High-Throughput Identification of Genes Promoting Neuron
Formation and Lineage Choice in Mouse Embryonic Stem Cells
ANNA FALK, TOBIAS E. KARLSSON, SANJA KURDIJA, JONAS FRIS´ EN, JOEL ZUPICICH
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden
Key Words. Developmental biology • Gene expression • Fluorescence-activated cell sorting analysis • Embryonic stem cells
Fluorescent protein reporter genes • Gene delivery systems in vivo or in vitro • Stem cell culture • Neural differentiation
The potential of embryonic stem cells to differentiate to all
cell types makes them an attractive model for development
and a potential source of cells for transplantation therapies.
Candidate approaches have identified individual genes and
proteins that promote the differentiation of embryonic stem
cells to desired fates. Here, we describe a rapid large-scale
screening strategy for the identification of genes that influ-
ence the pluripotency and differentiation of embryonic stem
cells to specific fates, and we use this approach to identify
genes that induce neuron formation. The power of the strat-
egy is validated by the fact that, of the 15 genes that resulted
in the largest increase in neuron number, 8 have previously
been implicated in neuronal differentiation or survival,
whereas 7 represent novel genes or known genes not previ-
ously implicated in neuronal development. This is a simple,
fast, and generally applicable strategy for the identification
of genes promoting the formation of any specific cell type
from embryonic stem cells. STEM CELLS 2007;25:1539–1545
Disclosure of potential conflicts of interest is found at the end of this article.
The pluripotency and unlimited growth of embryonic stem (ES)
cells make them an attractive source of differentiated cells for cell
therapy in a variety of human diseases. There are, however, several
hurdles for the realization of this prospect, with one of the greatest
being to find ways to differentiate ES cells to specific cell fates.
Our rapidly increasing understanding of the extracellular
signals that direct embryonic development has aided in the
improvement of protocols to differentiate ES cells to, for exam-
ple, motor neurons . However, in most cases, our knowledge
is incomplete regarding the molecular cascades that govern the
differentiation of specific cell fates, or they may potentially be
too complex to reproduce in vitro.
The orchestration of extracellular signals imposes the expres-
sion of a complement of transcriptional regulators, which direct
differentiation. In the case of motor neurons, for example, posi-
factor Mnr2/Hb9, which is necessary and sufficient to direct the
differentiation of spinal progenitors to motor neurons . Intrinsic
determinants necessary and sufficient for the generation of dopa-
minergic neurons have similarly been identified . Thus, in at
least some cases, single genes play a pivotal role in the induction of
specific cell types and can be used to drive the formation of a
desired cell type. Interestingly, there are several protocols that
promote the generation of dopaminergic neurons through the ad-
dition of extracellular factors [4, 5], but none are nearly as efficient
Identifying such genes may shed light on embryonic development
and allow the efficient generation of cells for therapy.
The often complex nature of the extracellular milieu, both
when it comes to the number of factors and their temporal
regulation, makes the strategy of identifying individual or small
numbers of key genes an attractive route for the directed differ-
entiation of ES cells. Unintended genetic manipulation of cells
for transplantation is undesirable due to the risk of cellular
transformation and tumor development. However, transient
gene expression or even administration of recombinant intracel-
lular proteins coupled to cell penetrable molecules may circum-
vent this problem. In addition, other promising gene therapy
approaches include human artificial chromosomes that can be
stably maintained without adversely affecting the host cell 
and targeted replacement of loci .
We report a method for the efficient gene transfer of an
expression library into monolayer murine ES cells carrying a
cell fate-specific fluorescent reporter. A functional screening
scheme was designed to identify genes which, when overex-
pressed, increase the proportion of neurons in a culture after 4
days of differentiation (Fig. 1). Unique cDNAs from an arrayed
expression library were combined into pools that were used to
transfect low-density cultures of ES cells carrying a fluorescent
marker (enhanced green fluorescent protein [eGFP]) under the
control of the T?1 early neuronal promoter. Following differ-
entiation, cultures were dissociated, and the frequency of eGFP-
positive cells was analyzed by flow cytometry. This method
allows efficient high throughput identification of genes that
promote cell differentiation to specific fates.
MATERIALS AND METHODS
Culture Conditions and Creation of Reporter
E14 ES cells were maintained without feeder cells, on 0.1% gelatin
(Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) coated
Correspondence: Jonas Frise ´n, M.D., Ph.D., Karolinska Institute, Cell and Developmental Biology, Box 285, Stockholm 17177, Sweden.
Telephone: ?46-8-52487562; Fax: ?46-8-324927; e-mail: firstname.lastname@example.org
12, 2007; first published online in STEM CELLS EXPRESS March 22, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/
Received August 1, 2006; accepted for publication March
EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES
plates (Costar, Lowell, MA, http:www.corning.com), in Glasgow
Minimum Essential Medium (Sigma) supplemented with 5% fetal
bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.
com), 5% KnockOut Serum Replacement (KSR; Gibco, Grand
Island, NY, http://www.invitrogen.com), 2 mM L-glutamine (In-
vitrogen, Carlsbad, CA, http://www.invitrogen.com), 0.1 mM non-
essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitro-
gen), 0.1 mM 2-mercaptoethanol (Sigma), and 1,000 units of
leukemia inhibitory factor (LIF; Chemicon, Temecula, CA, http://
www.chemicon.com). For minimal differentiation, the ES cells
were cultured in a 50/50 medium of Dulbecco’s modified Eagle’s
medium/F12 and neurobasal complemented with N2, B27 (contain-
ing transferrin, selenium, hormones, and vitamins important for
neural cell survival  [Invitrogen]), 20 ?g/ml human insulin
(Invitrogen), and 150 ?g/ml bovine serum albumin (Sigma). T?1-
eGFP ES cells were established with reporter construct T?1-eGFP
(kindly given by Oliver Bru ¨stle, Institute of Reconstructive Neuro-
biology, Life and Brain Center, University of Bonn and Hertie
Foundation, Bonn, Germany). T?1 ?-tubulin expression in early
neurons has been well documented , and the 1.1 kilobases (kb) of
the 5? flanking region direct expression of eGFP in developing
neurons. Following differentiation of picked ES cell colonies in
N2/B27 medium, cells were analyzed for eGFP fluorescence and
immunoreactivity against Nestin (Developmental Studies Hybrid-
oma Bank, Iowa City, IA, http://www.uiowa.edu/?dshbwww),
?III-tubulin (Berkeley Antibody, Princeton, NJ, http://www.crpinc.
com), tyrosine hydroxylase and peripherin (both Chemicon), glial
fibrillary acidic protein (GFAP) (Sigma), O4 (Chemicon), and
Oct-4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://
Transfection of ES Cells
ES cells were plated on gelatinized 6-well plates (Costar) at a
density of 1.75 ? 105cells per well. Twenty hours later, cells were
washed with Opti-MEM (Invitrogen) and transfected with a mixture
of 5 ?g of DNA and 10 ?l of Lipofectamine 2000 (Invitrogen)
diluted in a total volume of 1,000 ?l of Opti-MEM per well. The
transfection was allowed to continue for 4 hours before the trans-
fection mixture was removed, and ES cell medium containing 10%
FBS LIF KSR was added to the cells. Sixteen hours later, cells were
changed to N2/B27 medium, and medium was changed every day.
Cells were usually differentiated for 4 days in the N2/B27 medium
before flow analysis was performed on a BD Aria (BD Biosciences,
San Diego, http://www.bdbiosciences.com). The expression of the
transfected genes was controlled by either of the general promoters
of cytomegalovirus (CMV) or CMV-enhanced ?-actin promoter.
pEGFP-N1 (BD Biosciences), which contains the same promoter
(CMV) and termination signals as library cDNAs, was used to
analyze transfection efficiency. All cell fate-inducing activity of the
introduced cDNA was compared with the lineage choice of CMV-
?-galactosidase transfected cells.
IRAV MGC Mouse Verified Full-Length Ampicillin
Plates 1–84 were acquired from Geneservice (Cambridge, U.K., http://
www.geneservice.co.uk) and maintained as glycerol stocks in ?80°C.
For pooling, a multichannel pipettor was used to scrape and transfer
Escherichia coli to 2 ml deep-well 96-well plates containing 1.5 ml of
Luria-Bertani medium and carbenicillin. Plates were grown shaking for
20–24 hours at 37 degrees, and 32 wells were pooled for DNA
extraction. DNA was isolated using the PureYield Endotoxin Free
Midiprep System according to manufacturer’s directions (Promega,
Madison, WI, http://www.promega.com) and analyzed by spectropho-
tometry for purity.
Fluorescence-Activated Cell Sorting Analysis of
Cells were trypsinized after 4 days in minimal medium, and eGFP-
positive cells were detected by flow cytometry using BD FACS-
Calibur or FACSAria. Gates for the positive population were es-
tablished by placing square gates directly above the singlet
population (based on side-scatter width) defined as negative in
?-galactosidase control transfections. Listed genes were confirmed
by cycle sequencing. One clone, 58H05, was found by BLAST
analysis to be oxysterol binding protein 6, not RIKEN cDNA
2810475A17 gene, as seen in supplemental online data 2. Statistical
analyses were performed with GraphPad Prism and StatsDirect
using one-way analysis of variance and Dunnett’s post-test.
Polymerase Chain Reaction
Following 0–5 days in N2/B27 minimal differentiation medium, ES
cells were lysed in TRIzol (Invitrogen), and total RNA was prepared
according to directions. Random primed cDNA was synthesized
with SuperScript II (Invitrogen). Intron-spaced primer sequences
are available upon request.
Monolayer Culture Is Permissive for Diverse
To establish a screening procedure for genes affecting ES cell fate
rayed cDNAs were pooled into groups of 32 (A, B,
C) and transfected in duplicate into low-density ES
monolayer cultures containing a cell-specific pro-
moter driving a fluorescent reporter (T?1-eGFP).
After 4 days in minimal medium, cells were
trypsinized and analyzed by fluorescence-activated
cell sorting and compared with cultures plated at
the same time. High-scoring pools were retrans-
fected for verification and reduced for individual
clone analysis. See text for details. Abbreviations:
eGFP, enhanced green fluorescent protein; ES, em-
bryonic stem; FSC, forward scatter.
Functional screening schematic. Ar-
Identification of Genes Directing ES Cell Lineage
cells under minimally instructive conditions and differentiated low-
density cultures on a gelatin-coated surface in medium lacking LIF
and serum but in the presence of B27, N2, and insulin [8, 10].
Initially, all cells expressed Oct-4 (Fig. 2A), a marker of pluripo-
tency . ES cells cultured in defined medium at low density in
an adherent monolayer culture readily adopt neural characteristics
[10, 12–14]. After 2 days, Nestin-immunoreactive neural precur-
sors were found widely distributed throughout the culture (Fig.
2A). Over the course of 4 days, a small percentage (1%–2%) of the
ES cells differentiated to ?III-tubulin, a specific marker of neurons.
A subset of these neurons was immunoreactive to markers associ-
ated with mature cells, such as tyrosine hydroxylase, a marker of
dopaminergic neurons (Fig. 2A).
We assessed by reverse transcription-polymerase chain re-
action the expression of markers of the three different germ
layers over 5 days in the minimal differentiation medium (Fig.
2B). Otx2, a marker of anterior neurectoderm, increased with
time in culture. Brachyury, a mesoderm-specific marker, also
increased over 5 days, whereas ?-fetoprotein, a marker of vis-
ceral endoderm , was undetectable. These results show that
monolayer differentiation produces heterogeneous cell types
over the course of a few days.
In order to better define the composition of monolayer
cultures, we examined Oct-4 and ?III-tubulin localization by
immunohistochemistry. Examination of cultures over 4 days
showed accumulation of Oct-4-immunoreactive cells near the
center of ES cell clusters, whereas neurons appeared near the
periphery (Fig. 2C). The reduction of Oct-4-positive cells was
asynchronous in the culture and, following 4 days in differen-
tiation medium, there were still clusters of cells stained with
Oct-4 (Fig. 2C). Cells immunoreactive to antibodies directed
against peripherin (Fig. 2C), 5-hydroxytryptamine (serotonin),
and GABA (data not shown) were also observed near the border
of cell clusters, indicating diverse and mature neuronal cell
fates. Continued culture of the ES cells in the minimal differ-
entiation medium for up to 14 days favored the formation of
cells immunopositive for the astroglial cell marker GFAP and
the oligodendrocyte marker O4 (Fig. 2C).
Next, we examined if minimal conditions promoted neuroec-
monolayer cultures differentiated for 10 days, we detected beating
cells, indicating the presence of cardiomyocytes. Differentiation of
an ES cell line containing a GFP insertion in the Nkx2.5 locus, a
transcription factor specifically expressed in developing cardiomy-
ocytes , confirmed the presence of nascent heart tissue in the
beating cells (Fig. 2C and supplemental online data 1). These
results show that, whereas diverse neural markers are present under
minimal culture conditions, mesodermal fates are also possible.
Thus, differentiation of monolayer cultures under minimal condi-
Efficient Gene Delivery to ES Cells
Heterogeneous culture maturation over several days provides an
opportunity to influence several different steps in the differen-
tiation process from ES cells to mature, post-mitotic cells. In
order to examine if a particular cell fate could be promoted by
overexpression of genes, we procured a Mouse Genome Com-
mittee rearrayed IRAV library of approximately 8,000 full-
length mouse cDNAs in an expression vector under the general
CMV promoter. The prospect of introducing a library of full-
length cDNAs into ES cells confronted us with the requirement
for an efficient and easy gene delivery method. Using a modi-
fied liposome-based transfection procedure (Materials and
Methods), we were able to reliably achieve greater than 80%
transfection efficiency of monolayer cultures as detected by
flow cytometry for CMV-eGFP expression (Fig. 3A, 3B). After
4 days, eGFP expression decreased to approximately 40% of all
cells and decreased dramatically thereafter (Fig. 3A, 3B), con-
sistent with a loss of transient plasmids. This high rate of
transfection suggests that differentiating cells will be accessible
over the course of at least 4 days to exogenously expressed
genes from plasmid DNA that may influence their fate.
Measuring the differentiative capacity of each gene individ-
ually is laborious; therefore, we sought to refine our method to
include pooled plasmids at a frequency that still allows for the
detection of the effect of individual genes. Dilutions (1:100,
Figure 2. Immunocytochemical analysis of
low-density embryonic stem (ES) cells in
minimal differentiation medium. (A): Over 4
days, ES cell monolayers differentiated to
Nestin-positive neuronal precursors and then
to ?III-tubulin-immunoreactive neurons and
neuronal subtypes (tyrosine hydroxylase).
(B): Reverse transcription-polymerase chain
reaction of fate-specific genes showed the
lineage distribution of ES cell monolayers in
differentiation medium for 5 days. (C):
Monolayer cultures at 0, 4, and 10 days of
differentiation. The presence of Oct-4 immu-
noreactive cells decreased over time, but they
were present at the same time as differentiat-
ing neurons at 4 days. Differentiation to pe-
ripheral nerve cells, astrocytes, and oligoden-
drocytes in minimal differentiation medium
was visualized at 4 and 10 days by antibodies
directed against the markers peripherin,
GFAP, and O4, respectively. Differentiation
of ES cells containing a green fluorescent
protein insertion into the Nkx2.5 locus dis-
played fluorescence in beating cells. Cell nu-
clei were stained with DAPI. Abbreviations:
AFP, ?-fetoprotein; BF, bright field; Bra,
brachyury; DAPI, 4,6-diamidino-2-phenylin-
dole; eGFP, enhanced green fluorescent pro-
tein; GFAP, glial fibrillary acidic protein;
?RT, without reverse transcriptase.
Falk, Karlsson, Kurdija et al.
1:50, 1:32, 1:8) of the CMV-eGFP plasmid among other plas-
mids resulted in a preferable pool size of 32 full-length cDNAs,
based on observed eGFP expression. Using this dilution, we
were able to demonstrate that a single gene of a transfected
pool will be expressed by approximately 70% of cells, as indi-
cated by CMV-eGFP expression (Fig. 3C). The fluorescence
intensity was decreased for individual cells; however, the ma-
jority showed hundredfold higher expression than background
(Fig. 3C). This indicates that many genes per pool are likely to
be expressed in the same cell. Although most genes are not
expected to contribute directly to signaling pathways that influ-
ence cell fate in vitro, these results show that the expression of
a single gene in a pool is abundant in post-transfected ES cell
cultures, making it possible to assay its effect on lineage choice.
Neuronal-Specific ES Reporter Cell Line
In order to more readily detect specifically differentiated cell types,
we created an ES cell line containing a cell fate-specific promoter
driving the expression of a fluorescent reporter. This allows for a
live cell analysis and isolation of differentiated cells in a quantita-
tive manner by flow cytometry. For assaying neuronal differentia-
tion, we created an ES cell line expressing eGFP under the control
of the early neuronal promoter T?1-tubulin [9, 17]. Neuron-spe-
cific eGFP expression was validated by antibodies directed against
another neuronal marker, ?III-tubulin, in differentiated cells (Fig.
4A). Despite the apparent heterogeneity of differentiating mono-
layer cultures, the reporter cell line displayed a consistent fre-
quency of neurons following 4 days in differentiation monolayer
cultures, indicating that neurogenesis occurs similarly among cul-
tures plated at the same time. This reporter ES cell line therefore
provided us a simple tool to evaluate lineage choice in differenti-
ated living ES cells.
To test the ability of a single gene to promote lineage choice
in mouse ES cells, we introduced Mash1 into the T?1-eGFP ES
cell line by transient transfection. After 4 days of monolayer
differentiation, cells were trypsinized, and eGFP expression in
living cells was monitored by flow cytometry. A substantial
increase in neuron numbers was observed, from 1.3%–3.0% of
the total cells in cultures overexpressing Mash1 compared with
controls (Fig. 4B, 4C). To directly test if Mash1 was effective in
larger pools, we compared the effect of Mash1 on monolayer
neurogenesis at a dilution factor of 1:32 total CMV-containing
plasmid DNA transfected. Under these conditions, 1:32 Mash1
approximately doubled the number of neurons in the differen-
tiating ES cell cultures compared with the control, indicating
that reducing the 1:32 gene ratio further would diminish the
sensitivity of detection (Fig. 4B, 4C). Marker gene expression
confirmed ?III-tubulin immunoreactivity in eGFP positively-
sorted cells following flow cytometry (Fig. 4C). These results
indicate that a potent differentiation-promoting effect of a single
gene is sufficiently visible in a larger pool of other genes.
An Efficient Screen for Neuronal Inducers
Based on the above results, a schema was devised to test for
genes that could, upon overexpression, increase the proportion
Figure 3. Robust expression in transiently transfected embryonic stem
(ES) cells. (A): Expression of a cytomegalovirus (CMV)-eGFP plasmid
decreases over time, as revealed by reverse transcription-polymerase
chain reaction. (B): Flow cytometry results of average eGFP-positive
cells per transfected pool (n ? 3, ? SD). (C): Fluorescence-activated
cell sorting analysis of forward scatter versus eGFP of live ES cells
transiently transfected with a control CMV-?-galactosidase plasmid,
CMV-eGFP expression plasmid alone, or CMV-eGFP as a 1:32 fraction
of a pool of DNA with CMV-?-galactosidase (n ? 3, ? SD). Percent
eGFP expressing cells as well as intensity are indicated for transfected
ES cells. Abbreviations: ?gal, ?-galactosidase; eGFP, enhanced green
fluorescent protein; FSC, forward scatter.
Figure 4. Embryonic stem reporter cell line and neuronal differentia-
tion by Mash1 transfection. (A): Expression of eGFP under the T?1
promoter (T?1-eGFP) in differentiated cells is coincident with immu-
noreactivity to the ?III-tubulin protein. Panel 3 is a merger of panels 1,
2, and DAPI nuclear stain. (B): Transfection of a Mash1 expression
plasmid into the T?1-eGFP line significantly increases the percentage of
eGFP-expressing cells compared with control ?-galactosidase plasmid
when transfected singly (one-way analysis of variance, p ? .0004; n ?
2) or as pool diluted 1:32 with ?-galactosidase plasmid (p ? .0158; n ?
2) (? SD). (C): Fluorescence-activated cell sorting plots of (B) and
eGFP-positive and -negative sorted populations stained with antibodies
directed against ?III-tubulin and DAPI. Abbreviations: ?gal, ?-galac-
tosidase; DAPI, 4,6-diamidino-2-phenylindole; eGFP, enhanced green
fluorescent protein; FSC, forward scatter.
Identification of Genes Directing ES Cell Lineage
of neurons after 4 days in monolayer culture (Fig. 1). Wells of
E. coli containing unique IRAV cDNAs were grown individu-
ally to confluency then combined into pools of 32, and plasmid
DNA was extracted. The pooled cDNA was then used to trans-
fect low-density cultures of T?1-eGFP ES cells in duplicate.
After 4 days in minimal medium, cultures were trypsinized, and
the frequency of eGFP-positive cells was analyzed by flow
In total, 252 pools comprising approximately 6,500 unique
Unigene clusters (gene size 240–6,584 base pairs) were ana-
lyzed, and eGFP-positive values were recorded using gates
established in Figure 4 (supplemental online data 2). Normal-
ization of percent eGFP-positive cells to an average of transfec-
tions performed at the same time gave an indexed value for
comparison of all pools (Fig. 5A, mean set to 1). In order to
measure the reliability of individual pools, a score was devised
based on the standard deviation/index average (Fig. 5A, lower
value is more reliable). Seven pools gave approximately 50%
more neurons than the indexed average and were selected for
retransfection along with 18 pools scoring highly in their trans-
fection group (in red, Fig. 5A). A subset of eight pools with at
least 40% more neurons upon retransfection compared with
?-galactosidase transfected controls was chosen for reduced
pool transfections (4 cDNAs per pool) and individual gene
Figure 5. Neuronal lineage choice by transfected embryonic stem cells. (A): eGFP distribution of 252 IRAV MGC mouse verified full-length ampicillin
cDNA pools. Duplicate pools of 32 genes were normalized to an average of transfections performed at the same time (T?1-eGFP/AVG, mean ? 1), and
pool reliability was measured by standard deviation/average. Pools selected for retransfection are shown in red. (B): Fifteen high-scoring individual genes
were transfected, and mean percent increase in eGFP over ?-galactosidase controls was recorded (n ? 3, ? SD). Three randomly selected negative controls
transfected separately and represented by plate location are also shown (§). Four genes showed statistically significant differences, represented with ??
(analysis of variance, p ? .0029). A representative experiment is shown. References refer to a role in neuronal development or neuroprotection. ‡ Link to
Zellweger syndrome. Abbreviations: ?gal, ?-galactosidase; AVG, average; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; STD,
Falk, Karlsson, Kurdija et al.
transfections into T?1-eGFP ES cells. From these, 15 genes
were chosen for single gene transfections in triplicate, resulting
in 28%–97% greater eGFP-positive cells compared with ?-ga-
lactosidase controls (Fig. 5B). In contrast, three genes chosen
from the library at random failed to increase the percentage.
Eight of the fifteen genes have been implicated in nervous
system development or maintenance, suggesting they are bona
fide neural effectors.
In this study, we describe a sensitive method to uncover devel-
opmentally regulated inducers of cell fate choice and therapeu-
tically relevant genes whose transient application yields in-
creases in specific cell types. Monolayer differentiation presents
several unique advantages in discovering cDNAs that are able to
influence one or more steps of differentiation, from ES to
mature postmitotic cell. Under minimal differentiation condi-
tions, monolayers are highly transfectable, differentiate repro-
ducibly to diverse cell types, and only transiently alter cellular
DNA content. Culturing ES cells at a low density during dif-
ferentiation likely increases the effect of the introduced genes,
as it limits instructive signals from other cells in close contact.
This allows for subtle increases in eGFP positive cells to be
detected from several steps in a lineage. For example, cells near
the center of ES clusters may be susceptible to genes promoting
neural precursors, whereas neural precursors that form early at
cluster peripheries may be responsive to those directing neuro-
nal fates. Altogether, an overexpressed gene that promotes a
particular lineage is likely to result in a distinct increase over
background differentiation levels. The detection method we
employed, flow cytometry, sensitively detects even slight dif-
ferences among samples. Experimental variability in neuron
number was further limited by transfecting library pools in
duplicate and individual genes in triplicate.
In this report we have used an arrayed library of full-length
cDNAs and a cell fate-specific promoter for sensitive detection of
neurons. However, this method is also applicable for cells derived
from nonectodermal cell types. Permissive medium conditions
allow for the production of complex or developmentally late cell
types such as cardiomyocytes, peripheral nervous system cells, and
glia (Fig. 2C). Previously, these fates have been predominantly
attainable through the usage of embryoid bodies. Endoderm is
rarely formed from ES cells, consistent with our culture results, but
conditions are favorable for detecting endoderm formation, and the
screen could easily be adapted for the detection of genes inducing
endodermal derivatives. In order to examine several distinct fates,
combining additional spectrally separable promoter-fluorescent
markers to the same cell line would further allow for simultaneous
transfection and analysis.
The results of our analysis implicate several genes in a
neuroprotective capacity, an unsurprising discovery since apo-
ptosis is an important regulatory mechanism of neuronal num-
bers during development, and minimal differentiation medium
lacks important vitamins and growth factors. Whereas genes
such as the fragile X mental retardation gene 1, ring finger
protein 130, and nuclease sensitive element binding protein 1
(Ybx1) have established roles during neural development [18–
22, 27, 28], Ybx1 also plays a role in stress response ,
indicating both functions may contribute to its repeated discov-
ery in high scoring pools from separate plates (data not shown).
Mice mutant for heat shock factor 2 display abnormally large
ventricles and reduced areas of neurogenesis [30, 31], suggest-
ing newly born neurons may be particularly sensitive to stresses
inherent to both development and in vitro culture. This hypoth-
esis is also supported in our analysis by the identification of
ferritin light chain 1, whose function has been closely tied to
neuroferritinopathy in humans .
One potential caveat to this study is the potential for plasmid
integration into the genome where endogenous gene expression
may be affected. An analysis of the frequency of integration events
per well found approximately 300–500 integrants per initial
175,000 cells transfected. Although these integration events may
contribute to the total number of neurons formed after 4 days, no
significant differences were found between positive and negative
samples tested (data not shown). We also cannot exclude a bias
toward integration in loci containing homologous sequences found
in library plasmids or in loci that affect neuronal differentiation;
however, the CMV-promoter is also present in the control ?-ga-
lactosidase vector and would be equally subject to silencing or
positional effects upon integration .
Genetic screens such as the one described here are often
biased for or against a class of molecules. For transfections,
larger plasmids have a decreased chance of entry into lipo-
plexes than smaller plasmids, making it less likely to dis-
cover those clones. Under screening conditions, an 8–10-fold
difference in transfection efficiency can be expected between
the largest and smallest plasmids (, data not shown).
However, analysis of library plasmids revealed that 88.6% of
the constructs in the library were below 7.5 kb, the transfec-
tion of which is approximately 50% less efficient than that of
the smallest plasmid (data not shown). Hence, a twofold
difference in transfection efficiency exists for the large ma-
jority of the genes transfected. This effect is partially miti-
gated by the inclusion of fewer genes per pool. Although a
bias exists against large insert plasmids, one of the final 15
isolated, flk1 kinase insert domain protein receptor, is 9,860
bases, showing that, despite this bias, large genes may be
isolated from the library under these conditions.
Gene overexpression studies can circumvent loss-of-
function complexities arising from gene families that share
common function [37, 38]. Although in some cases canceling
effects of expressing multiple genes may limit detection,
synergies are also possible and can be identified through a
clone repooling strategy. Interestingly, many pools resulted
in potent decreases in neuron number (Fig. 5A), suggesting
they promote formation of other tissues or processes that
antagonize neuron formation. These pools effectively double
the breadth of our analysis and are available along with other
untested high scoring pools to the scientific community (sup-
plemental online data 2).
Embryonic stem cell differentiation research holds promise
for cell therapies that depend on a source of large numbers of
specialized cell types. The technique described here ad-
dresses two significant problems: the establishment of an
efficient and transient gene delivery method to embryonic
stem cells and the discovery of genes contributing to lineage
choice. Although generation of large absolute numbers of a
mature cell type was not a goal of this study (but rather to use
conditions sensitive for the detection of neuronal inducers),
rich medium conditions and gene delivery to ES cells of
molecules identified here and in similar studies may signif-
icantly increase purification of specialized cell types for cell
Identification of Genes Directing ES Cell Lineage
ACKNOWLEDGMENTS Download full-text
We are grateful to V. Wirta and J. Lundeberg for procuring the
cDNA library, M. Toro and K. Hamrin for help with flow
cytometry, C. K. Hidaka and T. Morisaki for generously pro-
viding the Nkx2.5-GFP ES cell line, S. Nystro ¨m for generous
technical assistance, and O. Hermanson and A. Simon for crit-
ical reading of the manuscript. This project was supported by
grants from the Swedish Research Council, the Karolinska In-
stitute, the Swedish Cancer Society, the Tobias Foundation, the
Foundation for Strategic Research, KOSEF, and by the Euro-
pean Commission through the FP6 project STEMS (LHSB-CT-
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1 Wichterle H, Lieberam I, Porter JA et al. Directed differentiation of
embryonic stem cells into motor neurons. Cell 2002;110:385–397.
Tanabe Y, William C, Jessell TM. Specification of motor neuron identity
by the MNR2 homeodomain protein. Cell 1998;95:67–80.
Andersson E, Tryggvason U, Deng Q et al. Identification of intrinsic
determinants of midbrain dopamine neurons. Cell 2006;124:393–405.
Yan Y, Yang D, Zarnowska ED et al. Directed differentiation of dopa-
minergic neuronal subtypes from human embryonic stem cells. STEM
Ohyama K, Ellis P, Kimura S et al. Directed differentiation of neural
cells to hypothalamic dopaminergic neurons. Development 2005;132:
Grimes BR, Monaco ZL. Artificial and engineered chromosomes: De-
velopments and prospects for gene therapy. Chromosoma 2005;114:
Zwaka TP, Thomson JA. Homologous recombination in human embry-
onic stem cells. Nat Biotechnol 2003;21:319–321.
Johe KK, Hazel TG, Muller T et al. Single factors direct the differenti-
ation of stem cells from the fetal and adult central nervous system. Genes
Gloster A, Wu W, Speelman A et al. The T alpha 1 alpha-tubulin
promoter specifies gene expression as a function of neuronal growth and
regeneration in transgenic mice. J Neurosci 1994;14:7319–7330.
10 Ying QL, Stavridis M, Griffiths D et al. Conversion of embryonic stem
cells into neuroectodermal precursors in adherent monoculture. Nat
11 Scholer HR, Dressler GR, Balling R et al. Oct-4: A germline-specific
transcription factor mapping to the mouse t-complex. Embo J 1990;9:
12 Smukler SR, Runciman SB, Xu S et al. Embryonic stem cells assume a
primitive neural stem cell fate in the absence of extrinsic influences.
J Cell Biol 2006;172:79–90.
13 Ginis I, Luo Y, Miura T et al. Differences between human and mouse
embryonic stem cells. Dev Biol 2004;269:360–380.
14 Tropepe V, Hitoshi S, Sirard C et al. Direct neural fate specification from
embryonic stem cells: A primitive mammalian neural stem cell stage
acquired through a default mechanism. Neuron 2001;30:65–78.
15 Dziadek MA, Andrews GK. Tissue specificity of alpha-fetoprotein mes-
senger RNA expression during mouse embryogenesis. EMBO J 1983;2:
16 Hidaka K, Lee JK, Kim HS et al. Chamber-specific differentiation of
Nkx2.5-positive cardiac precursor cells from murine embryonic stem
cells. FASEB J 2003;17:740–742.
17 Schmandt T, Meents E, Gossrau G et al. High-purity lineage selection of
embryonic stem cell-derived neurons. Stem Cells Dev 2005;14:55–64.
18 Reiss AL, Abrams MT, Greenlaw R et al. Neurodevelopmental effects of
the FMR-1 full mutation in humans. Nat Med 1995;1:159–167.
19 Jacquemont S, Hagerman RJ, Leehey M et al. Fragile X premutation
tremor/ataxia syndrome: Molecular, clinical, and neuroimaging corre-
lates. Am J Hum Genet 2003;72:869–878.
20 Willemsen R, Hoogeveen-Westerveld M, Reis S et al. The FMR1 CGG
repeat mouse displays ubiquitin-positive intranuclear neuronal inclu-
sions; implications for the cerebellar tremor/ataxia syndrome. Hum Mol
21 Castren M, Tervonen T, Karkkainen V et al. Altered differentiation of
neural stem cells in fragile X syndrome. Proc Natl Acad Sci U S A
22 Greco CM, Berman RF, Martin RM et al. Neuropathology of fragile X-as-
sociated tremor/ataxia syndrome (FXTAS). Brain 2006;129:243–255.
23 Cao L, Jiao X, Zuzga DS et al. VEGF links hippocampal activity with
neurogenesis, learning and memory. Nat Genet 2004;36:827–835.
24 Zachary I. Neuroprotective role of vascular endothelial growth factor:
signalling mechanisms, biological function, and therapeutic potential.
25 Lowndes HE, Beiswanger CM, Philbert MA et al. Substrates for neural
metabolism of xenobiotics in adult and developing brain. Neurotoxicol-
26 Baumgart E, Vanhooren JC, Fransen M et al. Molecular characterization
of the human peroxisomal branched-chain acyl-CoA oxidase: cDNA
cloning, chromosomal assignment, tissue distribution, and evidence for
the absence of the protein in Zellweger syndrome. Proc Natl Acad Sci U
S A 1996;93:13748–13753.
27 Borchers AG, Hufton AL, Eldridge AG et al. The E3 ubiquitin ligase
GREUL1 anteriorizes ectoderm during Xenopus development. Dev Biol
28 Ohba H, Chiyoda T, Endo E et al. Sox21 is a repressor of neuronal
differentiation and is antagonized by YB-1. Neurosci Lett 2004;358:
29 Lu ZH, Books JT, Ley TJ. YB-1 is important for late-stage embryonic
development, optimal cellular stress responses, and the prevention of
premature senescence. Mol Cell Biol 2005;25:4625–4637.
30 Kallio M, Chang Y, Manuel M et al. Brain abnormalities, defective
meiotic chromosome synapsis and female subfertility in HSF2 null mice.
EMBO J 2002;21:2591–2601.
31 Wang G, Zhang J, Moskophidis D et al. Targeted disruption of the heat
shock transcription factor (hsf)-2 gene results in increased embryonic
lethality, neuronal defects, and reduced spermatogenesis. Genesis 2003;
32 Batulan Z, Shinder GA, Minotti S et al. High threshold for induction of
the stress response in motor neurons is associated with failure to activate
HSF1. J Neurosci 2003;23:5789–5798.
33 Curtis AR, Fey C, Morris CM et al. Mutation in the gene encoding
ferritin light polypeptide causes dominant adult-onset basal ganglia dis-
ease. Nat Genet 2001;28:350–354.
34 Vidal R, Ghetti B, Takao M et al. Intracellular ferritin accumulation in
neural and extraneural tissue characterizes a neurodegenerative disease
associated with a mutation in the ferritin light polypeptide gene. J Neu-
ropathol Exp Neurol 2004;63:363–380.
35 Teschendorf C, Warrington KH, Jr., Siemann DW et al. Comparison of
the EF-1 alpha and the CMV promoter for engineering stable tumor cell
lines using recombinant adeno-associated virus. Anticancer Res 2002;
36 Kreiss P, Cameron B, Rangara R et al. Plasmid DNA size does not affect
the physicochemical properties of lipoplexes but modulates gene transfer
efficiency. Nucleic Acids Res 1999;27:3792–3798.
37 Grammer TC, Liu KJ, Mariani FV et al. Use of large-scale expression
cloning screens in the Xenopus laevis tadpole to identify gene function.
Dev Biol 2000;228:197–210.
38 Heidersbach A, Gaspar-Maia A, McManus MT et al. RNA interference
in embryonic stem cells and the prospects for future therapies. Gene Ther
See www.StemCells.com for supplemental material available online.
Falk, Karlsson, Kurdija et al.