HEMATOPOIESISAND STEM CELLS
Stephen B. Ting,1-3Eric Deneault,1Kristin Hope,1Sonia Cellot,1,4Jalila Chagraoui,1Nadine Mayotte,1Jonas F. Dorn,5
Jean-Philippe Laverdure,1Michael Harvey,2Edwin D. Hawkins,2Sarah M. Russell,2,6Paul S. Maddox,5Norman N. Iscove,7
and Guy Sauvageau1,8
1Molecular Genetics of Stem Cells Laboratory, Institute of Research in Immunology and Cancer (IRIC), University of Montreal, Montreal, QC;2Immune Signalling
Laboratory, Cancer Immunology, Research Division, Peter MacCallum Cancer Centre, East Melbourne,Australia;3Department of Clinical Hematology, Peter
MacCallum Cancer Centre, East Melbourne,Australia;4Hematology and Oncology and Division of Viral and Immune Disorders and Cancers, Centre Hospitalier
Universitaire Sainte-Justine, Montreal, QC;5Mitotic Mechanisms and Chromosome Dynamics Laboratory, IRIC, University of Montreal, Montreal, QC;6Centre
for Micro-Photonics, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn,Australia;7Department of Medical
Biophysics and Immunology, The Ontario Cancer Institute, University of Toronto, Toronto, ON; and8Division of Hematology and Leukemia Cell Bank of Quebec,
Maisonneuve-Rosemont Hospital, Montreal, QC
The stem cell–intrinsic model of self-
renewal via asymmetric cell division
(ACD) posits that fate determinants be
partitioned unequally between daughter
cells to either activate or suppress the
stemness state. ACD is a purported
mechanism by which hematopoietic stem
cells (HSCs) self-renew, but definitive evi-
dence for this cellular process remains
open to conjecture. To address this issue,
we chose 73 candidate genes that func-
tion within the cell polarity network to
identify potential determinants that may
concomitantly alter HSC fate while also
exhibiting asymmetric segregation at cell
division. Initial gene-expression profiles
of polarity candidates showed high and
differential expression in both HSCs and
leukemia stem cells.Altered HSC fate was
assessed by our established in vitro to in
vivo screen on a subcohort of candidate
polarity genes, which revealed 6 novel
positive regulators of HSC function:
Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1,
croscopy of the endocytic protein AP2A2
shows instances of asymmetric segrega-
tion during HSC/progenitor cell cytokine-
sis. These results contribute further evi-
dence that ACD is functional in HSC self-
renewal, suggest a role for Ap2a2 in HSC
activity, and provide a unique opportunity
to prospectively analyze progeny from
HSC asymmetric divisions. (Blood. 2012;
Self-renewal is inextricably linked to stem cell division, and
despite the premise that these processes in mammalian systems
likely involve asymmetric cell division (ACD), the molecular
details remain enigmatic. Our approach to addressing self-renewal
via ACD in the hematopoietic stem cell (HSC) is based on
increasing evidence that the mechanistic insights pertaining to
polarity molecular networks, which are integral to ACD and cell
fate in the invertebrate models of Drosophila melanogaster and
Caenorhabditis elegans, are functionally conserved throughout
Studies from invertebrate models support both extrinsic (niche)
and stem cell–intrinsic mechanisms of ACD. In relation to the cell
intrinsic machinery, polarity is initiated by asymmetrically localiz-
ing protein complexes to the cell membrane. Subcomponents of
these complexes act as cell fate determinants that are maintained
asymmetrically during mitosis and subsequently segregated differ-
entially into daughter cells. During this process, at the simplest
level and without factoring in other potential organelle4,5or cell
cycle component interactions,6,7these membrane complexes inter-
act with centrosomes and the cytoskeletal network to, respectively,
anchor and enable correct mitotic spindle orientation for an
ACD.8-10The distinct advantages of these invertebrate models
include the ability to follow the end fate of daughter cells during
successive rounds of ACD together with real-time video track-
ing to observe the clear segregation of established cell-fate
determinants during and after the ACD process. In contrast,
within the hematopoietic system, these advantages are attenu-
ated by the absence of definitive HSC markers or cell fate
determinants that could allow for investigations of successive
divisions of long-term repopulating HSCs (LT-HSCs). The
added factor of HSC motility outside of its niche further hinders
prospective daughter cell fate analysis.
Despite these limitations, important aspects of HSC self-
renewal with indirect implications for ACD as a mechanism have
been reported. For example, for many decades, single-cell manipu-
lations with more enriched HSC populations over time have
documented different in vitro11,12and in vivo13-15cell fates. Further,
asymmetric segregation of proteins within the hematopoietic
system has also been reported previously,16-18but did not confirm
alterations in cell fate because in vivo daughter cell assays were not
tenable in these studies. Other studies have used live-cell videomi-
croscopy of single cells derived from a population enriched for
HSCs together with clonal fate of progeny as measured by in vivo
repopulation assays to provide morphological clues as to the
There is an Inside Blood commentary on this article in this issue.
The online version of this article contains a data supplement.
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© 2012 by TheAmerican Society of Hematology
2510BLOOD, 15 MARCH 2012?VOLUME 119, NUMBER 11
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identity of LT-HSCs; however, this was done without confirming
ACD per se in this setting.19Far from being a critique, these
examples reflect the difficulty of directly connecting ACD to
daughter cell fate within the heterogeneity of the HSC system. The
present study was undertaken to provide further evidence that the
process of ACD is functional in HSC fate and to serve as a
foundation for ongoing future studies that may allow ACD, HSC
fate, and self-renewal to be directly and mechanistically linked.
Procedures for retroviral vectors, animal husbandry, HSC isolation, quanti-
tative RT-PCR expression studies, BM cell culture, retroviral infection and
transplantation, flow cytometric assessment of donor-derived hematopoi-
esis, Southern blot analysis of genomic DNA, competitive repopulating
units (CRU) assay of HSC-enriched cells, and cell proliferation and cell
death analyses were as detailed in Deneault et al.20All animal procedures
were performed with approval from the Animal Ethic Committees of
University of Montreal and Peter MacCallum Cancer Institute. For resource
information, please see http://www.bioinfo.iric.ca/self-renewal/.
Mouse adult BM and embryonic day 14.5 (E14.5) fetal liver (FL) cells were
isolated independently and lineage depleted by staining with allophycocya-
nin (APC)–conjugated primary Abs to Gr1, B220 and Ter119 (all from
BioLegend). Respective BM and FL cells were stained with anti-APC
magnetic microbeads according to manufacturer guidelines (Miltenyi
Biotec), and lineage positive (Lin?) cells were depleted using the AUTO-
MACS magnetic cell separator system (BD Biosciences). For the adult BM
cells, lineage negative (Lin?) cells were stained with PE-CD150, APC-
CD48, PE-Cy5-Sca, and PE-Cy7-Kit conjugated Abs, and the enriched
CD150?48?Lin?Sca?Kit?(LSK) subpopulation of cells collected by flow
cytometry using the FACSAria II (BD Biosciences). For the FL cells, Lin?
cells were stained with PE-CD150, APC-CD48, PE-Cy5-Sca, and FITC-
Mac–conjugated Abs and the enriched CD150?48?Sca?Mac1?Lin?sub-
population of cells was collected. These respective subpopulations of cells
were resuspended in DMEM with 15% FBS. Respective BM- and
FL-enriched subpopulations up to 50 000 cells per slide were seeded onto
poly-L-lysine–coated coverslips and incubated at 37°C for 60 minutes.
Cells were then fixed with 4% paraformaldehyde for 15 minutes at 4°C and
permeabilized with PBS, 1% BSA, 0.1% Tween and stained with primary
Abs to Ap2a2 (AP6, ab2730; Abcam) and/or Numb (ab4147; Abcam) and
revealed by Alexa Fluor 488– and/or Alexa Fluor 594–labeled secondary
Abs (Molecular Probes). Fluorescent images were obtained with a confocal
microscope (LSM510; Zeiss).
Freshly sorted PE-CD150?/APC-CD48?/PE-Cy5-Sca?/PE-Cy7-cKit?/
Lin?BM cells from histone H2B-GFPmice were transduced with retroviral
producers infected with MSCV-cherry-Ap2a2 and/or pCXIBSR-venus-
Numb vectors for 3 days. Cells were then sorted based on forward and side
scatter to differentiate between hematopoietic and feeder cells. Isolated
hematopoietic cells in BM medium20were seeded into 8-mm sterile cloning
discs (F378470100; Bel-Art Scienceware) placed on glass-bottom MakTek
dishes (number 1.5; MatTek) of a confluent and irradiated (1500 cGy of
137Cs ? radiation) layer of either NIH 3T3 or OP9 cells. Video imaging
comprised 3 colors (green fluorescence, red fluorescence, and transmitted
light) and 4 z-sections 2.5 ?m apart every 15 minutes for 24 hours with an
Olympus 60?/1.42 numerical aperture oil-immersion lens and a Photomet-
ric CoolSnap HQ2 camera on a DeltaVision video microscope fitted with a
37°C environmental chamber (Applied Precision). Video analyses were
performed with softWoRxExplorer Version 2.0 (Applied Precision) and
Imaris software (Bitplane Scientific Software).
Statistical significance was determined with the 2-tailed Student t test.
Candidate polarity screen for HSC expansion
Using existing expression databases21and literature analyses, we
compiled a candidate gene list of polarity cell-fate determinants for
initial gene-expression profiling. Given the heterogeneity of the
HSC compartment, we used 2 established enriched HSC subpopu-
lations, the CD150?48?41?Lin?and the CD49b?rhodaminelow
LSK cells, for assessment (Figure 1A-C and supplemental Table 1,
available on the Blood Web site; see the Supplemental Materials
link at the top of the online article). These gene-expression profiles
show that a broad range of polarity candidates are highly and
differentially expressed in both HSCs and leukemia cells (Figure
1A-C). To assess whether polarity genes would be altered by an
established HSC expansion factor,22we profiled gene-expression
levels of a subset of candidate genes after HSCs were transduced
with HoxB4 and with MSCV vector (Figure 1D). These data show
that HoxB4 does not affect this subset of polarity candidate genes at
Based on our gene-expression profiles (Figure 1), 43 (? 60%)
of the candidates were chosen to assess whether any of these genes
could alter the fate of LT-HSCs using an established gain-of-
function in vitro to in vivo assay (for full details, see Deneault
et al20; Figure 2A-B and http://www.bioinfo.iric.ca/self-renewal/).
The theoretical essence of this screen is based on the relative
inability of HSCs to preserve functional identity (ie, self-renewal)
during a period of in vitro culture when apoptosis or differentiation
are the predominant HSC fate, and as measured by the relative
absence of in vivo repopulation of sublethally irradiated recipients
after transplantation. In contrast, transduction by a candidate
fate-transforming gene to either maintain or expand the HSC
population after in vitro culture would result in a predominant
donor-derived in vivo transplantation output. Briefly, 1500 en-
riched CD150?48?Lin?HSCs (equivalent to ? 62 CRUs per well
or 8 CRUs per transplanted mouse20) from CD45.1 mice were
transduced with high titer retroviruses produced from transfections
with the candidate genes and cultured in vitro for 7 days before
transplantation into 3 recipient CD45.2 mice. Every 4 weeks up to
16 weeks, donor-derived WBC reconstitution was assessed. The
2 negative controls were HSCs transduced with vectors pKOF and
MSCV, and the 2 positive controls were transduced with the
NUP98-HOXA10 fusion (NA10HD)23and HoxB4,22,24with the
positive cutoff set at the latter’s mean reconstitution of 30% at
16 weeks. This primary screen revealed 6 (14%) positive candi-
dates that significantly increased hematopoietic reconstitution:
Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. See Figure 2B
and supplemental Table 2 for full results without (day 0) and with
because it was the most potent HSC expansion gene within the
screening experiments (Figure 2B). The other positive candidates
will be analyzed subsequently.
Validation and self-renewal of Ap2a2-transduced HSCs in vitro
To validate Ap2a2 as a genuine candidate, we performed 3 further
independent in vitro to in vivo assays with the mean reconstitution
levels from each assay supporting the initial screen results (Figure
Ap2a2 INASYMMETRIC HSC DIVISIONAND SELF-RENEWAL2511 BLOOD, 15 MARCH 2012?VOLUME 119, NUMBER 11
For personal use only.on October 26, 2015. by guest
N.M., M.H., and E.D.H.; S.B.T., J.F.D., and P.S.M. planned all
videomicroscopy experiments; S.B.T. performed all videomicros-
copy experiments; J.-P.L. constructed and maintains the IRIC
self-renewal website; N.N.I. and laboratory staff performed all
experiments and data analyses from cycling-quiescent microarrays;
S.M.R. provided significant intellectual input; S.B.T and G.S.
directed the research; S.B.T wrote the manuscript; and all authors
read and approved the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
The current affiliation for S.B.T. is Stem Cell Research Group,
Division of Blood Cancers, Australian Centre for Blood Diseases,
Department of Hematology, Central Clinical School, Monash
University-Alfred Health, Prahran, Australia. The current affilia-
tion for K.H. is McMaster Stem Cell and Cancer Research Institute,
McMaster University, Hamilton, ON.
Correspondence: Stephen B. Ting, Division of Blood Cancer,
Australian Centre for Blood Diseases, Monash University–Alfred
Health, The Alfred Centre, 99 Commercial Road, Melbourne,
Victoria 3004,Australia; e-mail: firstname.lastname@example.org.
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2522TING et alBLOOD, 15 MARCH 2012?VOLUME 119, NUMBER 11
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online December 14, 2011
2012 119: 2510-2522
Maddox, Norman N. Iscove and Guy Sauvageau
F. Dorn, Jean-Philippe Laverdure, Michael Harvey, Edwin D. Hawkins, Sarah M. Russell, Paul S.
Stephen B. Ting, Eric Deneault, Kristin Hope, Sonia Cellot, Jalila Chagraoui, Nadine Mayotte, Jonas
progenitor cells with endocytic Ap2a2
Asymmetric segregation and self-renewal of hematopoietic stem and
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