Cell Death and Differentiation (2012) 19, 1268–1276
& 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12
www.nature.com/cdd
Two-factor reprogramming of somatic cells to
pluripotent stem cells reveals partial functional
redundancy of Sox2 and Klf4
A Nemajerova*,1, SY Kim2, O Petrenko1 and UM Moll1
Ectopic expression of defined sets of transcription factors in somatic cells enables them to adopt the qualities of pluripotency.
Mouse embryonic fibroblasts (MEFs) are the classic target cell used to elucidate the core principles of nuclear reprogramming.
However, their phenotypic and functional heterogeneity represents a major hurdle for mechanistic studies aimed at defining the
molecular nature of cellular plasticity. We show that reducing the complexity of MEFs by flow cytometry allows the isolation of
discrete cell subpopulations that can be efficiently reprogrammed to pluripotency with fewer genes. Using these FACS-sorted
cells, we performed a systematic side-by-side analysis of the reprogramming efficiency with different two- and three-factor
combinations of Oct4, Sox2 and Klf4. We show that introduction of exogenous Oct4 with either Sox2 or Klf4 does not directly
convert MEFs to a pluripotent state. Instead, each combination of factors disrupts the normal cellular homeostasis and
establishes transient states characterized by the concurrent expression of mixed lineage markers. These cells convert into
induced pluripotent stem cells in a stochastic fashion. Our data suggest that there is a partial functional redundancy between
Sox2 and Klf4 in the disruption of cellular homeostasis and activation of regulatory networks that define pluripotency.
Cell Death and Differentiation (2012) 19, 1268–1276; doi:10.1038/cdd.2012.45; published online 27 April 2012
Reprogramming somatic cells into induced pluripotent stem
(iPS) cells represents a valuable resource for the development of general and patient-specific therapies. The crucial
issues facing the clinical translation of iPS technology are
(i) choosing the most appropriate cell type for induction of
pluripotency; (ii) improving the reprogramming efficiency; and
(iii) gaining a mechanistic understanding of the reprogramming process. To date, multiple cell types have been used for
iPS production, including embryonic and adult fibroblasts,
adipocytes, cardiomyocytes, epithelial, vascular, hematopoietic and neuronal progenitors.1,2 Reprogramming has
been achieved through the expression of various combinations of transcription factors, most often consisting of Oct4,
Klf4, Sox2 and c-Myc (OKSM).3–6 An important observation
from these studies is that the type of somatic cells chosen has
a significant effect on the efficiency of iPS generation and the
extent of manipulation required. Thus, less-differentiated cells
reprogram more efficiently than differentiated cells. Moreover,
partially differentiated cells, for example, neural stem cells,
can be reprogrammed with fewer factors.7–10
It is accepted that the ectopic expression of pluripotency
factors establishes an embryonic-like transcriptome that is
sustained by the subsequent reactivation of endogenous
gene-regulatory networks.1,2 However, determining the order
of critical events that take place during reprogramming and
their timing has proven difficult, in part because the process is
neither efficient (less than 0.1%), nor rapid (about 2–3 weeks),
nor homogeneous in terms of transgene integration. The
advent of a ‘secondary’ reprogrammable system based on
doxycycline-inducible lentiviruses circumvented some of
these problems.11–13 The system was further improved by
integration of a single inducible cassette with four reprogramming factors into mouse genome.14,15 Although the reprogramming efficiency of cells from such secondary mice is
higher (1–10%), several important questions regarding the
mechanistic underpinning of the process remain unanswered:
Why do only a small fraction of cells reprogram, while others
don’t? Which cell types can be effectively reprogrammed into
a pluripotent state? Is reprogramming a fundamentally
deterministic process that occurs in a well-defined temporal
order, or is it driven by random chains of stochastic events
triggered by the aberrant levels of transcription factors?16–18
To date, the majority of mechanistic studies of iPS cells
have used mouse embryonic fibroblasts (MEFs) as the
starting cell population, and it was assumed that MEFs are
rather well-defined and fairly homogeneous. The advantages
of fibroblasts are that they are relatively easy to derive and
reprogram. However, it has been shown that fibroblasts
represent a dynamic population of cells, exhibiting functional
heterogeneity among and within tissues.19,20 Moreover,
embryo-derived fibroblasts are expected to contain a wide
spectrum of progenitor cells with diverse properties that are
able to expand prior to terminal differentiation. The transcriptional profiles of partially reprogrammed cells, which exhibit
1
Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA and 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
*Corresponding author: A Nemajerova, Department of Pathology, Stony Brook University, 100 Nicolls Road, BST 9, L9, Room 136, Stony Brook, NY 11794, USA.
Tel: þ 1 631 444 3030; Fax: þ 1 631 444 2434; E-mail: [email protected]
Keywords: somatic cell reprogramming; induced pluripotent stem cells; iPSC
Abbreviations: FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; iPS cells, induced pluripotent stem cells; KSR, knockout serum replacement;
MEFs, mouse embryonic fibroblasts; OKSM, Oct4, Klf4, Sox2 and c-Myc
Received 30.12.11; revised 16.3.12; accepted 19.3.12; Edited by R De Maria; published online 27.4.12
Two-factor reprogramming of somatic cells
A Nemajerova et al
1269
similar features regardless of the starting cell type,21–24 may
support the notion of a stepwise process with well-defined
intermediate stages. However, the phenotypic complexity of
MEFs still has to be viewed as a confounding variable that
needs to be taken into account when evaluating highthroughput expression data from initially heterogeneous
populations.
In this study, we sought to address these questions and
determine whether different two- and three-factor combinations of Oct4, Sox2 and Klf4 induce the dedifferentiation of
MEFs into a unique progenitor-like state before they acquire
pluripotency. We also sought to prospectively identify and
isolate the most reprogrammable MEF populations. Our
results indicate that forced expression of Oct4 with either
Sox2 or Klf4 does not directly convert MEFs to a pluripotent
stem cell state. Instead, each combination of factors disrupts
the normal cellular homeostasis and establishes transient or
metastable states characterized by the concurrent expression
of mixed lineage markers. These cells convert into iPS cells in
a stochastic fashion.
Results
Efficient induction of iPS cells with two factors using
FACS (fluorescence-activated cell sorting)-sorted MEFs
and an optimized protocol. One of the biggest hurdles in
reprogramming somatic cells into pluripotent cells is the fact
that the process is inefficient and often incomplete. To
overcome this limitation, we generated REBNA episomal
retroviral vectors25 expressing human Myc, Klf4, Oct4 and
Sox2. With this optimized system, we were able to reprogram
into iPS cells up to 2% of freshly isolated MEFs using 4F
(OSKM) or 3F (OSK) combinations. We next assessed
whether the complexity of MEFs may influence the levels of
reprogramming. To this end, E12.5–E13.5 MEFs were
FACS-sorted into four phenotypically distinct subpopulations
based on the surface expression of Thy1 (CD90) and Sca1
(stem cell antigen 1) markers. Previous studies demonstrated that Thy1 expression defines lipofibroblastic or
myofibroblastic lineage,26 while Sca1 is a marker specifying
murine mesenchymal and epithelial progenitor cells.27 Our
microarray analyses confirmed that Sca1-single-positive
cells (SP) express genes broadly involved in embryonic
and tissue morphogenesis, thus indicating their immature
origin (Supplemental Table 1), whereas Thy1/Sca1-doublenegative (DN) cells selectively express markers of myofibroblast-committed progenitors (Supplemental Table 2).
Notably, the Thy1-positive fractions (composed of Thy1single positive (Thy1-SP) and Thy1/Sca1 double-positive
(DP) cells) represent the majority of MEFs (470%), while
Thy1-negative (Sca1-SP and DN) cells are a minority
(10–15% each, Figure 1a).
Freshly sorted MEFs were then seeded at equal densities
and transduced with different combinations of reprogramming
factors. The transduction efficiencies of the sorted MEF
fractions were equal, based on the levels of green fluorescent
protein (GFP) expression (data not shown) and ectopic factor
expression (Figure 1b). However, the frequency of iPS
colonies produced by each subpopulation differed
(Figure 1c). Thus, OSKM- or OSK-transduced DN and
Sca1-SP cells yielded significantly more iPS colonies than
the corresponding Thy1-SP or DP fractions (Figure 1c and
Supplemental Table 3). We found that Myc was fully
dispensable for the reprogramming of DN and Sca1-SP cells
(Figure 1c). Moreover, DN and Sca1-SP cells transduced with
2F combinations (OK or OS) also yielded significantly higher
numbers of iPS colonies compared to Thy1-SP and DP
fractions (Figure 1c). We calculated a reprogramming
efficiency of up to 2% for OSK-transduced, and B0.2% for
OK- and OS-transduced DN and Sca1-SP cells, respectively.
Colonies of iPS cells first appeared in OK- and 3F-transduced
MEFs (4 to 5 days post infection), followed by OS-transduced
cells (9 to 10 days post infection, Figures 1d and e). We
expanded over 100 independent OK and OS colonies for
further analysis. Genotyping of these iPS clones with
transgene-specific primers verified the integration of the
Oct4 and either Klf4 or Sox2 transgenes in OK and OS
infections, respectively (Supplemental Figure 1). None of the
clones examined (n ¼ 23) contained unwarranted integrated
transgenes (e.g., Myc), thereby ruling out the possibility of
cross-contamination (Supplemental Figure 1). Together,
these data indicate that DN and Sca1-SP cells represent
superior cell types for reprogramming and are accessible by
fewer genetic manipulations.
Two-factor-derived iPS cells are pluripotent. Most of our
2F colonies showed well-defined ES cell-like morphology
with sharp colony borders. The established iPS cells were
alkaline phosphatase-positive (100%), predominantly negative for Thy1 (90–100%), Sca1 (80–90%), CD34 (100%) and
CD133 (95–100%), and did express variable levels of ES cell
markers CDH1 (100%), EpCAM (100%), Kit (60–90%) and
SSEA1 (50–60%) (Figure 2a and data not shown). The
observed fractions of KIT-, Sca1- and SSEA1-negative iPS
cells likely reflect heterogeneous expression of these
markers, as previously reported for undifferentiated wild-type
ES cells.28,29 In a separate set of experiments, we generated
2F iPS cells from FACS-sorted MEFs containing the Oct4GFP reporter gene. These iPS cells homogeneously
expressed the GFP, confirming their undifferentiated status
(Figure 2b). Following subcutaneous injection into nude
mice, teratomas were formed by all clonal iPS cell lines
tested (n ¼ 20). Histological analysis revealed tissue
structures indicative of all three germ layers (Figure 2c).
We successfully obtained adult chimeric mice with independently produced OK (1 out of 1) and OS (1 out of 2) iPS cell
lines (Figures 2d and e). The degree of chimerism, as
assessed by coat color contribution, was wide-spread,
ranging from 50 to 90% in individual animals. Taken together,
these in vivo results indicate that 2F-derived iPS cells are
pluripotent.
Gene expression profiles of two-factor-transduced
MEFs. We next examined the expression profiles of each
Thy1/Sca1 subpopulation transduced with the reprogramming factors. Analysis of global gene expression patterns
demonstrated a strong correlation between transcriptomes of
OS-infected cells and those of OK-infected cells from the
Thy1-negative group (Figure 3a, top panels). In contrast, the
Cell Death and Differentiation
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A Nemajerova et al
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OSK
Sca1-SP
DN
Thy1-SP
DP
Sca1-SP
DN
Thy1-SP
DP
Control
18%
Oct4
58%
Thy1
Klf4
Sox2
11%
12%
MAPK
Sca1
OSKM
OSK
OK
OS
60
40
20
0
Unsorted
% iPS colonies
40
Thy1-SP
DP
OK
OSK
OS
30
% Cumulative number
Relative colony number
80
20
10
0
0
5
10
15
Days post infection
20
Sca1-SP
120
DN
OK
OSK
OS
100
80
60
40
20
0
0
5
10
15
Days post infection
20
Figure 1 Efficient induction of iPS cells with two factors. (a) Flow-cytomeric analysis of Thy1 and Sca1 expression by wild-type MEFs at passage 2. (b) Immunoblot
analysis of Oct4, Klf4 and Sox2 expression by sorted MEFs transduced with control or OSK retroviruses. MAPK is a loading control. (c) Relative efficiency of iPS cell
generation from total unsorted MEFs or Thy1-SP, DP, Sca1-SP and DN fractions transduced with the indicated retroviruses. The results represent the average of 10
experiments. Values are normalized relative to unsorted MEFs transduced with OK viruses. The error bars correspond to standard error. (d and e) Reprogramming kinetics of
DN cells measured as the percentage of newly produced iPS colonies per day (d) or the percentage of cumulative iPS colonies per day (e). Cells were transduced with the
indicated retroviruses. Colonies consisting of Z20 cells were counted daily. The results represent an average of two experiments performed in duplicate. The error bars
correspond to standard deviation
correlation was lower when the respective OS- versus OKinfections were compared (Figure 3a, bottom panels). As
expected, microarray analysis revealed that OK- versus OSmediated reprogramming elicited dissimilar effects on the
expression of many endogenous transcription factors. Thus,
combined expression of Oct4 and Klf4 altered the levels of
B120 endogenous transcription factors, while the combined
expression of Oct4 and Sox2 caused changes in the levels of
B90 endogenous transcription factors compared with the
respective uninfected controls (Figures 3b and c and
Supplemental Figures 2a and b). Only a small number of
endogenous transcription factors were similarly induced or
repressed in OK- and OS-transduced cells (Supplemental
Figure 2c). Notably, gene set enrichment analysis30 showed
Cell Death and Differentiation
that the OS combination of factors was more effective in
inducing genes associated with pluripotency (i.e., the previously identified Core, Myc and ESC-like gene modules31,32),
consistent with the notion that these two proteins bind
to DNA together (Supplemental Figure 3). However, expression of either OS or OK also resulted in the concurrent
induction of different or even conflicting lineage-specific
markers (Figure 3d). Thus, infection with OS viruses led to
upregulation of mesenchymal markers ESM1 (endothelial
cell-specific molecule 1), MSC (musculin) and SOSTDC1
(sclerostin domain containing 1, inhibitor of Wnt-mediated
signaling), but also neuronal genes, such as NES (Nestin),
RELN (reelin), SOX21 and TNN (tenascin N). Likewise, cells
infected with OK viruses upregulated mesenchyme-specific
Two-factor reprogramming of somatic cells
A Nemajerova et al
CD34
EpCam
EpCam
1271
CD133
CDH1
CD34
EpCam
EpCam
Oct4-GFP
Oct4-GFP
CD133
EpCam
N
N
C
N
P
P
G
C
R
G
P
N
Figure 2 Assessment of the quality of two-factor-produced iPS cells. (a) FACS analysis of two independent iPS clones derived from OS-transduced DN Oct4-GFP MEFs.
For the two panels on the right, each clone was gated on GFP-positive cells (R2). (b) GFP-positive ES-like colonies derived from OS-transduced Oct4-GFP-expressing MEFs.
(c) Teratoma formation by 2F iPS cell lines. Hematoxylin and eosin staining identified mature tissue derivatives of all three germ layers: squamous epithelium with keratotic
pearl (P, ectoderm); neuroepithelial cells (N, ectoderm); secretory epithelium (R, ciliated respiratory or G, glandular structures) (endoderm); and mature cartilage embedded in
fibrovascular stroma (C, mesoderm). (d and e) Chimeric mice with agouti coat color originating from an OS iPS clone (d) or OK iPS clone (e)
genes CD34, LY6A (Sca1), MSC and TEK (TEK tyrosine
kinase), the neuronal genes RELN and TNN, but also
the epithelial genes KERA, KRT14, KRT16 and
KRTDAP (keratocan, keratins 14, 16 and keratinocyte
differentiation-associated protein, respectively) (Figure 3d).
In sum, these data indicate that cells transduced with OSversus OK-expressing viruses differ in the molecular
mechanisms underlying the induction of pluripotency. The
observed alterations likely reflect the fact that, in addition to
their roles in stem cell maintenance, Oct4, Sox2 and Klf4 are
Cell Death and Differentiation
Two-factor reprogramming of somatic cells
A Nemajerova et al
DN OS
r = 0.989
Sca1-SP OS
r = 0.996
Sca1-SP OK
DN OK
Thy1-SP OS
r = 0.957
r = 0.896
DN OS
Sca1-SP OS
r = 0.944
DN OK
SOX21
NFE2L3
MSC
FOSB
NPAS4
FOXQ1
ETV1
EGR2
MEOX2
PROX1
MKX
MAFF
NR4A2
RB1
KLF4
ATF3
ETV4
CREB5
GLIS3
SIM2
EPAS1
NCOA2
OTX1
ESR1
CLOCK
ISL1
BHLHB5
BACH2
NOBOX
EBF4
MLX
SIX4
SATB2
FOXN1
BATF2
Thy1-SP OK
Thy1-/Thy1+
Sca1-SP OS
DN OS
DP OS
Sca1-SP OK
DN OK
Thy1-SP OK
Thy1-/Thy1+
Sca1-SP OK
DN OK
Thy1-SP OK
Thy1-/Thy1+
Sca1-SP OS
DN OS
DP OS
Sca1-SP OK
r = 0.977
Thy1-SP OS
Sca1-SP OS
1272
OTX1
MSC
CITED1
AHRR
TCFAP2B
MEOX2
EPAS1
TCF7
P42POP
LMX1A
TRP63
MLX
TCF3
GLIS2
TEAD3
ESR1
SOHLH1
NR5A2
NPAS4
NCOA2
ISL1
ZFP367
NFE2L3
ASCL2
CTNNB1
HOXC13
THRA
FOSB
DLX3
POU3F1
SIX4
KLF15
FOXA3
TBX4
TCFEB
MESDC1
ESM1
MSC
SOSTDC1
LY6A
TEK
CD34
CDH13
NGFR
NOTCH1
NES
SOX2
SOX21
TNN
TUBA8
RELN
CDH6
CDH1
TRP63
MET
KERA
KRT14
KRT16
KRTDAP
MYH1
MYH2
MYH11
MYL1
DES
TNNC2
TNNI2
Mesenchymal
Neuronal
Epithelial
Myogenic
Low
High
Expression (log2)
Figure 3 Gene expression profiles of two-factor-transduced MEFs. (a) Scatter plots of global gene expression patterns comparing Sca1-SP, DN and Thy1-SP cells 4 days
post infection with the indicated retroviruses. The degree of correlation was assessed using Pearson’s correlation coefficient (r). The central red line represents equal gene
expression. The outer red lines indicate twofold different expression. (b and c) Heat maps from microarray analyses showing induction of endogenous transcription factors
in Sca1-SP, DN, Thy1-SP and DP MEFs transduced with OS- (b) or OK-expressing retroviruses (c). Values are given relative to their own uninfected controls. Expression
profiles of the corresponding uninfected Sca1-SP and DN (summarily called ‘Thy1 # ’) versus Thy1-SP and DP (summarily called ‘Thy1 þ ’) cells are shown as controls.
(d) Simultaneous induction of lineage-specific genes in OS- and OK-transduced Sca1-SP, DN and Thy1-SP MEFs. Values are given relative to their own uninfected controls
lineage specifiers and promote mesodermal, neural and
epithelial differentiation, respectively.
Generation of iPS cells from Nestin-GFP MEFs. The
experiments described in Figures 1 and 2 show that DN and
Sca1-SP cells yield higher numbers of IPS colonies,
suggesting that Thy1-SP and DP cells represent inferior cell
types for nuclear reprogramming. In support of this, timecourse analysis of sorted MEFs showed that Thy1-SP and
DP fractions represent progressive differentiation stages
Cell Death and Differentiation
within MEF populations (Supplemental Figure 4). These
results suggest that in the course of reprogramming of total
unsorted MEFs, a significant proportion of 2F and 3F iPS
clones might be generated from immature DN and Sca1-SP
cells or their subsets thereof, whose reprogramming is more
efficient than that of Thy1-SP and DP cells. To test this idea,
we followed the reprogramming of embryonic fibroblasts
derived from mice expressing GFP under the control of the
endogenous Nestin promoter.33 Nestin has been extensively
studied as a marker for neural and mesenchymal stem/
Two-factor reprogramming of somatic cells
A Nemajerova et al
1273
progenitor cells.33,34 Our microarray analyses revealed that
both DN and Sca1-SP populations are enriched for Nestinpositive cells (Figure 3d and Supplemental Figure 5a). We
surmised that these Nestin-positive cells might represent a
virtually homogeneous subset of immature cells and thus be
tangible for mechanistic studies of reprogramming.
As reported,33 MEFs from Nestin-GFP embryos could be
sorted into three populations based on the levels of GFP
expression (GFP-bright, -dim and -negative, Figures 4a
and b). In several independent E12.5 MEF cultures, the
GFP-bright cells accounted for B2% of the total number of
cells (Figure 4a). A fraction of these cells expressed NGFR,
another surface marker that distinguishes Thy1-negative from
Thy1-positive cells (Figure 4a and Supplemental Figure 5b).
To test their respective reprogramming competence, each of
the Nestin-GFP fractions was infected with 2F or 3F
Sort Nestin-GFP MEFs
NGFR
Neg Dim Bright
R1
R2
R4
Infect with 3F or 2F viruses
R3
Maintain in culture;
Score and expand iPScolonies;
FACS analysis
IPS colonies/105
cells
2500
2000
1500
200
100
0
IPS colonies/ 105
cells
GFP
2000
total
negative
dim
bright
OSK
OK
OS
negative
GFP-bright
GFP-bright NGFR+
% GFP-positive
cells
1000
200
100
0
100
80
60
40
20
0
OSK
OK
OS
negative
dim
bright
1
OSK
2
OK
3
OS
Figure 4 Generation of iPS cells from Nestin-GFP MEFs. (a) FACS analysis of
GFP and NGFR expression by E12.5 Nestin-GFP MEFs. GFP-negative (R1), -dim
(R2) and -bright (R3 and R4) fractions are indicated. (b) Experimental design and
analysis of iPS clones derived from Nestin-GFP MEFs. (c) Reprogramming
efficiency of 3F- and 2F-transduced GFP-negative, -dim and -bright fractions. The
error bars correspond to standard error. (d) Reprogramming efficiency of 3F- and
2F-transduced GFP-negative, GFP-bright NGFR-positive and GFP-bright NGFRnegative MEFs. (e) Induction of GFP expression 7 days post transduction with OSK,
OK or OS viruses. Transduction with OSK induces strong expression of GFP
(i.e., Nestin) in both GFP-dim and GFP-negative cells. A representative of
two experiments is shown
retroviruses as a reference (Figure 4b). The iPS colonies
were detected based on their ES cell-like morphology and
alkaline phosphatase staining. Notably, the efficiency of 2F
reprogramming of GFP-bright cells was at least 5–10 times
higher than that of GFP-negative cells (Figure 4c). Furthermore, Nestin-SP cells were more reprogrammable compared
to their NGFR-positive counterparts (Figure 4d). In addition,
NGFR expression was virtually lost after transduction of GFPbright cells with OK or OSK retroviruses (see below), whereas
the Nestin-GFP transgene remained transcriptionally active
regardless of which reprogramming factors were used
(Figure 4e). Transduction with OSK retroviruses and, to a
lesser extent, with OS or OK viruses also induced strong
expression of GFP (i.e., Nestin) in both GFP-dim and GFPnegative cells (Figure 4e and Supplemental Figure 6). Thus,
the acquired expression of Nestin by cells mirrors their ability
to convert into iPS cells (Figure 4c).
We next assessed the temporal induction patterns of
lineage-specific markers during the reprogramming process.
To this end, GFP-bright, -dim and -negative cells were
infected with 2F or 3F retroviruses. On days 1, 4, 7 and 11
post infection and after becoming stable iPS cell lines, they
were stained with antibodies for the tissue- and lineagespecific surface proteins and scored by FACS on a single-cell
basis (Figure 5a and data not shown). Consistent with our
earlier results, this analysis again revealed that induction of
reprogramming by OK versus OS viruses generates phenotypically dissimilar cells. Thus, uninfected GFP-bright cells
were predominantly negative for Thy1 (80%) and Sca1 (70%),
and showed no significant expression of CD34, CD133, KIT,
SSEA1, CDH1 (E-cadherin) or EpCAM (Figure 5a). Transduction with OK or OSK viruses caused transient expression
of Thy1, Sca1, CD34 and CD133, but early loss of NGFR
(Figure 5a). Subsets of these cells concurrently expressed
CD34 and CD133 (Figure 5a). In contrast, cells transduced
with OS viruses retained significant NGFR expression but
showed no induction of CD34 or CD133 even after 11 days in
culture (Figure 5a). Only small fractions of these cells (1–5%)
showed expression of KIT, SSEA1, CDH1 and EpCAM prior to
their conversion into iPS cells (Figure 5a).
Western blot analysis confirmed these results. Thus,
transduction of sorted MEFs with OS, OK or OSK viruses
caused little or no changes in the expression levels of CDH1,
EpCAM or KIT genes during initiation of the reprogramming
event (Figure 5b). Whether the small amount of CDH1 protein
induced in OK and OSK cells (but not in OS cells) is present at
the cell surface rather than in the cytosol cannot be
determined with certainty, given the low score by FACS
(Figure 5a). Furthermore, quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) revealed that mRNAs
for E cadherin, EpCAM and KIT were only partially induced
compared to iPS cells (Figure 5c and Supplemental Figure 7).
On the other hand, the induced expression of pluripotency
transcription factors Sox2 and, to a lesser extent, Sall4 and
Zic3 either preceded or closely paralleled the later induction of
epithelial and ES cell markers (Figure 5c). Accordingly, OSand OSK-transduced cells expressed total (endogenous and
exogenous) Sox2 protein at levels similar to those present in
iPS and wild-type ES cells (Figure 5b). In sum, a side-by-side
comparison of 2F and 3F transductions demonstrates that
Cell Death and Differentiation
Two-factor reprogramming of somatic cells
A Nemajerova et al
1274
ES
1000
800
600
400
40
60
80
100 %
EpCam
CD34
CD133
20
0
300
20
CDH1
40
Relative expression
0
Relative expression
Nestin
Thy1
Sca1
CD34
CD133
NGFR
KIT
CDH1
EpCAM
SSEA1
Thy1+Sca1+
Thy1-Sca1CD34+CD133+
EpCAM+CD133+
KIT+SSEA1+
CD34+NGFR+
CD34+CDH1+
NGFR+CD133+
OSK
OK
OS
iPS
OSK
OK
OS
11d
OSK
OK
OS
Contr
4d
OS OK OSK
OS OK OSK
OS OK OSK
OS OK OSK
Sox2
Zic3
Oct4
Klf4
OS OK OSK
OS OK OSK
OS OK OSK
OS OK OSK
200
100
20
10
0
GFP-neg
GFP-neg (D1)
GFP-neg (D11)
GFP-bright
GFP-bright (D1)
GFP-bright (D11
iPS
Contr
OS
OK
OSK
iPS
ESC
Nestin-GFP Contr
GFP-neg
GFP-neg (D1)
GFP-neg (D11)
GFP-bright
GFP-bright (D1)
GFP-bright (D11
iPS
3000
EpCAM
KIT
Oct4
Relative expression
CDH1
Sall4
Tbx3
Myc
Nanog
2000
1000
20
10
GFP-neg
GFP-neg (D1)
GFP-neg (D11)
GFP-bright
GFP-bright (D1)
GFP-bright (D11
iPS
0
Sox2
OS OK OSK
OS OK OSK
OS OK OSK
OS OK OSK
< Klf4
MAPK
Figure 5 Multilineage pattern of gene expression parallels induction of pluripotency. (a) Schematic heat map showing the percentage of Nestin-GFP-bright cells with
co-expression of lineage-specific genes as determined by FACS analysis. The analysis before (Control) and after transduction with OS, OK or OSK viruses is shown at the
indicated time points. Expression profiles of iPS and WT ES cells are shown for comparison. Color scale indicates the percentage of cells that express the indicated markers.
(b) Immunoblot analysis of Nestin-GFP-bright MEFs before (Control) and 7 days after transduction with OS, OK or OSK viruses. Expression profiles of iPS and WT ES cells are
shown for comparison. MAPK is a control for equal loading. (c) Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR) analysis of the indicated
endogenous genes in Nestin-GFP-negative (yellow group) and Nestin-GFP-bright MEFs (blue group) before and 1 or 11 days after transduction with OS, OK or OSK viruses.
Data were normalized to the corresponding HPRT value to obtain relative changes in the indicated mRNAs. Mouse-specific primers that did not cross-react with the exogenous
human counterparts were used for Oct4, Sox2 and Klf4
forced expression of Oct4 with either Sox2 or Klf4 does not
directly convert MEFs to a pluripotent state. Instead, each
combination of factors disrupts the normal cellular homeostasis and establishes transient states characterized by the
concurrent expression of mixed lineage markers. These cells
convert into iPS cells in a stochastic fashion.
Discussion
Recent studies in fibroblasts suggest that reprogramming to
pluripotency follows a well-organized temporal sequence of
molecular and cellular events termed initiation, maturation
and stabilization.29,35,36 This sequence was proposed to entail
a rate-limiting step of mesenchymal-to-epithelial transition
(MET) that marks initiation.23,24 According to this model, the
acquisition of epithelial features is accompanied by the loss of
differentiated cell characteristics and massive downregulation
of somatic genes.23,24,29,35 A subset of cells will then initiate
the embryonic transcriptome, and, finally, upregulated
pluripotency genes establish an ES cell-like expression
Cell Death and Differentiation
program.23,24,29,35 Although this concept is appealing,
the question of why the majority of cells fail to reprogram
persists.
Several insights emerge from our study. We performed a
systematic molecular and functional dissection of primary
MEFs. First, we show that MEFs, independent of strain
background, are heterogeneous with respect to reprogramming competency and can be FACS-sorted into distinct
subpopulations based on differential expression of cell
surface markers. Importantly, these subpopulations yield
significantly different efficiencies in iPS generation with
different combinations of reprogramming factors. Specifically,
Thy1-SP and DP cells, which constitute the vast majority of
the unsorted MEF population, represent a poor target for iPS
production, whereas the preexisting DN and Sca1-SP
subpopulations represent the more reprogrammable cell
types. Thus, Nestin-GFP-positive cells, which represent a
distinct subpopulation of DN and Sca1-SP cells, can be
effectively reprogrammed with only two factors, Oct4/Klf4 and
Oct4/Sox2.
Two-factor reprogramming of somatic cells
A Nemajerova et al
1275
Second, we find that, compared to OKSM fibroblasts, our
three-partite system (OS, OK and OSK) enables a more
comprehensive and unbiased view of the reprogramming
process. We show that Sox2 stands out as the most
responsive endogenous pluripotency factor during early
reprogramming stages. Moreover, the sustained expression
of Sox2 precedes the acquisition of epithelial and ES cell-like
phenotypes in cells that undergo reprogramming. We find it
plausible that a subset of Nestin-GFP-positive cells expresses
sufficiently high levels of endogenous Sox2, and thus requires
fewer exogenous factors. However, it is unlikely that these
cells may still be heterogeneous and contain multiple
subpopulations, one responding to overexpression of Sox2
and the other responding to Klf4. Given that Klf4 is required for
the activation of epithelial gene expression during somatic cell
reprogramming,23,24 we speculate that a proportion of Sox2overexpressing cells are able to bypass an early Klf4dependent phase. Thus, notwithstanding the differences in
microarray profiling, our data suggest that there is a partial
functional redundancy between Sox2 and Klf4 in the disruption of cellular homeostasis and activation of pluripotencyrelated gene networks. The partial overlap between these
factors could explain why Oct4/Sox2 and Oct4/Klf4-mediated
reprogramming had the same degree of efficiency (B0.2%),
but was an order of magnitude higher with three factors
combined.
One important question is whether in our system all
transduced genes were expressed at levels sufficient to
achieve the optimal reprogramming efficiency. We addressed
this in the following way. The distribution of retroviralmediated gene expression is essentially stochastic.37 Simple
calculations show that for 2% of cells to be effectively
reprogrammed with three factors, at least 27% of them would
have contained an optimal amount of each of these factors
(i.e., 0.273 ¼ 0.02). Hence, B7% of cells (i.e., 0.272) would
have expressed optimal levels of each of the two factors,
Oct4/Klf4 or Oct4/Sox2. Given that 0.2% of cells became
reprogrammed, this corresponds to a reprogramming efficiency of B3% of cells with optimal expression of both factors
(i.e., 0.2 $ 100/7). Thus, we conclude that the expression
levels of exogenous factors did not have a rate-limiting role
during iPS induction in this system.
In sum, our results support a version of the stochastic model
in which cells of different degrees of differentiation have the
potential to generate iPS cells, albeit with very different
kinetics and the requirement of three rather than two factors
for reprogramming of more differentiated cells. The main
difference between undifferentiated and differentiated cells, in
this respect, is that the former are less stable at high levels of
internal or external stimuli.38,39 Our results infer that regardless of the cell type the net result of stochastic transitions is the
establishment of a precarious balance between ‘supply’ and
‘demand’ for transcription factors that have explicit lineagespecifying activities.39 Based on this, we conclude that forced
expression of Oct4 with either Sox2 or Klf4 does not directly
convert MEFs to a pluripotent stem cell state. Instead,
each combination of factors disrupts the normal cellular
homeostasis and establishes transient states characterized
by the concurrent expression of mixed lineage markers
(Supplemental Figure 8). Viewed in this light, the MET
observed in four-factor reprogramming might be just one of
many possible transient stages.
Materials and Methods
All cells were cultured on 0.1% gelatinized tissue culture plates. Wild-type mice
(Jackson Laboratory, Bar Harbor, ME, USA) were maintained on the 129/Sv
background. Oct4-GFP mice (Jackson Laboratory) and Nestin-GFP transgenic
mice33 were maintained on a mixed 129/B6 genetic background. MEFs were
derived from E12.5–E13.5 embryos using standard procedures. iPS induction was
performed according to Li et al.40 Retroviral vectors encoding human Oct4, Sox2,
Klf4 and c-Myc were transfected into packaging Phoenix E cells using Lipofectamine
reagent (Invitrogen, Grand Island, NY, USA). Viral supernatants were collected and
filtered through a 0.45-mm cellulose acetate filter. MEFs were plated at a density of
2 $ 105 cells per 6-cm plate and incubated with the viral supernatant overnight.
After four successive infections, cells were switched to knockout serum replacement
(KSR) medium consisting of DMEM/F12 supplemented with 10% KSR (Invitrogen),
1 $ nonessential amino acids, 1 $ Glutamine, 1 $ Pen/Strep, 0.1 mM
b-mercaptoethanol and 1000 U/ml leukemia inhibitory factor (Millipore, Billerica,
MA, USA). The transduction efficiency of MEFs was determined by extrapolation
from infection with a GFP-expressing control virus, which was conducted in parallel.
In order to improve transduction efficiencies, the proviral parts of pMX vectors
contained within the corresponding BspH1 sites were subcloned into episomal
vector REBNA.25 Molecular, histological, microarray and other analyses were performed
as described in Supplemental Experimental Procedures, Figures and Tables.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. This work was supported by NYSTEM contract no.
N08T-040 to UMM and OP. AN was supported by a fellowship from the Lymphoma
Research Foundation.
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Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Cell Death and Differentiation
Supplemental Experimental Procedures
Mice and cell culture
All animal studies were approved by the Institutional Animal Care and Use Committee at Stony
Brook University. Wild-type mice (Jackson Laboratory) were maintained on the 129/Sv
background. Oct4-GFP mice (Jackson Laboratory) Nestin-GFP transgenic mice were
maintained on a mixed 129/B6 genetic background. MEFs were derived from E12.5-E13.5
embryos using standard procedures. Mouse Oct4-GFP ES cells were kindly provided by
Antonella Galli (Columbia University). Primary MEFs were cultured in DMEM/10%FBS on 0.1%
gelatinized tissue culture plates. ES and iPS cells were cultured inKSR medium consisting of
F12 DME supplemented with 10% KSR (Invitrogen), 1 x nonessential amino acids, 1 x
Glutamine, 1 x Pen/Strep, 0.1 µM ȕ-mercaptoethanol, and 1000 µ/ml LIF (Millipore).
For teratoma assays, 1 x106 iPS cells were resuspended in Matrigel (Invitrogen) and
injected subcutaneously into CD1 athymic nude mice (Harlan). When tumors reached 1 cm3 in
size (6 to 8 weeks), they were harvested and processed histologically. For blastocyst injection,
4-week-old C57BL/6N (Taconic) female mice were superovulated by administration of 5 IU of
pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotrophin, and then
mated with C57Bl/6N males. All blastocysts were collected at day 3.5 after detection of vaginal
plugs and flushed in M2 medium (Millipore). The blastocysts were washed in M2 medium and
cultured in KSOM+AA medium (Millipore) at 370C, 5% CO2 incubator. Five days before
injection, the iPS cells were thawed and passaged once. Ten to fifteen iPS cells were injected
into each blastocyst for generation of chimeric mice.
Flow cytometry
Cells were harvested using Accutase (Sigma), stained with anti-Sca1, Thy1.2, CD34, CD133,
EpCAM, CDH1, SSEA1, NGFR and c-Kit antibodies (BD Pharmingen or eBioscience), and
analyzed using FACS Calibur (Becton-Dickinson) and CellQuest software. Hematopoietic cells
(Mac1- and CD45-positive) were excluded from the sorting gates.
Expression analyses
Total cellular RNA was extracted using the RNeasy Mini Kit (Quiagen). One-color hybridizations
of cRNAs (2 technical replicas) were performed against the mouse Ilumina MouseRef-8
Expression BeadChip 25K microarray according to the manufacturer’’s instructions by Empire
Genomics LLC (Buffalo NY). Microarray signals were processed with GenomeStudio Gene
Expression Module (GSGX) Version 1.6.0. Data were background corrected and quantile
normalized. Differentially expressed genes were defined as > 2-fold change. For scatter plot
analyses, Pearson correlation coefficients between two samples were calculated using
normalized signals. Scatter plots were generated using log2 transformed gene expression
values. The heatmaps were generated by calculating ratios of expression in each sample
versus control. The log2 values were then supplied to the heatmap function of the R statistical
package. Module expression analysis was conducted as describe in (Kim et al., 2010). Average
gene expression values (log2) of all genes were set as baseline 0. The gene expression values
(log2) of each module relative to the overall average were represented as mean ± SEM.
Definition of each module is as follows: the Core module is composed of genes co-occupied by
at least seven factors among nine factors shown in the Core cluster (Smad1, Stat3, Klf4, Oct4,
Nanog, Sox2, Nac1, Zfp281, and Dax1) (Kim et al., 2010); the ESC-like module (Wong et al.,
2008); the Myc module comprises targets of Myc, Max, N-Myc, Dmap1, E2F1, E2F4, and Zfx
(Kim et al., 2010); the PRC module is composed of targets of PRC cluster proteins, Phc1, Rnf2,
Eed and Suz12 (Kim et al., 2010). We confirmed reproducibility of our microarray by quantitative
real-time PCR, using RNA samples that had been independently isolated from newly infected
MEFs. RT-PCR was performed using total RNAs and gene-specific primers. The sequences of
primers are listed in Supplemental Table 4.
Western blotting
Western analyses were performed on a routine basis. Representative results are shown. Briefly,
cells were lysed in buffer containing 10ௗmM Tris (pH 7.4), 150ௗmM NaCl, 10% glycerol, 1%
Triton, 1ௗmM EDTA, 0.1% SDS and protease inhibitor cocktail (Roche). Aliquots of whole-cell
lysates (50––80 ȝg of protein) were separated on SDS––acrylamide gels and blotted onto BA85
nitrocellulose membranes (Schleicher & Schuell). They were then incubated with antibodies
specific for Oct4 (ab19857), Klf4 (ab34814, both from Abcam); Sox2 (AB5603, Millipore), c-Kit
(DI3A2, Cell Signaling), CDH1 (610404, BD Pharmingen), EpCam (E144, Novus), and Erk1,2
(05-157, Upstate).
Supplemental Figures
Suppl. Figure 1.
Genomic PCR to detect exogenous transgene integration in OS-generated (A) or OK-generated
(B) iPS clones. Mouse ES cells were used as negative controls.
Suppl. Figure 2.
A, B. Heat maps from microarray analyses showing induction of endogenous transcription
factors in Sca1-SP, DN, Thy1-SP and DP MEFs transduced with OS- (A) or OK-expressing
retroviruses (B). Values are given relative to their own uninfected controls. Expression in
corresponding uninfected Sca1-SP and DN (summarily called ‘‘Thy1-‘‘) versus Thy1-SP and DP
(summarily called ‘‘Thy1+’’) cells are shown as controls.
C. Heat maps of shared transcription factor genes in OS- and OK-transduced Thy1-negative
(Sca1-SP and DN) and Thy1-positive (Thy1-SP or DP) cells.
Suppl. Figure 3.
Average gene expression values of the ESC-like, Core pluripotency, Polycomb repressive
complex (PRC) and Myc transcriptional modules in control uninfected Thy1-negative (Sca1-SP
and DN) and Thy1-positive (Thy1-SP and DP) cells as well as in Sca1-SP, DN and Thy1-SP
subpopulations transduced with OS or OK retroviruses. Microarray analyses were performed 4
days post infection with the indicated retroviruses. Data are represented as mean +/- SEM.
Suppl. Figure 4.
The Thy1+ fractions (composed of Thy1+ single-positive, Thy1-SP; and Thy1+Sca1+, double
positive, DP cells) represent the majority of MEFs (>70%), while Thy1-negative fractions
(composed of Sca1+ single-positive, Sca1-SP; and Thy1-Sca1-, double negative, DN cells)
represent a relative minority (A). When sorted cells were maintained under standard culture
conditions for 7 days, Sca1-SP MEFs gradually lost their phenotype and converted to DN and
Thy1-SP cells (A). Likewise, DP cells largely lost the expression of Sca1, becoming Thy1-SP
cells, and the same drift was seen in DN cells (A). In contrast, the Thy1-SP fraction was stable
in retaining the corresponding phenotype over extended periods of time (A). Thus, DP, DN and
Sca1-SP cells spontaneously gravitate towards acquisition of Thy1 single positivity. Retroviral
expression of Klf4, alone or in combination with Oct4 and Sox2 rendered all fractions positive for
Sca1, thereby reducing the proportion of DN and Thy1-SP cells (B). On the other hand,
retroviral expression of Oct4 and Sox2 either alone or in combination with each other, produced
no significant changes (4B and data not shown). Thus, the sorted MEF fractions acquire
different phenotypic features depending on whether they were infected with OK versus OS
viruses or other factor combinations.
Suppl. Figure 5.
A. FACS analysis of Nestin-GFP+ MEFs. Nestin-GFP+ cells (left) were gated (R1) and analyzed
for Thy1 expression (right). The majority of Nestin-GFP+ cells are Thy1-negative (81%).
B. FACS analysis of NGFR+ MEFs. Nestin-GFP+NGFR+ cells (left) were gated (R1) and
analyzed for Thy1 expression (right). The majority of NGFR+ cells are Thy1-negative (79%).
Suppl. Figure 6.
A. Western blot analysis of GFP expression in FACS-sorted Nestin-GFP-negative MEFs. Cells
were analyzed 4 days post-infection with empty control retroviruses (Mock) or OS-, OK-, or
OSK-expressing viruses. Note that expression of GFP (i.e., Nestin) is significantly reduced upon
conversion of cells into iPS cells (OS-generated iPS colony is shown). ES cells derived from
Oct4-GFP transgenic mice are shown for control. MAPK is a control for equal loading.
B. Induction of GFP expression in FACS-sorted Nestin-GFP-negative MEFs transduced with
retroviruses as in (A). Cells were analyzed by FACS on days 4, 7, 11, and 14 post-infection.
Suppl. Figure 7.
A. Quantitative real-time RT-PCR analysis of gene expression levels in Nestin-GFP negative
(yellow group) and Nestin-GFP bright MEFs (blue group). Cells were analyzed before and 1 or
11 days after transduction with OS, OK or OSK viruses. Data were normalized to the
corresponding HPRT value to obtain relative changes in the indicated mRNAs.
B. Quantitative real-time RT-PCR analysis of NGFR expression levels in total unsorted NestinGFP MEFs and sorted populations of Nestin-GFP+NGFR+ and Nestin-GFP-NGFR- MEFs.
Cells were analyzed before and 1 or 11 days after transduction with OS, OK or OSK viruses.
Data were normalized to the corresponding HPRT value to obtain relative expression changes.
Suppl. Figure 8.
Schematic of reprogramming stages: disruption of cellular homeostasis (A), cell switch to
transient states characterized by concurrent expression of mixed lineage markers (B), activation
of pluripotency networks (C) and the conversion into IPS cells (D). The OSK and OS
combinations of factors are more efficient than OK in activation of pluripotency networks, while
the OSK and OK combinations are more efficient and faster than OS in inducing ES cell-specific
morphological changes.
Clone ID
B
mES cells
2 OSKM
1 OSKM
10 OK
9 OK
8 OK
7 OK
6 OK
5 OK
4 OK
3 OK
2 OK
H2O
mES cells
2 OSKM
1 OSKM
13 OS
12 OS
11 OS
10 OS
9 OS
8 OS
7 OS
6 OS
5 OS
4 OS
3 OS
2 OS
1 OS
Clone ID
1 OK
A
Oct4
Sox 2
Klf4
Myc
Oct 4
Klf4
Sox 24
Myc
Nemajerova et al., Suppl. Figure 1
Thy1-/Thy1+
Sca1-SP OS
DN OS
DP OS
Thy1-/Thy1+
Sca1-SP OK
DN OK
Thy1-SP OK
B
Shared factors
OK responsive factors
OS responsive factors
A
Thy1-/Thy1+
Sca1-SP OS
DN OS
DP OS
Sca1-SP OK
DN OK
Thy1-SP OK
C
Low
Expression (log2)
High
Nemajerova et al., Suppl. Figure 2
Thy1-SP/OK
DN/OK
Sca1-SP/OK
Thy1-SP/OS
DN/OS
Sca1-SP/OS
Thy1-positive
Thy1-negative
Log2 Average expression
ESC-like
Core
PRC
Myc
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
1
2
3
4
5
6
7
8
X Data
Control
Oct4 + Sox2
Oct4 + Klf4
Nemajerova et al., Suppl. Figure 3
Sorted
3%
7d culture
1%
24%
1%
Klf4
5%
Nemajerova et al., Suppl. Figure 4
A
Oct4 + Klf4
10%
8%
9%
DN
93%
72%
2%
3%
27%
58%
38%
46%
1%
2%
25%
2%
3%
14%
6%
12%
2%
95%
65%
7%
13%
70%
26%
56%
15%
82%
81%
9%
16%
74%
27%
64%
2%
1%
10%
0%
4%
6%
6%
3%
97%
1%
93%
2%
28%
50%
36%
42%
1%
0%
5%
0%
7%
15%
9%
13%
Sca1-SP
DP
B
Sorted
3%
7d culture
1%
24%
1%
Sca1
Oct4 + Sox2
17%
2%
DN
93%
72%
3%
2%
76%
6%
1%
2%
25%
2%
19%
2%
2%
95%
65%
7%
70%
8%
15%
82%
81%
9%
65%
19%
2%
1%
10%
0%
14%
2%
97%
1%
93%
2%
78%
7%
1%
0%
5%
0%
14%
1%
Sca1-SP
DP
Thy1
Thy1-SP
Sca1
Thy1
Thy1-SP
A
Thy1
Thy1
R1
6%
13%
68%
13%
GFP
Sca1
R1
GFP
NGFR
NGFR
B
79%
21%
Thy 1
Nemajerova et al., Suppl. Figure 5
Mock
OS
OK
OSK
iPS
ES Oct4GFP
A
GFP-negative
GFP
OSK
OK
OS
4
7
11
14
100
80
60
40
20
0
4
7
11
14
% GFP-positive cells
Mock
4
7
11
14
B
4
7
11
14
MAPK
1
2
3
Days post-infection
4
Nemajerova et al., Suppl. Figure 6
Relative expression
A
KIT
24
TCF3
p53
ARF
GFP-neg
GFP-neg (D1)
GFP-neg (D11)
GFP-bright
GFP-bright (D1)
GFP-bright (D11)
iPS
22
20
4
2
0
0
1
2
3
4
5
6
7
8
OS OK OSK
OS OK OSK
GFP+NGFR+
GFP-NGFR-
X Data
9
10
11
OS OK OSK
12
13
14
15
16
OS OK OSK
Relative expression
B
1.2
Unsorted
Mock
OS
OK
OSK
0.8
Sorted
0.4
0.0
1
D1
2
3
D11 X Data D1
4
D11
Nemajerova et al., Suppl Figure 7
Primary cell
OSK, OS, or OK
Disrupted homeostasis
A
OSK or OK
OS
Transient states
B
OK
Activation of pluripotency networks
C
Acquisition of ES cell properties
D
Nemajerova et al., Suppl. Figure 8
Nemajerova et al., Suppl. Table 1
Term
Count
%
P Value
------------------------------------------------------------------------------------------------------------GO:0048598~embryonic morphogenesis
8
6.1
0.016666071
GO:0048729~tissue morphogenesis
8
6.1
0.001862836
GO:0060284~regulation of cell development
8
6.1
1.69E-04
GO:0048736~appendage development
5
3.8
0.011506968
GO:0035107~appendage morphogenesis
5
3.8
0.010242797
GO:0060348~bone development
5
3.8
0.011181983
GO:0008544~epidermis development
4
3.0
0.064658192
GO:0001654~eye development
5
3.8
0.028622431
GO:0007507~heart development
7
5.3
0.006052495
GO:0003007~heart morphogenesis
5
3.8
0.002142245
GO:0001822~kidney development
4
3.0
0.044328062
GO:0035108~limb morphogenesis
5
3.8
0.010242
GO:0060173~limb development
5
3.8
0.011506968
GO:0014031~mesenchymal cell development
3
2.3
0.046788171
GO:0048762~mesenchymal cell differentiation
3
2.3
0.050424149
GO:0030182~neuron differentiation
10
7.6
0.002676603
GO:0045664~regulation of neuron differentiation
7
5.3
1.04E-04
GO:0007423~sensory organ development
8
6.1
0.002867616
GO:0001501~skeletal system development
9
6.8
0.001192193
GO:0014706~striated muscle tissue development
4
3.0
0.067142518
GO:0001655~urogenital system development
6
4.5
0.004445775
GO:0051216~cartilage development
6
4.5
2.69E-04
GO:0006928~cell motion
8
6.1
0.018575252
GO:0007155~cell adhesion
12
9.1
0.002739769
GO:0030574~collagen catabolic process
4
3.0
2.44E-04
GO:0030198~extracellular matrix organization
5
3.8
0.00653851
GO:0007167~receptor protein signaling pathway
7
5.3
0.015413984
GO:0042981~regulation of apoptosis
9
6.8
0.050942203
GO:0001558~regulation of cell growth
4
3.0
0.030321804
GO:0040008~regulation of growth
6
4.5
0.040578565
GO:0051252~regulation of RNA metabolic process
17
12.9
0.077021932
GO:0006355~regulation of transcription, DNA-dependent
17
12.9
0.068985832
GO:0006357~regulation of transcription from RNA polymerase II promoter
10
7.6
0.037471912
Supplemental Table 1.
Sca1-SP MEFs show increased expression of genes involved in embryonic morphogenesis, tissue morphogenesis and regulation of
cell development.
Nemajerova et al., Suppl Table 2
Fold Change
(DN vs. Thy1-SP)
--------------------------------------------------------------------------------------MYLPF
fast skeletal myosin light chain 2
27.6
MYL1
myosin, light chain 1, alkali; skeletal, fast
11.6
MYL4
myosin, light chain 4, alkali; atrial, embryonic
8.3
MYL6B
myosin, light chain 6B, alkali, smooth muscle and
non-muscle
5.1
MYH1
myosin, heavy chain 1, skeletal muscle, adult
2.48
MYH8
myosin, heavy chain 8, skeletal muscle, perinatal
4.24
MB
myoglobin
2.46
TNNC1
troponin C type 1
8.05
TNNC2
troponin C type 2
19.4
TNNI1
troponin I type 1
12.74
TNNI2
troponin I type 2
16.04
TNNT1
troponin T type 1
5.9
TPM2
tropomyosin 2
7.11
THY1
0.31
Sca1
1
NGFR
(CD271)
11.06
CD82
(Inducible membrane protein R2)
2.41
CD80
(T-lymphocyte activation antigen CD80)
2.37
CD68
(macrophage antigen CD68)
1
CD40
(TNF receptor superfamily member 5)
1
CD96
(T-cell surface protein tactile)
1
CD97
(heterodimeric receptor associated with inflammation)
1
CD55
(decay accelerating factor for complement)
1
Gene
Supplemental Table 2.
DN MEFs are enriched for myofibroblast-committed progenitors.
Supplemental Table 3.
Sorted Sca1-SP and DN MEFs yield significantly more iPS colonies
than the corresponding Thy1-SP or DP fractions.
MEF subpopulations
p value
(OSKM)*
p value
(OSK)*
p value
(OK)*
p value
(OS)*
Sca1-SP vs. Unsorted
<0.0001
<0.0001
<0.0001
<0.0001
Sca1-SP vs. Thy1-SP
<0.0001
<0.0001
<0.0001
<0.0001
Sca1-SP vs. DP
<0.0001
<0.0001
<0.0001
<0.0001
DN vs. Unsorted
<0.0001
<0.0001
<0.0001
<0.0001
DN vs. Thy1-SP
<0.0001
<0.0001
<0.0001
<0.0001
DN vs. DP
<0.0001
<0.0001
<0.0001
0.0002
0.17
0.97
0.039
0.42
DN vs. Sca1-SP
* Cells were transduced with OSKM, OSK, OK or OS combinations of reprogramming factors.
P values were calculated using Student’s t-test.
Nemajerova et al., Suppl Table 3
Supplemental Table 4
Primer sequences for quantitative real time RT-PCR:
Gene
name
HPRT
CDH1
EPCAM
CD34
CD133
c-KIT
Oct4
Sox2
Klf4
c-Myc
Nanog
Sall4
Tbx3
Tcf3
Zic3
Arf
p53
NGFR
Primer sequence 5’’-3’’
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
GGCTATAAGTTCTTTGCTGACC
CTCCACCAATAACTTTTATGTCC
CAGGTCTCCTCATGGCTTTGC
CTTCCGAAAAGAAGGCTGTCC
GCGGCTCAGAGAGACTGTG
CCAAGCATTTAGACGCCAGTTT
AAGGCTGGGTGAAGACCCTTA
TGAATGGCCGTTTCTGGAAGT
GTTGAGACTGTGCCCATGAAA
GACGGGCTTGTCATAACAGGA
GCCACGTCTCAGCCATCTG
GTCGCCAGCTTCAACTATTAACT
CCATGCATTCAAACTGAGGCACCA
AGCTATCTACTGTGTGTCCCAGTC
CGCCCAGTAGACTGCACAT
CCCTCCCAATTCCCTTGTAT
GTGCCCCGACTAACCGTTG
GTCGTTGAACTCCTCGGTCT
GCTCTCCATCCTATGTTGCGG
TCCAAGTAACTCGGTCATCATCT
TCTTCCTGGTCCCCACAGTTT
GCAAGAATAGTTCTCGGGATGAA
CCCTGGGAACTGCGATGAAG
TCAGAGAGACTAAAGAACTCGGC
GAACCTACCTGTTCCCGGAAA
CAATGCCCAATGTCTCGAAAAC
ACGAGCTGATCCCCTTCCA
CAGGGACGACTTGACCTCAT
TGCTGCCAGTTCAGGCTATG
CGAGAAGGGGTTTTAGTGGTATC
CTTGGTCACTGTGAGGATTCA
CTACGTGAACGTTGCCCATCA
ACAGCGTGGTGGTACCTTAT
GGTTCCCACTGGAGTCTTC
TGGGCTCAGGACTCGTGTT
CAGGGATCTCCTCGCATTCG
Amplicon
size (bp)
126
175
139
157
98
90
243
154
185
116
100
111
121
101
83
125
149
189
All primers are for mouse mRNA detection and have a hybridization temperature of 60°C. Oct4,
Sox2 and KLF4 primers are designed to amplify only the endogenous mRNA, not the transgene.
Relative expression of the target genes was calculated using the ǻCT method described
previously: Relative expression = 2-ǻCT, where ǻCT = CT (Target gene) - CT (HPRT).