Nucleofection Mediates High-Efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells

Article (PDF Available)inStem Cells 26(6):1436-43 · July 2008with54 Reads
DOI: 10.1634/stemcells.2007-0857 · Source: PubMed
Abstract
High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.
Nucleofection Mediates High-Efficiency Stable Gene Knockdown
and Transgene Expression in Human Embryonic Stem Cells
KRISTI A. HOHENSTEIN,
a,b
APRIL D. PYLE,
c
JING YI CHERN,
d
LESLIE F. LOCK,
b
PETER J. DONOVAN
b
a
Sue and Bill Gross Stem Cell Research Program, Department of Biological Chemistry, Developmental and Cell
Biology Department, Center for Molecular and Mitochondrial Medicine and Genetics, University of California
Irvine, Irvine, California, USA;
b
Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;
c
McKusick-Nathans Institute of Genetic Medicine, and
d
Department of Microbiology, Immunology and Molecular
Genetics, University of California, Los Angeles, California, USA
Key Words. Human embryonic stem cells Nucleofection Transfection Transgene expression RNA interference
Green fluorescent protein Oct4 Nanog
ABSTRACT
High-efficiency genetic modification of human embryonic
stem (hES) cells would enable manipulation of gene ac-
tivity, routine gene targeting, and development of new
human disease models and treatments. Chemical trans-
fection, nucleofection, and electroporation of hES cells
result in low transfection efficiencies. Viral transduction
is efficient but has significant drawbacks. Here we de-
scribe techniques to transiently and stably express trans-
genes in hES cells with high efficiency using a widely
available vector system. The technique combines nucleo-
fection of single hES cells with improved methods to select
hES cells at clonal density. As validation, we reduced
Oct4 and Nanog expression using siRNAs and shRNA
vectors in hES cells. Furthermore, we derived many hES
cell clones with either stably reduced alkaline phospha-
tase activity or stably overexpressed green fluorescent
protein. These clones retained stem cell characteristics
(normal karyotype, stem cell marker expression, self-
renewal, and pluripotency). These studies will accelerate
efforts to interrogate gene function and define the param-
eters that control growth and differentiation of hES cells.
S
TEM CELLS 2008;26:1436–1443
Disclosure of potential conflicts of interest is found at the end of this article.
I
NTRODUCTION
Human embryonic stem (hES) cells are potentially important
tools for studying human development and for identifying ge-
netic and environmental factors that affect developmental pro-
cesses [1– 4]. In addition, they are a potential resource for
developing models of disease and cell-based treatments. This
potential stems from their dual abilities to self-renew and dif-
ferentiate. A powerful method to address questions about regu-
lation of these dual abilities is to genetically manipulate hES
cells in culture. The ability to transfect these cells with high
efficiency would allow analysis of gene regulation using RNA
interference (RNAi), expression vectors, and targeting con-
structs and also enable large-scale, genome-wide screens to be
performed.
Two impediments to progress are the relatively poor
growth of hES cells in culture and the difficulty in transfect-
ing hES cells [4 6]. Recently, we demonstrated that addition
of neurotrophins (NTs) to hES cell cultures increases survival
of single cells and could consequently improve transfection
techniques and clonal selection [7]. Several methods have
been used to transfect hES cells, including chemical trans-
fection, electroporation, nucleofection, and viral transduc-
tion, with various efficiencies (reviewed in [6]). Chemical
and electrical methods of transfection result in transient
efficiencies from 1% to 40% and very low, stable efficien-
cies from one in 10
5
–10
7
cells [8–15]. Expression of genes
introduced into hES cells by retroviruses and lentiviruses is
more successful (85%) [16–18]. However, there are sig-
nificant drawbacks associated with viral transduction [6, 19,
20].
One promising technique for introducing DNA into cells
is nucleofection, which delivers DNA directly into the nu-
cleus with high efficiency [21–23]. Methods for nucleofect-
ing hES cells have been reported, with various transient
transfection efficiencies of 20%– 65% [10, 11]. The reported
stable transfection rate was low (1 in 10
6
cells), and little
characterization of stem cell markers, karyotype, and pluri-
potency was reported [11]. Here we describe improved meth-
ods for obtaining stably transfected hES cells via nucleofec-
tion of individual hES cells mediated by increased clonal
survival in the presence of NTs. We report a maximum
transient transfection efficiency of 85% (similar to viral
transduction) and a maximum stable efficiency of 1.2 in 10
4
cells (100 times better than current nucleofection rates). The
method described here provides an opportunity to generate
genetically modified diploid hES cell lines, which retain full
pluripotency with very high efficiency.
Correspondence: Leslie F. Lock, Ph.D., 2501 Hewitt Hall, University of California, Irvine, California 92697, USA. Telephone: 949-824-
3547; Fax: 949-824-6388; e-mail: llock@uci.edu; or Peter J. Donovan, Ph.D., 2501 Hewitt Hall, University of California, Irvine, California
92697, USA. Telephone: 949-824-3691; Fax: 949-824-6388; e-mail: pdonovan@uci.edu Received October 10, 2007; accepted for
publication February 23, 2008; first published online in S
TEM CELLS EXPRESS March 6, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0
doi: 10.1634/stemcells.2007-0857
E
MBRYONIC
S
TEM
C
ELLS
STEM CELLS 2008;26:1436–1443 www.StemCells.com
M
ATERIALS AND
M
ETHODS
hES Cell Culture
hES cell lines (H1, H9) were cultured as previously described [7,
24] on PMEFs (Chemicon, Temecula, CA, http://www.chemicon.
com) and passaged using collagenase IV (Invitrogen, Carlsbad, CA,
http://www.invitrogen.com). For nucleofection, cells were har-
vested using 0.05% trypsin-EDTA (Invitrogen), dissociated into
single cells, and, following addition of trypsin inhibitor (Invitro-
gen), passed through a 0.4-mm cell strainer (Becton Dickinson,
Franklin Lakes, NJ, http://www.bd.com; Falcon).
Vector and siRNA Design
Three cytomegalovirus (CMV)-immediate early (IE) promoter-
based vectors were used: pmaxGFP (Amaxa Biosystems, Gaithers-
burg, MD, http://www.amaxa.com; 589-base pair [bp] PCMV IE,
referred to as green fluorescent protein [GFP] vector) for transient
transfections, phrGFP II-1 (catalog no. 240143; Stratagene, La
Jolla, CA, http://www.stratagene.com; 665-bp CMV promoter, re-
ferred to as GFP-neo vector) for stable transfections, and pRNATin-
H1.2/Neo (catalog no. SD1223; GenScript, Piscataway, NJ, http://
www.genscript.com; 587-bp CMV promoter, referred to as shRNA
vector) for RNAi. shRNAs corresponding to target genes were
designed using GenScript’s Target Finder with the following se-
quences: alkaline phosphatase (AP; accession number: AB011406;
alkaline phosphatase, liver/bone/kidney [ALPL], also known as the
tissue-nonspecific alkaline phosphatase [TNAP]), 5-AAGAGCT-
TCAAACCGAGATA-3 and 5-GGACTATGCTCACAACAATA-
3. The Oct4 target was 5-AGCAGCTTGGGCTCGAGAA-3 [25],
the Nanog sequence was 5-AAGGGTTAAGCTGTAACATAC-3
[17], and the
2M target sequence was 5-GATTCAGGTTTACT-
CACGT-3 [25] Stem-loop structures for these targets were built into
pRNATin-H1.2/Neo. For RNAi by cotransfection, synthesized siRNAs
(Qiagen, Gaithersburg, MD, http://www1.qiagen.com) were trans-
fected with the pmaxGFP vector (5:1).
Nucleofection
All nucleofections were performed using the Nucleofector II
(Amaxa Biosystems). For optimization, six Nucleofector settings
(A-06, A-12, A-13, A-23, A-27, and B-16), two nucleofection
solutions (solution V and mouse embryonic stem [mES] solution),
and two cell harvesting methods (collagenase and trypsin) were
analyzed for effects on hES cell survival and transfection. GFP-
positive colonies were visualized using a Nikon Eclipse T100
microscope (Nikon, Tokyo, http://www.nikon.com) and counted
manually. For all subsequent transfections, hES cells were har-
vested by trypsinization and transfected using mES solution and
program A-23. A total of 2 10
6
trypsinized hES cells were
resuspended in 100
l of mES cell solution and incubated (37°C)
for 5 minutes. Two to three micrograms of DNA (or siRNA) was
added to the cell solution. The mixture was transferred to a cuvette,
nucleofected, and quickly transferred into 500
l of prewarmed
(37°C) RPMI medium (Invitrogen). Nucleofected cells were incu-
bated (37°C) for 5 minutes and plated at 1 10
6
cells per well in
a PMEF-Neo
r
-coated six-well plate containing hES medium sup
-
plemented with NTs (10 ng/ml of brain-derived neurotrophic factor,
NT-3, NT-4). To maximize viability and efficiency, the entire
procedure was completed within 20 minutes. To select for stably
transfected hES cell colonies, G418 sulfate (25
g/ml; Invitrogen)
was added 96 hours post-transfection. G418 concentration was
increased to 50
g/ml at 1 week post-transfection and finally to 100
g/ml at 10 days post-transfection.
Stem Cell Characterization
Flow cytometric analysis of GFP and SSEA-4 (Developmental
Studies Hybridoma Bank, Iowa City, IA, http://dshb.biology.uiowa.
edu) expression was performed using a FACSCalibur flow cytom-
eter (Becton Dickinson). Immunocytochemistry and AP staining
was performed as previously described [7] Antibodies to Oct4,
SSEA-3, and SSEA-4 were obtained from R&D Systems Inc. (Min-
neapolis, http://www.rndsystems.com), TRA1– 60 and TRA1–81
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.
scbt.com), and anti-GFP IgG-Alexa Fluor 488 and secondary anti-
bodies from Invitrogen.
Figure 1. Optimization of nucleofection in
hES cells. (A): The mean percentage of col-
onies (SEM; n 3) that were GFP-posi-
tive after nucleofection of collagenase-disso-
ciated hES cells in V solution (blue bars) or
mES solution (green bars). (B): The percent-
age of colonies (SEM; n 3) that were
GFP-positive after nucleofection of trypsin-
dissociated hES cells in V solution (yellow
bars) or mES solution (red bars). (C): The
mean percentage of GFP-positive cells (n
5) in each colony after nucleofection of col-
lagenase-derived (blue and green bars) and
trypsin-derived (yellow and red bars) hES
cells using program A-23 in either V solu-
tion (blue and yellow bars) or mES solution
(green and red bars). (D): BF (top) and GFP
expression (bottom) of transfected collage-
nase-dissociated hES cells and trypsin-disso-
ciated hES cells 4 days after nucleofection.
Abbreviations: BF, bright-field; GFP, green
fluorescent protein; hES, human embryonic
stem; mES, mouse embryonic stem.
1437Hohenstein, Pyle, Chern et al.
www.StemCells.com
Polymerase Chain Reaction Analyses
For reverse transcription (RT)-polymerase chain reaction (PCR),
RNA was prepared using the RNeasy Kit (Qiagen) and performed
using the Superscript One-Step Kit (Invitrogen) for 25–35 cycles.
For PCR analyses, DNA was prepared using the DNeasy Blood and
Tissue Kit (Qiagen) and performed using the Taq PCR Core Kit
(Qiagen) for 35 cycles. Supplemental online Table 1 has primers
and annealing temperature for each gene.
Karyotype Analysis
Analyses were performed by Johns Hopkins Cytogenetics Facility
(Baltimore, MD) or Genzyme Genetics (Orange, CA, http://www.
genzymegenetics.com).
Differentiation Analyses
Embryoid body and teratoma formation assays were carried out as
previously described [7, 24, 26]. Animal work was performed under
the Johns Hopkins University and University of California Irvine
Institutional Animal Care and Use Committee protocols. Tumors
were processed at the Johns Hopkins Comparative Medicine De-
partment (Baltimore, MD) and analyzed at University of California
Irvine Medical Center (Orange, CA).
R
ESULTS
To optimize conditions for high efficiency nucleofection of hES
cells, a 3.5-kilobase (kb) plasmid expressing GFP from the
CMV promoter (GFP vector) was transfected into H9 hES cells,
and the percentage of GFP-positive colonies was determined
visually 96 hours after nucleofection. Six nucleofection pro-
grams and two solutions were compared, and nucleofected col-
lagenase-derived clumps or trypsin-derived single hES cells
gave colonies with cells that expressed GFP (18%–35% or
11%–54%, respectively; Fig. 1A, 1B). Nucleofection of trypsin-
derived single cells using program A-23 and mES solution
resulted in the most GFP-positive colonies (61%). In some
transfections, NTs were omitted. Approximately 30% fewer
colonies were observed in the absence of NTs (data not shown),
suggesting that NTs enhance survival of clones after nucleofec-
tion. Notably, 70% of trypsin-derived colonies were entirely
GFP-positive, whereas 10% of collagenase-derived colonies
were uniformly GFP-positive (Fig. 1C, 1D). In fact, 90% of
the collagenase-derived colonies contained 50% GFP-positive
cells, suggesting that nucleofection does not effectively transfect
all cells in collagenase-derived hES cell clumps.
Figure 2. Nucleofection of hES cells with
DNA, shRNA vectors, or siRNAs alters
gene expression. (A): Flow cytometric anal-
ysis of GFP and SSEA-4 expression in H1
and H9 hES cells 4 days after transfection
with the GFP vector (middle and right) or no
DNA (left). (B): BF (top) and GFP expres-
sion (bottom) of hES cells 4 days after co-
transfection of the GFP vector and either
2M siRNA or Oct4 siRNA. GFP expres-
sion was used to identify transfected cells.
(C): BF (top) and SSEA-4 (bottom) immu-
nostaining of
2M KD and Oct4 KD hES
cells. (D): AP staining of
2M KD and Oct4
KD hES cells. (E): Reverse transcription-
polymerase chain reaction analysis of hES
cells transfected with either shRNA vectors
or siRNAs.
-Actin was used as a template
loading control. Abbreviations: AP, alkaline
phosphatase; BF, bright-field; DAPI, 4,6-
diamidino-2-phenylindole; GFP, green fluo-
rescent protein; hES, human embryonic
stem; KD, knockdown.
1438 Stable Nucleofection of hES Cells
To determine the efficiency of nucleofection, we analyzed
the number of cells transfected with either the GFP vector or a
4.9-kb vector containing hrGFP expressed from the CMV pro-
moter (GFP-neo vector). H1 or H9 hES cells were trypsin-
dissociated, nucleofected using program A-23 and mES solu-
tion, and cultured in hES cell medium supplemented with NTs
for 96 hours. Nucleofected hES cells were analyzed by flow
cytometry using SSEA-4, to distinguish hES cells from mouse
embryonic fibroblasts and differentiated cells. When either H1
or H9 hES cells were nucleofected, 66% of SSEA-4-positive
hES cells expressed GFP (Fig. 2A). In seven nucleofections
using the GFP vector in H9 hES cells, transfection efficiencies
ranged from 60% to 85%, with a mean of 76%. In 20 nucleo-
fections using the GFP-neo vector, transfection efficiencies
ranged from 47% to 81%, with a mean of 67% (data not shown).
An increase in transfection efficiency was observed over time as
the ability to handle and maintain single hES cells during
nucleofection improved. The vast majority of control hES cells
(nucleofected without DNA) continued to express SSEA-4, in-
dicating that nucleofection does not affect stem cell marker
expression (Fig. 2A).
Next we carried out a proof-of-principle experiment to dem-
onstrate the utility of nucleofection in studies of gene function.
Previous studies demonstrated that Oct4 and Nanog are required
to maintain the stem cell state [17, 25, 27–30]. To test the utility
of nucleofection in reducing Oct4 or Nanog expression, shRNAs
expressed from a CMV-based vector system (shRNA vector) or
siRNAs were introduced into either H1 or H9 hES cells. The
2M gene, which is expressed in hES cells but not required for
self-renewal [25], was used as a control (
2M knockdown
[KD]). shRNA vectors and siRNAs reduced expression of spe-
cific targets, as determined by RT-PCR (Fig. 2E). Because
siRNAs were cotransfected with the GFP vector and the shRNA
vector contains a GFP reporter gene, transfected cells could be
identified visually by fluorescence microscopy. Ninety-six
hours after nucleofection, all GFP-positive colonies transfected
with Oct4-specific siRNA (Oct4 KD; 500 colonies per well)
were markedly flattened and morphologically distinct from typ-
ical or
2M KD hES cell colonies (Fig. 2B, 2C). The stem cell
markers AP, SSEA-3, SSEA-4, TRA1–60, and TRA1– 81 were
reduced in the flattened, GFP-positive Oct4 KD colonies, in
contrast with
2M KD cells (Fig. 2C, 2D; data not shown).
Similar results were obtained when shRNA vectors were used to
reduce Oct4 or Nanog expression (Nanog KD; data not shown).
Interestingly, reduction of Oct4 or Nanog expression caused
reciprocal downregulation of these genes, consistent with our
understanding of the stem cell regulatory network (Fig. 2E) [31,
32]. To determine the identity of the differentiated cells, expres-
sion of trophectodermal genes GCM1 and hCG was assayed by
RT-PCR. In both Oct4 KD and Nanog KD cells, expression of
GCM1 and hCG increased, in comparison with
2M KD cells
(Fig. 2E). These data strongly support the idea that nucleofec-
tion of siRNAs/shRNA vectors effectively alters gene expres-
sion in hES cells.
For many applications, stable long-term alteration in gene
expression is necessary. To determine whether nucleofection
can produce hES cells with stable modifications in gene expres-
sion, the GFP-neo vector was introduced into hES cells, and
stable colonies were isolated by gradually increasing the G418
concentration. To isolate stable clones, G418 was added at a
concentration of 25
g/ml for 3 days, then 50
g/ml for 3 days,
and finally 100
g/ml for at least 2 weeks. The GFP vector was
used as a control in these experiments. GFP expression in
control cells was rapidly lost in the absence of G418, and all
cells grown in G418 (at only 25
g/ml) died after 3 days. After
2 weeks in the presence of G418, an average of 60 colonies per
well (n 12) and a maximum of 120 colonies per well were
observed in cultures transfected with the GFP-neo vector. GFP
was expressed in most of the cells of G418-resistant colonies
(Fig. 3A). G418-resistant, GFP-positive colonies were manually
passaged 3 weeks after nucleofection (2 weeks in selection)
and maintained for 17 months in culture (75 passages). Flow
cytometry of the stably transfected hES cells showed that 87%
of the cells expressed GFP (data not shown), suggesting that
some G418-resistant hES cells lose GFP expression. To monitor
the stability of transgene expression in prolonged culture, we
derived clonal cell lines from GFP-positive hES cell lines. A
total of 144 clones were isolated by dilution cloning, half in
G418 and half in the absence of G418. In both growth condi-
tions, approximately 85% of the derived clones expressed GFP
(Fig. 3B), consistent with the fluorescence-activated cell sorting
analysis of the nonclonal G418-resistant, GFP-positive cells
from which the clones were isolated. Genomic DNA from 14
clones was analyzed by PCR and demonstrated that the CMV
promoter and the hrGFP gene are present contiguously in all
clones, even those that do not express GFP (Fig. 3C), suggesting
that silencing of the GFP transgene occurs in some cells of the
stably transfected hES cell colonies [16, 18, 33].
Cell lines and clones derived by nucleofection that stably
express GFP retained an hES cell morphology and expressed
GFP in long-term culture. All stable cell lines expressed the
stem cell markers Oct4, SSEA-4, SSEA-3, TRA1–60, TRA1–
Figure 3. GFP expression in stable GFP-neo cell lines. (A): BF (left)
and GFP (right) expression of hES cells stably transfected with the
GFP-neo vector (GFP-neo cells). (B): Clonal cell lines derived from
GFP-neo cells grown with or without G418. (C): Polymerase chain
reaction analysis of genomic DNA isolated from clonal cell lines. Both
GFP-positive and GFP-negative clones contain the CMV promoter and
hrGFP transgene contiguously in their genomes. Untransfected hES
cells served as a control, and
-actin was used a template loading
control. Abbreviations: BF, bright-field; CMV, cytomegalovirus; GFP,
green fluorescent protein; hES, human embryonic stem; NT,
neurotrophin.
1439Hohenstein, Pyle, Chern et al.
www.StemCells.com
81, and AP (Fig. 4A, 4B; data not shown). Nucleofected hES
cells maintained a normal karyotype for 8 months of contin-
uous culture (Fig. 4B; data not shown), suggesting that nucleo-
fection does not induce major genomic instability. Stable cell
lines retained their ability to differentiate, as demonstrated by
formation of embryoid bodies (EBs) expressing GFP (Fig. 4C)
and markers of all three germ layers (Fig. 5C). GFP-positive
hES cells were also injected into NOD/SCID mice, producing
teratomas that, by comparison with tumors from GFP-negative
clones and nontransfected cells, expressed GFP broadly
throughout the tumor (data not shown). Histological examina-
tion of those tumors revealed that they contained derivatives of
all three germ layers, indicating that the cells were indeed
pluripotent (Fig. 4D). Fluorescence microscopy of sections in-
dicated that the GFP transgene continued to be expressed in
most of the tumor cells (Fig. 4D). Clonal cell lines also readily
formed teratomas expressing GFP (Fig. 4E), indicating long-
term, stable expression of the transgene in vivo.
The function of the stem cell marker ALPL (GenBank
accession no. AB011406) in hES cell growth and pluripotency
is not known. In mice, loss of function of TNAP (AP homolog)
has no effect on early development in vivo [34, 35]. We inves-
tigated the long-term effects of AP reduction in hES cells by
creating stable cell lines transfected with a vector encoding an
AP-specific shRNA (AP KD). As a control, we also created
2M-specific shRNA stable cell lines and maintained the cell
lines for more than 8 months of continuous culture. shRNA-
mediated reduction of AP in hES cells produced a marked
decrease in, but not complete loss of, AP activity, as judged by
RT-PCR and histochemical staining (Fig. 5A, 5B). The mor-
phology of AP KD cells was indistinguishable from untreated
and
2M KD hES cells (Fig. 5B). AP KD hES cells also
expressed Oct4 (Fig. 5A), SSEA-3, SSEA-4, TRA1– 60, and
TRA1– 81 (data not shown). AP KD hES cells were still pluri-
potent, since the cells differentiated into EBs containing cells
derived from all three germ layers and formed well-differenti-
ated teratomas (data not shown). Stem cell marker expression
and EB differentiation were performed approximately every 2
months and teratoma analyses were performed 7 months after
derivation. These data suggest that nucleofection of an AP-
specific shRNA reduces expression of the AP gene without
affecting hES cell characteristics.
D
ISCUSSION
In mice, ES cells are used to analyze many aspects of develop-
ment because of the relative ease with which they can be
Figure 4. Stably transfected clonal hES cell
lines retain stem cell characteristics, includ-
ing expression of stem cell markers, normal
karyotype, and the ability to differentiate in
vitro and in vivo. (A): DAPI (top), GFP
(middle), Oct4 (bottom left), and SSEA-4
(bottom right) staining of GFP-neo hES
cells. (B): AP staining (left) and karyotype
(right) of GFP-neo hES cells after 25 pas-
sages. (C): BF (left) and GFP expression
(right) of EBs derived from GFP-neo cells.
(D): Hematoxylin and eosin (H&E; top pan-
els), GFP, and DAPI staining (bottom pan-
els) of sectioned GFP-neo teratomas. (E):
Gross image of teratomas formed from GFP-
neo hES cell clones. Abbreviations: AP, al-
kaline phosphatase; BF, bright-field; DAPI,
4,6-diamidino-2-phenylindole; GFP, green
fluorescent protein.
1440 Stable Nucleofection of hES Cells
genetically modified. This ability facilitates studies to delineate
gene function, define lineage development, and identify genes
that function in self-renewal and pluripotency [36]. hES cells
provide the opportunity to carry out similar studies with human
cells. Differentiated human cells could have enormous potential
utility for disease treatment, drug development, and analysis of
development [1–3]. Although a few examples of genetic manip-
ulation of hES cells have been reported, major improvements in
the transfection efficiency will be necessary if hES cells are to
reach their full potential. Here we describe a method for repro-
ducible, high-efficiency production of genetically modified,
clonally derived hES cell lines. Using nucleofection combined
with culture conditions that improve the survival of hES cells as
single cells, we observed a transient transfection efficiency of up
to 85% and a stable transfection efficiency of up to 1.2 in 10
4
cells.
Previously, results using chemical methods of transfection
in hES cells were reported, with transient transfection efficien-
cies from 1% to 30%, consistent with our own observations
([8, 11, 13, 15]; data not shown). Addition of NTs following
chemical transfection (i.e., lipofection) had no effect on trans-
fection efficiency. The efficiency of deriving stably modified
hES cell lines appeared to be low, possibly because clumps of
hES cells were transfected [8, 9]. In addition to the low trans-
fection efficiency, chemical transfection may not be an effective
method for carrying out homologous recombination [37, 38], a
widely used technique in mouse genetics [36]. The low effi-
ciency of chemical transfection, in general, has hindered studies
of gene function in hES cells and made high-throughput screens
to identify genetic pathways all but impossible.
Viruses were also used successfully to introduce genes into
hES cells. Lentiviruses produce a very high rate of transient
transfection (70%– 85%) [16 –18]. Here we report similar tran-
sient transfection efficiencies using nucleofection (59%– 85%).
Lentiviral transduction also results in stable transgene expres-
sion during prolonged culture. Flow cytometric analyses of
stable cell lines demonstrated that 70%– 85% of hES cells
expressed eGFP from the EF1
promoter after 7–38 weeks in
culture [16, 18, 33]. Here we demonstrated similar stable ex-
pression of hrGFP from the CMV promoter using nucleofection
(87%). Importantly, the use of lentiviruses involves a significant
investment in vector development, and important safety con-
cerns must be considered [6]. Also, the DNA insert size in viral
vectors is limited, and host immune and inflammatory responses
to cells after transduction may occur [19]. The use of CMV-
based vectors eliminates many of these problems.
Electroporation is an attractive method for transfecting hES
cells because of the utility of this technique in mES cells,
relative ease of the method, and its utility for generating homol-
ogous recombination events [37, 38]. Electroporation of hES
cells resulted in slightly higher transient transfection efficiencies
than chemical methods (up to 40%) [8, 10, 11, 13]. However,
these reports involved electroporation of clumps of hES cells,
likely reducing the gene transfer efficiency and making clonal
selection difficult. In addition, such methods make it difficult to
determine the gene transfer efficiency because it is difficult to
determine the number of cells per clump. Indeed, the reported
rates of deriving stable clones were low. For example, only one
genetically modified hES cell line was produced using mES cell
electroporation conditions [12]. Recently, two stably modified
clones were derived following electroporation of 1.5–3.0 10
7
cells, giving an efficiency of one clone in 10
7
cells [14]. Here
we report a stable transfection efficiency of up to one clone in
10
4
transfected cells, a significant improvement over electropo
-
ration methods. Our data suggest that nucleofection of single
hES cells followed by growth in NTs is as efficient as lentivirus-
mediated transfection for transient modification of hES cells and
more efficient than other methods published to date for gener-
ating stably modified hES cell lines.
The utility of genetic modification of hES cells using
nucleofection was demonstrated in two sets of experiments.
First, we performed siRNA- and shRNA-mediated knockdown
of two genes involved in maintenance of the pluripotent state,
Oct4 and Nanog. In both cases, reduced gene expression re-
sulted in the differentiation of hES cells into trophectoderm-like
cells. Isolation of stable cell lines with constitutive reduction in
Oct4 and Nanog was not possible, consistent with the idea that
these genes are required for hES cell maintenance [17, 25,
27–30]. In the second experiment, the expression of the stem
cell marker gene AP was constitutively reduced in hES cells,
showing that stable downregulation of AP does not appear to
affect hES cell growth or developmental potency, consistent
with the conclusions from the mouse studies [34, 35].
An important feature of the work described here is the use of
a CMV IE-based vector system to drive gene expression. CMV
Figure 5. shRNA-mediated knockdown of AP does not affect the
expression of other stem cell genes. (A): Reverse transcription (RT)-
polymerase chain reaction (PCR) analysis of AP KD and
2M KD hES
cells. GFP-neo and untransfected hES cells were used as controls.
-Actin was used a template loading control. (B): BF (top), GFP
(middle), and AP (bottom) staining of AP KD and
2M KD hES cells.
(C): RT-PCR analysis of AP KD and
2M KD hES cells and EBs.
GFP-neo cells served as a control, and
-actin was used a template
loading control. Abbreviations:
MHC, alpha myosin heavy chain;
AFP, alpha fetoprotein; AP, alkaline phosphatase; BF, bright-field;
GCM1, glial cells missing-1; GFP, green fluorescent protein; hES,
human embryonic stem; KD, knockdown.
1441Hohenstein, Pyle, Chern et al.
www.StemCells.com
forms the basis of many widely available vector systems and
would enable use of many reagents that already exist (e.g.,
cDNA/shRNA libraries). Our studies suggest that CMV IE-
based systems are expressed effectively in hES cells and can
drive long-term gene expression in vitro and in vivo. The ability
to manipulate gene function with a commonly used vector
system is a major advantage in studies of gene function in hES
cells.
A central problem for all methods of genetic modification of
hES cells is the difficulty of clonal selection. In most studies,
transfection of hES cell clumps makes the process of clonal
selection difficult because transfected cells confer resistance to
nontransfected cells by communication through gap junctions,
and nontransfected hES cells may overgrow recovering, genet-
ically modified cells [25, 39, 40]. Nucleofection of a population
of single cells allows the development of true clonal cell lines
despite the difficulties normally seen when they are passaged as
single cells. The hES cell lines used in this study are routinely
passaged by enzymatic dissociation into clumps but gently
dissociated by trypsinization to yield a population of single cells
for nucleofection. We expect that hES cell lines passaged in a
similar manner will yield results equivalent to those reported
here. hES cell lines that are passaged exclusively by mechanical
dissociation may be more sensitive to gentle trypsinization and
may have a higher mortality rate [41, 42]. However, the actions
of NTs may ameliorate this effect. The development of true
clonal cell lines is important both for determining the effect of
regulating gene expression on cell physiology and for develop-
ment of robust methods for carrying out successful gene target-
ing. Increasing the efficiency of hES cell transfection methods
may also improve efficiency of deriving stable cell lines. Re-
cently, introduction of DNA into Accutase-dissociated cells by
nucleofection resulted in the derivation of four clones from
2– 4 10
6
cells, representing a stable clone derivation effi
-
ciency of one to two clones in 10
6
cells [11]. In conclusion, we
report the derivation of clonally derived stable cells lines with
an efficiency of up to one clone in 10
4
cells. The improved
efficiency should greatly facilitate some of the techniques of
genetic modification of hES cells, including homologous recom-
bination, gene trapping, and cDNA and siRNA/shRNA library
screens. These studies should accelerate efforts to interrogate
gene function in hES cells and to define the parameters that
control the growth and differentiation of these cells.
A
CKNOWLEDGMENTS
We thank Drs. John Gearhart, Mike Shamblott, Nicholas
Christoforou, David Valle, Erika Matunis, Daniel Shain, and
Grant MacGregor for helpful suggestions and comments; Scott
D’Andrea and Vicki Pomodoro (Amaxa Biosystems) and Rick
Marolt (Carl Zeiss Microimaging, Inc., Thornwood, NY, http://
www.zeiss.com) for technical help; Jorge Martin and Holly
Wellington for technical assistance; and Jeff Bonadio for tera-
toma analyses. We also thank members of the Donovan and
Lock laboratories for assistance and encouragement. We are
also grateful to Sean Donovan and Aaron Elliott for their patient
support. L.F.L. and P.J.D. dedicate this paper to Dr. Camilynn
Brannan (1963–2002). This work was supported in part by
funding from NIH (HD49488 and HD46765) (to P.J.D.), funds
from University of California Irvine (to P.J.D. and L.F.L.), and
by the Human Genetics and Molecular Biology Training Pro-
gram (NIH 2T32GM07814) at Johns Hopkins University School
of Medicine (to K.A.H.). L.F.L. and P.J.D. are co-senior au-
thors.
D
ISCLOSURE OF
P
OTENTIAL
C
ONFLICTS
OF
I
NTEREST
The authors indicate no potential conflicts of interest.
R
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1443Hohenstein, Pyle, Chern et al.
www.StemCells.com
    • "Transgenic stem cells using the homologous recombination technique were first reported in mouse embryonic stem cells by Thomas & Capecchi in 1987 [19] . Subsequently, researchers have successfully delivered transgenes into pluripotent stem cells using several methods, including electroporation [20], liposomal [21] and viral vectors [22,23], and nucleofection [24] . However, stably introducing transgenes in these cells has proven difficult because of the low efficiency and cytotoxic side effects. "
    [Show abstract] [Hide abstract] ABSTRACT: Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by in vitro differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes.
    Full-text · Article · Aug 2013
    • "For RNAi in hES cells, nucleofection was performed as previously described (Hohenstein et al., 2008). hES cells were transfected with 100 μM of short interfering RNAs (siRNAs) corresponding to SOX2 and β2M (Qiagen, Germantown, MD). "
    [Show abstract] [Hide abstract] ABSTRACT: Human embryonic stem (hES) cells have the dual ability to self-renew and differentiate into specialized cell types. However, in order to realize the full potential of these cells it is important to understand how the genes responsible for their unique characteristics are regulated. In this study we examine the regulation of the tropomyosin-related kinase (TRK) genes which encode for receptors important in hES cell survival and self-renewal. Although the TRK genes have been studied in many neuronal cell types, the regulation of these genes in hES cells is unclear. Our study demonstrates a novel regulatory relationship between the TRKC gene and the transcription factor SOX2. Our results found that hES cells highly express full-length and truncated forms of the TRKC gene. However, examination of the related TRKB gene showed a lower overall expression of both full-length and truncated forms. Through RNA interference, we knocked down expression levels of SOX2 in hES cells and examined the expression of TRKC, as well as TRKB. Upon loss of SOX2 we found that TRKC mRNA levels were significantly downregulated but TRKB levels remained unchanged, demonstrating an important regulatory dependence on SOX2 by TRKC. We also found that TRKC protein levels were also decreased after SOX2 knock down. Further analysis found the regulatory region of TRKC to be highly conserved among many mammals with potential SOX binding motifs. We confirmed a specific binding motif as a site that SOX2 utilizes to directly interact with the TRKC regulatory region. In addition, we found that SOX2 drives expression of the TRKC gene by activating a luciferase reporter construct containing the TRKC regulatory region and the SOX binding motif.
    Full-text · Article · Mar 2012
    • "Thus far, delivery of siRNA has relied either on stable transfection using viral means[10, 11] or transient transfection using commercially available reagents (Lipofectamin, HiperFect) [5–9, 13, 14]. Recently, nucleofection[15] and siRNA conjugated to peptide RNA-binding transduction domains was reported to facilitate siRNA uptake in a variety of primary cell lines including hESCs[12]; however, efficiency in hESCs was not reported. In vitro experiments involving RNAi to date have been performed in a 2D environment where the cells are seeded on a surface and the transfection reagents are added into the media. "
    [Show abstract] [Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.
    Full-text · Article · Nov 2011
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