Polycomb Group Protein Bmi1 Promotes
Hematopoietic Cell Development from Embryonic Stem Cells
Xiaolei Ding,1,2Qiong Lin,1,2Roberto Ensenat-Waser,3Stefan Rose-John,4and Martin Zenke1,2
Bmi1 is a component of the Polycomb repressive complexes and essential for maintaining the pool of adult stem
cells. Polycomb repressive complexes are key regulators for embryonic development by modifying chromatin
architecture and maintaining gene repression. To assess the role of Bmi1 in pluripotent stem cells and on exit
from pluripotency during differentiation, we studied forced Bmi1 expression in mouse embryonic stem cells
(ESC). We found that ESC do not express detectable levels of Bmi1 RNA and protein and that forced Bmi1
expression had no obvious influence on ESC self-renewal. However, upon ESC differentiation, Bmi1 effectively
enhanced development of hematopoietic cells. Global transcriptional profiling identified a large array of genes
that were differentially regulated during ESC differentiation by Bmi1. Importantly, we found that Bmi1 induced
a prominent up-regulation of Gata2, a zinc finger transcription factor, which is essential for primitive hemato-
poietic cell generation from mesoderm. In addition, Bmi1 caused sustained growth and a >100-fold expansion of
ESC-derived hematopoietic stem/progenitor cells within 2–3 weeks of culture. The enhanced proliferative ca-
pacity was associated with reduced Ink4a/Arf expression in Bmi1-transduced cells. Taken together, our ex-
periments demonstrate distinct activities of Bmi1 in ESC and ESC-derived hematopoietic progenitor cells. In
addition, Bmi1 enhances the propensity of ESC in differentiating toward the hematopoietic lineage. Thus, Bmi1
could be a candidate gene for engineered adult stem cell derivation from ESC.
potential. Lossofself-renewaland inductionofdifferentiation
are accompanied by specific changes in gene expression.
Additionally, there is increasing evidence that cell identity
and function are determined and maintained by epigenetic
status, including DNA methylation and histone modification
[1–3]. We have previously shown that chromatin modifying
. The Polycomb group (PcG) proteins act as epigenetic
modifiers and have received significant attention due to their
role in stem cell self-renewal, commitment, and differentia-
tion, and in cancer stem cell formation [5,6].
PcG proteins were initially described in Drosphila, where
they control embryonic development by repressing homeotic
gene expression [5–7]. PcG proteins are conserved from
Drosphila to humans, and most of them are involved in
maintenance of cellular memory by modifying chromosome
structure and silencing gene expression. PcG proteins occur in
large protein complexes to exert transcriptional repressor
tem cells are characterized by an exceptionally high
self-renewal activity and their multilineage differentiation
activity, referred to as Polycomb repressive complexes (PRC)
[6,7]. In mammals, 2 PRC complexes, PRC1 and PRC2, have
been described. PRC2, which contains Eed, Suz12, and Ezh2
proteins, is recruited to chromatin and trimethylates lysine
residue 27 of histone 3 (H3K27me3). PRC1 contains Bmi1,
Ring1A/B, Cbx, Mel18, and Mph and is recruited to specific
sites formed by PRC2, referred to as maintaining complex .
PRC and H3K27me3 co-occupy cis-regulatory elements
of a large number of development-associated genes [8,9].
Genome-wide mapping of histone modification in embry-
onic stem cells (ESC) identified regions that are modified
with both H3K27me3 and histone 3 lysine 4 trimethylation
(H3K4me3) [9–12]. H3K4me3 is catalyzed through trithorax
group proteins and associated with active transcription units
H3K27me3, referred to as ‘‘bivalent domain,’’ effectively
controls gene transcription and poise genes in a ready-for-
transcription status [10,11].
Down-regulation of the PRC2 component Eed and Suz12
caused silenced differentiation-associated genes to become
re-expressed in ESC [9,12]. Further, ESC could not be es-
tablished from Ezh2 deficient mice . For PRC1, it has
of H3K4me3 and
1Department of Cell Biology, Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany.
2Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
3Joint Research Centre, Institute for Health and Consumer Protection, In Vitro Methods Unit, European Commission, Ispra, Italy.
4Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
STEM CELLS AND DEVELOPMENT
Volume 21, Number 1, 2012
? Mary Ann Liebert, Inc.
been shown that RingA/B is directly involved in the tran-
scriptional network associated with ESC pluripotency .
All these studies indicate that PcG proteins are highly critical
for embryonic development, the establishment and mainte-
nance of ESC pluripotency. Recent studies demonstrated that
PRC2 components, such as Ezh2 and Suz12, also orchestrate
gene expression in adult stem cells [15–17].
Unlike other PcG components that are ubiquitously ex-
pressed during development, Bmi1, a key component of PRC1,
is selectively expressed in diverse postnatal adult stem cells,
including neural stem cells (NSC) and hematopoietic stem cells
(HSC) [18–22]. Bmi1 activity in maintaining the pool of adult
stem cells is mainly due to repression of the Ink4a/Arf locus,
which encodes inhibitors of the cell cycle kinase p16Ink4a
and p19Arf [18,19,23,24]. Accordingly, in Bmi1 deficient
(Bmi1-/-) mice, the number of adult HSC is markedly
decreased. Additionally, in competitive transplantation ex-
periments, the repopulation capacity of Bmi1-/- HSC was
significantly decreased, thus mirroring their deficiency in
self-renewal [19,20]. In fact, with HSC differentiation, Bmi1
expression declines gradually . In contrast, Bmi1 over-
expression enhances HSC self-renewal in both the human
and mouse system [26,27]. Thus, there is multiple evidence of
Bmi1 activity in maintaining the pool of adult stem cells, yet
its role in pluripotent stem cells and on transition toward
adult stem/progenitor cells has so far not been studied.
Here, we examined the activity of Bmi1 on ESC and on
hematopoietic stem/progenitor cells derived from ESC. We
present evidence for a novel activity of Bmi1 in enhancing
hematopoietic cell development from ESC.
Materials and Methods
Cells and cell culture
ESC culture was performed as previously described .
Briefly, R1 ESC were cultured on irradiated mouse embryonic
fibroblasts (MEF) feeder. E14 and CCE ESC were cultured on
0.1% gelatin (Sigma-Aldrich) precoated dishes with ESC me-
dium. ESC medium was Dulbecco’s modified Eagle’s medium
inactivated fetal calf serum (FCS; Lonza), 25mM HEPES and
1,000U recombinant leukemia inhibitory factor (LIF, a kind
gift of Anna M. Wobus, IPK, Gatersleben, Germany), 2mM
L-glutamine, 100 units penicillin/100mg streptomycin, 0.1mM
nonessential amino acids, and 50mM b-mercaptoethanol. ESC
differentiation medium was ESC medium but with 10% FCS
and without LIF. ESC were cultured at 37?C with 5% CO2.
Medium was refreshed daily, and cells were passaged every
2–3 days before reaching confluency. OP9 stroma cells (a kind
gift of Ursula Just, University of Kiel, Kiel, Germany) were
grown in a-MEM supplemented with 20% FCS (both from
PAN Biotech), 2mM L-glutamine, 100 units penicillin/100mg
streptomycin, 25mM HEPES, and 100mM b-mercaptoethanol.
293T human embryonic kidney cells were maintained in
DMEM medium containing 10% FCS, 2mM L-glutamine, and
100 units penicillin/100mg streptomycin. Unless stated differ-
ently, all reagents were purchased from Invitrogen.
Bmi1 lentivirus vector and infection of ESC
Bmi1 cDNA was obtained from mouse NSC  by reverse
transcriptase (RT)-polymerase chain reaction (PCR) (forward
reverse primer 5¢-GGGATCCCTAACCAGATGAAGTTGCT
GATGACCC) and cloned into pJET vector (Fermentas Life
Sciences). Bmi1 sequence was released from the construct by
XbaI and BamH1 digestion and subcloned into XbaI/BamH1
sites of FUGIE vector (a kind gift from Filip Farnebo, Kar-
olinska Institute, Stockholm, Sweden). The Bmi1 cDNA was
verified by sequencing. FUGIE vector contains the human
ubiquitin C promoter driving Bmi1 expression and an in-
ternal ribosome entry site (IRES) for expression of enhanced
green fluorescent protein (GFP).
For infection of ESC, lentivirus was produced from 293T
cells. Briefly, 293T cells were transfected with 10mg FUGIE or
FUGIE-Bmi1 plasmid DNA, 7.5mg pCMVDR8.74 packaging
plasmid, and 2.5mg envelope vector pVSV-G. Virus super-
natant was collected and used to infect ESC in the presence
of 8mg polybrene (Sigma-Aldrich). After 2 passages, infected
GFP+ESC were sorted by flow cytometry, and GFP+ESC
were further expanded. Alkaline phosphatase staining was
performed with Alkaline Phosphatase Staining Kit II (Stem-
gent) according to the manufacturer’s protocol.
ESC were subjected to differentiation by embryoid body
(EB) assay. Briefly, ESC were trypsinized into single-cell
suspensions. EB were generated in hanging drops at 100 cells
per 10mL drops in an inverted bacterial Petri dish for 2 days
with ESC differentiation medium. EB were then collected
and cultured in bacterial Petri dishes for an additional 4
days. For some experiments, ESC were directly subjected to
EB formation in mass culture as previously described .
Hematopoietic cell differentiation from ESC
Hematopoietic cell differentiation from ESC was adapted
from the protocol by Carotta et al. . Briefly, ESC were
differentiated by EB formation as just described. On day 6,
EB were dissociated into single cells with 0.05% trypsin/
ethylenediaminetetraacetic acid (EDTA) solution (Invitro-
gen), and cells were passed through a 40mm cell strainer.
Cells were then plated on gelatin-coated dishes at 2·106
cells/mL in serum-free medium (StemPro34 plus nutrient
supplement; Invitrogen) supplemented with 25ng/mL Flt3
ligand (Flt3L; PeproTech), 30U/mL murine SCF, 5ng/mL
IL-6/soluble IL-6R fusion protein (hyper-IL-6) , 40ng/
mL long-range IGF-1 (Sigma-Aldrich), 2ng/mL murine IL-3
(PeproTech), and 1mM dexamethasone (Sigma-Aldrich).
After 2–3 days, nonadherent cells were harvested, passed
through a 40mm cell strainer, and further cultured in the
same culture medium plus growth factors as just described.
The cell concentration was maintained at 2·106cells/mL,
and the medium was refreshed every 1–2 days. Cumulative
cell numbers were determined with a CASY-1 cell counter
and analyzer system (Scha ¨rfe Systems).
Colony-forming assay and cytospins
Day 6 EB were dissociated into single cells with 0.05%
trypsin/EDTA as just described. Cells were collected and re-
suspended in DMEM, 10% FCS at 1·106cells/mL; and
100mL of cell suspension was added to 3mL MethoCult GF
M3434 methylcellulose, containing insulin, transferrin, SCF,
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Address correspondence to:
Prof. Martin Zenke
Department of Cell Biology
Institute for Biomedical Engineering
RWTH Aachen University Medical School
Received for publication November 26, 2010
Accepted after revision April 28, 2011
Prepublished on Liebert Instant Online May 5, 2011
132 DING ET AL.
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