High-Efficiency Stem Cell Fusion-Mediated Assay Reveals
Sall4 as an Enhancer of Reprogramming
Connie C. Wong1,2.¤, Alexandre Gaspar-Maia2,3,4, Miguel Ramalho-Santos2,3.*, Renee A. Reijo Pera1,2¤*
1Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, California, United States of America, 2Institute
for Regeneration Medicine, University of California San Francisco, San Francisco, California, United States of America, 3Diabetes Center, University of California San
Francisco, San Francisco, California, United States of America, 4Doctoral Program in Biomedicine and Experimental Biology, Center for Neuroscience and Cell Biology,
University of Coimbra, Coimbra, Portugal
Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process
of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report
the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to
screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15–590
fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process.
Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a
regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The
reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome
remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that
improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular
characterization of enhancers of somatic cell reprogramming.
Citation: Wong CC, Gaspar-Maia A, Ramalho-Santos M, Reijo Pera RA (2008) High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of
Reprogramming. PLoS ONE 3(4): e1955. doi:10.1371/journal.pone.0001955
Editor: Thomas Zwaka, Baylor College of Medicine, United States of America
Received December 21, 2007; Accepted March 2, 2008; Published April 16, 2008
Copyright: ? 2008 Wong 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 research was supported by funds from the UCSF Institute for Regeneration Medicine and Juvenile Diabetes Research Foundation (to MRS) and
from the Stanford University Institute for Stem Cell Biology and Regenerative Medicine, and the California Institute of Regenerative Medicine (to RARP). The
funders were not directly involved in the study or the preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (MR); email@example.com (RR)
¤ Current address: Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, California,
United States of America
. These authors contributed equally to the work.
The developmental programs of somatic cells are characterized
by remarkably stable patterns of gene expression and repression.
Nonetheless, through nuclear reprogramming, the developmental
programs of somatic cells may be erased and redirected [1–6]. In
recent years, much attention has been given to nuclear
reprogramming of somatic cells in hopes of generating patient-
specific embryonic stem cells (ESCs) that might provide valuable
tools for basic science studies and potential novel therapeutics
Nuclear reprogramming was first demonstrated as an integral
part of mammalian development; following fusion of the egg and
sperm, the fused gametic nucleus must be reprogrammed,
through a series of changes that include DNA demethylation
and chromatin remodeling, to that of an embryonic cell if
development is to be successful [5,6,9]. In methods such as
somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell
is transferred to an enucleated oocyte for reprogramming to an
embryonic cell state, through the use of the endogenous
machinery [3,10,11]. Methods other than SCNT have also been
used to reprogram somatic cells including fusion with ESCs and
genetic reprogramming via co-expression of pluripotency-associ-
ated genes [12–16]. Each of these methods has advantages and
limitations. For example, although SCNT takes advantage of
endogenous programs, it requires the use of oocytes that may be
in short supply . In the case of cell fusion, although the cells
are in great supply, the procedure results in the formation of
tetraploid cells that are genetically unstable [12,18–20]. Finally,
although genetic reprogramming by co-expression of the stem cell
factors Oct4, Sox2, c-myc and Klf4 is remarkable in that it yields
ESCs capable of contributing to both the somatic and germ cell
lineages, use of the reprogrammed cells to generate offspring
results in increased tumorigenesis in progeny [13–16]. Moreover,
in all methods, the efficiency of reprogramming is very low,
suggesting that additional components of the reprogramming
pathways remain to be identified.
In this study, we sought to optimize cell fusion reprogramming
protocols, based on fusion of somatic cells and ESCs, in order to
screen for enhancers of somatic cell reprogramming. We
reasoned that if a factor functions in reprogramming, overex-
pression of that factor in somatic cells might increase the
efficiency with which the cells can be reprogrammed. Thus, we
tested whether overexpression of the following factors, individ-
ually, increased reprogramming efficiency of MEFs: Oct4,
Nanog, Sox2, and Sall4.
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Optimization of an Efficient Reprogramming Assay
Several different protocols have been developed to reprogram
somatic cells via cell fusion with ESCs, with protocol efficiencies
typically less than 0.001% (i.e. ranging from approximately 1
reprogramming event per 16105to 46106total somatic cells)
[12,20]. Such low efficiencies lead to technical difficulties in
screening for positive regulators of somatic cell reprogramming.
Thus, we sought to establish an efficient and quantitative
reprogramming assay via cell fusion between mouse ESCs and
G418-resistant (Rosa26) mouse embryonic fibroblasts (MEFs) that
carry the Oct4-gfp transgene [20,21]. We began by exploring
conditions required for efficient fusion. Traditionally, cells are
fused in suspension in 50% polyethylene glycol [12,18–20].
However, we found that the fusion efficiency was substantially
increased by both fusing the ESCs and MEFs in adherent cultures
and increasing polyethlyene glycol from 50 to 56%. FACS
(fluorescent-activated cell sorting) analysis of MEFs and ESCs,
which were fluorescently labeled with Vybrant DiD and Vybrant
DiO respectively, indicated that the fusion efficiency was 4.6 +/2
0.1% at 5 h post-fusion (Figure 1A).
The first visible, qualitative evidence of reprogramming (within
24 to 48 h post-fusion) was the expression of the Oct4-gfp
transgene, which was normally silent in MEFs . As time
progressed, the reprogrammed MEFs gradually obtained the
morphology of ESCs, as reflected by comparisons of forward and
side scatter profiles of the GFP-positive MEFs at 24, 48 and 72 h
post-fusion (Figure 1B).
Reprogramming efficiency was quantified by determining: 1)
the percentage of cells that expressed Oct4-gfp and 2) the number of
G418-resistant, stem cell-like colonies formed. The percentage of
GFP positive cells was measured by FACS at 24 h and 48 h post-
fusion, using wildtype MEFs (without the Oct4-gfp transgene) as a
negative control (Figure 1C). The number of GFP positive
particles from the wt MEFs was subtracted from that of Oct4-
GFP MEFs in order to eliminate any background fluorescence
from our calculations. Typical results indicated that the percentage
of GFP positive cells, among the total MEF population at 24 h
post-fusion, was 0.029 +/2 0.008% and that the number of
reprogrammed colonies obtained by this method was found to be
as many as 1 in 6.86103total MEF cells, a value 15 to 590 fold
higher than previously reported with other reprogramming assays
available [12,18–20]. These reprogrammed cells can be expanded
Figure 1. Establishment of an efficient fusion assay. A) Fusion efficiency of the assay. The MEFs and ESCs were stained with the fluorescent
dyes Vybrant DiD and Vybrant DiO respectively before fusion. Fusion efficiency was determined by FACS analysis at 5 h post-fusion, using an unfused
mixture of cells as a negative control. Note that previous studies have shown that cell surface dyes rarely diffuse across the cell membranes of stained
cells . The dual-labeled cells in the unfused population was most likely due to non-specific binding between the ESCs and MEFs. The green
fluorescent dye Vybrant DiO was only used in the determination of fusion efficiency but not in a typical reprogramming experiment (due to
interference with the observation of GFP signal). B) Morphology change of MEFs during reprogramming. The forward- and side-scatter profiles of the
GFP positive cells were FACS analyzed at 24, 48 and 72 h post-fusion. The morphology of the reprogrammed MEFs changed with time to resemble
that of the ESCs. C) Quantification of GFP expression. The number of GFP positive cells was FACS analyzed at 24 and 48 h post-fusion (right panel),
using wildtype MEFs that did not carry the Oct4-gfp transgene but had undergone identical fusion treatment with ESCs as a negative control (left
panel). The number of GFP positive cells from wt MEFs was subtracted from that of Oct4-gfp MEFs in all calculations.
Sall4 Enhances Reprogramming
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in ESC culture conditions, are tetraploid and express markers of
pluripotency (data not shown).
Potential Enhancers of Somatic Reprogramming
The significant improvements in reprogramming efficiency,
brought about by the modifications described above, allow for
scaling of the assay to multiple-well formats. We next tested
whether our protocol was suitable for quantitative analysis of
potential reprogramming factors, including those implicated in
maintaining pluripotency and in early embryo development: Oct4,
Sox2, Nanog and Sall4 [23–25]. Previously, Oct4, Sox2, and
Nanog have been shown to function in somatic cell reprogram-
ming, whereas the role of Sall4 in this process has not been
explored [14,18]. A schematic of the protocol is shown (Figure 2A).
Aliquots of G418-resistant MEFs, carrying the Oct4-gfp
transgene were infected with lentivirus constructs that expressed
one of the candidate factors 72 h prior to fusion. The
overexpression of candidate proteins was confirmed by Western
blotting (Figure 2B). 24 h prior to fusion, infected MEFs were
harvested and labeled with the fluorescent dye Vybrant DiD
(Figure 2C). The fluorescently-labeled MEFs and unstained ESCs
were then plated together in triplicate wells (Figure 2D); the visible
overexpression of the red fluorescent protein mCherry indicated
proper production and infection of the lentiviruses. Cells were
harvested at 24 h and 48 h post-fusion, and the percentage of
GFP-positive cells among the DiD-positive MEF population was
determined. G418 was then added to the remaining well of fused
cells 48 h post-fusion and subsequently at 10 days post-fusion, the
number of G418-resistant, GFP-positive colonies was determined.
The onset of Oct4-gfp expression provides an initial measure of
reprogramming. The percentage of GFP-positive cells, in the
population of MEFs that overexpressed each candidate gene, was
compared to that of uninfected MEFs and MEFs that overex-
pressed the negative control proteins, firefly luciferase and the red
fluorescent protein, mCherry. At 24 h post-fusion, GFP expression
in MEFs that overexpressed negative control proteins was similar
to that of the uninfected control, indicating that lentiviral-
mediated protein overexpression did not affect GFP expression
in MEFs carrying the Oct4-gfp transgene (Figure 3A). Unexpect-
edly, however, MEFs that overexpressed the known reprogram-
ming facilitators, Oct4, Nanog and Sox2, also did not show a
significant increase in Oct4-gfp expression relative to controls.
Figure 2. Screen of positive regulators of somatic cell reprogramming. A) Schematic of the screen. Candidate genes were overexpressed in
Oct4-gfp, G418-resistant MEFs via lentivirus infection 72 h before fusion. B) Successful lentiviral overexpression was verified by Western blotting, as
well as expression of the positive control mCherry. C) Infected MEFs were harvested at 24 h before fusion and stained with the fluorescent dye
Vybrant DiD. Prepared MEFs were plated with unstained ESCs. GFP expression was FACS analyzed at 24 and 48 h post-fusion. G418 was added to the
fused cells at 48 h post-fusion, and the formation of G418-resistant, GFP positive colonies was assayed 10 days post-fusion. D) The visible
overexpression of mCherry in infected MEFs indicated the effectiveness of our lentiviral overexpression system. Scale bar represents 50 mm.
Sall4 Enhances Reprogramming
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Although this was unexpected, the lack of enhanced GFP
expression when Oct4, Nanog and Sox2 were overexpressed in
MEFs might be attributed to several possibilities (this is further
described in the discussion section below). In contrast, we observed
that the percentage of GFP-positive cells in MEFs that
overexpressed the Spalt transcription factor, Sall4, increased 7-
fold relative to controls. The comparison of Oct4-gfp expression at
48 h post-fusion was similar to that at 24 h (Figure 3B). MEFs that
overexpressed Sall4 consistently demonstrated the highest per-
centage of GFP-positive cells compared to the other candidate
A second measure of reprogramming is colony formation. With
G418 drug selection, GFP-positive colonies began to appear
within 5 days post-fusion. The total number of colonies was
recorded 10 days post-fusion (Figure 3C). The number of
reprogrammed colonies formed by MEFs overexpressing negative
control proteins was again similar to the uninfected control,
confirming that the lentivirus itself did not affect reprogramming.
Nanog, Sox2 and Sall4 all showed a significant increase in the
number of reprogrammed colonies relative to controls (p,0.05).
However, the overexpression of Oct4 in MEFs did not
promote formation of reprogrammed colonies in these assays
Confirmation of Sall4 as an Enhancer of Reprogramming
by Cell Fusion
As described above, results indicated that Sall4 was likely a
positive regulator of somatic cell reprogramming, contributing to
both early activation of Oct4 in the somatic cells and formation of
reprogrammed colonies. However, given this data, we also
considered whether Sall4 might directly activate the Oct4-gfp
transgene in the absence of overall reprogramming. To examine
this alternative possibility, we tested whether increased GFP
expression at 24 h and 48 h post-fusion was due to transcription
activity of Sall4 alone. For this purpose, we overexpressed Sall4, as
well as the negative controls, in MEFs carrying the Oct4-gfp
transgene. Half of the infected MEFs were then cultured alone,
and the remainder was fused with ESCs. Then, when the 24 h
time point would typically be analyzed in a fusion experiment, cells
were harvested and the percentage of GFP-positive cells was
determined (Figure 4A). We found that the observed increase in
the number of GFP-positive cells was dependent on fusion with
ESCs; MEFs that overexpressed Sall4 but were not fused with
ESCs did not demonstrate increased Oct4-gfp expression. This
indicated that increased GFP expression in cells overexpressing
Sall4 is not a direct effect of Sall4 interacting with the Oct4
promoter of the Oct4-gfp transgene, but rather is a result of the
enhancement of reprogramming.
Next, we tested whether overexpression of Sall4 altered the
growth rate of MEFs, thus leading to an increased number of
colonies unrelated to reprogramming. For this purpose, we
overexpressed the negative control, mCherry, and Sall4 in MEFs,
plated the cells and determined cell number every 24 h as shown
(Figure 4B). An independent clone of Sall4 of identical sequence
was used in this experiment as a duplicate; clone 1 was the
construct used in all other experiments described in this study.
Results indicated that overexpression of Sall4 did not increase, but
instead slightly decreased, the growth rate of MEFs relative to the
We also addressed whether expression of Sall4 enhanced plating
efficiency of ESCs. For this purpose, we used both wildtype ESCs
and subcloned lines of tetraploid (4N, Figure 4C) reprogrammed
cells. In order to test the effect of Sall4 overexpression on ESC
colony formation efficiency, we infected ESCs and 4N cells with
constructs that expressed the negative control proteins and Sall4.
We observed under a microscope that the fluorescent intensity of
the negative control, mCherry, was significantly lower in infected
ESCs than in infected MEFs from previous experiments; Western
blotting also suggested that the expression level of Sall4 in infected
ESCs was not significantly increased relative to endogenous levels
(data not shown). This may reflect different activity, or
susceptibility to silencing, of the CMV promoter in MEFs relative
to ESCs . Nonetheless, we reasoned that the lower expression
levels in ESCs parallel observations during reprogramming: when
reprogrammed mCherry-infected MEFs gain ESC-like character-
istics after fusion, there appears to be a sharp decline of mCherry
expression during colony formation (data not shown). After
Figure 3. Overexpression of Sall4 enhanced Oct4-gfp expres-
sion and ES cell-like colony formation in MEFs during
reprogramming. The percentage of GFP positive MEFs at: A) 24 h
and B) 48 h post-fusion. C) The number of GFP positive, G418-resistant
colonies in 1 well of a 6-well plate, 10 days post-fusion. The
overexpression of Sall4 positively enhanced both Oct4-gfp expression
and colony formation of MEFs upon reprogramming.
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infection, we plated the infected ESCs and 4N cells with and
without feeders, and assayed colony formation after 7 days
(Figure 4D). The efficiency of forming colonies in uninfected
ESCs, or ESCs infected with constructs expressing the negative
controls mCherry and luciferase, was approximately 20% on
gelatin and 45% on feeder cells, similar to previously reported
values . We observed that cells infected with the Sall4
construct also did not demonstrate enhanced ability to form
colonies in either the presence or absence of feeders. Similar
results where obtained with expression of Sall4 in the 4N
reprogrammed ESCs (data not shown).
Structure-Function Studies of Reprogramming by Cell
Taken together, the data described above suggested that Sall4 is
a positive regulator of somatic reprogramming. Sall4 is a zinc
finger transcription factor expressed in cells of the early embryo
and the germ line, and is required for maintenance of pluripotency
[28–30]. Sall4 may act as both a positive transcriptional regulator
of genes such as Oct4  and as a transcriptional repressor .
The Sall family of proteins contains an N-terminal 12-amino acid
motif that recruits the nucleosome remodeling and deacetylase
corepressor (NuRD) complex, which is involved in global
transcriptional repression and regulation of specific developmental
processes [31,32]. The C-terminal region of Sall4 has also been
shown recently to contain weak transcription repression activity as
We sought to determine if our quantitative protocol for
reprogramming could be used to dissect the structure-function
relationships of factors implicated in reprogramming, such as
Sall4. Thus, we tested whether the N-terminal 12-amino acid
motif of Sall4 is required for somatic cell reprogramming. For this
purpose, we generated a truncated Sall4 mutant (Sall4 d12) that
lacked the N-terminal 12-amino acid motif and repeated the
fusion assays (Figure S1). We found that overexpression of Sall4
d12 resulted in both early activation of Oct4-gfp (Figure 5A) and in
increased numbers of ESC-like colonies (Figure 5B), similar to
results with wildtype Sall4. We also noted that overexpression of
Sall4 d12 did not alter the GFP expression pattern or growth rate
of MEFs carrying the Oct4-gfp transgene, nor did overexpression
increase colony formation efficiency (Figure S2, S3, S4). These
data show that the enhancement of somatic cell reprogramming
by Sall4 does not require the N-terminal domain of the protein
that has been implicated in recruiting the NuRD complex.
Figure 4. Sall4 is a bona fide positive regulator of reprogramming. A) Overexpression of Sall4 in Oct4-gfp MEFs did not induce GFP
expression. Sall4, mCherry and luciferase were overexpressed in Oct4-gfp MEFs via lentivirus infection. Half of the infected MEFs was fused to ESCs as
described, while the other half was not. Only MEFs overexpressing Sall4 and fused to ESCs showed an increased number of GFP positive cells when
compared to the negative controls, indicating that overexpression of Sall4 alone did not induce GFP expression. The numbers of GFP positive cells in
the infected cells relative to that of the uninfected cells were shown. B) Overexpression of Sall4 did not increase cell doubling time in MEFs. mCherry
and two different constructs of Sall4 were overexpressed in MEFs, which were plated onto 6 well plates and assayed for cell number every 24 h. Note
that another clone of Sall4 of identical sequence was used as a duplicate. C, D) Infection of Sall4-expressing lentiviruses did not increase the colony
formation efficiency in ESCs. Both ESCs and previously reprogrammed MEFs that were tetraploid were infected with lentiviruses expressing Sall4,
mCherry or luciferase. D) The infected cells were plated either on gelatin or on feeder cells. The number of colonies formed was assayed after 7 days.
Sall4 Enhances Reprogramming
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Furthermore, these data suggest that improvements in the fusion
assay may provide a useful platform for future structure-function
studies of regulators of reprogramming.
In this study, we optimized the cell fusion reprogramming assay.
The assay makes use of G418-resistant, Oct4-gfp MEFs and mouse
ESCs. Due to improved fusion and reprogramming efficiencies,
the assay is now potentially amenable to screening formats as
demonstrated here with the analysis of overexpression of the
pluripotency factors Oct4, Nanog, Sox2 and Sall4. Moreover, by
taking advantage of the fact that the Oct4-gfp transgene is activated
within the first 24–48 h of reprogramming, the assay allows for
further physical and molecular characterization of the reprogram-
ming process by microscopy and FACS.
Sall4: An Enhancer of Reprogramming
In this study, we demonstrate that the transcription factor Sall4
can enhance somatic cell reprogramming as evidenced by both
enhanced Oct4-gfp expression and colony formation. Previously,
Sall4 had not been shown to function in somatic cell reprogram-
ming. Sall4 is a member of the Spalt family of transcription factors
which was originally identified in Drosophila as a homeotic gene
required for head and tail development [28,34,35]. In mammals,
Sall4 is essential for early embryo development including
establishment and maintenance of the early cell lineages of the
inner cell mass . Sall4 is also essential for the maintenance of
pluripotency and self-renewal of ESCs and for their derivation
from blastocysts . Although Sall4 may act as a transcription
factor that regulates numerous genes, one of the few known target
genes is Oct4 . Recent studies show that Sall4 interacts with
Nanog to control the expression of Oct4 . Together, Oct4,
Nanog, Sox2 and Sall4 form a regulatory circuit to maintain
pluripotency of ESCs, prompting our exploration of these factors
[36–38]. Our results suggesting that Sall4 enhances reprogram-
ming in cell fusion prompts further analyses regarding whether it
may enhance reprogramming in other reprogramming strategies;
in addition, it is very likely that additional enhancers remain to be
Comparisons to Other Reprogramming Assays
A previous report by Silva and colleagues demonstrated that
overexpression of Nanog in mouse ESCs enhances reprogram-
ming of neural stem cells nearly 200-fold and reprogramming of
MEFs 10-fold as measured by colony formation . In the
current study, when we overexpressed Nanog in MEFs rather than
in ESCs, surprisingly, we achieved only a 3-fold increase in
reprogramming efficiency as judged by colony formation. Further
consideration and comparison of these studies is merited: First, it is
apparent from several studies, including that of Silva and
colleagues, that it is more difficult to reprogram somatic cells
such as MEFs than neural stem cells , perhaps due to the state
of differentiation of MEFs and/or epigenetic status of key
pluripotency genes. Second, we note that overexpression of Nanog
in ESCs resulted in greater enhancement of reprogramming
efficiency compared to overexpression in MEFs. This observation
might reflect fundamental differences between the two studies.
Since Nanog is an important pluripotency factor, it is highly likely
that the overexpression of Nanog in ESCs may reinforce the
pluripotency regulatory circuit, or stem cell properties, of ESCs. In
contrast, in our study, the overexpression of Nanog and other
positive regulators of reprogramming in MEFs most likely
enhances reprogramming by priming and preparing the somatic
cell genome for reprogramming. Thus, we suspect, from
comparisons of this data to that from other publications, that
the latter is a far less efficient process than reinforcing the
pluripotency regulatory circuit of ESCs.
Recently, several reports have demonstrated that MEFs can be
reprogrammed by co-overexpressing the pluripotency factors
Oct4, Sox2, C-myc and Klf4 [13–15]. In our study, neither
Oct4 nor Sox2 overexpression in the somatic compartment led to
early activation of Oct4 during reprogramming of MEFs, and only
Sox2 led to increased numbers of reprogrammed colonies. It is
possible that activation of Oct4 is not one of the earliest events to
occur during reprogramming, and clearly that not all factors that
facilitate reprogramming will lead to early activation of Oct4.
Thus, the lack of early Oct4 activation in our assay does not
preclude a factor from being an enhancer of reprogramming.
Furthermore, the expression level of Oct4 is regulated in a precise
manner in ESCs such that an increase in Oct4 expression level
leads to differentiation into primitive endoderm and mesoderm,
whereas a decrease results in trophectoderm formation . Thus,
we speculate that overexpression of Oct4 alone without other
reprogramming factors may actually inhibit reprogramming.
Finally we note that it is possible that the role of reprogramming
factors may differ depending on the method of reprogramming, be
it SCNT, cell fusion or over-expression of a subset of genetic
Figure 5. The N-terminal domain is not required for Sall4
function in reprogramming. A) Sall4 d12 mutant behaved similarly
to wt Sall4 in reprogramming. Sall4 d12, as well as two clones of wt
Sall4 of identical sequences, were overexpressed in Oct4-gfp MEFs,
which were then fused to ESCs and assayed for GFP expression as
described. B) The overexpression of Sall4 d12 resulted in a similar
increase in the number of Oct4-gfp cells as wt Sall4, as well as a similar
increase in the number of reprogrammed colonies.
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factors. The fusion reprogramming assay as optimized here is
useful for identification and characterization of new regulators or
enhancers of somatic reprogramming and may bypass some of the
difficulties with other methods. Together, the array of methods for
reprogramming holds great promise for the generation of patient-
specific stem cells for use in diverse basic and clinical studies in the
Materials and Methods
Mouse ESCs (E14) were cultured on plates coated with 0.1%
gelatin (Sigma-Aldrich, Steinheim, Germany) in ESC medium
[(Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
Carlsbad, CA) supplemented with 15% knockout serum replace-
ment (Invitrogen), 2 mM L-glutamine (Invitrogen), 1 mM sodium
pyruvate (Invitrogen), 1% non-essential amino acids (Invitrogen),
0.57 mM beta-mercaptoethanol (Sigma-Aldrich), 1% penicillin/
streptomycin (Invitrogen), and 0.3% leukemia inhibitory factor].
MEFs were harvested from (Rosa26 X Oct4-gfp) transgenic mice as
described [20,21] and cultured on gelatinized plates in MEF
medium [DMEM supplemented with 10% fetal bovine serum
(Hyclone Labs, Logan, UT), 2 mM L-glutamine, 1 mM sodium
pyruvate, 1% non-essential amino acids and 1% penicillin/
streptomycin]. 293 cells for lentivirus production were cultured
on gelatinized plates in MEF medium.
Lentiviral Vectors and Overexpression
The lentivirus overexpression vector pLove has been described
. The candidate genes were cloned individually into pEntr-1A
(Invitrogen), then subcloned into pLove using the GatewayH
technology (Invitrogen) according to the manufacturer’s protocol.
The companion vectors for lentivirus production, pMDL, pRSV
and pVSV-G, were gifts from Dr. Michael McManus (University
of California, San Francisco, CA).
The 293 cells were plated on 15-cm plates at 80000 cells/cm2
12–24 h before transfection. 4 mg of pLove and 1.3 mg each of
pMDL, pRSV and pVSV-G were transfected into 293 cells with
FuGENE 6 transfection reagent (Roche Applied Science,
Indianapolis, IN) according to the manufacturer’s protocol.
Supernatant containing mature lentivirus was harvested at 48 h
to 72 h after transfection and filtered with 0.45 mm PVDF syringe
filters (Millipore, Billerica, MA). For infection, 10 ml of the filtered
supernatant and 5 ml of fresh MEF medium was added to MEFs
cultured in 10-cm plates for 24 h. The cells were then rinsed
thoroughly 36 with DMEM and continued to culture in fresh
MEF medium for another 24 h. Overexpression of candidate
genes was verified by Western blotting. MEFs overexpressing the
candidate genes were harvested and homogenized in RIPA buffer
[50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholate, 1%
NP-40, 0.1% SDS, pH 8.0] at 100000 cells/ml. After a clarifying
centrifugation step at 12000 rpm for 20 min at 4uC, 30 ml of 66
Lammeli buffer [0.3 M Tris pH 6.8, 36% glycerol, 10% SDS,
120 mg/ml bromophenol blue] and 2 ml of betamercaptoethanol
were added to 60 ml of cell lysate, of which 20 ml was loaded per
lane on a 10% SDS-polyacrylamid gel. Western blotting was
performed using a goat anti-V5 antibody (Abcam, Cambridge,
MA) to detect the expression of all V5-tagged candidate genes, and
a goad anti-GAPDH antibody (Abcam) to detect the expression of
GAPDH as a loading control.
Cell Fusion Assay
At 24 h before fusion, G418-resistant, Oct4-gfp MEFs over-
expressing the candidate genes were stained with 0.5 ml/ml
Vybrant DiD (Invitrogen) in DMEM for 20 min at 37uC. The
cells were thoroughly rinsed 36 with phosphate buffered saline
(PBS) before trypsinized and replated on 6-well plates with
unstained ESCs; both MEFs and ESCs were seeded at 36105cells
per well in ESC medium. During fusion, the cells were first rinsed
16 with 2 ml PBS (pH 7.4) per well, then primed with 1 ml
50 mM sodium dodecyl sulphate (Sigma-Aldrich) in PBS for 3 min
at 37uC before incubating with 1 ml 56% PEG-3350 (Sigma-
Aldrich) resuspended in PBS for 1 min at 37uC . DMEM was
then added to the wells at 1 ml/min to dilute the PEG solution for
up to 5 ml. The cells were rinsed 16with 2 ml DMEM, 16with
2 ml ESC medium before returning to 3 ml ESC medium. The
medium was fully replaced daily post-fusion. At 24 h and 48 h
post-fusion, the fused cells were harvested and resuspended in
PBS-1% bovine serum albumin (Sigma-Aldrich) before assaying
for GFP expression with a FACSCalibur (BD Biosciences, San
Jose, CA). 200 mg/ml G418 solution (Invitrogen) was added at
48 h post-fusion to begin the selection for reprogrammed colonies.
In order to control for background fluorescence in our FACS
analysis, we fused both wt MEFs that did not contain any gfp
transgene, and MEFs that carried the Oct4-gfp transgene to ESCs
independently in our fusion experiments. We measured the
number of GFP positive cells in both populations, and we
subtracted the number of GFP positive particles of the wt MEFs
from that of Oct4-gfp MEFs in order to eliminate background
fluorescence from our calculations. All fusion experiments were
repeated between 3–6 times. The data were then pooled and the
average and standard deviation were calculated. Post-hoc tests
following a univariate analysis of variance (ANOVA) show that
average number of colony for Nanog, Sall4, and Sox2 are
significantly different from those of the uninfected, luciferase and
mCherry controls (p,0.05).
For the analysis of fusion efficiency described in Figure 1A,
MEFs were stained with 0.5 ml/ml Vybrant DiD (Invitrogen) and
ESCs were stained with 0.5 ml/ml Vybrant DiO (Invitrogen) in
DMEM for 20 min at 37uC before cell fusion. Cells were allowed
to recover in ESC medium for 5 h before FACS analysis.
d12 in Oct4-GFP MEFs was verified via Western blotting using
antibodies against Sall4 (gifts from Dr. Huck-Hui Ng from
Nanyang Technological University, Singapore). Sall4 was ex-
pressed in wildtype mouse ESCs but not uninfected MEFs.
Found at: doi:10.1371/journal.pone.0001955.s001 (0.11 MB TIF)
Sall4 d12 overexpression. Overexpression of Sall4
not induce GFP expression. Sall4 d12 was overexpressed in Oct4-
gfp MEFs and the activation of Oct4-GFP was measured as
described in the main text and Figure 4A.
Found at: doi:10.1371/journal.pone.0001955.s002 (0.21 MB TIF)
Overexpression of Sall4 d12 in Oct4-gfp MEFs did
doubling time in MEFs. Sall4 d12 was overexpressed in Oct4-gfp
MEFs and the doubling time of MEFs was measured as described
in the main text and Figure 4B.
Found at: doi:10.1371/journal.pone.0001955.s003 (0.26 MB TIF)
Overexpression of Sall4 d12 did not increase cell
colony formation efficiency in MEFs. Sall4 d12 was overexpressed
in E14 and the tetraploid reprogrammed MEFs, and the colony
forming efficiency of the infected ESCs was measured as described
in the main text and Figure 4D.
Found at: doi:10.1371/journal.pone.0001955.s004 (0.21 MB TIF)
Overexpression of Sall4 d12 did not increase the
Sall4 Enhances Reprogramming
PLoS ONE | www.plosone.org7April 2008 | Volume 3 | Issue 4 | e1955
Acknowledgments Download full-text
We thank Dr Michael McManus and the UCSF Lentiviral Core Facility
for valuable assistance and reagents (293 cells, pSicoR-GFP pMDL, pRSV
and pVSV-G). We also thank members of the Ramalho-Santos and Reijo
Pera laboratories for valuable discussions and comments on the
Conceived and designed the experiments: CW MR. Performed the
experiments: CW MR. Analyzed the data: CW MR RR. Contributed
reagents/materials/analysis tools: CW AG. Wrote the paper: CW RR.
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PLoS ONE | www.plosone.org8 April 2008 | Volume 3 | Issue 4 | e1955