Enhanced Generation of Induced Pluripotent Stem Cells
from a Subpopulation of Human Fibroblasts
James A. Byrne, Ha Nam Nguyen, Renee A. Reijo Pera*
Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology,
Stanford University, Palo Alto, California, United States of America
Background: The derivation of induced pluripotent stem cells (iPSCs) provides new possibilities for basic research and novel
cell-based therapies. Limitations, however, include our current lack of understanding regarding the underlying mechanisms
and the inefficiency of reprogramming.
Methodology/Principal Findings: Here, we report identification and isolation of a subpopulation of human dermal
fibroblasts that express the pluripotency marker stage specific embryonic antigen 3 (SSEA3). Fibroblasts that expressed
SSEA3 demonstrated an enhanced iPSC generation efficiency, while no iPSC derivation was obtained from the fibroblasts
that did not express SSEA3. Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3
expressing fibroblasts, suggesting a possible mechanistic explanation for the differential reprogramming.
Conclusions/Significance: To our knowledge, this study is the first to identify a pluripotency marker in a heterogeneous
population of human dermal fibroblasts, to isolate a subpopulation of cells that have a significantly increased propensity to
reprogram to pluripotency and to identify a possible mechanism to explain this differential reprogramming. This discovery
provides a method to significantly increase the efficiency of reprogramming, enhancing the feasibility of the potential
applications based on this technology, and a tool for basic research studies to understand the underlying reprogramming
Citation: Byrne JA, Nguyen HN, Reijo Pera RA (2009) Enhanced Generation of Induced Pluripotent Stem Cells from a Subpopulation of Human Fibroblasts. PLoS
ONE 4(9): e7118. doi:10.1371/journal.pone.0007118
Editor: Shukti Chakravarti, Johns Hopkins University, United States of America
Received July 31, 2009; Accepted August 21, 2009; Published September 23, 2009
Copyright: ? 2009 Byrne 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: All funding for this study was provided by California Institute of Regenerative Medicine (CIRM, http://www.cirm.ca.gov) grant #RL1-00670-1 to R. Reijo
Pera. 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: email@example.com
The generation of pluripotent stem cells that are genetically
identical to an individual provides unique opportunities for basic
research and for potential immunologically-compatible novel cell-
based therapies . Methods to reprogram primate somatic cells
to a pluripotent state include somatic cell nuclear transfer ,
somatic cell fusion with pluripotent stem cells  and direct
reprogramming to produce induced pluripotent stem cells (iPSCs)
[4–10]. These methodologies, however, are characterized by a low
reprogramming efficiency and a lack of knowledge regarding the
underlying mechanisms. While it has been demonstrated previ-
ously that more differentiated cells demonstrate a lower repro-
gramming efficiency  and different somatic cell types possess
differential reprogramming ability [12,13], no study to date, to our
knowledge, has identified subpopulations of cells within a primary
cell population possessing differential reprogramming potential. If
such subpopulations exist and can be identified and isolated, they
provide a method to significantly increase the efficiency of
reprogramming, enhancing the feasibility of the potential
applications based on this technology , and a tool for basic
research studies to understand the underlying reprogramming
We derived a fibroblast line from a skin biopsy from a healthy
adult male (HUF1) (Figure 1A) and used immunocytochemistry to
characterize the expression of cell surface markers commonly
expressed on pluripotent stem cells (Figure 1B, C and D).
Unexpectedly, we observed that, even prior to reprogramming,
the HUF1 line possessed cells that demonstrated heterogeneous
expression of stage specific embryonic antigen 3 (SSEA3;
Figure 1B). SSEA3 is a cell surface glycosphingolipid considered
an embryonic/pluripotency marker [14,15]. Overlaying phase
contrast and SSEA3 immunofluorescence images revealed that the
SSEA3 expression was detected across the entire cell surface
(Figure 1E) and using confocal microscopy we observed that the
SSEA3 expression was primarily localized to the cellular
membrane (Figure 1F). Additional small and localized regions of
SSEA3 fluorescence were also detected around the peri-nuclear
region, possibly reflecting the intracellular processing and
packaging of SSEA3 on peri-nuclear endoplasmic reticulum
and/or Golgi bodies (Figure 1F). Notably, in positive controls,
strong cell surface expression of SSEA3 was observed in H9
human embryonic stem cells (hESCs)(Figure 1G) and no
expression was observed in the negative controls (Figure 1H).
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We next examined whether the expression of SSEA3 in a subset
of fibroblasts was specific to HUF1 or a more general observation.
Eight additional primary adult human fibroblast lines were derived
from skin biopsies and immunoassayed. We observed that all eight
lines contained a subpopulation of cells that were positive for
SSEA3 (Figure 2A). Fluorescence activated cell sorting (FACS)
analysis of HUF1 cells stained with the SSEA3/488 antibody
complex, revealed a larger subpopulation of cells with little or no
SSEA3 expression and a smaller subpopulation with detectable
SSEA3 expression (Figure 2B). Subsequently, we isolated (through
FACS) and cultured the top 10% and bottom 10% of the SSEA3/
488 fluorescing cells as our SSEA3-positive and negative
populations respectively (Figure 2B). Immunofluorescence analysis
of the two populations, following overnight adherence to exclude
analysis of non-viable cells, revealed that .97% of the SSEA3-
positive population expressed detectable SSEA3/488 fluorescence
and 0% of the SSEA3-negative population expressed detectable
SSEA3/488 fluorescence (Figure 2C), demonstrating that the
fluorescence activated cell sorting process can purify viable
subpopulations of cells from a heterogeneous somatic population.
These subpopulations were then used for reprogramming to
Figure 1. Expression of SSEA3 from primary human dermal
fibroblasts. (A–B) Primary adult human fibroblast line HUF1 (A) Phase
contrast image (B) Immunocytochemical detection of SSEA3 expression
(green). (C–D) Immunofluorescence staining for (C) TRA-1-60 and (D)
TRA-1-81 on HUF1 cells. (E) Overlay of SSEA3 expression on phase
contrast image of HUF1 cells. (F) Confocal section through primary
human fibroblast (HUF1) cell demonstrating SSEA3/488 detection
primarily from the cell membrane in addition to localized peri-nuclear
detection. (G) SSEA3/488 detection on H9 human embryonic stem cells.
(H) 488 secondary antibody only negative control staining of HUF1 cells.
(C–H) DAPI staining in blue. Scale bars represent 100 microns.
Figure 2. FACS analysis and isolation of SSEA3-positive and
SSEA3-negative primary adult human fibroblasts. (A) Immuno-
cytochemical analysis for SSEA3 expression in eight additional primary
adult dermal human fibroblast (HUF) lines. DAPI staining in blue. (B)
Histogram of FACS analyzed HUF1 cells following live binding of SSEA3/
488 antibody complex and gating for SSEA3-positive (top 10%) and
SSEA3-negative (bottom 10%) populations. (C) Detection of SSEA3/488
fluorescence signal in FACS sorted SSEA3-positive and SSEA3-negative
populations following overnight adherence. (A & C) SSEA3 staining in
green. Scale bars represent 100 microns.
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Previous reprogramming work demonstrated that we could
reprogram the entire, unsorted population of HUF1 somatic cells
using retroviral vectors that express OCT4, SOX2, KLF4 and
cMYC to generate iPSCs that express the same pluripotency
markers as control H9 ESCs (Figure 3A). Reprogrammed cells
possessed a normal karyotype (Figure 3B), differentiated into
beating cardiomyocytes in vitro (Movie S1) and differentiated into
representatives of all three germ layers in vivo (Figure 3C). We
transduced our SSEA3-positive and SSEA3-negative populations
with the same retroviral vectors, under identical experimental
conditions, and seeded the transduced cells onto inactivated mouse
embryonic fibroblasts (MEFs). After three weeks of culture under
standard hESC conditions, plates were examined in a double-blind
analysis by three independent hESC biologists for iPSC colony
formation. Colonies with iPSC morphology were picked and
expanded. We observed that all three biological replicates with the
transduced SSEA3-negative cells formed many large background
colonies (10–27 per replicate, Figure 4A) but no iPSC colonies
emerged; in contrast, all three biological replicates with the
transduced SSEA3-positive cells resulted in the formation of iPSC
colonies (4–5 per replicate, Figure 4B) but very few large
background colonies (0–1 per replicate, Table 1). When we
further characterized the cell lines derived from the iPSC-like
colonies, we observed that they possessed hESC-like morphology,
growing as flat colonies with large nucleo-cytoplasmic ratios,
defined borders and prominent nucleoli (Figure 4C). When five
lines were further expanded and characterized, all demonstrated
expression of key pluripotency markers expressed by hESCs,
which included: alkaline phosphatase, Nanog, SSEA3, SSEA4,
TRA160 and TRA181 (Figure 5A). The SSEA3-selected iPSCs
also demonstrated a normal male karyotype (46, XY)(Figure 5B),
the ability to differentiate into functional beating cardiomyocytes
in vitro (Movie S2) and differentiate into representatives of all three
germ layers in vivo (Figure 5C). Most importantly, since we
observed no iPSC colony formation or line derivation from the
transduced SSEA3-negative cells, this suggests that these cells
possess significantly lower or even no reprogramming potential
relative to the SSEA3-expressing cells (Table 1). Additionally, a
10-fold enrichment of primary fibroblasts that strongly express
SSEA3 resulted in a significantly greater efficiency (8-fold increase)
Figure 3. Characterization of unsorted HUF1 derived induced pluripotent stem cells (HiPS-1 control). (A) Expression of pluripotency
markers from iPSCs (HiPSC-1 control) generated following retroviral transduction of unsorted HUF1 cells. DAPI staining in blue. Scale bar represents
100 microns. (B) SKY karyotype analysis of the HiPS-1 control line. (C) Histological analysis of teratoma derived from HiPS-1 control line.
PLoS ONE | www.plosone.org3 September 2009 | Volume 4 | Issue 9 | e7118
of iPSC line derivation compared to the control derivation rate
(p,0.05, Table 1).
We next examined the expression of genes that might
potentially confer the enhanced reprogramming to the SSEA3-
positive population, including Nanog , Sall4  and hTert
 as well as the control housingkeeping gene Gapdh. In addition
to the SSEA3-positive and negative populations of cells, which
represented the top 10% and bottom 10% of SSEA3 expressing
cells respectively, we also included the intermediary SSEA3-
expressing cells, which represented the remaining 80% of the total
HUF1 cell population. Three biological replicates for each of the
three subpopulations were analyzed. While no significant
differences in gene expression were observed for Sall4, hTert or
Gapdh (Figure 6), the analysis revealed that expression of Nanog
was significantly increased (p,0.05) in the SSEA3-positive cell
population compared to either the SSEA3-intermediate or
SSEA3-negative population (Figure 6).
In this study, we unexpectedly observed that SSEA3, a cell
surface marker detected on the surface of pluripotent cells, is
strongly expressed in a subpopulation of cells derived from a
human dermal biopsy. What exactly this subpopulation of cells
represents is currently unknown. The SSEA3-positive cells
appeared indistinguishable, morphologically, from the SSEA3-
negative fibroblasts; furthermore, expression of the SSEA3 antigen
is not considered a marker of other cell types such as mesenchymal
or epidermal adult stem cells [18,19]. We hypothesize that SSEA3
expression is either identifying another less differentiated and/or
tissue specific stem cell like population that is retrieved with the
donor skin biopsy or that with culture, a subpopulation of
fibroblasts may acquire the ability to express SSEA3.
Several recent studies have demonstrated that human iPSCs can
be generated without permanent integration of genetic factors into
the reprogrammed cell chromatin [7,8,20–22]. While these
integration-free human iPSCs avoid the possible oncogenic and
insertional mutagenesis issues that prevent the current generation
of integration-based iPSCs from being considered for use in
human clinical trials , the reprogramming efficiency is typically
very low. Methods to enhance the reprogramming efficiency
would significantly increase the feasibility of this approach,
Figure 4. Morphology of colonies and lines following retroviral transduction of HUF1 cells. (A) Large background colony with no ESC-like
attributes. (B) ESC-like iPSC colony. (C) Morphology of SSEA3-selected lines following derivation. (A–C) Scale bar represents 100 microns.
Table 1. Derivation of human iPSCs from SSEA3 sorted
primary dermal fibroblasts.
Control (unsorted cells)100 N/A
Control (unsorted cells)211 N/A
SSEA3-negative cells100 0%
SSEA3-negative cells200 0%
SSEA3-negative cells300 0%
SSEA3-positive cells25 4**800%
*Calculated as percentage compared to control derivation.
**HiPS-2E line demonstrated impaired proliferation and thus is not included.
Each biological replicate represented 100,000 transduced cells seeded onto
a 10 cm dish containing MEFs and cultured in hESC media for 3 weeks.
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especially for cell types which tend to be more difficult to
reprogram, such as the primary adult human fibroblasts used in
this study. Our control iPSC derivation efficiency using the HUF1
line was very low, with only 1 iPSC line derived from 200,000
cells. However, in this study we have demonstrated that a 10-fold
purification of the top SSEA3-expressing cells could increase the
efficiency of reprogramming 8-fold relative to unsorted cells and to
a much greater extent relative to the SSEA3-negative cells. This
result suggests that further investigation into the identification and
isolation of more purified subpopulations from patient-derived
somatic cell lines may result in further enhancement of the
reprogramming efficiency. In addition to identifying a cell
population with enhanced reprogramming efficiency, we also
identified an SSEA3-negative population with either significantly
reduced reprogramming efficiency or no reprogramming ability.
Comparison analysis between the SSEA3-positive and negative
populations may help us elucidate the currently poorly understood
mechanisms of reprogramming.
Our transcriptional analysis of the SSEA3-positive and negative
populations revealed a significantly increased expression of Nanog
in the SSEA3-positive population (p,0.05). As increased Nanog
expression has been demonstrated to enhance reprogramming
efficiency , this suggests Nanog may be playing a role in the
differential reprogramming observed. However, it should be noted
Figure 5. Characterization of SSEA3-selected HiPSCs. (A) Expression of pluripotency markers on H9 ESCs and SSEA3-selected HiPSC lines.
Alkaline phosphatase (AP) staining in dark red/purple. DAPI stained images are inset in blue. (B) Spectral karyotype (SKY) of SSEA3-selected HiPS-2C
line. (C) Histological analysis of teratoma derived from SSEA3-selected HiPS-2C line. (A & C) Scale bar represents 100 microns.
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that the level of Nanog expression is thousands of times higher in
hESCs and fully reprogrammed iPSCs than in the SSEA3-
expressing HUF1 cells, making it likely that other factors may also
be playing a role in the differential reprogramming observed.
Future studies using global transcriptional and epigenetic profiling
should assist in further identifying the differences between the
SSEA3-positive and negative subpopulations, and may help
elucidate the mechanisms of reprogramming.
In summary, we have reported the identification and isolation of
a subpopulation of human dermal fibroblasts that express the
pluripotency marker SSEA3, we have demonstrated an enhanced
efficiency of generation of iPSCs from these SSEA3-expressing
cells and observed no iPSC generation from the non-SSEA3-
expressing cells, and we have revealed significantly increased
Nanog expression in the SSEA3-expressing fibroblasts, suggesting
a possible mechanistic explanation for the differential reprogram-
ming. To our knowledge, this study is the first to identify a
pluripotency marker in a heterogeneous population of human
dermal fibroblasts, to isolate a subpopulation of cells that have a
significantly increased propensity to reprogram to pluripotency
and to identify a possible mechanism to explain this differential
Materials and Methods
Written approval for all somatic derivations and subsequent
iPSC generation performed in this study was obtained from the
Stanford University Institutional Review Board (IRB protocol
10368) and the Stanford University Stem Cell Research Oversight
Committee (SCRO protocol 40), and written informed consent
was obtained from each individual participant.
Isolation of Primary Adult Dermal Human Fibroblast
(HUF) Cell Lines
A somatic bank of nine primary adult dermal human fibroblast
(HUF) lines were derived and used inthis study. The gender, age and
disease status/phenotype of the participants were as follows: HUF1
male28healthy control, HUF2male 62 sporadicParkinson’s disease,
HUF3 female 30 healthy control, HUF4 male 42 Parkinson’s disease
(caused by triplication in the a-synuclein gene), HUF5 female 46
(sibling to HUF4), HUF6 female 60 Parkinson’s disease (homozygous
for G2019Smutationin the leucine-richrepeat kinase2 gene),HUF7
male 35 Parkinson’s disease (heterozygous for G2019S mutation in
HUF9 female 31 premature ovarian failure. For each HUF line
derivation, the adult donor was consented and an inner arm 4 mm
skin punch biopsy was obtained at the Stanford University
Dermatology Clinic by a qualified dermatologist. The skin biopsies
were washed in Ca/Mg-free Dulbecco’s Phosphate Buffered Saline
(PBS, Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and
minced into approximately 12 smaller pieces before being seeded
onto gelatin-coated 6-well cell culture plates (Corning Enterprises,
Corning, NY, http://www.corning.com) containing mouse embry-
onic fibroblast (MEF) media consisting of Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum (FBS,
Invitrogen) and 100 IU/ml penicillin-streptomycin (Invitrogen), and
cultured at 37uC in a 5% CO2incubator. The culture medium was
partially changed every two days until biopsy adhesion was observed
(usually day 4–6) and then completely changed every two days
afterwards. Once the fibroblasts migrated out (usually day 10-12) the
attached biopsy fragments and connected epithelial cells were
manually removed and the fibroblasts were allowed to expand up
to 80–90% confluence. This primary culture was passaged through
brief exposure to 0.05% trypsin-EDTA (Invitrogen) and seeded
onto gelatin coated 175-cm flasks with fresh culture medium. These
somatic cells were cultured until they reached 90% confluence and
then frozen down in MEF medium supplemented with 10%
dimethyl sulphoxide (DMSO, Sigma-Aldrich, St. Louis, http://
HUF cells were propagated in MEF media consisting of
DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen)
and 100 IU/ml Penicillin-Streptomycin (Invitrogen). When the
cells reached about 80–90% confluence, they were briefly treated
with 0.05% trypsin-EDTA (Invitrogen) and split at a 1:3 ratio into
a new dish. Human induced pluripotent stem cells (iPSCs) and H9
human embryonic stem cells (hESCs) were maintained in hESC
medium consisting of DMEM/F12 supplemented with 20%
Knockout Serum Replacer (KSR, Invitrogen), 2 mM L-glutamine
(Invitrogen), 0.1 mM non-essential amino acids (Invitrogen),
0.1 mM b-mercaptoethanol (Millipore, Billerica, MA, http://
www.chemicon.com), 100 IU/ml Penicillin-Streptomycin and
10 ng/ml recombinant human basic fibroblast growth factor (b-
FGF, Invitrogen). For passaging, individual colonies were
simultaneously cut and scraped off from the plate using a
customized hockey-style (half-loop) glass pipette tip and transferred
to a mitomycin C (Sigma) inactivated MEF seeded dish containing
fresh hESC media. All of the research in this study adhered to the
National Academy of Sciences guidelines.
Confocal images were collected with a Zeiss LSM510 Meta
laser scanning confocal microscope (Carl Zeiss, Jena, Germany,
http://www.zeiss.com) with a Zeiss 639 Plan-Apochromat objec-
tive (NA 1.4). For DAPI, excitation was at 405 nm, and a 420–
480 nm bandpass filter was used. For Alexa 488, excitation was at
488 nm, and a 505–530 nm bandpass filter was used. Both
detector pinholes were set at 1 Airy unit. Sampling was at
0.095 mm/pixel, 12-bits per pixel with a 2.18 ms pixel dwell time.
Figure 6. Transcriptional analysis of primary dermal fibroblast
subpopulations with differential SSEA3 expression. Relative
expression of Nanog, Sall4, hTert and Gapdh from three subpopulations
of HUF1 cells: SSEA3-negative cells (representing the bottom 10% for
SSEA3 expression/detection), SSEA3 intermediate cells (representing
the intermediate 80% of cells between the top and bottom 10% for
SSEA3 expression/detection) and SSEA3-positive cells (representing the
top 10% for SSEA3 expression/detection). Three biological replicates
were analyzed for each sample. The relative gene expression value
represents the level of gene expression for each sample compared to
the overall average for that gene across the three subpopulations.
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SSEA3 live cell staining and FACS cell sorting
Approximately 10 million HUF1 cells were trypsinized through a
5 min exposure to 0.05% trypsin-EDTA (Invitrogen), exposed to
MEF media to inactivate the trypsin and then washed twice with ice
cold PBS + 2% goat serum (PBS-G). After the first wash the cells
were passed through a 40 micrometer filter to remove cellular
clumps. For each wash the cells were centrifuged for 5 min at 80 g,
the supernatant was removed and the cells were gently resuspended
in ice-cold PBS-G. After the washes the cells were resuspended in a
1.5 ml Eppendorf tube in 1 ml of ice-cold PBS-G containing 1:100
SSEA3 antibody (Millipore, mab4303) and incubated for 45
minutes in the dark at 4uC with gentle rocking. After primary
antibody binding the cells were washed three times with ice-cold
PBS-G and then resuspended in a 1.5 ml Eppendorf tube in 1 ml of
ice-cold PBS-G containing 1:200 Alexa 488-conjugated goat anti-
rat IgM (Invitrogen, A21212) and incubated for 45 minutes in the
dark at 4uC with gentle rocking. After secondary antibody binding
the cells were washed three times with ice-cold PBS-G and then
resuspended in 2 ml of ice-cold PBS-G, passed again through a 40
micrometer filter and then immediately analyzed and sorted on a
FACSAria cell sorter (BD Biosciences, San Jose, CA, USA, http://
www.bdbiosciences.com) with blue laser excitation (488 nm). Data
was analyzed, doublet-exclusion gating was performed and the
relevant populations were sorted using BD FACSDiva Software
(BD Biosciences). Cells gated within the top 10% for SSEA3
expression were sorted into the ‘‘SSEA3-positive’’ population and
cells gated within the bottom 10% for SSEA3 expression were
sorted into the ‘‘SSEA3-negative’’ population. Both populations
were allowed to adhere, proliferate and recover for 3 days prior to
retroviral transduction. Cells used for immunofluorescence analysis
were fixed immediately following overnight adherence to remove
dead and non-viable cells and cells used for transcriptional analysis
were cultured for 6 days prior to analysis.
Retroviral Production, Infection and iPSC Generation
The following plasmids were obtained from Addgene: pMXs-
hOCT3/4 (17217), pMXs-hSOX2
(17219), pMXs-hc-MYC (17220), pUMVC (8449) and pVSV-G
(8454) (Addgene Inc., Cambridge, MA, USA, http://www.
addgene.org). 293FT cells (Invitrogen) were maintained in MEF
media supplemented with 0.5 mg/ml Geneticin (Invitrogen) and
cultured until reaching 90–95% confluence before transfection.
One day prior to transfection, fresh antibiotic-free culture media
was added to the cells. For each 175-cm flask, 293FT cells were
transfected with 10 mg of plasmid DNA carrying the transgene
(OCT4, SOX2, KLF4 or cMYC) along with 10 mg of the envelope
plasmid pVSV-G and 15 mg of the packaging plasmid pUMVC.
The transfection was facilitated by 120 ml of Lipofectamine 2000
(Invitrogen) and 15 ml opti-MEM (Invitrogen) for 6 hours and
then replaced with 18 ml of fresh MEF medium without
antibiotics. After 2 days, the viral supernatant was collected by
spinning and passing through a Millex-HV 0.45 um filter unit
(Millipore). The viral supernatants were concentrated to 100x by
ultracentrifugation (Beckman Coulter, Inc., Fullerton, CA, USA,
http://www.beckman.com) at 17,000 RPM for 2.5 hours at 20uC
and then resuspended overnight at 4uC in MEF media. These
100x concentrated viral stocks were either used fresh or frozen in
aliquots at 280C.
One day before transduction, HUF1 cells were seeded at 105
cells per well of a gelatin coated 6-well plate. On the following day
(considered day 0) the concentrated retroviral supernatants were
thawed and mixed at a 20x OCT4, 10x SOX2, 10x KLF4, 10x
cMYC ratio, supplemented with fresh MEF media up to 2 ml
volume (per well) and 8 ng/ml polyprene and then exposed to the
HUF1 cells at 37uC and 5% CO2. After 24 hours (on day 1) the
mixed viral supernatant was removed, the cells were washed twice
with PBS and then cultured in fresh MEF medium. On day 2 a
second transduction was performed at the same viral concentra-
tions. On day 3 the mixed viral supernatant was again removed,
the cells were washed twice with PBS and then cultured in fresh
MEF medium. Five days post-transduction (day 5), the cells were
resuspended with trypsin, counted and seeded onto 10-cm dishes
pre-plated with mitomycin C inactivated MEF feeders. 105
transduced HUF1 cells were seeded per biological replicate. After
overnight incubation, the MEF medium was replaced with hESC
medium, and thereafter, the medium was changed either every
day or every other day, as required. hESC-like colonies started to
appear among background colonies around 14 days post-
transduction. Large background colonies were classified as
colonies with no hESC-like characteristics and a diameter equal
or greater than 2.5 mm. Colonies with hESC-like morphology
were manually picked and transferred to 12 or 6-well plates pre-
plated with mitomycin C inactivated MEF feeders on day 21.
Colonies that continued to expand and maintained their hESC-
like morphology were further passaged; whereas, those that failed
to expand and/or spontaneously differentiated were discarded.
Alkaline Phosphatase Staining and Immunofluorescence
Alkaline Phosphatase (AP) staining was performed for 30 min at
room temperature in the dark using the Vector Red Alkaline
Phosphatase Substrate Kit I (Vector Laboratories, Burlingame,
CA, http://www.vectorlabs.com), according to the manufacturer’s
protocol. For immunofluorescence, the cells were fixed in 4%
paraformaldehyde/PBS for 20 minutes, washed twice with PBS,
and blocked with 4% goat serum in PBS for 30 min, with all
procedures performed at room temperature. For Nanog staining,
prior to blocking, the cells were permeabilized with 1% Triton-
X100 for 1 hour at room temperature. Subsequently, the primary
antibodies were added to PBS and incubated overnight at 4uC
with gentle shaking. The next day the cells were washed with PBS
before fluorescent-conjugated secondary antibodies were added
and incubated for an hour at room temperature. Finally, the cells
were rinsed with PBS three times and DAPI was used to label the
nuclei. Primary antibodies and their dilutions were used as follows:
SSEA3 (1:200, IgM, Millipore, mab4303), SSEA4 (1:200, IgG,
mab4360), TRA-1-81 (1:200, IgM, Millipore, mab4381), Nanog
(1:100, IgG, Abcam, Cambridge, MA, USA, http://www.abcam.
com, ab21603). Secondary antibodies used were: Alexa 594-
conjugated goat anti-rat IgM (1:500, Invitrogen, A21213), Alexa
488-conjugated goat anti-rat IgM (1:500, Invitrogen, A21212),
Alexa 488-conjugated goat anti-mouse IgM (1:500, Invitrogen,
A21042), Alexa 488-conjugated goat anti-mouse IgG (1:500,
Invitrogen, A11001), Alexa 594-conjugated goat anti-rabbit IgG
(1:500, Invitrogen, A11012).
Spectral karyotyping (SKY) was performed according to a
previously published protocol . Briefly, cells were treated with
0.03 mg/ml KaryoMAXH ColcemidH Solution (Invitrogen) over-
night, then treated with 0.05% trypsin (Invitrogen) for 5 minutes at
37uC to re-suspend the cells. The trypsin was inactivated by
adding DMEM medium containing 10% FBS. Pre-warmed
hypotonic solution containing equal amounts of 0.4% Potassium
Chloride and 0.4% Sodium Citrate was slowly added to the cells to
enhance swelling at 37uC for 7 minutes. Carnoy’s solution
(Methanol:Glacial Acetic Acid, 3:1 ratio) was used to fix the cells
for 30 min. The cells were then dropped onto a pre-cleaned slide
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(Fisher Scientific, Pittsburgh, PA, USA, http://www.fishersci.com)
and the metaphase spread quality was determined using a phase-
contrast microscope. After a 3–7 days of aging at room
temperature, the slide was hybridized with probes from the
SkyPaintTMDNA kit for human chromosomes (Applied Spectral
Imaging, Vista, CA, USA, http://www.spectral-imaging.com) for
2 days in a 37uC humidified chamber. The finished metaphase
spreads were visualized and analyzed using the SkyView spectral
imaging system (Applied Spectral Imaging).
In Vitro Differentiation to Beating Cardiomyocytes
For embryoid body formation, iPS cells were seeded into ultra
low attachment plates (Corning) containing DMEM + 20% FBS.
After 8 days growing in suspension, the cell aggregates were
transferred to gelatin-coated dishes containing the same medium
to allow the cells to attach. The medium was changed every 2–3
days for up to 3 weeks or until beating cardiomyocytes were
For each graft, approximately 106iPS cells were manually
harvested, washed and resuspended in a 1.5 ml tube containing
300 ml hESC medium and then injected subcutaneously into
female SCID mice (Charles River Laboratories International, Inc.,
Wilmington, MA, USA, http://www.criver.com). Any visible
tumors 4–8 weeks post-transplantation were dissected and fixed
overnight with 4% paraformaldehyde/PBS solution. The tissues
were then paraffin embedded, sectioned, stained with hematoxylin
and eosin, and examined for the presence of tissue representatives
of all three germ layers.
RNA Extraction and Real-time PCR Analysis
Total RNA was purified using RNeasy Mini Kit (Qiagen,
Valencia, CA, http://www1.qiagen.com) according to the manufac-
turer’s instructions. 500 ng of RNA was used in reverse transcription
with Superscript III (Invitrogen) and random hexamers. 1.25 ml of
cDNA from each sample was mixed with master mix consisting of
5 ml Cells Direct 2X reaction mix (Invitrogen), 2.5 ml 0.2X PPP mix
(48 genes, Taqman/Applied Biosystems Inc, Foster City, CA, USA,
http://www.appliedbiosystems.com), 0.5 ml Platinum Taq (Invitro-
gen) and 0.75 ml TE Buffer. The reactions were pre-amped using a
thermo cycler (Applied Biosystems) under the following conditions: 1
cycle at 95C, 10 minutes and 14 cycles at 95C, 15 seconds and at
60C, 4 minutes. Then the reactions were diluted with TE buffer to a
final volume of 20 ml. 2.25 ml of the pre-amplification products were
used in the downstream real-time PCR analysis using the Biomark
Fluidigm system (Fluidigm Corporation, San Francisco, CA, USA,
http://www.fluidigm.com) according to the company’s recommen-
dation. The Ct values for each sample and gene were normalized
relative to GAPDH, RPLPO and CTNNB1 by qBasePlus
(Biogazelle, Zulte, Belgium, http://www.biogazelle.com). The level
of gene expression for each sample was compared to the overall
(SSEA3-negative, SSEA3-intermediate and SSEA3-positive) to
produce a relative gene expression value.
Analysis of variance (ANOVA) statistical comparisons were
performed using Statview Software (SAS Institute, Inc., Cary, NC,
USA, http://www.jmp.com) with statistical significance set at
Beating cardiomyocytes observed following spontanous differenti-
ation of HiPS1-control embryoid bodies (EBs)
Found at: doi:10.1371/journal.pone.0007118.s001 (1.32 MB
Cardiomyocytes derived from HiPS1-control EBs.
2C EBs. Beating cardiomyocytes observed following spontanous
differentiation of SSEA3 selected HiPS-2C embryoid bodies (EBs)
Found at: doi:10.1371/journal.pone.0007118.s002 (2.50 MB
Cardiomyocytes derived from SSEA3-selected HiPS-
The authors thank T. Kalista Ladhardi, A. Chang, A. Olson, B. Schuele,
B. Byers, E. Chiao, A. Dominguez, B. Langston and S. Panula for their
assistance with this study.
Conceived and designed the experiments: JAB RARP. Performed the
experiments: JAB. Analyzed the data: JAB HNN. Contributed reagents/
materials/analysis tools: JAB. Wrote the paper: JAB HNN RARP.
Financial support: RARP.
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