Live Cell Monitoring of hiPSC Generation and
Differentiation Using Differential Expression of
Masakazu Kamata1, Min Liang1, Shirley Liu1, Yoshiko Nagaoka2, Irvin S. Y. Chen1*
1Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California,
United States of America, 2Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles,
California, United States of America
Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this
potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we
describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple
miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent
differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human
embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of
differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF
to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and
cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and
mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to
hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression
and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct
structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter
vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage
Citation: Kamata M, Liang M, Liu S, Nagaoka Y, Chen ISY (2010) Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of
Endogenous microRNAs. PLoS ONE 5(7): e11834. doi:10.1371/journal.pone.0011834
Editor: David S. Milstone, Brigham and Women’s Hospital, United States of America
Received November 19, 2009; Accepted June 29, 2010; Published July 28, 2010
Copyright: ? 2010 Kamata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the California Institute for Regenerative Medicine (CIRM) RS1-00172-1 and by the National Institutes of Health (NIH)
AI055281, AI069350, and AI028697. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Human embryonic stem cells (hESCs) have significant thera-
peutic potential for various diseases, but the generation of these
cells from individual patients raises ethical concerns. Recently, a
technological breakthrough where somatic cells from mouse and
human can be reprogrammed into hESC-like pluripotent cells,
termed induced pluripotent stem cells (iPSCs), was made possible
through ectopic expression of combinations of reprogramming
factors including OCT4, SOX2, KLF4, cMYC, LIN28 and NANOG
[1,2,3,4,5,6]. Like hESCs, hiPSCs can be self-renewed and have
been proven to differentiate into a variety of cell types.
Furthermore, iPSCs generated from patient-derived cells can
serve as useful tools for potential therapies, drug screening, or to
study pathogenesis outside of patients [7,8,9]. In order for this
potential to be achieved, it is necessary to efficiently monitor the
differentiation of these iPSCs into specific lineages.
MicroRNAs (miRNAs) are small non-coding RNAs which
regulate gene expression post-transcriptionally (see recent review
[10,11,12]). miRNAs can be expressed differentially during
development and in a tissue-specific fashion [13,14]. Since the
target sequences for miRNAs are small (21 to 25 in size)  and
act in a relatively context-independent fashion, they can readily be
incorporated into vectors together with reporter genes, resulting in
reporter expression that is downregulated only in the presence of
the endogenous miRNA within cells.
Previous studies demonstrated the utility of such miRNA
regulated reporter vectors to distinguish between somatic cells in
distinct differentiation lineages and throughout the course of
differentiation [13,16,17]. The miRNA target sequence for miR-
142-3p was inserted after the transgene and expressed in the same
mRNA transcript in the context of a lentiviral vector. This vector
expression was specificallysuppressed inhematopoietic lineages and
successfully used to eliminate off-target expression of transgenes
. They further showed the effectiveness of regulation of
transgene expression by cell type dependent miRNA expression
using hESC in pre- and post-differentiated conditions .
Here, we use a similar reporter vector sensitive to differentiation
specific miRNAs to show that a single vector bearing multiple
miRNA target sequences conjugated to different reporters can be
used to monitor hiPSC formation from human fibroblasts and
subsequent differentiation of the hiPSC.
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Characterization of a miRNA dependent reporter vector
that distinguishes pluripotent cells from differentiated
We constructed a bidirectional vector whereby the reporter
gene mCherry is conjugated with perfectly complementary
miRNA target sites for miR-223, miR-155, miR-142-3p and EGFP
expressed in the anti-sense direction conjugated with miR-302a
and miR-302d (miR-302 a/d). The miR-302 gene encodes a cluster
of eight miRNAs on chromosome 4 (miR-302b*-b-c*-c-a*-a-d-367)
that are preferentially expressed in embryonal carcinoma cells,
hESCs and hiPSCs [19,20,21]. Whereas miR-223, miR-155, and
miR-142-3p are enriched in differentiated cells [14,22], miR-223
and miR-142-3p are found in cells of primarily hematopoietic
origin . miR-155 is found in hematopoietic cells as well as in
many types of lymphoma and solid cancers [24,25,26,27]. miR-155
is also expressed 20–50 fold higher in fibroblasts than in hESCs
and hiPSCs . Thus, endogenous expression of the miRNAs
segregated by differentiation state would result in ablation of
EGFP, but not mCherry in pluripotent stem cells and, conversely,
ablation of mCherry, but not EGFP in differentiated cells of
hematopoietic or fibroblast lineage, or in various malignant cells.
We first demonstrated that this reporter construct is responsive
to the endogenous miRNAs as predicted (Fig. 1). Both EGFP and
mCherry are detected in 293T cells which express the relevant
miRNAs at very low levels  (Fig. 1B). Ectopic expression of the
miRNAs by co-transduction, either miR-302a, miR-302b, miR-302c,
and miR-302d or miR-155 results in ablation of EGFP or mCherry
expression, respectively, demonstrating sensitivity of the vector to
specific miRNAs (Fig. 1B, miR-302a-d and miR-155, respectively).
In hematopoietic lineage cells (U937, monocyte lymphoma cell
line) mCherry expression is entirely ablated whereas EGFP is
maintained (Fig. 2A, mCherry miR-T and EGFP miR-T/
mCherry miR-T). Similar results are seen in Ramos (B-cell
lymphoma cell line) and CEM (T-cell lymphoma cell line) (Fig. 2B,
EGFP miR-T/mCherry miR-T). We further tested the expression
of EGFP miR-T/mCherry miR-T vector in CD34+ hematopoi-
etic progenitor/stem cells (HPSCs) isolated from the fetal liver (FL-
CD34+) (Fig. 2C). As expected, mCherry expression was strongly
diminished, whereas EGFP was detectable in CD34+ HPSCs. In
contrast, following transduction of hESC (H1 cell line), EGFP
expression is fully ablated, whereas mCherry expression is
maintained (Fig. 2D). We confirmed the phenotype by isolating
and propagating the colonies and subjecting the cells to analysis by
flow cytometry. The cells were maintained mCherry positive and
EGFP negative for over 20 generations without notable adverse
effects on their growth. Therefore, this reporter construct is
suitable for assaying differentiation of hESC into particular
lineages in a quantitative fashion utilizing a relatively small
number of cells.
miRNA dependent reporter expression during
reprogramming of human fetal fibroblast (HFF) to hiPSC
The reprogramming of HFFs to hiPSCs can be achieved by the
introduction of four transcription factors (OCT4, SOX2, KLF4 and
cMYC). After a prolonged period of culture on feeder cells, a
fraction (,0.01%) of the cells reprograms and appears as hiPSC
colonies [3,6]. These hiPSCs have the characteristics of hESCs,
including prolonged growth in culture and differentiation to
multiple tissue lineages. We tested the ability of the reporter vector
to distinguish between the terminally differentiated fibroblast
starting population and hiPSC throughout the course of
reprogramming (Fig. 3).
We first transduced HFFs derived from dermal skin with the
reporter construct to assess its characteristics (Fig. 3A). Since the
fibroblasts do not express miR-302 [19,20,21], the cells were
positive for EGFP (Fig. 3A, EGFP miR-T and EGFP miR-T/
mCherry miR-T). We observed some ablation of mCherry
expression due to low level expression of miR-155 in fibroblasts
 (Fig. 3A mCherry miR-T and EGFP miR-T/mCherry miR-
T). EGFP expression was robust and would be predicted to be
extinguished during reprogramming to hiPSC.
We transduced HFFs with the reporter vector concomitantly
with vectors expressing the four hiPSC reprogramming factors
(OCT4, SOX2, KLF4, and cMYC). Expression levels of EGFP and
mCherry were monitored over the four week course of
reprogramming to assess the activity of the reporter vector during
generation of hiPSC (Figs.3B and 3C). We observed a decrease in
EGFP expression over time, presumably reflecting the induction of
the hESC specific miRNAs, including miR-302a/d. In addition,
mCherry was partially extinguished in the HFF (Fig. 3B, Day0)
and over time we observed an increase in mCherry expression
(Fig. 3B, Days 7, 14, 21, and 28), presumably reflecting loss of miR-
155 which is expressed only in the latest stages of differentiation
and not in cells with characteristics of pluripotent stem cells .
Interestingly, our results show that many cells expressed the
hESC-specific miR-302a/d as evidenced by reduction of EGFP
expression from day 0 to day 21 (70% to 27%, Fig. 3B), but only a
small fraction of those cells actually formed hESC-like colonies. In
addition to these colonies, a greater number of colonies of
transformed phenotype, characterized by large granulated colo-
nies, were observed, similar to a previous report . Among these
colonies, approximately 90% were EGFP negative and mCherry
positive, indicating expression of miR-302a/d and no expression
of the differentiation specific miRNAs, suggesting a partially
reprogrammed state for these transformed cells as previously
reported [19,28,29]. A fewer number of transformed colonies were
positive for both EGFP and mCherry (see example Fig. 3C, left
side colony in bottom panels of Day21). Colonies with the
distinctive morphologic appearance of hiPSC, characterized by
small and tightly packed colonies with smooth borders, were
isolated from the culture on day 21–25. The frequency of hiPSC
formation induced by four factors plus the reporter vector was
approximately 0.03%. Nearly 100% of the morphologically
distinct hiPSC colonies were EGFP negative and mCherry
positive (Fig. 3D). We isolated and propagated 13 hiPSC clones
with transduction of EGFP miR-T/mCherry miR-T lentiviral
vector and 20 hiPSC clones without transduction of the reporter
We analyzed three out of 13 hiPSC clones transduced with the
reporter vector (E/m #1, E/m #5, and E/m #101) and one
hiPSC clone that was not transduced with this reporter vector
(hiPSC #19) for characteristics of pluripotent stem cells. These
hiPSC clones transduced with the reporter vector stably
maintained mCherry expression and lack of EGFP expression
for over 20 generations (Fig. 4A). Although all the clones were
EGFP negative, there were some differences in the expression
levels of mCherry. For example, E/m #101 clone had reduced
mCherry expression compared to other iPSC clones (Fig. 4A).
However, by cell surface staining, they were almost all negative for
SSEA1 and mostly positive for SSEA3, TRA-1-60, and TRA-1-81
consistent with the phenotype of hESC (Fig. 4B). Furthermore, all
clones including the remaining 10 clones transduced with the
reporter vector and 19 clones without transduction of the reporter
vector (data not shown), were Nanog positive confirmed by
indirect immunofluorescent staining (Fig. 4C). Reverse transcrip-
tase PCR (RT-PCR) analysis of these clones confirmed expression
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Figure 1. Ectopic expression of the miRNAs specifically suppresses expression of the reporter vector containing miRNA targets in
293T cells. (A) Map of transcriptional units of the reporter vector used in this study. CMVmini: CMV minimal promoter. UbiC: ubiquitin C promoter.
CCR5 target: siRNA against CCR5 target sequence (59-GAGCAAGCTCAGTTTACACC-39) . (B) 293T cells were infected with lentiviral vectors encoding
various reporters shown in (A). Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. 293T cells infected with
a lentiviral vector encoding EGFP miR-T/mCherry miR-T were super-infected by a lentiviral vector encoding either miR-302a, miR-302b, miR-302c, and
miR-302d (miR-302 a-d) or miR-155 2 days post-infection. Cells were then further cultured for 4 days and analyzed for EGFP and mCherry expression
by flow cytometry. The number (%) in each quadrant is listed on each plot.
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of hESC-specific mRNAs (Fig. 5). The clones were positive for
NANOG, REX1, LIN28, UTF1, DPPA5, hTERT, DNMT3B, OCT4
and SOX2 similar to the control hESC H1 line. In contrast, HFFs
were positive for KLF4 and cMYC as reported [1,30]. These results
indicate that ectopic expression and the expression levels of this
reporter vector do not grossly affect either hiPSC induction or the
expression of hESC-specific markers.
The reporter vector indicates differentiation of hiPSC into
EBs and into neural lineages
hESC as well as hiPSC can be differentiated in vitro into EBs
comprising the three embryonic germ layers [3,31]. We first tested
whether the hiPSC clones transduced with the reporter vector can
be differentiated into EBs and whether expression of the reporter
vector is dependent upon the miRNA expression profile (Fig. 6).
Our results show that upon differentiation of the hiPSC into EBs
for 25 days, the majority of the cells harboring the reporter vector
now expressed EGFP, presumably reflecting the loss of miR-302a/
d expression as the cells differentiated. Concomitantly, mCherry
expression was slightly reduced in the cells, reflecting expression of
one or more of the differentiation specific miRNAs miR-223, miR-
155, or miR-142-3p. However, since the EBs represent multiple
lineages of differentiated cells, we were unable to conclude which
cells and to what extent these miRNAs are being expressed.
We further tested the reporter expression in neural lineages.
Differentiation into neural lineages from hESCs and hiPSCs can
be induced by culturing them in the presence of Noggin and
transforming growth factor-b inhibitor, SB431542, both of
which are inhibitors of SMAD signaling . Under this condi-
tion, hESCs and hiPSCs organize into neural tube-like rosettes
identified as neural progenitor cells [33,34,35]. With further
differentiation, neural rosettes produce neural crest-progenitor
cells which give rise to diverse derivatives, such as the peripheral
nervous system, melanocytes, and cranial mesenchymal cells
[36,37,38,39]. We generated EBs from hiPSCs transduced with
the reporter vector and induced differentiation into neural lineages
by culturing them in the presence of Noggin and SB431542. The
differentiation status of the EBs into neural lineages was monitored
by the expression of SOX1, SOX3 and PAX6, markers of
neuroectodermal differentiation , whereas the undifferentiated
iPSCs were monitored by the expression of DNMT3B, REX1 and
endogenous OCT4 which are assumed to be expressed in fully-
reprogrammed hiPSCs . One month after induction of
differentiation, DNMT3B, REX1 and endogenous OCT4 were
downregulated, whereas SOX1, SOX3 and PAX6 were upregulated
in the differentiated population compared to the undifferentiated
population (Fig. 7A). Neural tube-like rosettes were observed
throughout the culture plate, most of which were both EGFP and
mCherry positive (Fig. 7B), indicating that they do not express any
miRNA that recognizes the targets in the EGFP miR-T/mCherry
miR-T reporter vector. We observed dark pigmented melanocyte-
like cells surrounding neural tube-like structures as previously
reported  (Fig. 7C). Interestingly, the fluorescence of EGFP
and mCherry allowed a clear demarcation of boundaries between
apparently different structures within the neural tube-like structure
and the surrounding dark pigmented area. The cells located
around the edge of the neural tube-like structure were mostly
double positive for EGFP and mCherry. In contrast, the cells
located at the inner side of the neural tube-like structure were
EGFP-single positive. The majority of melanocyte-like cells
Figure 2. The reporter vectors containing miRNA targets show the lineage-specific expression. (A) U937 cells were infected with
lentiviral vectors encoding various reporters showed in Fig. 1 A. Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-
infection. (B and C) CEM and Ramos cells (B) and CD34+ HPSCs derived from 3 independent donors (C) were infected with a lentiviral vector encoding
EGFP miR-T/mCherry miR-T. Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. (D) hESCs (H1) were
infected with a lentiviral vector encoding EGFP miR-T/mCherry miR-T. Single cell clone was isolated by culturing transduced cells in the presence of
10 mM Y27632 for 14 days. Expression levels of EGFP and mCherry were analyzed by fluorescence microscopy and by flow cytometry. The number (%)
in each quadrant is listed on each plot.
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surrounding neural tube-like structures were mCherry-single
positive. Expression of the specific miRNAs tested here is
unknown within the neural tube-like structures, however there
appears to be expression of one or more differentiation specific
miRNAs in the interior whereas miR-302a/d appears to be
expressed in the surrounding dark pigmented area. Therefore,
these results demonstrate the use of the reporter vector to provide
real-time observation of hiPSC differentiation.
Our results demonstrate the utility of a miRNA dependent
reporter vector system to monitor reprogramming of human
fibroblasts to hiPSC and subsequent differentiation of the hiPSC
into multi-lineage embryoid bodies. Loss of EGFP expression that
is dependent upon expression of the hESC-specific miR-302a/d, is
highly specific to the pluripotent cells. There is robust EGFP
expression observed in HFFs and significant levels in differentiated
EBs and neural-lineage cells, but no detectable expression in hESC
or the hiPSC. In contrast, the hiPSCs express mCherry at high
levels similar to hESCs, reflecting the absence of miR-223, miR-
155, or miR-142-3p in the hiPSCs. Differentiated cells, including
HFF and some cells in EBs express some of these miRNAs,
resulting in downregulation of mCherry expression.
The miRNA dependent reporter vector can be used to study
stages of reprogramming and differentiation since it allows for
assay and/or isolation of cells based upon fluorescence intensity. It
is noteworthy that the hESC-specific miRNAs, miR-302a/d, are
expressed in a significant proportion of the cells during the process
of reprogramming. Consistent with previous studies [19,42], these
results indicate that expression of hESC-specific factors, including
miRNAs, occurs in the majority of cells as a result of the ectopic
introduction of reprogramming factors, but a much smaller
percentage of those cells reprogram properly to form hiPSC. In
our studies, more than 50% of the cells express miR-302a/d based
upon loss of EGFP during reprogramming at day 14, but only
0.03% of the starting HFF result in hiPSC. These results indicated
that miR-302a/d is not sufficient for reprogramming and therefore
cannot be used solely as a reporter to identify true hiPSC. Future
more selective choice of miRNAs in combination with miR-302 a/
d may be utilized to fractionate hiPSC from partially repro-
grammed cells based upon their expression profile of fluorescence
and to further investigate reprogramming mechanisms. Similarly,
differentiated cells can be fractionated for further investigation
based upon the lineage restricted expression of specific miRNAs.
Ectopic expression of miRNA target sequences may be of
concern in potentially interfering with the endogenous miRNA
Figure 3. Reprogramming state specific expression of the reporter vector containing miRNA targets during hiPSC formation from
human fetal fibroblasts (HFFs). (A) HFFs were infected with lentiviral vectors encoding various reporters shown in Fig. 1A. Expression levels of
EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. (B and C) HFFs were infected with lentiviral vectors encoding 4 different
hiPSC factors (OCT4, SOX2, KLF4, and cMYC) and with or without a lentiviral vector encoding EGFP miR-T/mCherry miR-T. Cells were then cultured for 3
days and replated on irradiated mouse embryonic fibroblast (iMEF) feeder cells at 56104cells/60 mm plate. Expression levels of EGFP and mCherry
were analyzed by flow cytometry on days 0, 7, 14, 21, and 28 (B), and by fluorescence microscopy on days 0, 7, 14, and 21 (C). iMEF feeder cells were
labeled with PE-Cy7 conjugated mouse CD29 antibody and excluded from the flow cytometry analysis in (B). (D) hiPSC colonies expressing mCherry
were picked on Matrigel coated plate on days 21–25 and propagated in mTeSR medium. Expression levels of EGFP and mCherry were analyzed by
fluorescence microscopy and by flow cytometry. The number (%) in each quadrant is listed on each plot. hiPSC #1: hiPSC clone without transduction
of EGFP miR-T/mCherry miR-T reporter vector. E/m#8: hiPSC clone with transduction of EGFP miR-T/mCherry miR-T reporter vector.
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Figure 4. Transduction of the reporter vector containing miRNA targets does not grossly affect expression of hESC-specific
markers. (A and B) Single-cell suspensions of hESC (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/
m#5, and E/m#101) or untransduced (hiPSC#19) were analyzed for the expression of EGFP and mCherry (A) and that of hESC-specific markers
(SSEA1, SSEA3, TRA1-60, and TRA-1-81) (B) by flow cytometry. The number (%) in each quadrant is listed on each plot. (C) hESCs (H1), hiPSCs
transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were plated on
poly-L-lysine and Matrigel coated glass coverslips and expanded for a week. Cells were then fixed with 1% formaldehyde, permeabilized with 0.2%
Triton X-100 for 5 min on ice, and stained with anti-Nanog antibody and DyLight488 conjugated anti-rabbit IgGs. 7-amino-actinomycin D (7-AAD)
was used for nuclear staining.
Figure 5. Molecular characterization of hiPSCs transduced with the reporter vector containing miRNA targets. Total RNA was isolated
using QIAGEN’s RNeasy Mini kit from HFFs transduced with (4Fs/HFF) or without 4 reprogramming factors (HFF), hESCs (H1), and 4 different hiPSC
clones transduced with (E/m#1, E/m#5, and E/m#101) or without (hiPSC#19) the reporter vector encoding EGFP miR-T/mCherry miR-T. Total RNA
(250 ng) was reverse-transcribed using QIAGEN’s Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME’s
HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used
as an internal control.
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machinery. Other investigators have not noted alterations in
cellular miRNA metabolism with relatively high copy number
. Nevertheless, the use of vectors and coding miRNA targets
should ideally be utilized at low copy number per cell and should
be monitored for any effects upon cellular differentiation and/or
function. In our case, all hiPSC clones transduced with the
reporter vector were well-maintained under hESC culture
conditions similar to non-transduced hESC or hiPSC. These
hiPSCs expressed multiple hESC-specific mRNAs and antigens,
like Nanog, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. Further-
more, these cells were able to differentiate into neural cells and
beating cardiomyocytes (data not shown), identically to control
cells, indicating that the expression of miRNA targets did not
grossly affect their differentiation abilities.
Interestingly, one of the hiPSC clones (E/m #101) had reduced
mCherry expression compared to two other hiPSC clones
transduced with the reporter vector (Fig. 4A). Although this clone
resembled the other hiPSC clones and hESCs in regards to
expression of hESC-specific markers, SSEA3 expression was
slightly lower than that of other clones or hESCs (Fig. 4B).
Figure 6. hiPSCs transduced with the reporter vector containing miRNA targets show differentiation-specific reporter expression in
EBs. hESC (H1) and 4 different hiPSC clones (E/m#1, E/m#5, E/m#101, and hiPSC#19) were differentiated into EBs and maintained 25 days in IMDM
containing 10% FBS. EBs were then dissociated with 0.25% trypsin/EDTA and the reporter expression was analyzed by flow cytometry. Histograms
filled with black are undifferentiated controls. Histograms filled with blue (EGFP) and pink (mCherry) are differentiated cells, respectively. The
numbers indicated in histogram show percentage of positive cells (EGFP) and negative cells (mCherry). MFI: mean fluorescence intensity. U: MFI of
undifferentiated cells. D: MFI of differentiated cells.
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Furthermore, this clone had some morphological differences - the
colonies of this clone were flatter compared to those of hESCs or
hiPSCs and the boundaries between cells which are indistinguish-
able for hESCs and other hiPSCs all over the colonies were
distinct especially at the outer edge of the colonies (data not
shown). Growth and propagation of this clone also resulted in a
greater level of spontaneous differentiation than that of the other
clones. Moreover, this clone showed a weaker shift of EGFP and
mCherry expression upon differentiation into EBs (Fig. 6). These
results suggest that functional assays based upon endogenous
miRNA expression may be another means to assess properties of
The development of this vector adds to the tools available to
monitor hiPSC generation and subsequent differentiation of the
hiPSC into different lineages. In addition to taking advantage of
differential miRNA expression, other investigators have utilized
differential promoters/enhancer expression in different lineages
[43,44]. Whether one utilizes miRNA or promoter/enhancer
expression of reporter constructs for live-cell tracking of repro-
gramming and differentiation will depend on the particular
experimental setting and application.
The work presented here indicates the potential great utility and
flexibility of miRNA-regulatable lentiviral vectors to monitor
various stages of reprogramming and the subsequent differentia-
tion into lineage specific cells and tissues. For example, miR-223,
miR-142-3p, and miR-155 are enriched primarily in cells of
hematopoietic origin [22,23]. The expression of mCherry
conjugated with these miR targets was strongly suppressed in
CD34+ HPSCs (Fig. 2 C). These results suggest that our reporter
vector can be used to monitor the differentiation into CD34+
HPSCs from hESCs/hiPSCs, and to isolate low frequency
populations of cells based upon the differential expression of
EGFP and mCherry. Further selective use of miRNA targets
would be predicted to preferentially suppress expression in specific
lineages or specific stages of differentiation within a given lineage.
Thus, the activity of the reporter vector can be readily modulated
depending upon the miRNA target sequence incorporated and
such a vector can be used for studies involving specific
differentiation lineages where the miRNA expression profile is
known. Conversely, a vector with a specific target sequence can be
used to determine the temporal and lineage specific expression of
the corresponding miRNAs during differentiation.
Figure 7. hiPSCs transduced with the reporter vector containing miRNA targets indicate differentiation-specific reporter
expression in neural lineages. EBs generated from pooled hiPSCs were differentiated into neural lineages using Noggin and SB431542. EBs were
then transferred onto fibronectin coated 6-well plates and further differentiated in N2 medium. (A) Total RNA was isolated using QIAGEN’s RNeasy
Mini kit from the hiPSCs on day 0 (undifferentiated population: Undiff.) and day 30 (differentiated population: Diff.) after induction of differentiation.
Total RNA (250 ng) was reverse-transcribed using QIAGEN’s Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-
PRIME’s HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. GAPDH was used as an internal control. (B) Neural tube-
like rosettes observed after the differentiation. (C) Dark pigmented melanocyte-like cells surrounding neural tube-like structures.
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Materials and Methods
Construction of lentiviral vector
For construction of the reporter vector, whole sequences of
hESC specific-miRNA targets (CCCGGGCGAGCAAGCTCA-
TGGAAGCACTTAGATATCGTCGAC) and differentiated cell
specific-miRNA targets (CCCGGGTCGAATTCGGTACCAG-
GCCC) were synthesized by Genescript (Piscataway, NJ). The
synthesized fragment of differentiated cell specific-miRNA targets
was conjugated to mCherry coding sequence amplified by PCR
from pmCherry (Clontech Laboratories, Inc. Mountain View, CA)
with primers (sense: ACGCACCGGTGGATCCAAGCTTGC-
CACCATGGTGAGCAAGGGCGAGGA and reverse: CTGC-
G) and exchanged with EGFP coding sequence in FG12 lentiviral
vector  (mCherry-T/FG12). For bidirectional expression from
one promoter , EGFP coding sequence was amplified by PCR
from the pEGFP-N1 (Clontech Laboratories) with primers (sense:
CTCGTCCATG), and cloned into a pAAV-MCS vector (Strata-
gene, La Jolla, CA) (pAAV-EGFP). The synthesized fragment of
hESC specific-miRNA targets was inserted into the pAAV-EGFP
between stop codon of EGFP and hGH poly A signal. The
fragment containing CMV minimal promoter, b-globin intron,
EGFP, hESC specific-miRNA targets, and hGH poly A signal was
amplified by PCR with primers (sense: GTACTCTCGAGCCC-
CATTGACGCAAATGGGCGGTAGG and reverse: CTCGTC-
TAGAAGGACAGGGAAGGGAGCAGTGGT) and cloned into
the FG12 lentiviral vector deleted EGFP (EGFP-T/FG12) or into
the mCherry-T/FG12 (EGFP-T/mCherry-T/FG12).
For reprogramming of HFFs, we substituted the ubiqutin C
promoter of FG12 lentiviral vector with the RhMLV promoter
(FRh11). The RhMLV promoter is derived from the long
terminal repeat (LTR) region of Moloney murine leukemia virus
(MLV) in the serum of one rhesus macaque monkey that
developed T-cell lymphoma following autologous transplantation
[47,48]. This promoter shows around 5–10 fold stronger
promoter activity in HFFs compared to that of the parental
MLV LTR (unpublished observation). Furthermore, this pro-
moter activity is strongly silenced in hESC or hiPSC as well as
that of the parental MLV LTR (unpublished observation).
cDNAs encoding human OCT4, SOX2, KLF4, and cMYC
(Addgene) were substituted with EGFP coding sequence in the
FRh11. The infectious titer was determined in 293T cells by
infecting with the FRh11 encoding EGFP in the presence of
8 mg/ml polybrene. Reporter gene expression was monitored by
For ectopic expression of miRNAs, we purchased miRNA
expressing lentiviral vectors for miR-302a, miR-302b, miR-302c, and
miR302-d (PMIRH302abcdPA-2) and miR-155 (PMIRH155PA-1)
from System biosciences (Mountain View, CA). CopGFP sequence
was eliminated from the vector.
293T , Ramos , U937  and CEM  cells were
maintained with Dulbecco’s Modified Eagle Medium (DMEM)
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine
serum (FBS) (Omega Scientific, Tarzana, CA) and 2 mM
GlutaMax (Invitrogen). All cells were incubated at 37uC in 5%
hESCs (H1 clone)  and hiPSCs were maintained in
mTeSRTM1 (StemCell Technologies, Inc., Vancouver, Canada)
on hESC-qualified MatrigelTM(BD Biosciences, San Jose, CA)
coated plates. Differentiated colonies were removed daily through
aspiration, and the medium was replaced on a daily basis. Cells
were passed upon confluency (typically 7–10 days), using 1 mg/ml
dispase (StemCell Technologies, Inc.). All work with hESC and
hiPSC was approved by the UCLA Embryonic Stem Cell
Research Oversight committee.
HFFs were isolated from the skin of 16 week-old fetus with
DMEM supplemented10% FBS and 2 mM Glutamax (fibroblast
medium) as reported previously .
CD34+ HPSCs were prepared from the liver of 16 weeks-old
fetus as previously described .
Lentiviral vector stocks were generated using a vector plasmid, a
packaging plasmid pCMV R8.2 DVpr, and a VSV-G envelope
protein-coding plasmid by calcium phosphate-mediated transient
transfection as previously described . After 48 and 72 hr,
lentiviral vector particles were harvested and concentrated by
ultracentrifugation and resuspended in a 150-fold lower volume of
Hanks’ balanced salt solutions and stored at 280uC. The viral titer
was measured by anti-p24 Gag ELISA.
Induction of hiPSC
The day before lentiviral vector transduction, HFFs (passage 1–
3) were seeded at 56104cells per well of 6-well plates and infected
with vectors encoding each reprogramming factor (OCT4, SOX2,
KLF4, and cMYC) with or without a lentiviral vector encoding
EGFP-T/mCherry-T at 300 ng (around multiplicity of infection
of 3–5) of p24 per each virus. The cells were cultured for 3 days in
fibroblast medium and replated at 56104cells per 60 mm dish on
irradiated mouse embryonic fibroblast (iMEF) feeder cells. On the
next day, the medium was replaced with KO-DMEM (Invitrogen)
supplemented with 20% Knockout Serum Replacer (KSR,
Invitrogen), 2 mM Glutamax (Invitrogen), 0.1 mM non-essential
amino acids (Invitrogen), 0.1 mM b-mercaptoethanol (Sigma-
Aldrich, St. Louis, MO), and 50 ng/ml of recombinant human
basic fibroblast growth factor (Invitrogen) (hiPSC medium). The
medium was changed on a daily basis. To increase a reprogram-
ming efficiency, the cells were treated with 0.5 mM valproic acid
(VPA; Sigma-Aldrich) and 10 mM Y27632 (Tocris Bioscience,
Ellisville, MO) for first 14 days [57,58]. On day 21–25, hiPSC
colonies were identified based upon hESC-like morphology as
described previously  and picked out into wells of 48-well plates
coated with Matrigel and expanded in mTeSR medium.
Reprogramming efficiency was calculated as the number of hiPSC
colonies formed per number of seeded HFFs with transduction of
Live Cell Monitoring of iPSC
PLoS ONE | www.plosone.org9 July 2010 | Volume 5 | Issue 7 | e11834
Embryoid body formation
Size-controlled EBs (3000 hESCs/EB) were formed using
AggreWellTM400 plates (StemCell Technologies, Inc.) following
the manufacturer’s protocol. Briefly, hESCs and hiPSCs were
incubated with 10 mM Y-27632 for 24 hrs before EB formation.
Cells were harvested with Accutase (Innovative Cell Technologies,
San Diego, CA) as a single-cell suspension and used for EB
formation. EBs were harvested into ultra low attachment plates
(Corning, Corning, NY) and maintained in Iscove’s Modified
Dulbecco’s Medium (IMDM, Sigma-Aldrich) containing 10%
FBS, 2 mM Glutamax, and 0.1 mM b-mercaptoethanol for
differentiation into EBs. The medium was changed every 3 days.
Differentiation into neural lineages
Induction of differentiation into neural lineages was performed
as previously described with some modifications . Briefly, EBs
were maintained in the hiPSC medium containing 500 ng/ml of
Noggin (R&D) and 10 mM SB431542 in ultra low attachment
plates. SB432542 was withdrawn on day 5 and increasing amounts
of N2 medium (Stem cell technologies) (25%, 50%, 75%) was
added to the hiPSC medium every 2 days while maintaining
500 ng/ml of Noggin. Upon day 12 of differentiation, EBs were
transferred onto fibronectin-coated 6-well plates and maintained
in 100% N2 medium without Noggin for further 18 days.
For detection of EGFP and mCherry expression, single-cell
suspensions from 293T, HFF, hESC, and hiPSC were prepared
using 0.25% trypsin-EDTA and collected in FACS buffer (2%
FBS and 0.01% sodium azide in PBS). U937, Ramos and CEM
cells were also collected in FACS buffer. For detection of hESC-
specific markers, hESCs and hiPSCs were dissociated with 0.25%
trypsin-EDTA into a single cell suspension. Cells were adjusted to
100,000 per sample in 100 ml of FACS buffer and then labeled
with monoclonal antibodies conjugated with a fluorescent dye
[SSEA1 and SSEA3: Alexa488 purchased from eBioscience (San
Diego, CA); TRA-1-60 and TRA-1-81: PE purchased from
BioLegend (San Diego, CA)]. For detection of iMEFs, cells were
stained with an antibody specific with mouse CD29 conjugated
with PE-Cy7 (eBioscience). Data were collected on a Cytomics
FC500 (Beckman Coulter, Fullerton, CA) and analyzed using FCS
express (De Novo Software, Los Angeles, CA).
hESC and hiPSC colonies were grown on poly-L-lysine and
Matrigel coated glass coverslips. Cells were fixed with 1.0%
formaldehyde/PBS and permeabilized with 0.2% Triton-X 100
for 5 min on ice. Cells were then incubated with anti-human
Nanog antibody (Abcam Inc., Cambridge, MA) and subsequently
Table1. Primers for RT-PCR.
Genes Forward (59 to 39) Reverse (59 to 39) Size (bp)
NANOG CAGCCCTGATTCTTCCACCAGTCCC GGAAGGTTCCCAGTCGGGTTCACC 390
REX1 CAGATCCTAAACAGCTCGCAGAAT GCGTACGCAAATTAAAGTCCAGA305
UTF1 CCGTCGCTGAACACCGCCCTGCTG CGCGCTGCCCAGAATGAAGCCCAC147
hTERT TGTGCACCAACATCTACAAGGCGTTCTTGGCTTTCAGGAT 165
DNMT3B ATAAGTCGAAGGTGCGTCGT GGCAACATCTGAAGCCATTT121
KLF4 GATGAACTGACCAGGCACTA GTGGGTCATATCCACTGTCT144
GAPDHGAAGGTGAAGGTCGGAGT GAAGATGGTGATGGGATTTCC 225
KLF4TCTCAAGGCACACCTGCGAA TAGTGCCTGGTCAGTTCATC 104
KLF4 AGCATTTTCCAGGTCGGACCACC GGAGCAACATAGTTAAGAATACCAGTC328
Live Cell Monitoring of iPSC
PLoS ONE | www.plosone.org10 July 2010 | Volume 5 | Issue 7 | e11834
with DyLight488 conjugated donkey anti-rabbit IgG (BioLegend)
and 7-amino-actinomycin D (7-AAD) (Invitrogen) for nuclear
staining. After washing, cells were visualized with a LEICA DM
IRB (Leica Microsystems Inc. Bannockburn, IL) equipped with a
SPOT camera and software (Diagnostic Instruments, Sterling
RNA extraction and RT-PCR
RNA extraction from hESC and hiPSC was performed using
QIAGEN’s RNeasy Mini kit following the manufacture’s protocol
(QIAGEN, Valencia, CA). Total RNA (250 ng) was reverse-
transcribed using QIAGEN’s Omniscript RT-kit with a 0.5 ng/ml
oligo dT primer (Invitrogen) in 20 ml reaction. PCR was
performed with the HotMaster Taq DNA polymerase (5 PRIME,
Inc., Gaithersburg, MD), using 0.5 ml of cDNA template and
primers at a concentration of 3 pmol/ml. Five ml of PCR products
was loaded in a 2% agarose gel containing ethidium bromide. All
primer sequences were listed in Table 1.
We are grateful to Dr. Betty Poon for proofreading the manuscript and to
Si-Hua Mao for technical help. We also greatly appreciate the generous
help from Rina Lee for typing and editing this manuscript.
Conceived and designed the experiments: MK ISYC. Performed the
experiments: MK ML SL YN. Analyzed the data: MK. Wrote the paper:
1. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, et al. (2008)
Generation of human induced pluripotent stem cells from dermal fibroblasts.
Proc Natl Acad Sci U S A 105: 2883–2888.
2. Park IH, Lerou PH, Zhao R, Huo H, Daley GQ (2008) Generation of human-
induced pluripotent stem cells. Nat Protoc 3: 1180–1186.
3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007)
Induction of pluripotent stem cells from adult human fibroblasts by defined
factors. Cell 131: 861–872.
4. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:
5. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al. (2007) In vitro
reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:
6. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al.
(2007) Induced pluripotent stem cell lines derived from human somatic cells.
Science 318: 1917–1920.
7. Lee G, Papapetrou EP, Kim H, Chambers SM, Tomishima MJ, et al. (2009)
Modelling pathogenesis and treatment of familial dysautonomia using patient-
specific iPSCs. Nature 461: 402–406.
8. Raya A, Rodriguez-Piza I, Guenechea G, Vassena R, Navarro S, et al. (2009)
Disease-corrected haematopoietic progenitors from Fanconi anaemia induced
pluripotent stem cells. Nature 460: 53–59.
9. Ye L, Chang JC, Lin C, Sun X, Yu J, et al. (2009) Induced pluripotent stem cells
offer new approach to therapy in thalassemia and sickle cell anemia and option
in prenatal diagnosis in genetic diseases. Proc Natl Acad Sci U S A 106:
10. He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene
regulation. Nat Rev Genet 5: 522–531.
11. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-
transcriptional regulation by microRNAs: are the answers in sight? Nat Rev
Genet 9: 102–114.
12. Gangaraju VK, Lin H (2009) MicroRNAs: key regulators of stem cells. Nat Rev
Mol Cell Biol 10: 116–125.
13. Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, et al. (2007) A
microRNA-regulated lentiviral vector mediates stable correction of hemophilia
B mice. Blood 110: 4144–4152.
14. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, et al. (2007) A
mammalian microRNA expression atlas based on small RNA library
sequencing. Cell 129: 1401–1414.
15. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell
16. Brown BD, Sitia G, Annoni A, Hauben E, Sergi LS, et al. (2007) In vivo
administration of lentiviral vectors triggers a type I interferon response that
restricts hepatocyte gene transfer and promotes vector clearance. Blood 109:
17. Brown BD, Venneri MA, Zingale A, Sergi Sergi L, Naldini L (2006) Endogenous
microRNA regulation suppresses transgene expression in hematopoietic lineages
and enables stable gene transfer. Nat Med 12: 585–591.
18. Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, et al. (2007)
Endogenous microRNA can be broadly exploited to regulate transgene
expression according to tissue, lineage and differentiation state. Nat Biotechnol
19. Chin MH, Mason MJ, Xie W, Volinia S, Singer M, et al. (2009) Induced
pluripotent stem cells and embryonic stem cells are distinguished by gene
expression signatures. Cell Stem Cell 5: 111–123.
20. Suh MR, Lee Y, Kim JY, Kim SK, Moon SH, et al. (2004) Human embryonic
stem cells express a unique set of microRNAs. Dev Biol 270: 488–498.
21. Wilson KD, Venkatasubrahmanyam S, Jia F, Sun N, Butte AJ, et al. (2009)
MicroRNA profiling of human-induced pluripotent stem cells. Stem Cells Dev
22. Ramkissoon SH, Mainwaring LA, Ogasawara Y, Keyvanfar K, McCoy JP, Jr.,
et al. (2006) Hematopoietic-specific microRNA expression in human cells. Leuk
Res 30: 643–647.
23. Chen CZ, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate
hematopoietic lineage differentiation. Science 303: 83–86.
24. Eis PS, Tam W, Sun L, Chadburn A, Li Z, et al. (2005) Accumulation of miR-
155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci U S A 102:
25. Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, et al. (2005) BIC and
miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large
B cell lymphomas. J Pathol 207: 243–249.
26. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, et al. (2005) MicroRNA
gene expression deregulation in human breast cancer. Cancer Res 65:
27. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, et al. (2006) A microRNA
expression signature of human solid tumors defines cancer gene targets. Proc
Natl Acad Sci U S A 103: 2257–2261.
28. Marion RM, Strati K, Li H, Tejera A, Schoeftner S, et al. (2009) Telomeres
acquire embryonic stem cell characteristics in induced pluripotent stem cells.
Cell Stem Cell 4: 141–154.
29. Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, et al. (2008) Dissecting
direct reprogramming through integrative genomic analysis. Nature 454: 49–55.
30. Zhao Y, Yin X, Qin H, Zhu F, Liu H, et al. (2008) Two supporting factors
greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3:
31. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, et al. (2000)
Differentiation of human embryonic stem cells into embryoid bodies
compromising the three embryonic germ layers. Mol Med 6: 88–95.
32. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, et al.
(2009) Highly efficient neural conversion of human ES and iPS cells by dual
inhibition of SMAD signaling. Nat Biotechnol 27: 275–280.
33. Li XJ, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, et al. (2005)
Specification of motoneurons from human embryonic stem cells. Nat Biotechnol
34. Shin S, Mitalipova M, Noggle S, Tibbitts D, Venable A, et al. (2006) Long-term
proliferation of human embryonic stem cell-derived neuroepithelial cells using
defined adherent culture conditions. Stem Cells 24: 125–138.
35. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro
differentiation of transplantable neural precursors from human embryonic stem
cells. Nat Biotechnol 19: 1129–1133.
36. Huang D, Gao Q, Guo L, Zhang C, Jiang W, et al. (2009) Isolation and
identification of cancer stem-like cells in esophageal carcinoma cell lines. Stem
Cells Dev 18: 465–473.
37. Lee G, Kim H, Elkabetz Y, Al Shamy G, Panagiotakos G, et al. (2007) Isolation
and directed differentiation of neural crest stem cells derived from human
embryonic stem cells. Nat Biotechnol 25: 1468–1475.
38. Motohashi T, Aoki H, Chiba K, Yoshimura N, Kunisada T (2007) Multipotent
cell fate of neural crest-like cells derived from embryonic stem cells. Stem Cells
39. Thomas S, Thomas M, Wincker P, Babarit C, Xu P, et al. (2008) Human neural
crest cells display molecular and phenotypic hallmarks of stem cells. Hum Mol
Genet 17: 3411–3425.
40. Pankratz MT, Li XJ, Lavaute TM, Lyons EA, Chen X, et al. (2007) Directed
neural differentiation of human embryonic stem cells via an obligated primitive
anterior stage. Stem Cells 25: 1511–1520.
41. Chan EM, Ratanasirintrawoot S, Park IH, Manos PD, Loh YH, et al. (2009)
Live cell imaging distinguishes bona fide human iPS cells from partially
reprogrammed cells. Nat Biotechnol.
42. Sridharan R, Tchieu J, Mason MJ, Yachechko R, Kuoy E, et al. (2009) Role of
the murine reprogramming factors in the induction of pluripotency. Cell 136:
Live Cell Monitoring of iPSC
PLoS ONE | www.plosone.org 11July 2010 | Volume 5 | Issue 7 | e11834
43. Hotta A, Cheung AY, Farra N, Vijayaragavan K, Seguin CA, et al. (2009) Download full-text
Isolation of human iPS cells using EOS lentiviral vectors to select for
pluripotency. Nat Methods 6: 370–376.
44. Kita-Matsuo H, Barcova M, Prigozhina N, Salomonis N, Wei K, et al. (2009)
Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent
tracking and drug resistance selection of cardiomyocytes. PLoS One 4: e5046.
45. An DS, Donahue RE, Kamata M, Poon B, Metzger M, et al. (2007) Stable
reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-
human primates. Proc Natl Acad Sci U S A 104: 13110–13115.
46. Amendola M, Venneri MA, Biffi A, Vigna E, Naldini L (2005) Coordinate dual-
gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters.
Nat Biotechnol 23: 108–116.
47. Kung SK, An DS, Chen IS (2000) A murine leukemia virus (MuLV) long
terminal repeat derived from rhesus macaques in the context of a lentivirus
vector and MuLV gag sequence results in high-level gene expression in human T
lymphocytes. J Virol 74: 3668–3681.
48. Vanin EF, Kaloss M, Broscius C, Nienhuis AW (1994) Characterization of
replication-competent retroviruses from nonhuman primates with virus-induced
T-cell lymphomas and observations regarding the mechanism of oncogenesis.
J Virol 68: 4241–4250.
49. Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer
helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90:
50. Klein G, Giovanella B, Westman A, Stehlin JS, Mumford D (1975) An EBV-
genome-negative cell line established from an American Burkitt lymphoma;
receptor characteristics. EBV infectibility and permanent conversion into EBV-
positive sublines by in vitro infection. Intervirology 5: 319–334.
51. Ralph P, Moore MA, Nilsson K (1976) Lysozyme synthesis by established
human and murine histiocytic lymphoma cell lines. J Exp Med 143: 1528–1533.
52. Foley GE, Lazarus H, Farber S, Uzman BG, Boone BA, et al. (1965) Continuous
Culture of Human Lymphoblasts from Peripheral Blood of a Child with Acute
Leukemia. Cancer 18: 522–529.
53. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al.
(1998) Embryonic stem cell lines derived from human blastocysts. Science 282:
54. Normand J, Karasek MA (1995) A method for the isolation and serial
propagation of keratinocytes, endothelial cells, and fibroblasts from a single
punch biopsy of human skin. In Vitro Cell Dev Biol Anim 31: 447–455.
55. Liang M, Kamata M, Chen NK, Pariente N, An DS, et al. (2010) Inhibition of
HIV-1 infection by a unique shRNA to CCR5 delivered into macrophages
through hematopoietic progenitor cell transduction. Journal of Gene Medicine:
56. Kamata M, Nagaoka Y, Chen IS (2009) Reassessing the role of APOBEC3G in
human immunodeficiency virus type 1 infection of quiescent CD4+ T-cells.
PLoS Pathog 5: e1000342.
57. Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, et al. (2008)
Induction of pluripotent stem cells by defined factors is greatly improved by
small-molecule compounds. Nat Biotechnol 26: 795–797.
58. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, et al. (2008) Reprogramming
of human somatic cells to pluripotency with defined factors. Nature 451:
59. Shimizu S, Kamata M, Kittipongdaja P, Chen KN, Kim S, et al. (2009)
Characterization of a potent non-cytotoxic shRNA directed to the HIV-1 co-
receptor CCR5. Genet Vaccines Ther 7: 8.
Live Cell Monitoring of iPSC
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