ORIGINAL ARTICLE
Reprogramming fibroblasts 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: office@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
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 hedge-
hog 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 differ-
entiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data sup-
port 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.
Keywords: reprogramming; transdifferentiation; neural stem cells; induced pluripotent stem cells; Bmi1; Oct4
Cell Research advance online publication 28 June 2011; doi:10.1038/cr.2011.107
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 inefficient.
Recently, a number of groups have demonstrated that the
inactivation of p53 markedly increases the efficiency 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 first identified 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) :1-11.
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Reprogramming of fibroblast into iPSCs with Bmi1
2
npg
Cell Research | www.cell-research.com
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 efficiently 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
fibroblasts 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 fibroblasts into iPS cells. To answer
this question, we first determined the expression levels
of reprogramming-related genes (p16Ink4a, p19Arf, Sox2,
N-Myc, p53, and Klf4) in parental mouse embryonic
fibroblasts (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 fibroblasts 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 efficiency.
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 first 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 first transdifferentiated into NSC-like cells and then