Vol 460 | 27 August 2009 | doi:10.1038/nature08248
LETTERS
L1 retrotransposition in human neural progenitor cells
Nicole G. Coufal1, José L. Garcia-Perez2,3, Grace E. Peng1, Gene W. Yeo1{, Yangling Mu1, Michael T. Lovci1{,
Maria Morell4, K. Sue O’Shea4, John V. Moran2,5 & Fred H. Gage1
Long interspersed element 1 (LINE-1 or L1) retrotransposons have
markedly affected the human genome. L1s must retrotranspose in
the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic
cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus
progenitor cells in vitro and in the mouse brain in vivo1. Here we
demonstrate that neural progenitor cells isolated from human
fetal brain and derived from human embryonic stem cells support
the retrotransposition of engineered human L1s in vitro.
Furthermore, we developed a quantitative multiplex polymerase
chain reaction that detected an increase in the copy number of
endogenous L1s in the hippocampus, and in several regions of
adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor.
These data suggest that de novo L1 retrotransposition events may
occur in the human brain and, in principle, have the potential to
contribute to individual somatic mosaicism.
The human nervous system is complex, containing approximately
1015 synapses with a vast diversity of neuronal cell types and connections that are influenced by complex and incompletely understood
environmental and genetic factors2. Neural progenitor cells (NPCs)
give rise to the three main lineages of the nervous system: neurons,
astrocytes and oligodendrocytes. To determine whether human NPCs
can support L1 retrotransposition, we transfected human fetal brain
stem cells (hCNS-SCns) (Fig. 1a)3 with an expression construct containing a retrotransposition-competent human L1 (RC-L1) driven
from its native promoter (L1RP). The RC-L1 also contains a retrotransposition indicator cassette in its 39 untranslated region (UTR),
consisting of a reversed copy of the enhanced green fluorescent protein
(EGFP) expression cassette, which is interrupted by an intron in the
same transcriptional orientation as the RC-L1 (refs 4–7). The orientation of the cassette ensures that EGFP-positive cells will only arise if the
RC-L1 undergoes retrotransposition (Supplementary Fig. 1a).
A low level of L1RP retrotransposition, averaging 8–12 events per
100,000 cells, was observed in three different human fetal brain stem
cell lines (BR1, BR3 and BR4; Fig. 1d). By comparison, an L1 containing two missense mutations in the open reading frame 1 (ORF1)encoded protein (JM111/L1RP)5,7 did not retrotranspose (Fig. 1b, d).
Controls demonstrated precise splicing of the intron from the retrotransposed EGFP gene (Fig. 1b and Supplementary Figs 1 and 4), and
indicated that L1 retrotransposition events were detectable by both
PCR and Southern blotting 3 months after transfection (Fig. 1c).
Moreover, reverse transcriptase PCR (RT–PCR) revealed that
hCNS-SCns express endogenous L1 transcripts and that some transcripts are derived from the human-specific (L1Hs) subfamily4,9,10
(Supplementary Fig. 6a, b and Supplementary Tables 4 and 5).
To determine whether L1 retrotransposition occurred in undifferentiated cells, we conducted immunocytochemical localization of
cell-type-restricted markers in EGFP-positive hCNS-SCns. These
cells expressed neural stem cell markers, including SOX2, Nestin,
Musashi-1 and SOX1 (Fig. 1e and Supplementary Fig. 2a, b), and
some co-labelled with Ki-67, indicating that they continued to proliferate (Supplementary Fig. 2c). EGFP-positive hCNS-SCns could
also be differentiated to cells of both the neuronal and the glial
lineages (Fig. 1f, g). Notably, L1RP did not retrotranspose using our
experimental conditions in primary human astrocytes or fibroblasts,
although a low level of endogenous L1 expression was detected in
both cell types (Fig. 1d and Supplementary Figs 2d, e and 6a, b).
We next used two different protocols to derive NPCs from five
human embryonic stem cell lines (hESCs; Fig. 2a). As in our previous
study1, NPC differentiation led to a ,25-fold increase in L1 promoter
activity over a 2-day period, and then a decline (Fig. 2c); there was also
a ,250-fold increase in synapsin promoter activity during differentiation (Supplementary Fig. 4b). H13B-derived NPCs expressed both
endogenous L1 RNA and ORF1 protein8, although the level of ORF1
protein expression was less than in the parental H13B hESC lines
(Fig. 2d). HUES6-derived NPCs also expressed endogenous L1 RNA
(Supplementary Fig. 6a, b), and sequencing indicated that some transcripts are derived from the L1Hs subfamily (Supplementary Tables 4
and 5). Similar studies performed with fetal brain, liver, and skin
samples showed evidence of endogenous L1 transcription
(Supplementary Fig. 6c, d and Supplementary Tables 4 and 5).
RC-L1 retrotransposition was readily detected at varying efficiencies
in hESC-derived NPC lines (Supplementary Table 1 and Supplementary Figs 1 and 4f, g). Again, we determined that JM111/L1RP could
not retrotranspose (Supplementary Table 1), that EGFP-positive
NPCs expressed canonical neural stem cell markers (Fig. 2b, e and
Supplementary Fig. 3c, d), and that EGFP-positive HUES6-derived
NPCs could be differentiated to cells of both the neuronal and
glial lineages (Fig. 2f and Supplementary Fig. 3e, f). The variability in
retrotransposition efficiencies in hESC-derived NPCs probably
depended on several factors (see Supplementary Table 1 for specific
details).
Characterization of EGFP-positive neurons showed that some
expressed subtype-specific markers (tyrosine hydroxylase (Fig. 2g)
and GABA (c-aminobutyric acid; data not shown)) and whole-cell
perforated patch-clamp recording demonstrated that some HUES6derived NPCs are functional (Fig. 2h–k; n 5 4 cells). Furthermore, we
demonstrated that an RC-L1 tagged with neomycin or blasticidin
retrotransposition indicator cassettes could retrotranspose in NPCs
(Supplementary Figs 1 and 4c–e)5,11. Some G418-resistant foci also
expressed SOX3 and could be differentiated to a neuronal lineage
(Fig. 2b).
1
Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 2Departments of Human Genetics and Internal
Medicine, 1241 East Catherine Street, University of Michigan Medical School, Ann Arbor, Michigan 48109-5618, USA. 3Andalusian Stem Cell Bank, Center for Biomedical Research,
Avda Conocimiento s/n, University of Granada, 18100, Spain. 4Department of Cell and Developmental Biology, 109 Zina Pitcher, University of Michigan Medical School, Ann Arbor,
Michigan 48109-2200, USA. 5Howard Hughes Medical Institute, Chevy Chase, Maryland 20815-6789, USA. {Present address: Stem Cell Program, Department of Cellular and
Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-5004, USA.
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©2009 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 460 | 27 August 2009
Neurosphere
a
Analysis, day 2–25
b
1 2 3 4 5 6 7 8 9
Dissociate
1,243 bp
c
RP
+FGF2
+EGF
+LIF
Ct
rl
FACS sort
342 bp
50
ng
L1
Fetus
Green cell
+ NGN
retrovirus
– Growth factors
Transfect with
L1 pCEP plasmid
3
Neurons
2,547 bp
2
1,645 bp
1.6
CD133+
CD45–
5E12+ CD34–
GFP events per 100,000
d
f
e
12
10
BR3 L1RP
8
g
GFP
Nestin
SOX2
GFP
MAP2
βIII tubulin
GFP
GFAP
βIII tubulin
DAPI
6
BR4 L1RP
4
2
0
JM111
12
15
18
21
Time (days)
25
BR1 L1RP
Fibro L1RP
Astro L1RP
DAPI
Figure 1 | L1 retrotransposition in hCNS-SCns. a, Experimental rationale.
b, PCR of genomic DNA. The 1,243-bp product contains the intron, the 342bp product indicates intron loss and retrotransposition. Lane 1, standards;
lane 2, hCNS-SCns transfected with JM111/L1RP; lanes 3–5, three human
fetal brain stem cell lines transfected with L1RP; lanes 6–7, primary astrocytes
and fibroblasts transfected with L1RP; lane 8, positive control; lane 9, water.
c, Southern blot of hCNS-SCns (line FBR-BR3). The 2,547-bp band
represents plasmid, the 1,645-bp band is diagnostic for genomic insertion.
Ctrl, control. d, Time course of L1 retrotransposition. Astro, astrocytes;
fibro, fibroblasts. e, EGFP-positive cells express Nestin and SOX2. Arrows
indicate co-labelled cell body, arrowheads indicate co-labelled processes.
f, EGFP-positive cells can differentiate to neurons (bIII tubulin and MAP2ab
positive). g, EGFP-positive cells can differentiate into glia (GFAP-positive,
bIII-tubulin-negative). Scale bars, 25 mm.
We next characterized 19 retrotransposition events from EGFPpositive NPCs (Supplementary Fig. 7b and Supplementary Table 2).
Comparison of the pre- and post-integration sites demonstrated that
retrotransposition occurred into an actual or inferred L1 endonuclease consensus cleavage site (59-TTTT/A and derivatives). Five
of eight fully characterized events were flanked by target site duplications, and no large deletions were detected at the insertion site5,9,12
(Supplementary Fig. 7b and Supplementary Table 2). Interestingly,
16 out of 19 retrotransposition events were fewer than 100 kilobases
(kb) from a gene and some occurred in the vicinity of a neuronally
expressed gene1,12,13.
Notably, we consistently observed higher L1 retrotransposition
efficiencies in hESC-derived NPCs when compared to fetal NPCs.
A Euclidian distance map on the basis of exon-array expression analysis14 indicated that hCNS-SCns cluster closer to HUES6 cells,
whereas HUES6-derived NPCs cluster closer to fetal brain
(Supplementary Fig. 11a). Thus, hESC-derived NPCs and hCNSSCns may represent different developmental stages in progenitor
differentiation. That being stated, we conclude that engineered
human L1s can retrotranspose in human NPCs.
Several studies have reported an inverse correlation between L1
expression and the methylation status of the CpG island in their
59 UTRs15,16. Thus, we performed bisulphite conversion analyses on
genomic DNAs derived from matched brain and skin tissue samples
from two 80–82-day-old fetuses (Fig. 3a, one male/one female sample).
We then amplified a portion of the L1 59 UTR containing 20 CpG sites
and sequenced the resultant amplicons. Notably, the L1 59 UTR
exhibited significantly less methylation in both brain samples when
compared to the matched skin sample (two-sample Kolmogorov–
Smirnov test, P # 0.0079 day 80 female, P # 0.0034 day 82 male;
Fig. 3b). The analysis of individual L1 59 UTR sequences demonstrated
the greatest variation between the brain and skin at CpG residues
located near the 39 end of the amplicon, and six amplicons from the
brain samples were unmethylated (Fig. 3e and Supplementary Fig. 8a,
b). Restricting this analysis to ten L1s from brain and skin with the
highest sequence homology to an RC-L1 showed that 19 out of 20
sequences were derived from the L1Hs subfamily (data not shown),
and that one L1Hs element from the brain was completely unmethylated (Fig. 3c). In all cases, control experiments showed that the
bisulphite conversion efficiency was .90% (Supplementary Fig. 8c).
Previous data indicated that SOX2 and MECP2 could associate
with the L1 promoter and repress L1 transcription under some
experimental conditions1,17. Two putative SRY-binding sites are
located in the L1 59 UTR immediately 39 to the CpG island (Fig. 3a
and Supplementary Fig. 11b)18. Thus, we performed chromatin
immunoprecipitation (ChIP) for SOX2 and MECP2 in hCNSSCns, HUES6-derived NPCs, and HUES6-derived neurons. SOX2
associated with the L1 59 UTR in a pattern that correlates with the
decrease in SOX2 expression observed during neural differentiation
(Fig. 3d and Supplementary Fig. 4h). MECP2 expression was lower in
both hCNS-SCns and HUES6-derived NPCs than in neurons
(Supplementary Fig. 4h), and both hCNS-SCns and HUES6-derived
NPCs expressed similar levels and types of L1 transcripts (Supplementary Fig. 6a, b). However, higher levels of MECP2 were
detected in association with the L1 promoter in hCNS-SCns than
in HUES6-derived NPCs (Fig. 3d). We propose that less L1 promoter
methylation in the developing brain may correlate with increased L1
transcription and perhaps L1 retrotransposition, and the differential
interaction of SOX2 and MECP2 with L1 regulatory sequences may
modulate L1 activity in different neuronal cell types.
Although NPCs are useful to monitor L1 activity, they only allow
monitoring of a single L1 expressed from a privileged context. By
comparison, the average human genome contains ,80–100 active
L1s, the expression of which may be affected by chromatin structure4.
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©2009 Macmillan Publishers Limited. All rights reserved
LETTERS
c
d
60.4
47.2
EB
BF
Luciferase activity
25
L1-GFP
15
24.9
SOX1
47.2
35.1
0
1
2
7
12
Differentiation (days)
47.2
35.1
e
+FGF2
AntiORF1
RNPs
AntiS6
AntiSOX3
Nestin
2,000
1,000
SOX2
–1,000
*
200 ms
j
g
f
Analysis,
day 2–25
3,000
WCLs
AntiSOX1
SOX3
βIII tubulin
Hoescht
Neural
progenitor cell
200 ms
i
0
GFP
Transfect with
L1.3 pCEP
plasmid
35.1
35.1
5
0
Neural rosette
L1 5 UTRluciferase
h
pA
b
ESC
GFP
TH
βIII tubulin
+ Differentiation
factors,
BDNF, GDNF
DAPI
80
60
40
20
0
–20
–40
–60
–80
mV
a
H
13
B
H
7
N
H SC
13 s
B
NATURE | Vol 460 | 27 August 2009
20 mV
k
1s
FACS
analysis
βIII tubulin
Neurons
GFP
DAPI
Figure 2 | L1 retrotransposition in hESC-derived NPCs. a, Experimental
rationale. BDNF, brain-derived neurotrophic factor; EB, embryoid body;
GDNF, glial-cell-derived neurotrophic factor. b, L1 retrotransposition in
H13B (top, LRE3 with EGFP reporter) and H7 (bottom, LRE3 with neomycin
reporter)-derived NPCs (BF, bright field). G418-resistant foci can express
progenitor (SOX3) and neuronal (bIII tubulin) markers. c, L1 59 UTR is
induced upon differentiation. Error bars denote s.d. d, H13B-derived NPCs
express endogenous ORF1 protein. RNP, ribonucleoprotein particle samples;
WCL, whole cell lysates. Lane markers denote kDa. e–g, EGFP-positive,
HUES6-derived NPCs express SOX2 and Nestin and can differentiate to be
tyrosine hydroxylase (TH) positive. Arrows indicate cell soma co-localization,
arrowheads indicate co-labelled processes. Scale bars, 25 mm. h, An LRE3EGFP positive neuron. Scale bar, 10 mm. i–k, Data are derived from the neuron
in h. i, Transient Na1 (asterisk) and sustained K1 (arrow) currents in response
to voltage step depolarizations. j, Suprathreshold responses to somatic current
injections. k, Spontaneous action potentials (Vm 5 250 mV).
Therefore, we developed a quantitative multiplexing PCR strategy to
investigate endogenous L1 activity in the human brain, proposing
that active retrotransposition would result in increased L1 content in
brain genomic DNA as compared to other tissues (Fig. 4a).
In brief, we designed Taqman probes against a conserved 39 region
of ORF2 (conjugated with the VIC fluorophore), as well as several
control probes (conjugated with the 6FAM fluorophore). Controls
were designed against the L1 59 UTR and other non-mobile DNA
sequences in the genome that have copy numbers that are higher
(such as a satellite19) or lower (such as HERVH and the 5S ribosomal
DNA (rDNA) gene) than ORF2. Furthermore, because most L1
retrotransposition events are 59 truncated9,20,21, we reasoned that
the L1 59 UTR probes should detect a smaller copy number increase
than the L1 ORF2 probes. Each probe set amplified a single product of
the predicted size (Supplementary Fig. 10b). Moreover, sequencing
PCR products derived from both ORF2 probe sets showed enrichment for members of the L1Hs subfamily (Supplementary Table 3).
We next isolated genomic DNA from the hippocampus, cerebellum,
liver and heart from three adult humans. We consistently observed a
statistically significant increase in L1 ORF2 content in the hippocampus
when compared to heart and liver samples from the same individual
(Fig. 4b, c and Supplementary Figs 9a and 10a). Notably, two individuals (1079 and 1846) showed more marked copy number differences
a
5′ UTR
L1
3′ UTR
CpG
ORF2
ORF1
b
SRY
SRY
c
Brain
Brain 1
Brain 2
Skin 1
Skin 2
50
0
0
50
Methylated CpGs (%)
ns
SC
d
N
S-
hC
Anti-SOX2
Anti-MECP2
Anti-IgG
DNA input
PC
N
ES
eu
N
n
ro
Skin
P = 0.0079
Brain 1 v. skin 1
P = 0.0034
Brain 2 v. skin 2
100
O
H2
1
e
20
100
Methylation (%)
Sequences (%)
100
48
492
1
41
428
4
44
441
5
25
251
5
26
9
28
4
29
3
30
5
31
327
320
7
35
1
36
363
379
7
23
2
Methylation CpG sites
Skin
Brain
50
0
1
5
10
15
CpG position in L1 5′ UTR
20
Figure 3 | Methylation analysis and ChIP for the endogenous human L1
59 UTR. a, Schematic illustrating the L1 CpG island, and SRY-binding sites.
b, Cumulative distribution function plot, comparing overall methylation and
collapsing CpG sites into a single data point (two-sample
Kolmogorov–Smirnov test). c, Individual methylation of sequences showing
highest sequence similarity to consensus RC-L1s. Open and closed
circles denote unmethylated and methylated CpG dinucleotides, respectively.
Dash indicates mutated CpG site. d, ChIP identifying MECP2 and SOX2
occupying the endogenous human L1 promoter, extracts were analysed by
PCR towards the L1 59 UTR SRY-binding region (SOX2
immunoprecipitation) or CpG island region (MECP2 immunoprecipitation).
e, CpG dinucleotides exhibited higher methylation at the 59-end of the CpG
island; higher methylation overall was observed in skin samples.
1129
©2009 Macmillan Publishers Limited. All rights reserved
LETTERS
a
NATURE | Vol 460 | 27 August 2009
Hippocampus
Cerebellum
Genomic DNA
extraction
Brain-specific
qPCR 80 pg/reaction
de novo L1 insertions
Taqman multiplex
Germline or
quantitative PCR parental L1 insertion
Heart
Liver
C
L
H
Hi
f
1.2
5s rDNA per SATA
1.1
1.0
1.0
Hi
C
L
H
C
L
1.3
H
P = 0.0001
1.1
METHODS SUMMARY
1.0
1.2
1.1
1.0
H
ip
po
ca
m
Relative 5sRNA DNA
content
e
Hi
1.1
Relative ORF2 DNA content
1.0
*
ORF2 per HERVH
1.2
DG
C
A1
C
Fr A3
on
Pa tal
rie
ta
l
C SVZ
au
da
C P te
er o
e n
Sp be s
in llum
al
co
r
H d
ea
r
Li t
ve
r
1.1
ORF2 per HERVH
pu
s
Li
ve
r
+
L 10
+
L 10
+
0
1
L+ ,00
10 0
,0
00
H
ea
r
H t
+1
H 0
+1
H 00
+1
H ,00
+1 0
0,
00
0
*
1.2
d
1.2
L
Relative ORF2 DNA content
c
ORF2 per SATA
ORF2 per 5sRNA DNA
content
Relative ORF2 DNA content
b
The large degree of variability in L1-ORF2 copy numbers between
brain regions and individuals may represent unsystematic rates of L1
retrotransposition, or another level of regulation that requires
further determination. That being stated, our in vitro findings in
NPCs coupled with the observed L1-ORF2 copy number changes
in the brain make it tempting to speculate that somatic retrotransposition events occur during the early stages of human nervous system
development. This study contributes to a growing body of evidence
indicating that engineered L1s can retrotranspose during early
development, and in select somatic cells1,6,23–25. Future experiments
will determine whether endogenous L1s truly retrotranspose in the
brain and whether these events are simply ‘genomic noise’ or have the
potential to affect neurogenesis and/or neuronal function.
Figure 4 | Multiplex quantitative PCR analyses of L1 copy number in human
tissues. a, Experimental schematic. b, c, Relative quantity of L1,
standardized such that the lowest liver value was normalized to 1.0.
C, cerebellum; H, heart; Hi, hippocampus; L, liver. Further L1 ORF2 assays
with other internals controls are shown in Supplementary Figs 9 and 10. All
error bars are s.e.m. *P , 0.05 (repeated measures one-way analysis of
variance (ANOVA) with Bonferroni correction, n 5 3 individuals, with three
repeat samples from each tissue). SATA, a satellite. d, Ten samples from
various brain regions (n 5 3 individuals) compared to somatic liver and
heart. DG, dentate gyrus; SVZ, subventricular zone. One-way t-test,
P # 0.0001 with 34 degrees of freedom. e, Multiplexing of 5S rDNA with
a-satellite indicated no significant change, P # 0.5054. f, Hippocampal
tissue compared to liver (L) and heart (H) spiked with estimated plasmid
copy numbers of L1 (10, 100, 1,000 and 10,000 copies).
than a third individual (4590) (Supplementary Fig. 10a). Controls
demonstrated that the ratio of the 5S rDNA gene to a satellite DNA
between each tissue remained relatively constant (Fig. 4e).
We extended this analysis to ten brain regions from three other
individuals (Fig. 4d and Supplementary Fig. 9b). The samples were
derived from the frontal and parietal cortex, spinal cord, caudate,
CA1 and CA3 areas of the hippocampus and pons, as well as from the
hippocampal dentate gyrus and the subventricular zone22. As
described earlier, there was marked variation between different brain
areas and between individuals (Supplementary Fig. 9c). However, an
unpaired t-test comparing all the grouped brain samples to the heart
and liver DNA again showed a small, but statistically significant
increase in ORF2 content in the brain (Fig. 4d).
To independently corroborate the observed increase in L1 copy
number in the hippocampus and cerebellum samples, we spiked
80 pg of liver and heart genomic DNA (approximately 12 genomes)
from individual 1846 with a calculated quantity of L1 plasmid, then
we repeated the multiplexing approach to assay ORF2 quantity relative to the 5S rDNA internal control (Fig. 4f). Three replications of
this experiment indicated that the hippocampus samples contained
approximately 1,000 more L1 copies than the heart or liver genomic
DNAs, suggesting a theoretical increase in ORF2 of approximately
80 copies per cell. The spiked L1 copies were in the form of a plasmid,
which probably affects the copy number estimates, providing an
estimate of relative change and not precise quantification of the
absolute number of L1s per cell. Therefore, ultimate proof that endogenous L1s are retrotransposing in the brain requires identification of
new retrotransposition events in individual somatic cells.
Cell culture, transfection and analysis. Fetal hCNS-SCns lines3 and hESCs24,26
were cultured as previously described. Neural progenitors were derived from
hESCs as previously described14,27. NPCs were transfected by nucleofection
(Amaxa Biosystems), and either maintained as progenitors in the presence of
FGF2 or differentiated as previously described14. Cells were transfected with L1s
containing an EGFP retrotransposition cassette in pCEP4 (Invitrogen) that lacks
the CMV promoter and contains a puromycin-resistance gene7.
Cell lysates. Ribonucleoprotein particles were isolated and analysed as previously described8. Luciferase assays were performed as previously described1.
ChIP was performed using primers towards the L1 59 UTR and a ChIP assay
kit (Upstate/Millipore) as per the manufacturer’s protocol.
Bisulphite analysis. Fetal tissues were obtained from the Birth Defects Research
Laboratory at the University of Washington. Bisulphite conversions were performed by manufacturer’s instructions using the Epitect kit (Qiagen). BLASTN
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to align sequences to a database of full-length L1s.
PCR. Adult human tissues were obtained from the NICDH Brain and Tissue Bank
for Developmental Disorders (University of Maryland). Taqman probes and
primers were designed using L1 Base (http://l1base.molgen.mpg.de/) and copy
number estimates were based on the UCSC genome browser (http://genome.
ucsc.edu). Experiments were performed on an ABI Prism 7000 sequence detection
system (Applied Biosystems). For each tissue, three separate tissue samples were
extracted and considered as repeated measures. Whole genome size was
estimated based on the equation: cell genomic DNA content 5 3 3 109(bp) 3
2(diploid) 3 660(molecular mass of 1 bp) 3 1.67 3 1012 (weight of 1 Da), (in
which bp denote base pairs), resulting in the approximation that one cell contains
6.6 pg genomic DNA28. Therefore, the 80 pg of genomic DNA used per reaction is
derived from approximately 12 cells. Inverse PCR was performed as previously
described1,24.
Received 31 March; accepted 1 July 2009.
Published online 5 August 2009; corrected 27 August 2009 (see full-text HTML
version for details).
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank J. Simon for excellent schematic drawings,
M. L. Gage, J. Kim and H. Kopera for editorial comments, B. Miller and R. Keithley
for cell culture assistance, C. T. Carson for hESC advice, D. Chambers and J. Barrie
for flow cytometry assistance, L. Randolph-Moore for molecular advice, B. Aimone
for statistics advice, T. Liang for microarray assistance, and Y. Lineu and J. Mosher
for helpful comments. We also thank T. Fanning and M. Klymkowsky for the ORF1
protein and SOX3 antibodies, respectively. F.H.G. and N.G.C. are supported by the
Picower Foundation, G. Harold and Leila Y. Mathers Charitable Foundation,
Lookout Fund (MH082070), and the California Institute for Regenerative
Medicine (CIRM). J.L.G.-P. is supported by Plan Estabilizacion Grupos SNS ENCYT
2015 (EMER07/56, Instituto de Salud Carlos III, Spain) and through the
IRG-FP7-PEOPLE-2007 Marie Curie program. K.S.O. was supported by grants
GM069985 and NS048187 from the National Institutes of Health (NIH). J.V.M.
was supported by grants GM082970 and GM069985 from the NIH and by the
Howard Hughes Medical Institute. Work in the laboratories of K.S.O. and J.V.M.
only used NIH-approved stem cell lines.
Author Contributions N.G.C. and F.H.G. directed the project. J.V.M. and J.L.G.-P.
directed aspects of the project conducted at Michigan. N.G.C., J.L.G.-P., J.V.M. and
F.H.G. designed experiments and drafted the manuscript. N.G.C., F.H.G., J.L.G-P.
and G.E.P. performed the experiments. G.W.Y. and M.T.L. carried out
bioinformatics data analysis. Y.M. performed electrophysiology experiments.
M.M. and K.S.O. provided hESC culture and NPC differentiation assistance. All
authors commented on or contributed to the current manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Correspondence and requests for materials should be
addressed to F.H.G. (gage@salk.edu).
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