Reprogramming somatic cells into iPS cells
activates LINE-1 retroelement mobility
Silke Wissing1, Martin Mun ˜oz-Lopez5,9, Angela Macia5,9, Zhiyuan Yang1, Mauricio Montano1,
William Collins2, Jose Luis Garcia-Perez5,6,9, John V. Moran6,7,8and Warner C. Greene1,3,4,∗
1Gladstone Institute of Virology and Immunology,2Gladstone Institute of Cardiovascular Disease,3Department of
Medicine and4Department of Microbiology and Immunology, University of California, San Francisco, CA, USA,
5Andalusian Stem Cell Bank, Center for Biomedical Research, University of Granada, Spain,6Department of Human
Genetics,7Department of Internal Medicine and8Howard Hughes Medical Institute, University of Michigan Medical
School, Ann Arbor, MI, USA and9Department of Human DNA Variability, GENYO (Pfizer - University of Granada and
Andalusian Government Centre for Genomics and Oncology), Spain
Received August 9, 2011; Revised and Accepted September 28, 2011
Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA
and represent a major evolutionary force that has reshaped the structure and function of the human genome.
However, questions remain concerning both the frequency and the developmental timing of L1 retrotranspo-
sition in vivo and whether the mobility of these retroelements commonly results in insertional and post-inser-
tional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially
at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically
relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as
well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading
frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing
analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed
that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in
the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more
efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is asso-
ciated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition,
which could potentially impact the genomic integrity of the resultant iPSCs.
Human embryonic stem cells (hESCs) are pluripotent cells
derived from the inner cell mass of human blastocysts (1).
Recent studies have shown that the introduction of three or
four defined transcription factors into lineage-restricted
somatic cells (e.g. fibroblasts) leads to cellular reprogramming
culminating in induced pluripotent stem cells (iPSCs). iPSCs
share a similar transcriptional profile and potential for differen-
tiation into three germ layers with hESCs (2–4). Both hESCs
and iPSCs hold promise for regenerative therapies for a
variety of diseases. Indeed, iPSCs may hold greater promise
than hESCs as they represent a potential source of autologous
cells compatible with the host immune system. However, the
therapeutic utility of iPSCs and hESCs could be limited by
adverse changes in genomic integrity that occur during repro-
gramming or subsequent expansion in vitro (5,6). For
Thus, it is important to understand processes that may impact
genomic integrity in both iPSCs and hESCs.
Long interspersed element-1 (LINE-1 or L1) sequences are
abundant retrotransposons in the human genome (7). Although
(reviewed in 8,9), it is estimated that the average human
genome harbors ?80–100 retrotransposition-competent L1s
∗To whom correspondence should be addressed at: Gladstone Institute of Virology and Immunology, 1650 Owens Street, San Francisco, CA 94158,
USA. Tel: +1 4157342000; Fax: +1 4153550855; Email: email@example.com
# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
Human Molecular Genetics, 2012, Vol. 21, No. 1
Advance Access published on October 11, 2011
(RC-L1s) (8–11) that can impact genome integrity by insert-
ing into new genomic locations via the reverse transcription
of an RNA intermediate (reviewed in 8,9). Human RC-L1s
are ?6 kb and contain two open reading frames (ORF1 and
ORF2) whose protein products (ORF1p and ORF2p) are
required for retrotransposition (12,13). The majority of these
RC-L1s belong to a human-specific subfamily of L1s
(L1Hs), and a small number of these elements (termed hot
L1s) are responsible for the bulk of retrotransposition activity
in modern day humans (10,11,14). In addition, the L1-encoded
proteins also can act in trans to facilitate the retrotransposition
of short interspersed elements, certain non-coding RNAs, and
certain messenger RNAs to new genomic locations (15–20).
Ongoing L1-mediated retrotransposition events contribute to
inter-individual human genetic diversity (11,21–24) and
have been implicated in a broad range of sporadic diseases, in-
cluding hemophilia A, Duchenne muscular dystrophy, X-
linked retinitis pigmentosa, b-thalassemia and colon cancer
(25; reviewed in 8,26,27). Therefore, RC-L1 ongoing mobility
have the potential to adversely impact genome integrity.
In principle, heritable L1-mediated retrotransposition events
must occur incells that giverise to gametes, during gametogen-
esis, or during early embryonic development. Indeed, previous
studies revealed that endogenous L1s are expressed in male
and female germ cells, in hESCs and in select somatic tissues
(28–32,34,36,37). Consistently, genetic studies, as well as
studies conducted with engineered human RC-L1s, have
revealed that L1 retrotransposition can occur in the germ line,
during early embryonic development, and in select somatic
rotransposition in vivo and whether L1 retrotransposition is
induced as a consequence of cellular reprogramming. We now
describe studies assessing L1 mRNA expression and the retro-
transposition efficiency of engineered human L1 retrotranspo-
sons in hESCs, iPSCs derived from human dermal fibroblasts
(HDFs) as well as parental HDFs. We demonstrate that L1
expression is reinstated upon somatic cell reprogramming and
that the resultant iPSCs support levels of engineered L1 retro-
transposition similar to those of hESCs.
Reprogramming HDFs into iPSCs induces L1 retroelement
Previous studies demonstrated that mRNAs from both human-
specific (L1Hs) and older L1 subfamiles are expressed in
hESCs (31,37). Here, we determined the relative levels of
L1 mRNA expression in hESCs, iPSCs derived from HDFs
and parental HDFs. Adult HDFs from skin biopsies of two
subjects were reprogrammed with retroviruses expressing
four factors (Sox2, Oct3/4, Klf4 and c-Myc) (3) to generate
46X,del(x)(q24)] and iPS 8.11 (karyotype: 46,XY). Another
t(1;17); karyotype HDFs: 46,XY], was generated by repro-
gramming newborn foreskin fibroblasts with a polycistronic
lentiviral vector (41) (see also Supplementary Material,
Table S1). A fourth previously derived iPSC line from
HDFs was also used in the study (MSUH001, generated
with Sox2, Oct3/4, Klf4 and Lin28) (41). The iPSC phenotype
in new derived lines was verified by analyzing the transcrip-
tion profiles of specific pluripotency genes, the silencing of
retroviral/lentiviral expression vectors, karyotyping and the
ability of the cells to differentiate into each of the three
germ cell layers (W. Collins et al., manuscript in preparation
and Supplementary Material, Figs S1–S3).
To determine whether pluripotent cells express full-length
L1 mRNA, we isolated cytoplasmic poly(A)+RNA from
hESCs and iPSCs and performed northern blot analyses with
an RNA probe complementary to the 5′UTR of an RC-L1
(Fig. 1A). The ?6-kb full-length sense-strand L1 mRNA
was detected in H9, H13, iPS-F and a human embryonic car-
cinoma cell line (Ntera2.D1) (14), but not in parental HDFs or
human HeLa cells (Fig. 1B). Several smaller L1 RNA species
also were evident. These shorter L1 RNAs may arise from the
use of alternative polyadenylation signals and/or cryptic splice
sites in the coding strand of L1 (42–44).
To quantify L1 RNA produced in iPSCs and hESCs, we
generated Taqman primer/probe sets that recognize specific
regions in the L1 5′UTR, ORF1 and ORF2 regions of a con-
sensus L1Hs element (10) (Fig. 1A). The probe sets were
designed to discriminate between full-length L1 RNAs and
truncated L1 RNAs, which may be fortuitously transcribed
from other genomic sites. In addition, the reverse-transcription
reaction was performed with a strand-specific primer comple-
mentary to the 3′UTR of L1Hs/L1P1, allowing the detection of
only sense-strand L1 RNA transcripts (Fig. 1A). Real-time
reverse transcriptase polymerase chain reaction (RT–PCR)
analyses revealed similar L1 RNA levels in iPSCs and
hESCs (Fig. 1C–E). However, iPSCs had on average
18.5+5.4-fold higher L1 RNA levels for all three primer/
probe sets when compared with parental fibroblasts. The
larger amount of L1 mRNA detected with the L1 ORF2
primer/probe pair when compared with the L1 ORF1 and L1
5′UTR primer/probe pairs may be due to the presence of
5′-truncated L1s located in the introns of unspliced mRNAs
or non-coding RNAs (45,46). Consistent with the above ana-
lyses, a different set of primers directed against sequences in
either the L1 5′UTR or ORF2 revealed ?15-fold higher
levels of L1 mRNA in iAND-4 iPSCs when compared with
the parental fibroblast cell line (Fig. 1F–G). Using the same
set of primers, we also observed elevated expression of L1
mRNA (Fig. 1H–I) in an iPSC line generated with Lin28
instead of c-Myc (MSUH001, karyotype: 46,XX) (41)
(Supplementary Material, Table S1 and Fig. S3).
We next tested for the expression of the L1 ORF1p protein
in iPSCs. Immunoblotting of ribonucleoprotein particles
(RNPs) isolated by sucrose gradient centrifugation from the
cytosolic fraction of cells with a polyclonal L1 ORF1p anti-
body (47) revealed detectable ORF1p protein expression in
iPSCs (Fig. 1J), but not in HDFs or other differentiated cell
types (i.e. HeLa cells and HEK293T cells; Fig. 1J). As
described previously, L1 ORF1p also was detected in RNPs
derived from H9 and H13 hESCs as well as in Ntera2.D1
cells (Fig. 1J) (31,36,48). Similarly, L1 ORF1p expression
was observed in the RNP fraction isolated from the iPSC
lines iPS-iAND4 and MSUH001 utilizing a different ORF1p
antibody (Fig. 1K). Thus, reprogramming of fibroblasts into
Human Molecular Genetics, 2012, Vol. 21, No. 1209
iPSCs is associated with a striking increase in the expression
of both endogenous L1 mRNA and L1 ORF1p.
Increased expression of L1 is associated with
hypomethylation of the L1 promoter region
L1 expression appears to be regulated by the methylation of
CpG islands in the L1 promoter region (28,49). To determine
whether the L1 methylation pattern differs between iPSCs and
HDFs, we performed bisulfite conversion analysis on genomic
DNA corresponding to a region of the L1 5′UTR promoter
containing 20 CpG dinucleotides from five pluripotent cell
lines (H9 and H13 hESCs as well as iPS-F, iPS 8.11 and
iPS-iAND-4 iPSCs) and five HDFs (HDF-F, HDF 8.11,
HDF iAND-4, HDF 2134 and a neonatal HDF sample) (36).
In general, hESCs and iPSCs exhibited similar levels of
Figure 1. Endogenous full-length sense-strand L1 mRNA is transcribed in pluripotent cells and is up-regulated during the reprogramming of adult HDFs. (A) A
schematic depiction of an L1 retrotransposon. Depicted below the cartoon are the approximate locations of the RNA probe used in the L1 northern blot analysis
in (B) (triangles) as well as the Taqman primer/probe pairs (small convergent arrows) used in RT–PCR analyses shown in (C)–(E) (large backward arrows
indicate the position of the RT primer). (B) Northern blot analysis of cytoplasmic poly(A+) mRNA with a strand-specific RNA probe corresponding to the
L1 5′UTR. The signal at ?6.0 kb corresponds to full-length L1 mRNA (marked by an arrow). Endogenous expression of L1 mRNA in hESCs, iPSCs and
HDFs as assessed by real-time RT–PCR using probe sets that correspond to the L1 5′UTR (C), ORF1 (D) or ORF2 (E). Standard curves were prepared
using known L1 DNA concentrations and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to control for the amount of RNA input per reaction.
Values are RNA copies per 10 pg of total RNA, which corresponds approximately to the total RNA per cell. Values are the mean+SEM (n ¼ 3).∗P ≤ 0.05,
∗∗P ≤ 0.01. L1 mRNA expression levels in iAND-4 (F and G) and MSUH001 iPSCs (H and I) determined by quantitative RT–PCR with a set of primers against
the 5′UTR (F and H) or ORF2 (G and I) sequences as described (37). The amount of L1 RNA was determined in H9 hESCs and parental HDFs for iAND-4 and
HeLa for MSUH001 as controls. The graphs show the fold change in L1 expression compared with H9 hESCs and are normalized to GAPDH RNA. (J) Western
blot analysis of RNPs with an L1 ORF1p antibody (47). 293T cells transfected with a full-length L1 construct were used as a positive control. The ribosomal S6
protein (RPS6) served as a loading control. (K) Western blot analysis using a polyclonal antibody against ORF1p (kind gift of Dr G. Cristofari) in ribonucleo-
proteins isolated from iAND-4 and MSUH001 iPSCs. b-Actin served as a loading control. Numbers on the left side, molecular weight standards (kD).
210Human Molecular Genetics, 2012, Vol. 21, No. 1
methylation that were lower than those detected in HDF cells
(Fig. 2A and B). Direct comparison of three iPS/parental HDF
pairs showed significantly fewer CpG methylation events in
iPSCs when compared with HDFs (36.8% reduction; P ¼
0.029, 3.87 × 107and 1.18 × 104; Fig. 2A and B). Analysis
of the 20 CpG dinucleotides in the L1 5′UTR revealed signifi-
cant differences between pluripotent cells and HDF cells, par-
ticularly in the 3′region of the analyzed CpG island (∗∗∗P ≤
0.001; Fig. 2C and Supplementary Material, Fig. S4).
Notably, fully unmethylated sequences were detected in pluri-
potent cells, but not HDFs (iPSCs 6 of 31 sequences versus
HDFs 0 of 30 sequences; Fig. 2A). Thus, the increase in L1
expression in pluripotent cells appears to correlate with an
overall decreased CpG methylation in the 3′region of the
L1 promoter. These results are in general agreement with a
recently published study (36) where higher expression of L1
mRNA was demonstrated in brain compared with skin
samples and correlated with a decreased CpG methylation in
the L1 promoter in the brain. Although the above experiments
suggest a global hypomethylation of L1 promoter sequences
upon reprogramming HDFs into iPSCs, it is worth mentioning
that reprogramming of other somatic cells might result in
slightly different CpG methylation changes in L1 promoters,
especially as variable methylation patterns between different
iPSC lines were recently reported (50).
CpG methylation is significantly decreased in hot L1
We next investigated whether alleles of known RC-L1s were
hypomethylated during the establishment of iPSCs. Previous
studies suggested that the bulk of L1 retrotransposition ema-
nates from a small group of hot RC-L1s (10,11). To explore
the CpG methylation state of the promoter region of alleles
of known hot RC-L1s, we performed bisulfite conversion ana-
lysis on genomic DNA from the iPS-F iPSCs and parental
HDFs. We chose two hot RC-L1 alleles [AL512428
(6p22.3) and AL137845 (Xq22.33)] as well as an RC-L1
allele that is the progenitor of a mutagenic insertion in man
[M80343.1, L1.2A (22q11-q12)] for analysis (10,25). To
study the CpG islands in these specific L1s, we performed
nested PCR, first amplifying a ?900–1200-bp region includ-
ing the first 700 bp of L1 and a 200–500-bp region upstream
of the specific L1, followed by the internal amplification of the
L1 CpG island. In agreement with the prior results (Fig. 2), the
level of CpG methylation in iPSCs was significantly lower
when compared with parental HDFs [Fig. 3A and B,
AL512428 (61% decrease), AL137845 (83% decrease) and
M80343.1 (L1.2) (81% decrease), P ≤ 0.001]. These findings
suggest that the 5′UTR of these hot L1 alleles becomes
hypomethylated upon somatic cell reprogramming.
Figure 2. Analysis of the L1 5′UTR promoter reveals significant hypomethylation in iPSCs. (A) Bisulfite sequencing results from three iPSC/parental HDF cell
lines. Shown are the 10 clones with the highest sequence similarity to a hot L1 element (L1.3, accession number L19088.1) (53). Each line reflects an independ-
ent clone. For iPS 8.11, 11 clones are shown, as the two most divergent clones had the same sequence similarity. White and black circles represent unmethylated
and methylated CpG dinucleotides, respectively. Mutated CpG islands are skipped (no circle). (B) Bisulfite analysis of different HDF and pluripotent cell lines
show similar percentages of methylated CpG dinucleotides in hESCs and iPSCs and an overall increase in CpG methylation in HDFs. Each bar represents data
from the clones shown in (A) presented as methylated CpG dinucleotides in percent of total CpG dinucleotides (mean+SEM,∗P ≤ 0.05,∗∗∗P ≤ 0.001,
unpaired two-tailed t-test). (C) Analysis of each single CpG dinucleotide between five grouped HDF lines (HDF-F, HDF 8.11, HDF iAND-4, HDF 2134
and neonatal HDFs) and five grouped pluripotent cell lines (H9 hESCs, H13 hESCs, iPS-F, iPS 8.11 and iPS-iAND-4) reveals significant methylation differences
in the 3′region of the CpG island. Values are the mean+SEM.∗P ≤ 0.05,∗∗P ≤ 0.01,∗∗∗P+0.001, unpaired two-tailed t-test. Values for each single cell line
are shown in Supplementary Material, Fig. S4.
Human Molecular Genetics, 2012, Vol. 21, No. 1211
iPSCs express young human-specific L1 transcripts
To determine whether L1 mRNAs expressed in iPSCs are
derived from RC-L1 alleles, we isolated RNA from iPS-F
cells and used it as an RT–PCR template to analyze a
?750-bp region of ORF1. We then analyzed the sequences
of 37 independent clones using RepeatMasker (http://repea
tmasker.org) (51). Approximately 84% (31 of 37) of the L1
cDNAs were derived from human-specific L1s (Fig. 3C).
Similar analyses conducted on a 236-bp RT–PCR amplified
region of ORF1 revealed that ?50% L1 cDNAs derived
from iAND-4 (12 of 28) or MSUH001 iPSCs (12 of 27)
were derived from human-specific L1s (Fig. 3D). Notably,
the two different primer sets used in these studies had
various specificities for the distinct L1 subtypes; the primers
used to analyze the iPSC lines iAND-4 and MSUH001
could amplify L1Hs and L1P1–L1P5 subfamilies, while the
primers used to analyze the iPS-F iPSCs were specific for
L1Hs and L1P1. The difference in primers could explain the
difference in the proportions of L1Hs cDNAs detected in the
different iPSCs. Nevertheless, these data clearly reveal that
L1Hs elements are expressed in iPSCs and may have the
potential to retrotranspose in these cells.
Engineered human L1 retrotranspose more efficiently
in iPSCs than HDFs
Since human-specific L1s are expressed in iPSCs, we next
assessed the mobility of engineered human L1s in hESCs,
iPSCs and parental HDFs. To measure L1 retrotransposition
efficiency, we tagged the 3′UTR of hot RC-L1s with a retro-
transposition indicator cassette that contains an antisense
copy of a reporter gene interrupted by an intron in the same
transcriptional orientation as the L1 (13,38,52,53). This con-
figuration results in expression of the reporter only when the
tagged L1 completes a successful cycle of retrotransposition
We first used a modified L1 retrotransposition assay utiliz-
ing an enhanced green fluorescent protein (EGFP) retrotrans-
position indicator cassette (mEGFPI) (52). Briefly, cells
(pLRE3-EF1-mEGFPI), and the L1 retrotransposition effi-
ciency (i.e. EGFP-positive cells) was quantified by flow cyto-
metry 4 days post-transfection (Supplementary Material,
Fig. S5A). To control for variations in transfection efficiency
between different cell lines, we divided the number of EGFP-
pLRE3-EF1-mEGFPI by the number of EGFP-positive cells
obtained after nucleofection
(pLRE3-EF1-mEGFP(△intron)) lacks the intron in the
mEGFPI gene, and thus, the expression of the EGFP protein
occurs in all successfully nucleofected cells (Supplementary
Material, Fig. S5B). To further increase assay sensitivity, auto-
fluorescent cells were eliminated by plotting against an empty
channel as described previously (54).
Consistent with previous findings, control experiments
conducted in H9 and H13 hESCs revealed ?30+5 EGFP-
positive cells per 10 000 nucleofected cells (Fig. 4D). The
retrotransposition efficiency in H9 and H13 cells was
highly reproducible and was ?10% of the level of retrotrans-
position detected in HeLa cells (Fig. 4D). PCR analyses on
genomic DNA derived from clonal H9 hESC lines revealed
precise splicing of the intron from the mEGFPI indicator
Figure 3. Methylation analysis of the promoter region of hot RC-L1s in iPSCs. (A) Bisulfite sequencing results of the promoter region of three hot RC-L1 loci in
unmethylated and methylated CpG dinucleotides, respectively. Mutated CpG islands are skipped (no circle). (B) Bisulfite analysis of the iPS-F/HDF-F cell lines
reveals a significant decrease in CpG methylation in iPSC lines (M80343.1/(L1.2): 81%, AL512428: 61%, AL137845: 83%). Each bar represents data from the
clones shown in (A). Values are the mean+SEM of the percentage of methylated CpGs.∗∗∗P ≤ 0.001 (unpaired two-tail t-test). (C) Analysis of cloned ORF1
cDNA sequences (?750 bp) from iPS-F cells revealed that ?84% (31 of 37 sequences) are derived from L1Hs elements. Analysis was performed using Repeat-
Masker (51). Div., diversity; Del., deletion; Ins., insertion in respect to the reference sequences. (D) Analysis of cloned ORF1 cDNA sequences (236 bp) from
iAND-4 and MSUH001 iPSCs revealed that ?50% are derived from L1Hs elements. Analyses were performed using RepeatMasker (51).
212Human Molecular Genetics, 2012, Vol. 21, No. 1
cassette gene, which is indicative of retrotransposition
(Supplementary Material, Fig. S6C) (31,36,55). Further, treat-
ment with lamivudine (3TC, 100 mM), a reverse transcriptase
inhibitor that potently suppresses L1 retrotransposition (56),
Material, Fig. S5A and D), but did not affect the expression
We next used the same strategy to examine the level of
engineered L1 retrotransposition in iPSCs and parental
fibroblasts. The level of L1 retrotransposition in the iPS-F
cells was similar to that detected in H9 and H13 hESCs.
Strikingly, L1 retrotransposition was ?10-fold higher in
iPSCs than in the parental HDFs (Fig. 4D and E). Addition-
al controls revealed robust EGFP expression in iPS-F clones
(Fig. 4B) and precise splicing of the engineered intron as
demonstrated by PCR using genomic DNA isolated from
(Fig. 4C). In addition, experiments conducted with an L1
containing an mneoI retrotransposition indicator cassette
(pKUB102/L1.3-sv+, Supplementary Material, Fig. S6A)
(13,57) indicated that iAND-4 cells support ?20–30-fold
more L1 retrotransposition than parental fibroblasts (an
average of 32 foci in iAND-4 versus 0–2 foci in the
parental HDFs; Fig. 5A). Notably, we did not detect any
L1 retrotransposition events from an RT-defective L1
allele (data not shown). In addition, PCR using genomic
DNAderived from four
iAND-4 clonal lines revealed precise splicing of the
intron from the mneoI cassette (Fig. 5B). Together, these
findings demonstrate that reprogramming fibroblasts to
iPSCs is associated with an approximate one log10increase
in the retrotransposition efficiency of an engineered human
L1 element, at least when the L1 mRNA is produced by a
strong exogenous promoter. Further, comparable levels of
retrotransposition were detected in hESCs and iPSCs, indi-
cating that reprogramming does not lead to elevated levels
of retroelement mobility beyond that found in hESCs. In
addition, these data confirm that primary human fibroblasts
only accommodate very low levels of retrotransposition
Figure 4. The retrotransposition efficiency of an engineered human L1 is higher in pluripotent stem cells than in adult HDFs. (A) An overview of the L1 retro-
transposition assay. An RC-L1 is tagged with a retrotransposition indicator cassette (mneoI or mEGFPI). The indicator cassette contains an antisense copy of a
selectable (NEO) or detectable (EGFP) reporter gene that is disrupted by an intron in the same transcriptional orientation as the L1 (13,52). The reporter gene
contains a heterologous promoter (SV40 for the mneoI and UBC for the mEGFPI) and a poly (A) signal. An EF1a or UBC promoter (Enh, enhancer) drives L1
expression. The reporter gene can only be activated when L1 RNA is reverse transcribed, integrates into genomic DNA and is expressed from its own promoter
(13). (B) An undifferentiated iPS-F colony harboring a L1 retrotransposition event from the pLRE3-EF1-mEGFPI reporter. (C) L1 retrotransposition events are
stably integrated into the genome. Genomic DNA isolated from EGFP-positive colonies after nucleofection served as a PCR template. The 343-bp PCR product
indicates the spliced tagged L1 (insertion); the 1243-bp product contains the intron (vector). (D) L1 retrotransposition efficiencies in different cell lines. Cells
were transfected with pLRE3-EF1-mEGFPI reporter or the pLRE3-EF1-mEGFP(△intron) plasmid as a control. Cells nucleofected with pLRE3-EF1-mEGFPI
were harvested 4 days after nucleofection; cells nucleofected with pLRE3-EF1-mEGFP(△intron) were harvested 2 days after nucleofection at the peak of EGFP
expression. The frequency of new integrations (EGFP-positive cells) was analyzed by flow cytometry. For hESCs or iPSCs, ?1–2 × 106cells/sample were
analyzed; for HDF cells, ?0.25–0.5 × 106cells/sample were assayed. The retrotransposition frequency was calculated as the number of EGFP-positive cells
pLRE3-EF1-mEGFP(△intron) control plasmid. The experiments were repeated at least three times. Values are the mean+SEM.∗∗P ≤ 0.01. (E) Flow cyto-
metry plots of iPS-F iPSCs and HDF-F cells either untransfected or transfected with pLRE3-EF1-mEGFPI reporter (harvested 4 days post-transfection) or
the pLRE3-EF1-mEGFP(△intron) control plasmid (harvested 2 days post-transfection at the peak of EGFP expression). For iPSCs, ?1 × 106cells/sample
were analyzed; for HDFs, ?0.5 × 106cells/sample were assayed. Cells were plotted against an empty channel (FL-2) to eliminate background autofluorescent
cells and to increase sensitivity.
thenumberof EGFP-positive cellsafternucleofectionwith the
Human Molecular Genetics, 2012, Vol. 21, No. 1213
Retrotransposed L1s in iPSCs exhibit hallmarks
To determine whether L1 retrotransposition events occurring
in iPSCs exhibited bona fide structural hallmarks, we used
inverse PCR to characterize four retrotransposition events
from iAND-4 iPSCs. Three insertions (iAND4-2, iAND4-6
and iAND4-7) were completely characterized by DNA
sequencing. A fourth insertion (iAND4-E) could not be com-
pletely analyzed because it was located in a-satellite DNA
and was truncated at the restriction enzyme site used in the
inverse PCR (data not shown). The iAND4-2 and iAND4-6
insertions were full length and contained part of the exogenous
UBC promoter used to drive L1 expression (Fig. 5C), while
the iAND4-7 insertion was 5′-truncated. Each insertion exhib-
ited typical L1 structural hallmarks. They ended in poly(A)
tails, were flanked by target-site duplications that ranged in
size from 8 to 14 bp and appeared to integrate into a sequence
resembling a consensus L1 endonuclease cleavage site
(12,19,59) (Fig. 5C and Supplementary Material, Fig. S6B–
D). Notably, the iAND4-2 and iAND4-7 insertions occurred
within the introns of known genes (CDKAL1 and DAPK2),
which is consistent with previous studies (31,32,36,55).
While iPSCs might one day be of great value in the treatment
of currently incurable human diseases (2,3), several challenges
posed by these cells must be overcome. Chief among these is
ensuring that the genomic integrity of iPSCs is not compro-
mised during their generation or ex vivo culture.
In this study, we demonstrate for the first time that cytoplas-
mic full-length L1 mRNAs are expressed in hESCs and iPSCs,
but are not detectable in HDFs and HeLa cells under our
experimental conditions. In addition, we detected endogenous
L1 ORF1 protein in ribonucleoprotein fractions from iPSCs
and hESCs. Both cytoplasmic full-length L1 RNA and RNPs
containing the L1 ORF proteins are required for L1 retrotran-
It is thought that L1 transcription is suppressed by the CpG
methylation of its 5′UTR in most somatic cells (49). We have
detected decreased DNA methylation in CpG islands located
within the L1 5′UTR promoter region in hESCs and iPSCs.
Notably, sequencing revealed that L1 cDNAs in iPSCs are
derived from human-specific L1s. These findings suggest
that L1 expression is a feature of pluripotent cells and indicate
that hot L1s might actively retrotranspose in iPSCs. Genomic
(21,22,63) and fosmid-based, paired-end DNA sequencing
(11) approaches have recently been used to detect and/or
quantify new L1 integration sites in various cell types.
Future adaptations of these approaches could be used to
examine whether the retrotransposition of endogenous L1s is
increased in iPSCs compared with HDFs.
We also demonstrated that an engineered human L1 can ret-
rotranspose with ?10-fold higher efficiency in iPSCs than in
parental HDFs, and the characterization of three retrotranspo-
sition events in iPSCs revealed canonical L1 structural hall-
marks. Thus, reprogramming of somatic fibroblasts not only
reinstates L1 transcription, but also appears to create an intra-
cellular milieu that supports at least a 10-fold higher level of
engineered L1 retrotransposition than observed in parental
fibroblasts. Interestingly, a recent study suggested engineered
L1 retrotransposition events could be epigenetically silenced
in certain human embryonic carcinoma cells and that this
silencing was attenuated in differentiating cells (55). Thus,
Figure 5. Engineered L1 retrotransposition events in pluripotent stem cells
exhibit hallmarks of retrotransposition. (A) An mneoI-based L1 retrotransposi-
tion assay shows higher levels of retrotransposition in the iPSC line iAND-4
than in parental HDFs. Cell were transfected with the pKUB102/L1.3-sv+
engineered L1 plasmid and treated with G418 for 2 weeks. G418-resistant
foci were stained with crystal violet for visualization. In parental HDFs, typ-
ically 0–2 foci/experiment were observed. (B) G418-resistant clones harbor a
stable integrated event derived from pKUB102/L1.3-sv+. Genomic DNA iso-
lated from four G418-resistant clones served as a PCR template. The 468-bp
PCR product represents the spliced product (insertion); the 1361-bp product
contains the intron (vector). (C) The cartoons show schematic representations
of the L1 retrotransposition events in iAND-4 iPSCs. Names of the insertions
are indicated at the tops of the schematics. Red lines indicate the genomic site
of insertion and whether the insertion occurred within a gene. Blue arrowheads
indicate target site duplications. The approximate length of the L1 poly(A) tail
and the position of the L1 where 5′truncation occurred also are indicated and
are based on the L1.3 reference sequence (L19088.1) (53). Additional details
can be found in Supplementary Material, Fig. S6.
214Human Molecular Genetics, 2012, Vol. 21, No. 1
our studies may actually underestimate the efficiency of engi-
neered human L1 retrotransposons in iPSCs. In addition, high
levels of L1 expression and higher engineered L1 retrotranspo-
sition efficiencies in pluripotent cells may explain why some
L1 insertions in humans seem to be of early embryonic
Two recent reports demonstrated that somatic coding muta-
tions (64) and copy number variations (5) during iPSC repro-
gramming could lead to genetic mosaicism. Genomic
instability can also occur during the culturing of hESCs (6).
Thus, it is tempting to speculate that L1-mediated processes
may occasionally lead to the generation of genomic variability
in cultured pluripotent cells by a variety of mechanisms
L1-mediated retrotransposition events affect genomic stability
in hESCs and iPSCs is warranted before these cells, or more
likely their progeny, are utilized as cellular therapies.
MATERIAL AND METHODS
Cell lines and culture conditions
iPSC lines were derived as described (3,41). HDFs, H9 and
H13B (hereafter referred as H13) hESCs and iPSCs were cul-
tured and expanded under standard conditions (Supplementary
Material and Methods).
Cloning strategies are available upon request. L1 retrotranspo-
sition assays using the neomycin indicator cassette (mneoI)
were performed with the pKUB102/L1.3-sv+ plasmid, which
contains an L1.3 element (53) without its 5′UTR, the mneoI
indicator cassette (57) and the SV40 late polyadenylation
signal 3′of the engineered L1. The engineered L1 was
expressed from a modified version of pBSKS-II (Stratagene)
that contains a human UBC promoter upstream of the engi-
neered L1 (36). pLRE3-EF1-mEGFPI contains a full-length
LRE3 element (38) under the control of a heterologous
EF-1a promoter and the internal 5′UTR promoter, and the
EGFP retrotransposition indicator cassette is under the
control of an ubiquitin promoter and the SV40 late polyadeny-
lation signal; the construct was cloned into pBSKS-II (Strata-
gene). The positive control pLRE3-EF1-mEGFP(△intron) is
identical to pLRE3-EF1-mEGFPI but lacks the intron in the
mEGFPI indicator cassette.
Nucleofection, retrotransposition assay and flow cytometry
L1 retrotransposition assays using the L1 mneoI indicator cas-
sette were performed as follows. Cells (4 × 106) were nucleo-
fected with an Amaxa nucleofector, using 4 mg of plasmid
DNA, hESC solution 2 (Amaxa) and the A-23 program. The
transfected cells were cultured in iPSC medium supplemented
with 10 mM Rho-associated coiled kinase (ROCK) inhibitor
(73). Cells were selected with 50 mg/ml G418 for 7 days
and 100 mg/ml G418 for an additional 7 days, fixed in 0.2%
glutaraldehyde and 2% formaldehyde and stained with
crystalvioletto visualizefoci. For
retrotransposition assays, cells were nucleofected with an
Amaxa nucleofector, using the V-Kit solution (Amaxa) and
the A-23 program. Transfected cells were cultured in condi-
tioned hESC medium supplemented with 10 mM ROCK inhibi-
tor (73). Two [pLRE3-EF1-mEGFP(△intron)] or 4 days
(pLRE3-EF1-mEGFPI) after nucleofection, cells were ana-
lyzed by flow cytometry (hES or iPS, ?1–2 × 106cells/
sample; HDF, ?0.25–0.5 × 106cells/sample). Data were ana-
lyzed with FlowJo software (Tree Star).
L1 promoter methylation studies
Bisulfite analysis was performed as described (36). Briefly,
genomic DNA was extracted with a DNeasy kit (Qiagen).
Bisulfite conversion was performed with the Epitect kit
(Qiagen), according to the manufacturer’s instructions. For
whole-genomic L1 CpG analysis, PCR was performed to
amplify a 363-bp region in the 5′UTR harboring 20 CpG dinu-
cleotides (2 min at 958C, 35 cycles of 30 s at 948C followed by
30 s at 548C and 60 s at 728C, and a final extension of 5 min at
728C; primers are listed in Supplementary Material, Table S2).
To analyze hot L1 promoter regions, we first amplified a
?900–1200-bp region including the first 700 bp of L1 and a
200–500-bp region, upstream (3 min at 958C, 35 cycles of
30 s at 948C followed by 30 s at 508C and 2 min at 728C,
and a final extension of 7 min at 728C; primers are listed in
Supplementary Material, Table S2). Two microliters of the
PCR product were used for the second round of PCR, using
the same primer and condition as for the whole-genomic bisul-
fite analysis. The PCR products were subcloned and analyzed
with online software (QUantification tool for Methylation
Analysis, QUMA; quma.cdb.riken.jp) (74). Only sequences
with a CpG conversion rate .95% were accepted for analysis;
the final analysis included the clones with the highest sequence
similarity with a hot L1 element (L1.3, accession number
Total RNA was extracted with Trizol (Invitrogen), treated
with a Turbo DNA-free kit (Ambion), enriched with an
RNeasy kit (Qiagen) and reverse transcribed with the Super-
script III first-strand synthesis system, using sense-strand L1
primers. PCR and real-time RT–PCR were performed accord-
ing to standard procedures (Supplementary Experimental Pro-
cedures). Sequences and specifications of primers are shown in
Supplementary Material, Table S2.
Northern blot analysis
Northern blot analysis was performed by standard procedures
(Supplementary Experimental Procedures). Briefly, 2.5 mg of
cytoplasmic poly(A)-selected RNA were fractionated on a
1% agarose-formaldehyde gel. The RNA was transferred
onto a Hybond-N+-Nylon membrane (Amersham), hybri-
dized with alpha32P-UTP-labeled RNA probes overnight at
688C and subjected to two 10-min low-stringency washes at
208C and two 20-min high-stringency washes at 688C.
b-Actin mRNA served as the loading control.
Human Molecular Genetics, 2012, Vol. 21, No. 1 215
Isolation of genomic DNA for integration analysis
Fully spliced L1 reporter constructs in the genomic DNA were
detected as described (13,52).
Inverse PCR analysis
Inverse PCR on clonal iPSC lines was conducted as described
Analysis of expressed L1 mRNAs
Briefly, total RNA was used in RT–PCRs using ORF1p
primers as described (31,37,55). Amplified products were
cloned and at least 25 independent clones sequenced.
Sequences were analyzed with RepeatMasker (http://repeat
Statistical analysis was performed with the unpaired two-tailed
This study was approved by the Human Gamete, Embryo and
Stem Cell Research Committee UCSF (GESCR numbers
7396-29609 and H51338-32135-03) and the Andalusian and
Spanish Embryo and Cellular Reprogramming Ethical Institu-
tional Review Board.
Supplementary Material is available at HMG online.
We thank Drs J.L. Goodier and G. Cristofari for polyclonal L1
ORF1 antibodies, Dr H.H. Kazazian for the LRE3 construct,
Drs Jose Cibelli and Steve Suhr for the lentiviral construct
ThOKSIM and the Gladstone Stem Cell Core for technical
assistance. Wealso thank
Gutierrez-Aranda, Rene Rodriguez, Paola Leone and Pablo
Menendez (Andalusian Stem Cell Bank) for assistance with
S. Richardson for editorial assistance, and S. Cammack,
R. Givens and J. Carroll for assistance in preparation of the
manuscript and graphics.
Ordway, G. Howardand
Conflict of Interest statement. None declared.
This work was supported by funding from the California Insti-
tute for RegenerativeMedicine
(RS1-00210-1 and TRI-01227) and a CIRM scholarship
(TG2/01160) and byafellowship
Academy of Sciences Leopoldina (BMBF-LPD 9901/8-144)
to S.W.J.L.G.-P.’s laboratory
ISCIII-CSJA-FEDER (EMER07/056), a Marie Curie IRG
from the German
4980), Proyectos en Salud PI-002 from Junta de Andalucia
(Spain) and the Spanish Ministry of Health (FIS-FEDER
PI08171). J.V.M. is supported by NIH (GM082970 and
GM060518) and is an Investigator of the Howard Hughes
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