrolled growth,
resulting in the formation of a teratoma.
The problem of insertional mutagenesis will be ubiquitous
with any gene transfer strategy that is designed to replicate with
the progeny of hESCs. A therapeutic dose of genetically modified
stem cells is likely to be in the tens of millions of cells, represent
ing a comparable number of potentially damaging mutations. It is
likely that insertions are most likely to occur in transcriptionally
active DNA, increasing the likelihood of an insertional mutation
in a critical stretch of coding sequence.8386
Finally, there is a risk of transfer of a genetic disorder that
would be undetected in the hESC line, particularly if the disorder
did not manifest in the early stages of differentiation. Although
it is possible to test for mutations in hESCs and their derivatives,
the large number of possible genetic abnormalities presents
a daunting challenge to ensuring the normalcy of the hESC
source.
Future strategies
In the context of the challenges to developing hESC-based thera
pies, it is apparent that strategies capable of mitigating risks related
to the therapeutic use of hESCs should be pursued alongside
the development of the therapies per se. These strategies might
include several nonmutually exclusive approaches, including
careful selection of the hESC before administration and a failsafe mechanism for ablating all genetically modified cells while
sparing most endogenous cells. The introduction of a step in the
development of the therapy at which a single genetically modi
fied cell would be isolated, expanded, and characterized with
respect to the location of the mutation would allow an analysis
of the relative risk of the insertion site. Similar limiting dilution
cloning strategies are now routinely performed during the origi
nal isolation of a stem cell line to ensure that only one karyo
type is represented. Progress in understanding how insertional
mutagenesis can lead to uncontrolled growth of stem cells is an
essential prerequisite for this analysis and is currently an active
area of research.
Genetic modification can be used to enhance our ability to
conduct such an isolation step by adding a convenient ligand
for cell isolation. Genetic modification is also potentially use
ful for solving the problem of uncontrolled cell growth. Incor
porating the genes for an ablation strategy at the same time as
the genes for the therapeutic strategy would give the best chance
of ensuring that the safety mechanism will be present when and
if needed. Initial applications of genetically modified hESCs are
likely to occur where the risk/benefit ratio tilts in favor of ben
efit, as in fatal disorders for which there is no therapy. The risks
of the hESC therapies will have to be understood and probably
reduced to maintain an appropriate risk/benefit ratio before these
technologies can be applied to diseases that are inherently less
dangerous to the patient. Gene therapy should prove to be valu
able in reducing the risks associated with making hESC therapy
a reality.
864
Acknowledgments
We thank Ralf-Harto Hbner and Maija Kiuru for assistance in the
hESC gene transfer studies, Neil Hackett and Julie Boyer for helpful
discussions, and Nahla Mohamed and Tienne Virgin-Bryan for help in
preparing this manuscript. These studies were supported, in part, by
the Will Rogers Memorial Fund, Los Angeles, CA; The Malcolm Hewitt
Wiener Foundation, Greenwich, CT; and Nathans Battle Foundation,
Greenwood, IN.
Supplementary Material
Supplementary Materials and Methods
Table S1. Human embryonic cell lines eligible for NIH funding.
Table S2. Human embryonic cell lines not eligible for NIH funding
not disease related.
Table S3. Human embryonic cell lines not eligible for NIH funding
disease related.
Figure S1. Human embryonic stem cells grown on different feeder
layers.
Figure S2. Genetically modified human embryonic stem cells.
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