Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

Abstract

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.

Full text

ORIGINAL ARTICLE
Reprogramming broblasts into induced pluripotent stem
cells with Bmi1
Jai-Hee Moon1, June Seok Heo1, Jun Sung Kim1, Eun Kyoung Jun1, 2, Jung Han Lee1, 2, Aeree Kim3,
Jonggun Kim4, Kwang Youn Whang4, Yong-Kook Kang5, Seungeun Yeo5, Hee-Joung Lim4, Dong Wook Han6,
Dong-Wook Kim7, Sejong Oh8, Byung Sun Yoon1, Hans R Schöler9, 10, Seungkwon You1
1Laboratory of Cell Function Regulation, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic
of Korea; 2Division of Stem Cell Research Institute, Stemmedience Corp., Seoul, Republic of Korea; 3Department of Pathology,
College of Medicine, Korea University Guro Hospital, Seoul, Republic of Korea; 4Division of Biotechnology, College of Life Sci-
ences and Biotechnology, Korea University, Seoul, Republic of Korea; 5Development and Differentiation Research Center, KRIBB,
Daejeon 305-333, Republic of Korea; 6Department of Stem Cell Biology, SMART Institute of Advanced Biomedical Science,
Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea; 7Department of Physiology, Center for Cell
Therapy, Yonsei University College of Medicine, Seoul, Republic of Korea; 8Division of Animal Science, Chonnam National Uni-
versity, Gwangju 500-757, Republic of Korea; 9Department of Cell and Developmental Biology, Max Planck Institute for Molecu-
lar Biomedicine,Röntgenstraße 20, Münster D-48149, Germany; 10Medical Faculty, University of Münster, Domagkstr. 3, Münster
D-48149, Germany
Correspondence: Seungkwon Youa, Hans R Schölerb, Byung Sun Yoonc
aTel: +82-2-3290-3057; Fax: +82-2-3290-3507
E-mail: bioseung@korea.ac.kr
bTel: +49-251-70365-300; Fax: +49-251-70365-399
E-mail: ofce@mpi-muenster.mpg.de
cTel: +82-2-3290-3493; Fax: +82-2-3290-3507
E-mail: biosun302@korea.ac.kr
Received 9 February 2011; revised 28 March 2011; accepted 4 May 2011;
published online 28 June 2011
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4,
Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram broblasts and Oct4
alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1
leads to the transdifferentiation of mouse broblasts into NSC-like cells, and, in combination with Oct4, can replace
Sox2, Klf4 and c-Myc during the reprogramming of broblasts into iPS cells. Furthermore, activation of sonic hedge-
hog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with
Oct4, reprograms mouse embryonic and adult broblasts into iPS cells. One- and two-factor iPS cells are similar to
mouse embryonic stem cells in their global gene expression prole, epigenetic status, and in vitro and in vivo differ-
entiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data sup-
port that converting broblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type
that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.
Keywords: reprogramming; transdifferentiation; neural stem cells; induced pluripotent stem cells; Bmi1; Oct4
Cell Research (2011) 21:1305-1315. doi:10.1038/cr.2011.107; published online 28 June 2011
Introduction
The potential of induced pluripotent stem (iPS) cell
technology is enormous, but comprehension of the mo-
lecular mechanisms that underlie reprogramming is mea-
ger, largely because the procedure is still very inefcient.
Recently, a number of groups have demonstrated that the
inactivation of p53 markedly increases the efciency of
iPS cell generation [1-6]. Furthermore, by reducing the
expression of both p16Ink4a and p19Arf (both of which are
encoded by alternative reading frames of the Ink4a/Arf
locus, also known as the Cdkn2a locus), iPS cell forma-
tion was increased relative to that achieved by reducing
the expression of p19Arf alone.
Bmi1 was rst identied as a proto-oncogene that co-
operates with c-Myc to promote the formation of B- and
T-cell lymphomas [7, 8] by inhibiting c-Myc-induced
npg
Cell Research (2011) 21:1305-1315.
© 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00
www.nature.com/cr

Reprogramming of broblast into iPSCs with Bmi1
1306
npg
Cell Research | Vol 21 No 9 | September 2011
apoptosis through repression of the Ink4a/Arf locus [9,
10]. Bmi1 is also required for the self-renewal of stem
cells and the Ink4a/Arf locus is the main target of Bmi1
stem cell proliferation activity [11-13]. Furthermore, ac-
tivation of the sonic hedgehog (Shh) signaling pathway
induces Bmi1, Sox2, and N-Myc expression, resulting
in increased proliferation of neural precursors [14-16].
In this study, we hypothesized that cells with reduced
expression of p16Ink4a and p19Arf as well as increased N-
Myc, Klf4, and Sox2 expression [13, 16] mediated by
Bmi1 overexpression could be efciently converted into
iPS cells with either two factors (Oct4 and Sox2: hereaf-
ter designated as 2F-Bmi1-iPS cells (OSB)) or only one
factor (Oct4: hereafter designated as BO-iPS cells). Fur-
thermore, we also asked whether Shh or activation of the
Shh signaling pathway by oxysterol or purmorphamine
can regulate the downstream target genes of Bmi1 in
the generation of BO-iPS cells. Our study demonstrates
that Bmi1 has dual effects on iPS cell generation from
broblasts by both suppressing p16 Ink4a and p19 Arf and
augmenting Sox2 and N-Myc. By inducing Bmi1 with
some chemicals, we have also shown that only one
transcription factor (Oct4) is required to reprogram fi-
broblasts into pluripotent cells, which have the ability to
differentiate into all three germ layer cell types and are
capable of germline transmission.
Results
Bmi1 replaces the function of Klf4 and c-Myc, and
increases reprogramming efficiency
Considering that Bmi1 is essential for the self-renewal
of stem cells through repression of the p53 and Rb path-
ways, we investigated whether Bmi1 could enhance the
reprogramming of broblasts into iPS cells. To answer
this question, we rst determined the expression levels
of reprogramming-related genes (p16Ink4a, p19Arf, Sox2,
N-Myc, p53, and Klf4) in parental mouse embryonic
broblasts (MEFs) and Bmi1-transduced cells. p16Ink4a,
p19Arf, and p53 were significantly repressed in Bmi1-
transduced cells compared to MEFs; however, consistent
with a previous report [16], Sox2, N-Myc, and Klf4
were abundantly expressed (Figure 1A and 1B). Next,
we tested whether Bmi1 could replace Oct4, Sox2, Klf4,
or C-Myc. The four transcription factors (Oct4, Sox2,
Klf4, and C-Myc; OSKM) or a combination of three
of the transcription factors with Bmi1 (OSKB, OSBM,
OBKM, and BSKM) were introduced into MEFs. Bmi1
was able to replace Sox2, Klf4, or C-Myc in inducing
Nanog-positive colonies that resemble embryonic stem
(ES) cells (Figure 1C and Supplementary information,
Figure S1). However, we found that in the absence of
Oct4, Nanog positive colonies were not formed (data not
shown), indicating that Bmi1 is not able to replace Oct4
for reprogramming MEFs. We also tested whether Bmi1
was able to replace both Klf4 and C-Myc (Figure 1A).
We reprogrammed mouse broblasts with Oct4 and Sox2
or with the two factors plus Bmi1 (2F-Bmi1-iPS (OSB)).
Overexpression of Bmi1 in these two-factor experiments
increased the number of Nanog-positive colonies, con-
sistent with a role of Bmi1 as a replacement for Klf4 and
C-Myc, as well as a limited role in regulating the p53
and Rb pathways during reprogramming. The OSB colo-
nies were very similar to those comprised of mouse ES
cells and expressed pluripotency-associated transcription
factors as well as pluripotent cell surface markers (Figure
1D and Supplementary information, Figure S1). Taken
together, these data suggest that Bmi1-mediated regula-
tion of N-Myc, Klf4, p16Ink4a, and p19Arf activity mark-
edly increases reprogramming efciency.
Induction of fibroblasts into iPS cells with Bmi1 plus
Oct4
Methods designed to reduce the number of factors
necessary for reprogramming have taken advantage of
endogenously expressed reprogramming factors such as
Sox2 [17]. For example, studies show that adult mouse
neural stem cells (NSCs), which exhibit endogenous
Sox2 expression, can be reprogrammed by Oct4 alone [17,
18]. However, methods reprogramming somatic cells
that lack endogenous Sox2 expression to pluripotency
with Oct4 alone need to be explored. Based on previ-
ous results [16], we hypothesized that cells transduced
by Bmi1 could be transdifferentiated into NSC-like
cells and then converted into iPS cells by Oct4 (Figure
2A left panel). We rst determined whether Bmi1 could
transdifferentiate MEFs into NSC-like cells. Bmi1-
transduced MEFs, but not empty vector-transduced
MEFs, formed colonies exhibiting an NSC-like morphol-
ogy within 3-7 days in NSC culture (Figure 2A and 2B).
Of the 45 colonies generated, four were selected and
grown using standard mouse NSC culturing methods.
All four selected colonies expressed genes and cell sur-
face markers characteristic of mouse NSCs, including
Nestin and Sox2, as well as AP activity (Figure 2C). In
addition, Bmi1-transduced spheres gave rise to neurons,
oligodendrocytes, and astrocytes (Figure 2D). Next, we
investigated whether Bmi1-transduced NSC-like cells
could be reprogrammed into iPS cells by transduction
with Oct4 alone. Indeed, we succeeded in generating ES-
like colonies within 10-14 days. We refer to these repro-
grammed cells as transdifferentiated BO-iPS cells (dBO-
iPS cells). These cells were generated from MEFs that
were rst transdifferentiated into NSC-like cells and then

www.cell-research.com | Cell Research
Jai-Hee Moon et al.
1307
npg
further reprogrammed into iPS cells (Figure 2E). We also
determined whether Bmi1 could replace Sox2, Klf4, and
C-Myc and, in combination with Oct4, reprogram MEFs
into iPS cells (Figure 2A right panel). We introduced
Bmi1 and Oct4 into MEFs and were able to induce the
formation of ES-like cells, hereafter designated as BO-
iPS cells. The estimated reprogramming efciency was
calculated at 0.01% and 0.17% for dBO- and BO-iPS
cells, respectively. The reprogramming efficiency of
dBO-iPS cells was approximately 6% of that obtained
Figure 1 Increased generation of iPS cells by overexpression of Bmi1. (A) Hypothesis of Bmi1’s function in the course of re-
programming. (B) MEFs were infected with a retrovirus encoding Bmi1 or an empty vector. Three days after infection, protein
levels of Bmi1, p16Ink4a, p19Arf, and Sox2 were analyzed by western blot. Actin was used as a loading control. (C) Reprogram-
ming efciencies of factor combinations on iPS cell induction. Bmi1 replaces Sox2, Klf4, and C-Myc in the reprogramming of
MEFs. Reprogramming efciency was quantied by determining the percentage of Nanog-positive colonies. (D) MEFs were
infected with retroviruses encoding two factors (Oct4 and Sox2; OS) or with the two factors plus Bmi1 (OSB). Seven days af-
ter infection, Nanog-positive colonies formed from cells infected with OSB, but not OS (left and lower panel). Scale bars, 200
µm. Protein levels of Oct4, Sox2, c-Myc, Klf4, and p53 were analyzed by western blot. α-tubulin was used as a loading control
(right panel).

Reprogramming of broblast into iPSCs with Bmi1
1308
npg
Cell Research | Vol 21 No 9 | September 2011
Figure 2 Characterization of iPS cells generated from MEFs by retroviral transduction with Oct4 and Bmi1. (A) Hypothesis
of Bmi1’s function in the course of reprogramming MEFs into dBO- (left) and BO-iPS cell (right). (B) Phase contrast images
of vehicle- (upper) and Bmi1-transduced (lower) MEFs cultured in proliferation medium (left) and in NSC medium (right).
Bmi1-transduced MEFs cultured in NSC medium for 3-7 days rapidly changed morphology, resulting in bipolar and expanded
NSC-like cells (right). Scale bars, 200 µm. (C) Characterization of NSC-like cells was conducted by AP staining and immuno-
cytochemistry for Nestin and Sox2, as well as RT-PCR analysis of NSC marker genes (right). Scale bars, 200 µm. (D) NSC-
like cells exhibit multipotency and thus can give rise to cells expressing neuronal and glial markers for oligodendrocytes (O4,
CNPase, and GalC), neurons (Tuj1 and Map2a), and astrocytes (GFAP and S100). Scale bars, 200 µm. (E) Phase contrast
images showing mES cells (upper left) and BO-iPS cells (clone 1; lower left) on feeder cells. The reprogramming efciency of
dBO- and BO-iPS cells was quantied by determining the percentage of Nanog-positive colonies (right). (F) Characterization
of BO-iPS-1 cells. AP staining, as well as SSEA1, Oct4, Sox2, and Nanog immunoreactivities, were measured in mES cells
(upper panels) and BO-iPS cells (lower panels). Scale bars, 200 µm. (G) Scatter plots of the global gene expression compar-
ing BO-iPS cells with MEFs (right) and BO-iPS cells with mES cells (left). Red and green lines indicate 2-fold changes in gene
expression levels. The pluripotency genes Oct4, Sox2, Nanog, c-Myc, and Klf4 are shown in red. (H) Bisulte genomic se-
quencing of Oct4 and Nanog promoter regions in mES cells, MEFs, and BO-iPS clones (1, 2, and 3). Open and lled circles
indicate unmethylated and methylated CpG dinucleotides, respectively.

www.cell-research.com | Cell Research
Jai-Hee Moon et al.
1309
npg
by BO-iPS cells. The reason for the lower reprogram-
ming efficiency of dBO-iPS than BO-iPS cells is not
clear; however, it may be due to the poor transduction
efciency of Oct4 into the NSC-like spheres. To further
characterize the BO-iPS cells, six colonies that were
morphologically indistinguishable from mouse ES (mES)
cell colonies were selected and grown under standard
conditions (Figure 2E). BO-iPS cells expressed marker
genes at levels typical of mES cells (Figure 2F and Sup-
plementary information, Figures S2A, B, and S3A, B).
Furthermore, scatter plots of the global gene expression
proles showed that the BO-iPS cell prole was similar
to mES cells and was different from MEFs (Figure 2G).
To further compare BO-iPS cells with mES cells, the
methylation state of CpG dinucleotides in the Oct4 and
Nanog promoter regions was analyzed. Bisulte genomic
sequencing analyses showed that the Oct4 and Nanog
promoter regions were demethylated in BO-iPS cells
relative to the parental broblasts and showed a similar
pattern to that of mES cells (Figure 2H). Chromatin im-
munoprecipitation (ChIP) analyses showed that the Oct4,
Sox2, and Nanog promoters had increased acetylation
of histone H3 (AcH3) and decreased dimethylation of
lysine 9 of histone H3 (diMeK9H3) (Supplementary in-
formation, Figure S2C), consistent with the epigenetic
remodeling that occurs during reprogramming. Oct4
mRNA was transcribed from the endogenous Oct4 locus,
which can be distinguished from the virally expressed
human Oct4. Genomic integration of the Bmi1 and Oct4
transgenes was conrmed (Supplementary information,
Figure S3C), and the expression of both transgenes was
efciently silenced in all iPS cell lines examined (Sup-
plementary information, Figure S3D) [19].
We next investigated the differentiation potential of
BO-iPS cells in vitro with the embryoid body (EB) as-
say. EBs derived from BO-iPS cells expressed markers
of the three germ layers, including the endoderm marker
GATA4, the mesoderm markers smooth muscle actin
and Brachyury, and the ectoderm marker Nestin (Figure
3A). To investigate the differentiation potential of BO-
iPS cells in vivo, we analyzed teratomas that formed after
subcutaneous injection of BO-iPS cells into nude mice.
These teratomas contained derivatives of the three em-
bryonic germ layers, including neural epithelium, mus-
cle, cartilage, and various glandular structures (Figure 3B
and Supplementary information, Figure S3E). To assess
their developmental potential, we tested whether BO-
iPS cells could generate chimeric mice after injection
into blastocysts. According to coat color, BO-iPS clones
generated chimeric mice with germline transmission
(Figure 3C). Taken together, these results demonstrate
that Bmi1 can either transdifferentiate MEFs into NSC-
like cells and/or replace Sox2, Klf4, and C-Myc during
reprogramming of MEFs into iPS cells in the presence
of Oct4. In addition, our results suggest that chemically
induced activation of Bmi1 may be a useful strategy for
enhancing reprogramming efficiency without genetic
manipulation of the potential oncogenic Bmi1.
Shh induces Bmi1 and stimulates the generation of iPS
cells from fibroblasts by transduction with Oct4 alone
Shh, the most prominent member of the Hedgehog
family, plays an essential role during development. The
Shh signaling pathway involves the activation of Gli
transcription factors, which regulate the transcription
of target genes including Gli1 and Ptch1. Furthermore,
Bmi1, Sox2, and N-Myc expression was upregulated in
response to Shh treatment and parallels the expression of
Gli1, suppressor of fused (Sufu), and cyclin D2, which
is indicative of the activation of the Shh pathway and in-
duction of proliferation [14-16, 20, 21]. Moreover, over-
expression of Gli1 induces Bmi1 expression, suggesting
that Bmi1 is a downstream target in the Shh pathway [15].
Therefore, we tested whether Shh could replace Bmi1
in the generation of dBO- and BO-iPS cells. Bmi1, Sox2,
N-Myc, Klf4, and Gli1 mRNAs were upregulated (in
contrast to p16Ink4a and p19Arf mRNAs, which were sup-
pressed) in response to Shh treatment as early as 72 h
after incubation, indicating the activation of the Shh
pathway (Figure 4A and 4B). Moreover, cells developed
into colonies exhibiting an NSC-like morphology within
3-7 days of Shh treatment in standard NSC culture condi-
tions (Figure 4C). These NSC-like cells expressed genes
and cell surface markers characteristic of mouse NSCs,
including Sox2, Nestin, and SSEA1, as well as AP activ-
ity (Figure 4D). Shh-treated NSC-like cells were then
transduced with Oct4 to reprogram them into iPS cells
(1F combination of Shh and Oct4, hereafter designated
as ShO-iPS cells). ShO-iPS colonies obtained within 14
days in culture were further analyzed by the same tests
described above for BO-iPS cells to conrm reprogram-
ming to pluripotency (Figure 4E-F and Supplementary
information, Figure S4). Taken together, these results
demonstrate that Shh can induce Bmi1, and together with
Oct4, can reprogram MEFs into iPS cells that are very
similar to mES cells (Supplementary information, Figure
S4I).
Recently, it was demonstrated that specic oxysterol
and purmorphamine not only stimulate the Shh pathway
but also activate Shh target gene transcription through
the protein Smo [22, 23]. Similar to ShO-iPS cells, treat-
ment of MEFs with either oxysterol or purmorphamine
activated the Shh pathway, reprogramming MEFs into
NSC-like cells that exhibited gene expression profiles

Reprogramming of broblast into iPSCs with Bmi1
1310
npg
Cell Research | Vol 21 No 9 | September 2011
Figure 3 In vitro and in vivo differentiation of BO-iPS cells. (A) In vitro differentiation of BO-iPS cells. Micrographs show EBs
generated from BO-iPS-1 clones and their in vitro differentiation into ectodermal, mesodermal, and endodermal cell types,
as revealed by immunoreactivity to typical markers Nestin, Brachyury, SMA, and GATA4, respectively. Nuclei were counter-
stained with DAPI (blue). Scale bars, 200 µm. RT-PCR analysis shows that cDNAs from EBs exhibit expression of represen-
tative lineage markers in differentiating cells. (B) The in vivo developmental potential of BO-iPS cells. Teratomas generated
by BO-iPS cells differentiated into neural rosettes (ectoderm), muscle and fat (mesoderm), and epithelium (endoderm). He-
matoxylin and eosin-stained sections of teratomas derived from BO-iPS cells in a nude mouse after 8-10 weeks are shown.
Scale bars, 200 µm. (C) Chimeric mouse (upper panels) and germline contribution of BO-iPS cells in adult chimera gonads
(lower panels). Established iPS cells give rise to live chimeras (upper left) after an injection of BO-iPS cells (Balb/c genetic
background) into a C57BL6 blastocyst and contribute to the germline (lower left). PCR was performed on genomic DNA to
detect exogenous and endogenous transgene integration in chimeric (upper right) and germline transmission mice (lower
right) produced with BO-iPS cells.

www.cell-research.com | Cell Research
Jai-Hee Moon et al.
1311
npg
Figure 4 Generation and characterization of 1F ShO-iPS cells. (A) Hypothesis of induction of Bmi1 in the course of
reprogramming. (B) Induction of sonic hedgehog target genes by Shh treatment. RT-PCR and qPCR of mRNAs from MEFs
treated with vehicle (con) or Shh were analyzed for the induction of Shh target genes (Gli1, Bmi1, Sox2, and N-Myc, as well
as p16Ink4a, p19Arf, and Klf4). Data are from a representative experiment (left) and are shown as the means and SD’s of rela-
tive values compared to control MEFs (n = 3). *P < 0.05 compared to MEFs. (C) Phase contrast images of vehicle- (shh(−))
and Shh-treated (shh(+)) MEFs cultured in proliferation medium (left) or NSC medium (right). Shh-treated MEFs cultured
in NSC medium for 7 days rapidly changed morphology, resulting in bipolar (right) and expanded neurosphere-like cells
(right). Scale bars, 200 µm. (D) Characterization of neurosphere-like morphology from Shh-treated MEFs was conducted by
AP staining, immunocytochemistry, and RT-PCR for Nestin, Sox2, SSEA1, Musashi1, and CD133. Scale bars, 200 µm. (E)
Timeline for ShO-iPS cell induction using Shh treatment with retroviral transduction of Oct4 (upper and left, panel). Phase
contrast images showing the ESC-like morphology of ShO-iPS cells on feeder cells and the characterization of ShO-iPS
cells. AP staining, as well as SSEA1, Oct4, Sox2, and Nanog immunoreactivity, was detected in ShO-iPS cells (lower panel).
The reprogramming efciency of BO- and ShO-iPS cells in reprogramming MEFs. Reprogramming efciency was quantied
by determining the percentage of Nanog-positive colonies. Scale bars, 200 µm (upper and right panel). (F) The in vivo devel-
opmental potential of ShO-iPS cells. Teratomas of ShO-iPS cells differentiated into epithelium (endoderm; left), muscle and
fat (mesoderm; middle), and neural rosettes (ectoderm; right). Hematoxylin and eosin-stained sections of teratomas derived
from ShO-iPS cells in a nude mouse host after 8 weeks are shown. Scale bars, 200 µm.

Reprogramming of broblast into iPSCs with Bmi1
1312
npg
Cell Research | Vol 21 No 9 | September 2011
characteristic of NSCs (Supplementary information,
Figure S5A-S5C). Furthermore, the treatment of MEFs
with oxysterol and/or purmorphamine enhanced the
reprogramming of MEFs to pluripotency by the forced
expression of Oct4 (1F combinations of oxysterol and/or
purmorphamine and Oct4, hereafter designated as OxyO-
iPS, PO-iPS, or POxyO-iPS cells) (Supplementary in-
formation, Figure S5D). Again, the tests described above
were successfully conducted with PO-iPS and OxyO-iPS
cells (Supplementary information, Figures S5E-M and
S6A-H). Furthermore, PO-iPS cells were germline com-
petent, as demonstrated by the generation of albino off-
spring from crossing chimeric mice with wild-type mice
(Supplementary information, Figure S6I). These results
demonstrate that MEFs can be reprogrammed to pluripo-
tency by Oct4 alone when the Shh pathway is activated.
Given that Bmi1 is an important regulator of repro-
gramming-related genes (Figure 1A and 1B) [13], the
transdifferentiation of MEFs into NSC-like cells, and the
generation of iPS cells with Oct4, we studied whether
Figure 5 Decreased generation of iPS cells by the knockdown of Bmi1. (A, B) Inhibition of Bmi1 expression prevents neu-
rosphere formation. Scale bars, 200 µm. (C) qPCR analysis of Bmi1 target genes in vehicle and Bmi1 siRNA-transfected
purmorphamine- or oxysterol-treated MEFs. *P < 0.05 compared to control. (D) The reprogramming efciency of vehicle and
Bmi1 siRNA-transfected POxyO-iPS cells in reprogramming MEFs. Reprogramming efciency was quantied by determining
the percentage of Nanog-positive colonies. (E) Model summarizing the presented data. Bmi1 and Bmi1 inducers (Shh, purmor-
phamine, and oxysterol) enhance Oct4-induced reprogramming of broblasts by downregulating p16Ink4a and p19Arf expression
and upregulating Sox2, N-Myc, and Klf4 expression.

www.cell-research.com | Cell Research
Jai-Hee Moon et al.
1313
npg
knocking down Bmi1 expression would blunt neural
sphere formation. Transdifferentiation was performed
in the presence of oxysterol and/or purmorphamine to
induce the transdifferentiation of MEFs into NSC-like
cells. The formation of neurospheres and reprogram-
ming-related gene expression were signicantly altered
by the knock down of Bmi1 (Figure 5A-5C). Moreover,
when cells treated with both oxysterol and purmor-
phamine were transduced with Oct4 in the presence of
Bmi1 siRNA, the formation of Nanog-positive colonies
was signicantly decreased compared to control (Figure 5D).
We next tested the combination of oxysterol and Oct4
on the reprogramming of tail-tip broblasts (TTFs) from
10-week-old male mice (hereafter designated as OxyO-
iPS-TTF cells) and established two independent iPS
cell lines. These cells were very similar to ES cells with
an identical differentiation potential (Supplementary
information, Figure S7). Growth rates and hierarchial
clustering analysis of the global gene expression proles
showed that 1F (Oct4) and 2F (Oct4 and Bmi1) iPS cells
clustered close to mES cells and were distinct from the
parental MEFs and Bmi1-transduced neurospheres (Sup-
plementary information, Tables S1-S2 and Supplemen-
tary information, Figure S8). The chromosomal stability
of 1F and 2F iPS cells was confirmed by metaphase
spread (Supplementary information, Figure S9). Taken
together, these results show that Bmi1 promotes iPS cell
generation from broblasts; Bmi1 likely has dual effects
on iPS cell generation from broblasts, by both suppress-
ing p16Ink4a and p19Arf and augmenting Sox2 and N-Myc
(Figure 5E).
Discussion
In summary, our data support two main conclusions.
First, Bmi1 can replace Sox2, Klf4, and C-Myc during
reprogramming and enhances the reprogramming effi-
ciency of mouse broblasts infected with Oct4 and Sox2.
This suggests that not only p16Ink4a and p19Arf [1-4, 6],
but also Sox2 and C-Myc [17-19, 24] are rate-limiting
determinants in the reprogramming process. The second
conclusion relates to the number of factors applied to
somatic cells for iPS cell generation. We demonstrated
previously that mouse and human NSCs, which express
endogenous Sox2, can be reprogrammed with Oct4 alone
[17, 18]. Here, we expand upon these results by showing
that Oct4, together with Bmi1 or activators of the Shh
signaling pathway (Shh, oxysterol, and purmorphamine
in this study), is sufcient for the generation of iPS cells
from mouse embryonic and adult broblasts. This is es-
pecially crucial because adult broblasts do not express
Sox2. This study, together with others is an important
step forward in dening the critical determinants for the
generation of iPS cells from differentiated fibroblasts
[25-27]. Future studies will determine if we can combine
our direct reprogramming procedure with small molecule
compounds to activate endogenous expression of Oct4,
knock down Oct4-specific suppressor(s), or achieve
reprogramming with Oct4 recombinant protein alone.
Materials and Methods
Generation of 4F and 2F iPS cells
pBabe-based retroviral vectors encoding Oct4, Sox2, Klf4, or
C-Myc (from Gou Young Koh), and Bmi1 (from Goberdhan P.
Dimri) were transfected into PT67 amphotropic packaging cells
(Clontech) using Turbofect (Fermentas) according to the manu-
facturer’s protocol. The cells were subjected to drug selection
with 3 µg/ml puromycin (Bmi1, Klf4 and C-Myc) for 4 days or 1
000 ng/ml G418 (Sox2 and Oct4) for 14 days. The viruses were
collected after 24 h and ltered through a 0.45-µm lter before
transduction. Induction of iPS cells was performed as described
previously [24] with some modication. In brief, MEF cells (2 ×
105) were seeded in one well of a six-well plate and 2 ml of each
retroviral supernatant (Bmi1 and Oct4) was added to the cells in
the presence of 6 µg/ml of polybrene (Sigma). Two days after the
initial transduction, cells were subcultured on mitomycin C-treated
CF1 mouse feeder layers and maintained in mES cell medium
consisting of DMEM supplemented with 15% FBS (Hyclone),
β-mercaptoethanol, 1% penicillin-streptomycin, and 1 000 units/
ml leukemia inhibitory factor (LIF; Millipore). ES and iPS cells
were passaged every 3-5 days using 0.05% trypsin-EDTA and
seeded at 2 × 105 cells/well in a six-well plate. The induction of
dBO-iPS cells was performed in two steps. Generation of NSC-
like cells was achieved with MEFs infected with retroviruses
containing Bmi1, as described previously [16], and these NSC-like
cells were then further infected with retroviruses containing Oct4,
as above. Two days after Oct4-transduction, cells were grown in
mES-culture conditions as described above. For the quantication
of transduction efciency, we introduced GFP to MEF as above
and more than 70% of cells expressed GFP. To quantify efciency
of iPS generation, total number of Nanog-positive colonies were
counted and calculated as percentage of infected cell numbers,
which was calculated as GFP+ cells [28].
Generation of 1F iPS cells
To induce ShO-, PO-, OxyO-iPS, and OxyO-iPS-TTF cells,
cells were cultured in NSC medium [16] with either Shh (500 ng/
ml; R&D systems), purmorphamine (0.5, 1 µM; Calbiochem),
or 25-hydroxycholesterol (oxysterol; 0.1, 0.5 µM; Sigma) for 1
day. Next, retroviral supernatants (Oct4) in NSC medium con-
taining 6 µg/ml polybrene were added to the cells with Shh,
purmorphamine, or oxysterol. A third round of transduction was
performed and the cells were incubated for 2 days. Two days after
the initial transduction, cells were transferred and grown on mi-
tomycin C-treated MEF layers in mES cell medium. For siRNA
experiments, cells were transfected with lipofectamine RNAiMAX
(Invitrogen). Bmi1 siRNAs and control siRNAs were purchased
from Bioneer.

Reprogramming of broblast into iPSCs with Bmi1
1314
npg
Cell Research | Vol 21 No 9 | September 2011
Microarray analysis
Total RNA from MEF, mES, and iPS cells was labeled with
Cy3. Labeled RNA was hybridized to the Agilent mouse whole-
genome array (G4122F) according to the manufacturer ’s instruc-
tions. Arrays were scanned with the G2565BA Microarray Scanner
System (Agilent Technologies). All data normalization and gene
selection was performed using GeneSpring GX 7.3 (Agilent Tech-
nologies) [24].
Bisulte sequencing analysis
DNA from MEF, mES, and iPS cells was isolated using the
Genomic DNA Purification Kit (Promega). DNA was prepared
for bisulte sequencing with the EpiTect Bisulte Kit (QIAGEN).
Treated DNA was used to amplify the sequences of interest. The
primers used for promoter fragment amplification are listed in
Supplementary information, Table S3. The resulting fragments
were cloned using the pGEM-T Easy Vector (Promega) for se-
quencing and sequenced with T7 forward and SP6 reverse primers.
ChIP assay
The ChIP assay was performed on MEFs, ES cells, and iPS
cells using the EZ ChIP Kit (Millipore) according to the manufac-
turer’s instructions. Anti-acetyl H3 and anti-dimethyl K9 H3 an-
tibodies were used in this experiment. PCR primers used for real-
time PCR are listed in Supplementary information, Table S3.
Differentiation of iPS cells
iPS cells were examined by the in-vitro differentiation of EBs.
iPS cells were trypsinized and single cells were cultured in suspen-
sion with EB medium without LIF for 7 days, and EBs were then
replated onto 0.1% gelatin-coated plates. Spontaneous differentia-
tion was examined by immunostaining and RT-PCR for represen-
tative lineage-specic markers with the indicated antibodies and
primers at various timepoints (5-7 days).
Teratoma formation
iPS cells (1 × 106/mouse) were injected under the kidney cap-
sule or subcutaneously into the dorsal ank of nude mice. Eight
to ten weeks later, the mice developed teratomas, which were re-
moved, immediately rinsed with PBS, xed in 10% formalin, and
embedded in parafn. Tissue sections 5-6 µm thick were cut and
processed for hematoxylin-eosin staining.
Chimera formation and germline transmission
Four- to ve-week-old female mice (C57BL/6) were induced to
superovulate by intraperitoneal injection of 7.5 IU PMSG followed
48 h later by intraperitoneal injection of 7.5 IU hCG and mated
with a stud male mouse (C57BL/6). Blastocysts were collected
3.5 days after vaginal plug check and ushing in H-CZB medium.
Approximately 10 iPS cells then were expelled from the injection
pipette against the inner cell mass of the blastocyst. Injected blas-
tocysts were transferred into the uterine horn of 2.5 days post co-
itum (dpc) pseudopregnant CD1 female mice that had been mated
with vasectomized male mice. Chimerism was ascertained by the
contribution rate of albino coat color (from iPS cells) in black host
pups. High-contribution chimeras were mated with C57BL/6 mice
to test for germline transmission.
Statistical analysis
Data were analyzed by analysis of variants using the general
linear model procedures of the Statistical Analysis System (SAS,
9.13 PACKAGE). Data were expressed as the means ± SD. P < 0.05
was considered signicant.
Accession codes
Gene Expression Omnibus (GEO): GSE24208
Further details and other methods can be found in the supple-
mentary information, Data S1.
Acknowledgments
We are grateful to Drs Goberdhan P Dimri (NorthShore Univer-
sity HealthSystem Research Institute, USA)and Gou Young Koh
(Korea Advanced Institute of Science and Technology, Republic of
Korea) for kindly providing the pBabe-Bmi1, pMX-Oct4, pMX-
Sox2, pMX-Klf4, and pMX-C-Myc constructs. We thank Jihyun
Kim, Jihye Hwang, and Suhyun Kwon for technical support. This
research was supported by a grant (SC-5150) from the Stem Cell
Research Center of the 21st Century Frontier Research Program
funded by the Ministry of Education, Science and Technology,
Republic of Korea, a grant (09172KFDA653) from the Korea
Food and Drug Administration, and a grant (2010-0020347)
from National Research Foundation (NRF) funded by the Korea
government (MEST). Work in Germany was supported by the
Max Planck Society and the Federal Ministry of Education and
Research (BMBF) on Cell-Based Regenerative Medicine (Grant
01GN0539).
References
1 Hong H, Takahashi K, Ichisaka T, et al. Suppression of indu-
ced pluripotent stem cell generation by the p53-p21 pathway.
Nature 2009; 460:1132-1135.
2 Kawamur a T, Suzuki J, Wang YV, et al. Linking the p53
tumour suppressor pathway to somatic cell reprogramming.
Nature 2009; 460:1140-1144.
3 Li H, Collado M, Villasante A, et al. The Ink4/Arf locus is a
barrier for iPS cell reprogramming. Nature 2009; 460:1136-
1139.
4 Marion RM, Strati K, Li H, et al. A p53-mediated DNA dama-
ge response limits reprogramming to ensure iPS cell genomic
integrity. Nature 2009; 460:1149-1153.
5 Pei D. Regulation of pluripotency and reprogramming by
transcription factors. J Biol Chem 2009; 284:3365-3369.
6 Utikal J, Polo JM, Stadtfeld M, et al. Immortalization eli-
minates a roadblock during cellular reprogramming into iPS
cells. Nature 2009; 460:1145-1148.
7 Haupt Y, Alexander WS, Barri G, Klinken SP, Adams JM. No-
vel zinc nger gene implicated as myc collaborator by retro-
virally accelerated lymphomagenesis in E mu-myc transgenic
mice. Cell 1991; 65:753-763.
8 van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der
Gulden H, Berns A. Identication of cooperating oncogenes
in E mu-myc transgenic mice by provirus tagging. Cell 1991;
65:737-752.
9 Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen
M. The oncogene and Polycomb-group gene bmi-1 regulates
cell proliferation and senescence through the ink4a locus. Na-

www.cell-research.com | Cell Research
Jai-Hee Moon et al.
1315
npg
ture 1999; 397:164-168.
10 Jacobs JJ, Scheijen B, Voncken JW, Kieboom K, Berns A, van
Lohuizen M. Bmi-1 collaborates with c-Myc in tumorigenesis
by inhibiting c-Myc-induced apoptosis via INK4a/ARF. Ge-
nes Dev 1999; 13:2678-2690.
11 Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF,
Morrison SJ. Bmi-1 dependence distinguishes neural stem
cell self-renewal from progenitor proliferation. Nature 2003;
425:962-967.
12 Park IK, Qian D, Kiel M, et al. Bmi-1 is required for mainte-
nance of adult self-renewing haematopoietic stem cells. Natu-
re 2003; 423:302-305.
13 Valk-Lingbeek ME, Bruggeman SW, van Lohuizen M.
Stem cells and cancer; the polycomb connection. Cell 2004;
118:409-418.
14 Hatton BA, Knoepfler PS, Kenney AM, et al. N-myc is an
essential downstream effector of Shh signaling during both
normal and neoplastic cerebellar growth. Cancer Res 2006;
66:8655-8661.
15 Leung C, Lingbeek M, Shakhova O, et al. Bmi1 is essential
for cerebellar development and is overexpressed in human
medulloblastomas. Nature 2004; 428:337-341.
16 Moon JH, Yoon BS, Kim B, et al. Induction of neural stem
cell-like cells (NSCLCs) from mouse astrocytes by Bmi1.
Biochem Biophys Res Commun 2008; 371:267-272.
17 Kim JB, Sebastiano V, Wu G, et al. Oct4-induced pluripoten-
cy in adult neural stem cells. Cell 2009; 136:411-419.
18 Kim JB, Greber B, Arauzo-Bravo MJ, et al. Direct reprogra-
mming of human neural stem cells by OCT4. Nature 2009;
461:649-643.
19 Huangfu D, Osafune K, Maehr R, et al. Induction of pluri-
potent stem cells from primary human broblasts with only
Oct4 and Sox2. Nat Biotechnol 2008; 26:1269-1275.
20 Dahmane N, Ruiz i Altaba A. Sonic hedgehog regulates the
growth and patterning of the cerebellum. Development 1999;
126:3089-3100.
21 Wechsler-Reya RJ, Scott MP. Control of neuronal precursor
proliferation in the cerebellum by Sonic Hedgehog. Neuron
1999; 22:103-114.
22 Corcoran RB, Scott MP. Oxysterols stimulate Sonic hedge-
hog signal transduction and proliferation of medulloblastoma
cells. Proc Natl Acad Sci USA 2006; 103:8408-8413.
23 Lipinski RJ, Gipp JJ, Zhang J, Doles JD, Bushman W. Unique
and complimentary activities of the Gli transcription factors
in Hedgehog signaling. Exp Cell Res 2006; 312:1925-1938.
24 Takahashi K, Yamanaka S. Induction of pluripotent stem cells
from mouse embryonic and adult broblast cultures by de-
ned factors. Cell 2006; 126:663-676.
25 Chen J, Liu J, Yang J, et al. BMPs functionally replace Klf4
and support efcient reprogramming of mouse broblasts by
Oct4 alone. Cell Res 2011; 21:205-212.
26 Li Y, Zhang Q, Yin X, et al. Generation of iPSCs from mouse
broblasts with a single gene, Oct4, and small molecules. Cell
Res 2011; 21:196-204.
27 Yuan X, Wan H, Zhao X, Zhu S, Zhou Q, Ding S. Combined
chemical treatment enables Oct4-induced reprogramming
from mouse embryonic broblasts. Stem Cells 2011; 29:549-
553.
28 Shao L, Feng W, Sun Y et al. Generation of iPS cells using
dened factors linked via the self-cleaving 2A sequences in a
single open reading frame. Cell Res 2009; 19:296-306.
(Supplementary information is linked to the online version of
the paper on the Cell Research website.)