Abcg2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side
Population Progenitor Cells
Konstantina-Ioanna Sereti1,2, Angelos Oikonomopoulos1, Kazumasa Unno1, Xin Cao1, Yiling Qiu1,
Ronglih Liao1
1
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical
School, Boston, MA, USA, and 2University of Crete-Medical School, Heraklion, Greece.
Running title: Abcg2 Regulates the Division Mode of CSP Cells
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Subject codes:
[137] Cell biology/structural biology
[154] Myogenesis
Address correspondence to:
Dr. Ronglih Liao
Cardiac Muscle Research Laboratory
Cardiovascular Division
Department of Medicine
Brigham and Women’s Hospital
Harvard Medical School
77 Avenue Louis Pasteur, NRB 431
Boston, MA 02115,
Tel: (617) 525-4854
Fax: (617) 525-4868
[email protected]
In October 2012, the average time from submission to first decision for all original research papers submitted
to Circulation Research was 12.5 days.
DOI: 10.1161/CIRCRESAHA.111.300010
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ABSTRACT
Rationale: Following cardiac injury, cardiac progenitor cells are acutely reduced, and are replenished in
part by regulated self-renewal and proliferation, which occurs through symmetric and asymmetric cellular
division. Understanding the molecular cues controlling progenitor cell self-renewal and lineage
commitment is critical towards harnessing these cells for therapeutic regeneration. We have previously
found that the cell surface ATP binding cassette (ABC)-transporter, Abcg2, influences the proliferation of
cardiac side population (CSP) progenitor cells, though through unclear mechanisms.
Objective: To determine the role of Abcg2 on cell cycle progression and mode of division in mouse CSP
cells.
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Methods and Results: Herein, using CSP cells isolated from wild-type and Abcg2-knockout mice, we
find that Abcg2 regulates G1-S cell cycle transition by FUCCI cell cycle indicators, cell cycle-focused
gene expression arrays and confocal live cell fluorescent microscopy. Moreover, we find that modulation
of cell cycle results in transition from symmetric to asymmetric cellular division in CSP cells lacking
Abcg2.
Conclusions: Abcg2 modulates CSP cell cycle progression and asymmetric cell division, establishing a
mechanistic link between this surface transporter and cardiac progenitor cell function. Greater
understanding of progenitor cell biology, and in particular the regulation of resident progenitor cell
homeostasis, is vital for guiding the future development of cell-based therapies for cardiac regeneration.
Keywords:
ABC transporter, cardiac side population cells, asymmetric division, adult stem cell, cell cycle
Non-standard Abbreviations:
CSP
Abcg2
FUCCI
PI
Cardiac Side Population
ATP-Binding Cassette G-subfamily transporter 2
Fluorescence Ubiquitination Cell Cycle Indicator
Propidium Iodide
INTRODUCTION
Adult cardiac stem cells have recently been introduced in the treatment of cardiovascular diseases
with encouraging results.1 During the course of tissue repair, stem/progenitor cells self-renew to expand
their pool, and differentiate to create a specialized progeny. Stem/progenitor cells modulate their cell fate
decision through their modality of replication, since they can divide symmetrically and asymmetrically.
With symmetric stem cell division, two identical daughter cells are formed that retain both stem cell
properties or become both early committed cells. With asymmetric stem cell division, two daughter cells
with divergent fate are generated, one capable of self-renewal and the other committed to differentiation.2
While during physiological tissue homeostasis progenitor cells divide asymmetrically, these progenitor
cells revert to symmetric division and rapid proliferation following tissue injury, a pattern of cell growth
postulated for the damaged human heart and in neuronal progenitor cells.3-5 Similarly, following cardiac
injury, we have previously found that endogenous cardiac progenitor cell populations are acutely
DOI: 10.1161/CIRCRESAHA.111.300010
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reduced, and are replenished within days by self-renewal and proliferation.6 The molecular cues that
dictate the mechanisms of stem cell division, however, remain unclear.
The cell surface ABC-transporter, Abcg2, is highly expressed in several reported stem/progenitor
cell populations and is responsible for the DNA-binding dye extrusion that marks the population
phenotype of cardiac side population (CSP) progenitor cells.7, 8 Moreover, we have found that expression
of Abcg2 may influence CSP proliferation.9 Herein, we provide evidence demonstrating that Abcg2
directly regulates cell cycle progression in CSP cells with loss of Abcg2 resulting in delayed G1-S
transition. Additionally, Abcg2 regulates in CSP cells the switch between symmetric and asymmetric cell
division, determining progenitor cell fate decisions. These findings establish that the Abcg2 transporter is
a critical determinant of cardiac progenitor cell function and may be essential for cardiac regeneration.
METHODS
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Cell cycle analysis using the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) lentiviral
system.
FUCCI-expressing CSP cells were synchronized in G1 and analyzed by flow cytometry or confocal livecell imaging for up to 40hrs following synchronization for the expression of Cdt1 and geminin.
An expanded Methods section describing all procedures and protocols is available in the Online Data
Supplement at http://circres.ahajournals.org.
RESULTS
Abcg2 controls cell cycle progression of CSP cells.
To dissect the role that Abcg2 has in the growth of CSP cells, the progression of the cell cycle
was measured in CSP cells isolated from transgenic mice lacking Abcg2 (Abcg2-KO) and wild-type mice
(WT). Deletion of Abcg2 was associated with a reduction in the number of CSP cells in S and G2/M
phases of the cell cycle, while the fraction of cells in G0/G1 increased (Figure 1A-C). To characterize
the length of the cell cycle in WT CSP cells and Abcg2-KO CSP cells, a lentivirus-based fluorescent
ubiquitination-dependent cell cycle indicator (FUCCI) was utilized. Flow cytometric analysis of presorted FUCCI-expressing WT and Abcg2-KO CSP cells 24 hours post-synchronization revealed striking
differences in the distribution of cells within the various phases of the cell cycle. WT CSP cells were
actively cycling with the vast majority of cells residing within the G1-S transition. In contrast, most
Abcg2-KO CSP cells were in G1 and only a small fraction was in G1-S or S-G2-M phases (Figure 2A
and B). Greater than 50% of Abcg2-KO CSP cells were in early G1.
Abcg2 regulates G1-S transition in CSP cells.
To elucidate the effects of Abcg2 on cell cycle progression, we monitored cell cycle kinetics in
WT and Abcg2-KO CSP cells. WT CSP cells entered G1-S transition as early as 14 hours post
synchronization (data not shown). Within 24 hours, WT cells in G1 reached a peak followed by a
progressive decrease and concomitant entry into S-G2-M. Entry in G2-M was rapidly followed by
evidence for cell division (Figures 2C and E). In contrast, Abcg2-KO CSP cells were unable to progress
through the cell cycle and remained largely in G1 40 hours post-synchronization (Figures 2D and F).
Moreover, only 4% of Abcg2-KO CSP cells were in G1-S transition and 12% in S/G2/M at 40 hours;
DOI: 10.1161/CIRCRESAHA.111.300010
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these values were negligible at 18 hours (Figures 2D and F). Modeling of cell exit from the G1 phase
using a “plateau followed by exponential decay” model10 revealed that the T50 for WT CSP cells was 20.2
hours while Abcg2-KO CSP cells had a T50 of 32.6 hours (Figure 2G and H). Confocal time-lapse, livecell imaging documented that WT CSP cells cycled normally, while Abcg2-KO CSP cells remained
predominantly confined to G1 (Figures 2I, J and Online Videos I, II). Collectively, these data strongly
suggest that Abcg2 regulates the cell cycle in CSP cells.
Abcg2 and cell cycle gene expression profile of CSP cells.
Based on these observations, we next determined the expression of cell cycle regulatory genes in
WT and Abcg2-KO CSP cells. Following shRNA-lentivirus mediated silencing of Abcg2 (sh-Abcg2),
the expression of cyclin C, cyclin D1 and cyclin E was downregulated (Figure 3A and Online Table I).
Moreover, negative regulators of the cell cycle, including p27, were up-regulated (Figure 3A and Online
Table I). Strikingly, the absence of Abcg2 increased significantly the expression of DNA-damage related
genes such as Atm, Msh2, p53, Smc1a, Rad51, Brca1 and Brca2 (Figure 3B and Online Table I).
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Abcg2 and asymmetric stem cell division.
The dual fundamental property of stem cells is to self-renew and differentiate.2 Immunolabeling
for the cell fate determinant α-adaptin demonstrated that sh-Abcg2 CSP cells preferentially divided
asymmetrically (65.3±5.4% vs. scramble 27.9±6.6%) (Figure 4A-D). Moreover, in comparison to shAbcg2 CSP cells, a higher number of scramble CSP cells expressed the cell-fate determinant, numb
(Figure 4E-G), supporting the concept that during division in Abcg2-deficient cells, the fate determinants
segregate asymmetrically to one daughter cell.
DISCUSSION
Despite accumulating evidence as to the therapeutic benefits of stem cell therapy, little is known
regarding the molecular mechanisms that dictate progenitor cell homeostasis. The identification of novel
regulators of cardiac stem/progenitor cell self-renewal is of paramount importance for the development of
new strategies for treatment of chronic heart failure. Such regulators may allow for manipulation of
endogenous progenitors enhancing tissue regeneration following injury.
The expansion of
stem/progenitor cells through symmetric divisions, followed by a switch towards a differentiated progeny
via asymmetric division would greatly enhance myocardial repair. In this context, the identification of
the molecular variables that regulate the balance between symmetric and asymmetric stem cell division
are of great relevance for the expansion and lineage specification of resident stem cells. In this report, we
have identified that the cell surface transporter, Abcg2, is a critical determinant of cell cycle progression
and transition between symmetric and asymmetric stem cell division in cardiac progenitor cells.
Regulation of cardiac progenitor cells either by direct modulation of Abcg2 expression or indirectly
through manipulation of Abcg2 substrates may represent a promising approach for cell-based therapy.
Abcg2 activation following cardiac injury could allow the restoration and increase of progenitor cell
numbers while inhibition of the transporter and activation of asymmetric cell division of cardiac
progenitors would promote tissue regeneration.
Stem cell fate decisions such as proliferation, quiescence and differentiation are influenced by the
cell cycle.11 Interestingly, an association may be present between the length of G1 and asymmetric cell
division, further highlighting the significance of the effects of Abcg2 in CSP cell growth and
commitment.11-14 Cells exhibiting high self-renewal ability, such as embryonic stem cells, have a short
G1 phase, which is significantly prolonged with differentiation.11 Similarly, inhibition of G1 in adult
DOI: 10.1161/CIRCRESAHA.111.300010
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neural stem cells results in neurogenesis, while shortening of G1 promotes their expansion through
symmetric divisions.12, 15 By a number of techniques and approaches, our data show that the majority of
Abcg2-KO CSP cells reside in the G1 phase of the cell cycle. In addition to the prolonged G1 phase and
delayed entry in S phase, crucial cell fate determinants such as α-adaptin and numb were asymmetrically
segregated in Abcg2-deficient CSP cells. These results strengthen the notion that the length of the cell
cycle is linked to the mode of cell division in cardiac progenitor cells. We cannot exclude the possibility
that a fraction of CSP cells negative for both probes could exit the cell cycle and reside in G0. These
cells would correspond to daughter cells acquiring a committed fate or cells undergoing apoptosis. In
fact, lack of Abcg2 increases apoptotic and necrotic cell death in basal conditions.9 Evidence of apoptotic
cells can also be seen in the live cell imaging data. Interestingly, the role of Abcg2 in the regulation of
the mode of division may extend beyond progenitor cells to transformed cancer cells, in which high
expression of Abcg2 and unchecked symmetric division have been described.16-19
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Importantly, it remains to be determined whether loss of Abcg2 influences the replication of CSP
cells and the replenishment of endogenous progenitor cell niches following cardiac injury. Abcg2 is a
membrane bound transporter that is primarily responsible for the cytoplasmic clearance of a number of
substrates.8 We have previously shown that Abcg2 expression is regulated in a developmental manner in
CSP cells.9 Abcg2 is highly expressed in neonatal CSP cells and its expression decreases with age.
Moreover, during hypoxia, Abcg2 has been shown to be regulated by HIF-1α signaling,20 as well as
upregulated by EGF 21, 22 and EGFR23 in transformed cells. The regulation of Abcg2 expression may, in
part, be modulated by use of alternative 5’UTR leader exons.24 Abcg2 downstream signaling, however,
remains completely undefined and the exact mechanisms by which Abcg2 regulates cell cycle and mode
of division are unclear at the present time. It is likely that levels of certain molecules are altered in cells
lacking Abcg2, and such yet to be defined molecules may subsequently reprogram the cell cycle mode of
CSP cells directly or indirectly toward asymmetric division. For instance, Abcg2 has been shown to
regulate embryonic stem cell function through maintenance of porphyrin homeostasis.25 A number of
reports suggest that p53 inhibits proliferation while promoting asymmetric cell divisions.26, 27 Notably,
lack of Abcg2 in CSP cells increases the expression of several cell cycle inhibitors such as p27 as well as
p53, a well-known stress sensor. At the present time, we cannot exclude the possibility that Abcg2 may
directly interact with other signaling cascades to influence cell division. Further investigation is required
to address the exact role of Abcg2 in the regulation of progenitor cell mode of division. Stem/progenitor
cell function, including both proliferation and mode of division, is highly sensitive to external signals
from the stem cell microenvironment, or stem cell niche,28 including those effector pathways originating
from neighboring progenitors, tissue stromal and parenchymal cells, and potentially extra-tissue
infiltrating cells. Given that Abcg2 is a cell surface transporter, it may function to link internal and
external signals and thereby regulate the function of progenitor cell populations via the
microenvironment.
ACKNOWLEDGEMENTS
We thank G. Losyev at the Cardiovascular FACS Core at Brigham and Women’s Hospital and Harvard
Medical School for assistance with CSP cells sorting.
SOURCES OF FUNDING
This study was supported in part by NIH grants (HL086967 and HL093148) to R.L. K.-I.S. was
supported by the Manasaki fellowship from the University of Crete-Greece.
DISCLOSURES
No disclosures.
DOI: 10.1161/CIRCRESAHA.111.300010
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Figure legends
Figure 1: Abcg2-KO CSP cells exhibit altered cell cycle profile. Representative flow cytometric
analysis of (A) WT and (B) Abcg2-KO CSP cells stained with PI. (C) Quantification of propidium iodide
(PI) staining.
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Figure 2: Lack of Abcg2 prolongs cell cycle duration in CSP cells. Representative flow cytometric
analysis of Fucci+ (A) WT and (B) Abcg2-KO CSP cells 24 hours post-synchronization. Time course
+
analysis of Fucci (C) WT and (D) Abcg2-KO CSP cells over a period of 10 and 22 hours respectively,
starting at 18 hours post-synchronization. Bar graphs indicate the percentage of (E) WT and (F) Abcg2KO CSP cells residing in each cell cycle phase during the specified time course. (G-H) Mathematical
calculation of the G1-phase T50 value following a “plateau followed by exponential decay” model. The
T50 for (G) WT and (H) Abcg2-KO CSP cells is 20.2hr and 32.6hr respectively. (I-J) Sequential (2 hours
interval) DIC and fluorescent confocal microscopy images of Fucci+ (I) WT and (J) Abcg2-KO CSP cells
18-40 hours following synchronization. Cells were infected with lentiviruses expressing both Cdt1Kusabira Orange probe (red) and Geminin-Azami Green (green) present in the G1 and the S-G2-M
phases respectively. Cells residing in the G1-S transition phase appear as orange. Cells immediately after
cytokinesis or in early G1 phase lose the fluorescence. Scale bars 20μm. Yellow box indicates the
position of representative (I) WT and (J) Abcg2-KO CSP cells.
Figure 3: Abcg2 deficiency alters the gene expression profile of CSP cells. Gene expression analysis
of (A) cell cycle regulators and (B) DNA-damage responsive genes in sh-Abcg2 CSP cells compared to
Scramble cells (n=3). Data are mean ± s.e.m. *p<0.05.
Figure 4: Lack of Abcg2 favors asymmetric cell division in CSP cells. Representative immunofluorescent images of (A) WT and (B) Abcg2-KO CSP cells stained for phospho-Histone-H3 (pH3,
green), α-adaptin (red) and DAPI (blue) (Scale bars 10μm). Quantification of symmetric and asymmetric
cell division mode of pH3+ (C) scramble and (D) sh-Abcg2 CSP cells, based on the distribution of αadaptin. Representative flow cytometric analysis of (E) scramble and (F) sh-Abcg2 CSP stained with
numb. (G) Quantification of numb-expressing scramble and sh-Abcg2 CSP cells. Data are mean ± s.e.m.
*p<0.05.
DOI: 10.1161/CIRCRESAHA.111.300010
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Novelty and Significance
What Is Known?

Stem/progenitor cells modulate their cell fate decision in part through a balance between
symmetric stem cell division, in which two identical daughter cells are formed, versus
asymmetric stem cell division, in which two daughter cells with divergent fates are generated, one
capable of self-renewal and one committed to differentiation.

Abcg2 regulates cardiac side population (CSP) progenitor cell homeostasis by promoting
proliferation and survival while inhibiting differentiation, but mechanisms underlying this
relationship remain unknown.
What New Information Does This Article Contribute?
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
Abcg2 regulates cell cycle duration and CSP proliferation through G1-S transition.

Abcg2 deficiency promotes asymmetric division in CSP cells.
Adult cardiac stem/progenitor cells represent a promising therapeutic option for the treatment of heart
failure. Understanding the molecular mechanisms regulating adult progenitor cell self-renewal and
lineage commitment is key towards their therapeutic utilization. The maintenance of progenitor cell
pools and the production of differentiated progeny are ensured by a balance between symmetric and
asymmetric division. Progenitor cell homeostasis is regulated by expression of Abcg2 cell surface
transporter through unresolved mechanisms. This study finds that Abcg2 controls cell cycle
progression and CSP cell proliferation as well as the balance between symmetric and asymmetric
division. Manipulation of Abcg2 could represent a novel therapeutic strategy for enhancing efficacy
of cell-based therapy for cardiovascular diseases.
DOI: 10.1161/CIRCRESAHA.111.300010
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Figure 1: Abcg2-KO CSP cells exhibit altered cell cycle profile.
Representative flow cytometric analysis of (A) WT and (B) Abcg2-KO CSP
cells stained with PI. (C) Quantification of propidium iodide (PI) staining.
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Figure 2 continued
Fo
Figure 2: Lack of Abcg2 prolongs cell cycle duration in CSP cells. Representative flow
cytometric analysis of Fucci+ (A) WT and (B) Abcg2
Abcg2-KO
KO CSP cells 24 hours post
postsynchronization. Time course analysis of Fucci+ (C) WT and (D) Abcg2-KO CSP cells over a
period of 10 and 22 hours respectively, starting at 18 hours post-synchronization. Bar graphs
indicate the percentage of (E) WT and (F) Abcg2-KO CSP cells residing in each cell cycle
phase during the specified time course. (G-H) Mathematical calculation of the G1-phase T50
value following a “plateau followed by exponential decay” model. The T50 for (G) WT and (H)
Abcg2-KO CSP cells is 20.2hr and 32.6hr respectively. (I-J) Sequential (2 hours interval) DIC
and fluorescent confocal microscopy images of Fucci+ (I) WT and (J) Abcg2-KO
Abcg2 KO CSP cells 1818
40 hours following synchronization. Cells were infected with lentiviruses expressing both Cdt1Kusabira Orange probe (red) and Geminin-Azami Green (green) present in the G1 and the SG2-M phases respectively. Cells residing in the G1-S transition phase appear as orange. Cells
immediately after cytokinesis or in early G1 phase lose the fluorescence. Scale bars 20μm.
Yellow box indicates the position of representative (I) WT and (J) Abcg2-KO CSP cells.
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Figure 3: Abcg2 deficiency alters the gene expression profile of CSP cells.
Gene expression analysis of (A) cell cycle regulators and (B) DNA-damage
responsive genes in sh-Abcg2 CSP cells compared to Scramble cells (n=3).
aaa
are
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p 0 05
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Figure 4: Lack of Abcg2 favors asymmetric cell division in CSP cells. Representative immunofluorescent images of (A) WT and (B) Abcg2-KO
Abcg2 KO CSP cells stained for phospho-Histone-H3
phospho Histone H3 (pH3,
(pH3
green), α-adaptin (red) and DAPI (blue) (Scale bars 10μm). Quantification of symmetric and
asymmetric cell division mode of pH3+ (C) scramble and (D) sh-Abcg2 CSP cells, based on the
distribution of α-adaptin. Representative flow cytometric analysis of (E) scramble and (F) sh-Abcg2
CSP stained with numb. (G) Quantification of numb-expressing scramble and sh-Abcg2 CSP cells.
Data are mean ± s.e.m. *p<0.05.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Abcg2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side
Population Progenitor Cells
Konstantina-Ioanna Sereti, Angelos Oikonomopoulos, Kazumasa Unno, Xin Cao, Yiling Qiu and
Ronglih Liao
Circ Res. published online November 6, 2012;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2012/11/06/CIRCRESAHA.111.300010
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2012/11/06/CIRCRESAHA.111.300010.DC1
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SUPPLEMENTAL MATERIAL
Detailed Methods
Animals: WT FVB and Abcg2 deficient1 (cat. #002767) male mice (8 weeks old) were
purchased from Taconic and bred in our animal facility. All animal studies were performed
according to the guidelines of the Harvard Medical School, the Longwood Medical Area’s
Institutional Animal Care and Use Committee (IACUC) and the National Society for Medical
Research.
CSP cell isolation and expansion: CSP isolation was performed as previously described.2
Briefly, mouse hearts were enzymatically and mechanically digested with a mixture of 0.1%
collagenase B (Roche), 2.4U/ml dispase II (Roche) and 2.5mmol/L CaCl2. Mononuclear cells
were stained with Hoechst 33342 (Sigma Cat #B2261) (5μg/106 cells/ml) at 37oC for 90min.
Verapamil (50 mmol/L) (Sigma, #V4629) was used as a negative control. Cells were further
incubated with fluorochrome-conjugated monoclonal rat anti-mouse antibodies against Sca-1,
CD31 and CD45 (Pharmigen). 7-Aminoactinomycin-D (7-AAD) was used for dead cell
detection. Sca1+/CD31-/CD45- CSP were used for all experiments. Sorted CSP cells were
cultured in expansion medium (α-ΜΕΜ, 20% FBS HI, 2 mM L-Glutamine and 1%
penicillin/streptomycin). All experiments were performed with CSP cells from passages 4-6.
Propidium iodide staining (PI): PI staining and analysis were performed as previously
described.3 Briefly, CSP cells were fixed in 70% ethanol and incubated with PI staining solution
(0.1% Triton-X100, 2μg/ml PI, 400ng/ml RNase A) for one hour at 37oC protected from light.
Cells were analyzed by flow cytometry and data processing was performed by the ModFit Lt
software (Verity House).
Flow cytometric analysis and sorting: Flow cytometric analysis was performed with the
DXP11 analyzer (Cytek) and the Accuri C6 analyzer (BD Biosciences). FACS was performed
with a FACSAria sorter (BD Biosciences). Hoechst 33342 dye was excited by an UV (355-nm)
laser. Data were analyzed by the FACSDIVA software (BD Biosciences) and FlowJo (Tree
Star).
Cell Cycle Analysis Using the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI)
Lentiviral System: CSP cells were infected with lentiviruses expressing the FUCCI reporter
system and selected by FACS.4 Cells were synchronized in G1 (24 hours in α-ΜΕΜ, 0.1% FBS
HI, 2 mM L-Glutamine and 1% pen/strep) and cultured in CSP expansion medium for up to 40
hours. Sampling of the cells was performed at several time points and cells were analyzed by
flow cytometry with the DXP11 analyzer (Cytek) or by confocal live imaging (Zeiss LSM 510
inverted live-cell confocal system) for Cdt1 and geminin reporters. Data were analyzed with the
FlowJo and LSM510 software programs respectively. For cell cycle analysis, expression of the
Cdt1 reporter in G1 phase, geminin reporter in S-G2-M cell cycle phases, expression of both
protein reporters during the G1-S transition, and lack of expression of either reporter shortly
after cytokinesis and in early G1 were monitored, as previously described.4 T50 values were
calculated according to a “plateau followed by exponential decay” model.5 The T50 value
represents the required time for G1-residing CSP cells to be decreased by 50% from their initial
value.
Abcg2 shRNA: shRNA lentiviral constructs were generated according to the instructions
provided by Addgene. Scramble (5’CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAG
GGCGACTTAACCTTAGGTTTTTG3’) or Abcg2 (5’CCGGGCAACACTTCTCATGACAATCC
TCGAGGATTGTCATGAGAAGTGTTGCTTTTTG3) shRNA sequences were cloned in the
pLKO.1-puro lentiviral vector under the control of the U6 promoter. Infected CSP selection was
achieved through selection with puromycin (4μg/ml).
Cell cycle-focused RT-PCR gene array: mRNA from scramble and sh-Abcg2 expressing CSP
cells was used to synthesize cDNA (RT2-First strand kit, SABiosciences). Cell cycle gene
expression profile was established using the Cell Cycle RT-PCR array (Cat #PAMM-020,
SABiosciences) and the MyiQ cycler (Bio-Rad) according to the manufacturer’s guidelines.
Data were obtained from three independent experiments. Data analysis was carried out
according to the manufacturer’s instructions and as previously described.3 The full list of the
analyzed genes is provided in table 1.
Asymmetric division assessment: Unsynchronized CSP cells on coverslips were fixed with
4% paraformaldehyde, permeabilized with methanol followed by blocking with 1% BSA. Cells
were then incubated with antibodies against phospho-Histone H3 (pH3) (1/200, Abcam Cat
#ab32107) and α-adaptin6 (1/50, Santa Cruz cat # sc-17771) followed by secondary antibodies
(1/200, anti-rabbit Alexa-488 and anti-mouse Alexa-555, Molecular probes). Coverslips were
mounted on slides with DAPI-containing mounting medium (Vector Vectashield). Visualization
was performed with an epi-fluorescent microscope (Zeiss, Axiovert 200M). Staining for numb 6, 7
was performed in CSP cells fixed with 4% paraformaldehyde and permeabilized with BD
Perm/Wash solution (BD biosciences) according to the manufacturer’s instructions. Cells were
then blocked with 1% BSA and incubated with anti-numb antibody (1/500, Abcam Cat #ab4147)
followed by a secondary goat-anti-rabbit 488 antibody (Molecular probes). Data were acquired
with the Accuri C6 analyzer.
Statistical analysis: Statistical differences were evaluated using one-way ANOVA analysis
and Student’s unpaired t-test, using GraphPad Prism (Version 5.03). Data are presented as
mean ± s.e.m. A p-value ≤ 0.05 was considered statistically significant.
Online Table I: Cell cycle-focused RT-PCR array of scramble and sh-Abcg2 expressing CSP cells
Description
C-abl oncogene 1, receptor tyrosine kinase
Adenylate kinase 1
Amyloid beta (A4) precursor protein-binding, family B,
member 1
Ataxia telangiectasia mutated homolog (human)
Breast cancer 1
Breast cancer 2
Calcium/calmodulin-dependent protein kinase II alpha
Calcium/calmodulin-dependent protein kinase II, beta
Caspase 3
Cyclin A1
Cyclin A2
Cyclin B1
Cyclin B2
Cyclin C
Cyclin D1
Cyclin E1
Cyclin F
Cell division cycle 25 homolog A (S. pombe)
Cyclin-dependent kinase 2
Cyclin-dependent kinase 4
CDK5 regulatory subunit associated protein 1
Cyclin-dependent kinase inhibitor 1A (P21)
Cyclin-dependent kinase inhibitor 1B
Cyclin-dependent kinase inhibitor 2A
Checkpoint kinase 1 homolog (S. pombe)
CDC28 protein kinase 1b
DNA-damage inducible transcript 3
DnaJ (Hsp40) homolog, subfamily C, member 2
Dystonin
E2F transcription factor 1
E2F transcription factor 2
E2F transcription factor 3
E2F transcription factor 4
Growth arrest and DNA-damage-inducible 45 alpha
G protein-coupled receptor 132
Hus1 homolog (S. pombe)
Inhibin alpha
Symbol
(ΔCt)
Abl1
Ak1
Scramble sh-Abcg2
11.106178 11.420862
1.8289
2.147363
Apbb1
Atm
Brca1
Brca2
Camk2a
Camk2b
Casp3
Ccna1
Ccna2
Ccnb1
Ccnb2
Ccnc
Ccnd1
Ccne1
Ccnf
Cdc25a
Cdk2
Cdk4
Cdk5rap1
Cdkn1a
Cdkn1b
Cdkn2a
Chek1
Cks1b
Ddit3
Dnajc2
Dst
E2f1
E2f2
E2f3
E2f4
Gadd45a
Gpr132
Hus1
Inha
8.367457
5.850675
7.734002
6.90382
8.726875
11.106178
4.375578
11.106178
4.476715
3.212888
5.263591
6.091513
1.426526
7.093276
4.612443
4.979034
5.539315
2.786019
7.099793
1.11014
6.03447
2.639825
6.8862
3.106571
2.194981
3.320207
4.176003
7.65494
11.106178
6.080322
3.772541
4.521487
11.106178
6.020265
7.611555
8.377834
5.30991
6.825685
6.069443
8.514801
11.635309
3.339269
11.635309
3.571836
2.579624
3.677749
7.270975
2.148219
7.731017
3.817572
4.111663
5.067542
1.978295
7.425954
1.840963
4.990849
3.229998
6.34775
2.649848
3.402252
3.546802
4.096243
6.996614
11.635309
6.247647
3.754677
5.196044
11.635309
6.168142
7.451969
Up-Down
Regulation
(comparing to
Scramble)
-1.2437
-1.247
-1.0072
1.4547
1.8769
1.7831
1.1584
-1.4431
2.051
-1.4431
1.8724
1.5511
3.0018
-2.2649
-1.6491
-1.5559
1.7349
1.8243
1.3868
1.7504
-1.2537
-1.6596
2.0614
-1.5054
1.4524
1.3724
-2.309
-1.1701
1.0568
1.5783
-1.4431
-1.123
1.0125
-1.5961
-1.4431
-1.1079
1.117
Integrin beta 1 (fibronectin receptor beta)
Microtubule-actin crosslinking factor 1
MAD2 mitotic arrest deficient-like 1 (yeast)
Minichromosome maintenance deficient 2 mitotin (S.
cerevisiae)
Minichromosome maintenance deficient 3 (S. cerevisiae)
Minichromosome maintenance deficient 4 homolog (S.
cerevisiae)
Transformed mouse 3T3 cell double minute 2
Antigen identified by monoclonal antibody Ki 67
Meiotic recombination 11 homolog A (S. cerevisiae)
MutS homolog 2 (E. coli)
Mdm2, transformed 3T3 cell double minute p53 binding
protein
Myeloblastosis oncogene
NIMA (never in mitosis gene a)-related expressed kinase 2
Nuclear factor of activated T-cells, cytoplasmic,
calcineurin-dependent 1
Notch gene homolog 2 (Drosophila)
Nucleophosmin/nucleoplasmin 2
Proliferating cell nuclear antigen
Pescadillo homolog 1, containing BRCT domain
(zebrafish)
Polycystic kidney disease 1 homolog
Peripheral myelin protein 22
Protein phosphatase 1D magnesium-dependent, delta
isoform
Protein phosphatase 2 (formerly 2A), regulatory subunit
B'', alpha
Protein phosphatase 3, catalytic subunit, alpha isoform
Protamine 1
RAD17 homolog (S. pombe)
RAD21 homolog (S. pombe)
RAD51 homolog (S. cerevisiae)
RAD9 homolog (S. pombe)
RAN, member RAS oncogene family
Retinoblastoma-like 1 (p107)
Retinoblastoma-like 2
Sestrin 2
Stratifin
Src homology 2 domain-containing transforming protein
C1
S-phase kinase-associated protein 2 (p45)
Schlafen 1
Itgb1
Macf1
Mad2l1
-1.705923
2.085609
4.865167
-1.351013
2.332797
4.094512
-1.2789
-1.1869
1.706
Mcm2
Mcm3
5.947158
4.530528
4.838795
3.72946
2.156
1.7424
Mcm4
Mdm2
Mki67
Mre11a
Msh2
2.64628
2.738524
3.514161
6.648424
4.539862
2.084709
1.620668
2.721685
5.72085
3.850688
1.4759
2.1702
1.732
1.9021
1.6124
Mtbp
Myb
Nek2
8.635183
8.600524
11.106178 11.635309
11.106178 10.252296
1.0243
-1.4431
1.8074
Nfatc1
Notch2
Npm2
Pcna
3.728189
3.840408
8.373932
6.396327
11.106178 11.350593
2.143046
1.263756
-1.0809
3.9384
-1.1846
1.8395
Pes1
Pkd1
Pmp22
4.101918
4.02754
2.233625
4.226599
4.328398
2.111629
-1.0903
-1.2319
1.0882
Ppm1d
5.607756
5.029499
1.493
Ppp2r3a
Ppp3ca
Prm1
Rad17
Rad21
Rad51
Rad9
Ran
Rbl1
Rbl2
Sesn2
Sfn
10.805114
9.865458
3.788133
4.481805
11.106178 11.635309
6.058783
6.062272
4.681669
4.442202
9.395378
8.334477
5.869122
5.696741
-1.819387 -1.503806
7.246548
6.439949
6.16683
5.920503
6.770173
7.372871
10.925972 11.333543
1.9181
-1.6174
-1.4431
-1.0024
1.1806
2.0862
1.1269
-1.2445
1.7491
1.1862
-1.5186
-1.3265
Shc1
Skp2
Slfn1
3.27843
3.011812
6.762598
7.075956
11.106178 11.635309
1.203
-1.2426
-1.4431
Structural maintenance of chromosomes 1A
Stromal antigen 1
SMT3 suppressor of mif two 3 homolog 1 (yeast)
Smc1a
Stag1
Sumo1
TAF10 RNA polymerase II, TATA box binding protein
(TBP)-associated factor
Telomeric repeat binding factor 1
Transcription factor Dp 1
Proteasome (prosome, macropain) assembly chaperone 2
Transformation related protein 53
Transformation related protein 63
Tumor susceptibility gene 101
WEE 1 homolog 1 (S. pombe)
Taf10
Terf1
Tfdp1
Psmg2
Trp53
Trp63
Tsg101
Wee1
4.750962
5.081462
2.225602
3.845469
4.207143
2.041863
1.8732
1.8331
1.1358
2.538463
3.137654
6.846584
5.840404
1.425901
1.350236
6.058063
5.13353
3.339588
2.5548
11.106178 11.635309
5.633285
6.251244
5.28807
5.570576
-1.5149
2.0086
1.0538
1.8981
1.7228
-1.4431
-1.5347
-1.2163
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Online Video I: Representative video of time-lapse live imaging (40X) monitoring of Fucci+ WT
CSP cells 18-40 hours following synchronization.
Online Video II: Representative video of time-lapse live imaging (40X) monitoring of Fucci+
Abcg2-KO CSP cells 18-40 hours following synchronization.