A Functional Screen
to Identify Novel Effectors
of Hematopoietic Stem Cell Activity
Eric Deneault,1Sonia Cellot,1Ame ´lie Faubert,1Jean-Philippe Laverdure,1Me ´lanie Fre ´chette,1Jalila Chagraoui,1
Nadine Mayotte,1Martin Sauvageau,1Stephen B. Ting,1and Guy Sauvageau1,2,*
1Molecular Genetics of Stem Cells Laboratory, Institute of Research in Immunology and Cancer (IRIC), University of Montreal,
Montreal, Quebec H3C 3J7, Canada
2Divisionof Hematology andLeukemia Cell Bank of Quebec(BCLQ), Maisonneuve-Rosemont Hospital, Montreal,Quebec H1T2M4, Canada
Despitetremendous progress made towardtheiden-
tification of the molecular circuitry that governs cell
fate in embryonic stem cells, genes controlling this
process in the adult hematopoietic stem cell have
proven to be more difficult to unmask. We now report
the results of a novel gain-of-function screening
approach, which identified a series of 18 nuclear
factors that affect hematopoietic stem cell activity.
Overexpression of ten of these factors resulted in an
increased repopulating activity compared to unma-
nipulated cells. Interestingly, at least four of the 18
factors, Fos, Tcfec, Hmgb1, and Sfpi1, show non-
cell-autonomous functions. The utilization of this
base enriched for potential determinants of hemato-
poietic stem cell self-renewal will serve as a resource
to uncover regulatory networks in these cells.
The mature cell contingent of adult hematopoietic tissue is
continuously replenished during the life span of an animal by
the periodic supplies from hematopoietic stem cells (HSCs)
that reside in a niche. To maintain blood homeostasis, these
primitive cells rely on two critical properties, namely multipo-
tency and self-renewal. The former enables differentiation into
multiple lineages, while the latter ensures preservation of HSC
fate upon cellular division. By definition, a self-renewal division
implies that an HSC is permissive to cell cycle entry, while
restrained from engaging in differentiation, apoptosis or senes-
cence pathways. The transcriptional regulatory network of
HSC self-renewal still remains largely undefined, an observation
self-renewal is increasingly dissected molecularly (reviewed in
Jaenisch and Young, 2008). Only a few nuclear factors have
been documented as promoters of HSC expansion (reviewed
in Hai-Jiang et al., 2008). Of these factors, Hoxb4 and its deriva-
tives (Hoxa9, NA10HD) are among the most potent and best
documented (Ohta et al., 2007; Thorsteinsdottir et al., 2002).
The differential pace of progress propelling the fields of ESC
and HSC research reflects, at least in part, seminal discoveries
that rendered ESCs more amenable to large-scale experiments.
First, although only observable as a transient state in vivo, ESCs
derived fromthe innercell masscan bemaintained invitro ascell
lines by the addition of serum (as a source of bone morphogenic
protein [BMP]) and leukemia inhibitory factor (LIF). Attempts to
maintain or expand HSCs ex vivo as homogenous populations
have been modest, and successful development of cell lines
have not been reported, hampering harvest of large numbers
of HSCs. Second, a stringent surrogate marker to follow the
HSC multipotent state, comparable to the pluripotency tags of
Oct4, Nanog, AP, or SSEA1 for ESCs, is still lacking. Albeit
yielding small numbers, current cell sorting strategies allow
isolation of HSC populations to near purity (Kiel et al., 2005).
However, shortly upon facing the selective pressures of in vitro
culture conditions, changes in cell phenotype are observed
(Uchida et al., 2004), impeding HSC tracking in this context.
Thegold standardtoconfirmHSCactivityforcellskeptin culture
remains the in vivo competitive repopulation assay. Importantly,
generation of retroviral vectors provides highly efficient tools to
infect and thereby modulate gene expression in both ESCs
and HSCs (Root et al., 2006).
The demonstration thatin mousefibroblasts a givennucleocy-
toplasmic configuration, or state, can be reverted to a stem cell
phenotype by the enforced overexpression of four defined
nuclear factors, i.e., Oct4, Sox2, c-Myc, and Klf4, stands as
a conceptual breakthrough (Takahashi and Yamanaka, 2006).
Indeed, the ability to create induced pluripotent stem cells, or
iPS cells, suggests that a putative role for as yet unidentified
nuclear factors in orchestrating HSC fate is probable. With this
mindset, interrogation of stem cell expression profile databases
was undertaken. From this data set, a listing and ranking of
nuclear factors whose transcripts were abundant in stem cell-
enriched subpopulations was generated. Over 100 of the high-
est-scoring candidates were then functionally tested in HSCs,
using a high-throughput overexpression in vitro to in vivo assay
tailored to circumvent current limitations imposed by the biology
of HSCs. As detailed below, these studies serve as a further step
Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc. 369
forward into the exploration of the molecular circuitry that
governs HSC self-renewal.
Selection and Ranking of Candidate Genes
As acorollary of ESCstudies,it canbeproposed thatHSC fate is
also controlled by a series of master regulators analogous to
Oct4 and several subordinate effectors, providing sound basis
for the generation of a stem cell nuclear factors database.
Toward this end, we created a database consisting of 689
nuclear factors (Figure 1A; Table S1 available online; see also
http://www.bioinfo.iric.ca/self-renewal/) considered as candi-
date regulators of HSC activity. This list was predominantly
derived from microarray gene expression profiling of normal
and leukemia stem cells, including our recently generated
FLA2 leukemia (1 in 1.5 cells is a leukemia stem cell, A.F. and
G.S., unpublished data). Genes obtained after a review of the
literature on stem cell self-renewal were also added to the list
(see legend of Table S1 for references). Genes in this database
were then ranked from 1 (lowest priority) to 10 (highest priority)
on the basis of three factors: differential expression between
primitive and more mature cell fractions (e.g., HSC-enriched),
expression level (high levels were given highest priority), and
the consistency of findings between data sets. Genes with a
score of 6 and above (n = 139) were selected for functional
studies, of which 104 were tested (Figure 1A; see also asterisks
inTable S1).Interestingly, thelistof selected candidates ishighly
Gata2, Sfpi1 (PU.1), Foxo1, Meis1, Myb, Hoxa9, and Runx1 (Min
et al., 2008; reviewed in Lessard et al., 2004). However, the
majority of the other candidates have no reported function in
primitive hematopoietic cells.
Design and Principle of the Screen
The screening protocol is outlined in Figure 1B. In brief, high-titer
retroviruses were produced in 96-well plates seeded with viral
producer cells using an optimized procedure. Protein extracts
derived from producer cells in each of the 104 wells were
analyzed by western blotting, which confirmed the presence of
a FLAG protein in 89% of the cases (Figure 1C provides eight
representative candidates; details for all 104 genes are listed in
Table S2, sixth column), with 92% of these proteins showing
the expected molecular size (Table S2, compare the fifth and
sixth columns). CD150+CD48?Lin?mouse bone marrow (BM)
cells were infected during 5 days and transplanted at two
different time points (i.e., day 0 and day 7 in Figure 1B). Under
these conditions, the average gene transfer to the cultured
CD150+CD48?Lin?cells was at 49% ± 31% (Figure 1D provides
eight representative candidates; details for all 104 genes are
Figure 1. Experimental Design of Nuclear Factors
(A)A listofcandidategenes(seeTable S1forthecomplete list)
was generated as described in the Results. The 689 nuclear
factors were subsequently ranked on the basis of an algorithm
that stratifies them according to properties predictive of self-
renewal regulation. The highest scoring candidates (n = 139)
were further selected for functional assessment with a retro-
viral overexpression approach. Of these, 104 were tested
(see ‘‘*’’ in Table S1), and the remaining 35 genes were
excluded for technical reasons.
(B) The coding sequence of each tested candidate was subcl-
oned into one out of three modified MSCV vectors, each
containing a different reading frame (pKOF-1, -2 and -3).
Respective retroviral producers were seeded in a single well
of a 96-well plate and cocultured for 5 days with 1500
CD150+CD48?Lin?freshly sorted bone marrow CD45.1+
cells. Immediately upon infection (day 0), one-eighth of each
well was transplanted into two congenic recipient mice along
with 23 105total BM cells (CD45.2+).A similarassay, this time
with three recipient mice, was performed after an additional
week of ex vivo culture (day 7), on which the screen was
(C) Expression of candidate proteins in retroviral-producing
cells was tested by western immunoblotting and revealed
with an anti-FLAG antibody. A list of predicted and observed
molecular weights for most proteins tested in this screen is
available in Table S2. NS, nonspecific signal; *, example of
a protein that could not be detected by western blot analysis
(see also Table S2).
(D) Range of retroviral gene transfer efficiencies of sampled
candidate genes on the basis of EGFP expression assessed
at day 4 of HSC culture (only eight representatives shown;
dashed line represents average on all 104 genes).
370 Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc.
listed in Table S3, second column). Harvested cells from each
well were transplanted into irradiated recipients together with
cell reconstitution was assessed after short (4 and 8 weeks) and
long (12 and 16 weeks) periods of time after transplantation.
Previous results obtained from several in vivo transplantation
experiments, using freshly transduced CD150+CD48?Lin?cells,
revealed marked interrecipient heterogeneity in hematopoietic
tissue reconstitution for a given candidate gene, thereby raising
the critical issue of signal-to-noise discrimination. Optimization
of this parameter was crucial for increasing the specificity of
the screen while limiting to a minimum the number of mice that
would be required. Toward this goal, we confirmed previous
findings (Antonchuk et al., 2002) showing that the activity of
Hoxb4-overexpressing HSCs is enhanced during short-term
cultures (see red line in Figure 2A). For control (i.e., vector-trans-
duced) HSCs, we also confirmed a noticeable decline in their
activity during the 7 day culture (black line in Figure 2A). Interest-
ingly, this HSC activity was preserved during the infection period
(Figure 2A). Importantly, we found that recipients of these
cultured control CD150+CD48?Lin?cells showed much less
variation in blood cell reconstitution levels than those trans-
planted with ‘‘day 0’’ cells (compare error bars at day 0 versus
day 7 on the black line in Figure 2A). In search for additional
ratio is thus substantially enhanced by keeping cells in such
cultures (Figure 2A). For this reason, the primary screen was
performed with cells harvested at day 7 of the culture (see
‘‘Screen’’ in Figure 1B).
Primary Screen and Validation
primary screen was set on the basis of the standard deviation of
the mean reconstitution level observed in multiple recipients of
Hoxb4-transduced CD150+CD48?Lin?cells (Figure 2B, see
also shaded area in Figure 2A). We therefore expected the newly
identified candidates to be equivalent to, or more potent than,
Figure 2. Identification of Positive Candidates with Hoxb4-like
(A) Estimation of HSC activity during viral transduction and culture. Lines show
estimation of HSC activity in recipients of day ?5, day 0,or day 7 bone marrow
(BM) cells transduced with Hoxb4 (red) or control vector (black). Two indepen-
dent experiments were performed with purified and whole bone marrow cells
for Hoxb4, and three with purified BM cells for controls. Extrapolation of HSC
numbers in recipients (y axis) was based on relative reconstitution levels
observed in the CRU assay performed with our starting HSC subpopulation
in Figures S2B–S2C. In all experiments, each mouse received eight equivalent
day ?5 HSCs (representing 100% HSC activity and evaluated by CRU assay)
and 20 equivalent fresh competitor HSCs (see legend of Figure S2 for details).
Cutoff for identification of Hoxb4-like candidates used in the primary screen/
validation experiments (shaded area) is set on the basis of the standard devi-
ation/error of the mean reconstitution level observed in multiple recipients of
Hoxb4-transduced HSCs. Cutoff for other less potent candidates (area
between dotted lines) is used only in validation experiments and is based on
statistical difference between candidates and vector control.
(B) Graft-derived hematopoiesis was evaluated at 4 week intervals in recipi-
ents of cultured HSCs during the primary screen. As a set of reference values,
the left panel indicates peripheral blood reconstitution levels from mice trans-
planted with cells from cultures initiated with a positive regulator of self-
renewal (Hoxb4) in relation to values observed with control vectors (mean of
pKOF-1, -2 and -3), all after 7 days of culture. Mean reconstitution level values
for each of the 104 tested candidates (at day 7) are compiled and presented in
the middle panel, with the established cutoff level for a gain-of-function
readout. Candidates clustering above the cutoff level for identification of
Hoxb4-like genes (shaded area in Figure 2A), corresponding to 30%
CD45.1+donor-derived cells in the primary screen, were selected for the vali-
dation experiments (upper-right panel), while those below were disregarded
(lower-right panel). One candidate (Hes1) was eliminated on the basis of the
marked reduction in repopulation noted between early and late time points
(upper line in lower-right panel). Values are presented as mean ±SEM of inde-
pendent experiments (n) for the left panel (n = 2 for Hoxb4 and n = 3 for control
vector; mean of three mice per experiment) and as mean ±SD for the middle
and right panels (n = 3 mice for each candidate cDNA). Note that several
mice were eliminated at 12 or 16 weeks after transplantation because they
did not meet our criteria for hit selection (see also Table S3, ninth and tenth
(C) Validation experiments confirming ten Hoxb4-like genes and eight other
less potent candidates. p values were established at the 16 weeks after trans-
plantation time point. Values are shown as mean ±SEM. The number of inde-
pendent experiments (n) per candidate gene equals four, except for control
vectors (n = 8); Sox4, Zfp472, Xbp1, and Hnrpdl (n = 5); and Hoxb4, Cnbp,
Ski, and Prdm16 (n = 3). For each experiment, a mean of three mice per
gene was evaluated. cand., candidates.
Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc. 371
criterion, a total of 18 hits were identified for a frequency of
17% (18/104; Figure 2B, upper-right panel; see also Table S3,
tenth column). These 18 hits included Cnbp, Erdr1, Fos,
Hdac1, Hmgb1, Hnrpdl, Klf10, Pml, Prdm16, Sfpi1 (PU.1), Ski,
Smarcc1 (Baf155), Sox4, Tcfec, Trim27, Vps72, Xbp1, and
in inducingenhanced HSC activity. With this
To validate the primary screen, we repeated the procedure
described in Figure 1B in several independent experiments
in Table S3). From left to right and top to bottom, genes are pre-
sented onthe basisof the levelof statistical significance reached
in these experiments, in comparison to control vector: Hoxb4
(positive control, gray in Figure 2C), Smarcc1, Vps72, Fos,
Trim27, Sox4, Klf10, Ski, Prdm16, Erdr1, and Sfpi1, for a positive
predictive value (PPV) of 56%. Although the other eight candi-
dates failed to demonstrate Hoxb4-like activity (i.e., shaded
area in Figure 2A), it is noteworthy that they all significantly
enhanced HSC activity to level above that detected with
vector-transduced cells (see ‘‘other less potent candidates’’ in
Figures 2A and 2C).
We then explored whether the ten newly identified Hoxb4-like
genes are endogenously expressed in populations highly en-
riched in HSCs. To test this, we sorted cells from hematopoietic
tissues isolated from unmanipulated mice sacrificed at three
different developmental stages including E14.5 fetal liver (HSC-
enriched subset number 1) and postnatal bone marrow, where
a switch between cycling (3 weeks, subset number 2) and quies-
cent (4 weeks, subset number 3) HSCs has been described
(Bowie et al., 2006). In line with the selection of these factors in
our database, all ten genes are highly expressed in HSC-
enriched populations with endogenous levels exceeding those
of TATA binding protein (TBP), used as endogenous control
(see relative threshold cycle [Ct] values for all HSC-enriched
subsets in Figure S1A). Interestingly, most of these genes are
expressed at higher levels in HSC-enriched populations than in
total tissues (total bone marrow or fetal liver, Figure S1B). This
observation is most prominent in the fetal liver-derived HSC-
enriched subset (upper panel Figure S1B). We next verified the
level of overexpression achieved for each of these genes after
retroviral infection in sorted CD150+CD48?Lin?cKit+Sca1+BM
cells and documented relative increases in mRNA levels
that could be as low as 3-fold (e.g., Smarcc1) to as high as
1000-fold and above (e.g., Fos; Figure S1C).
Most Genes Identified Confer Enhancement
in HSC Activity
One important question is whether the newly identified genes
enhance or simply maintain input stem cell activity. To address
this point, we compared the reconstitution levels by donor cells
which received an equivalent number of freshly purified
CD150+CD48?Lin?cells. As shown in Figure 3A, baseline long-
term HSC (LT-HSC) activity (Sauvageau et al., 2004), measured
at ?12%–16% reconstitution level (see left black bars in Fig-
ure 3A), was essentially preserved during the 5 day infection
period and set the baseline values for maintenance of input
stitution activity above that determined for fresh cells (Figure 3A).
In order to further quantitate the impact of our validated genes
on HSC activity, we used the mean activity of stem cell (MAS)
provides a measure of the proliferative output per LT-HSC, is
easily applicable to our culture condition since they were all
Figure 3. HSC Activity Is Enhanced by Overexpression of the Newly
Identified Hoxb4-like Genes
(A) Percentages of donor-derived blood cells at 16 weeks after transplantation
in primary mice recipients of day 7 culture cells (experiments in Figure 2C) for
the ten identified hits. Bars at the far left show values for freshly purified
CD150+CD48?Lin?cells, day 0 control cells (empty vector), and day 7 control
reconstitution levels observed in mice transplanted with day 0 control-trans-
duced HSCs, ±1 SEM. Reconstitution values falling within this range are
considered to reflect HSC activity derived from an injected dose of HSCs
equivalent to culture input numbers, i.e., 8 competitive repopulation units
(CRUs) (see Figure S2C). d-5, day ?5; d0, day 0; d7, day 7. Note that two
different cDNA were tested for Trim27 (see Figure S5).
(B) The mean activity of stem cells (MAS) was calculated from the data ob-
tained in (A). As previously established, the MAS is equivalent to repopulation
units (RUs) divided by CRUs (Ema and Nakauchi, 2000), where RU represents
the donor-derived reconstitution level divided by the competitor-derived
reconstitution level. MAS index was normalized to 1 for freshly sorted cells
and excluded the gene transfer efficiency in its estimation for the candidate
genes. In cases where gene transfer is low, this could lead to underestimated
values. Grafts overexpressing genes shown in red and marked by an asterisk
giverisetoaMAS significantlyhigherthanday0controlgrafts(p%0.05). Note
thatvaluesarepresented astheMAS relative tothatoffreshcells (d?5), which
was set at a value of 1. d-5, day ?5; d0, day 0; d7, day 7.
from control vectors (day 0 or 7) and validated genes. Day 0 p values framed in
red (penultimate column) mirror the order and color code of candidate genes
presented in (B) and are %0.05. In comparison, day 7 p values are listed in
the adjacent column.; n, number of independent experiments; d0, day 0; d7,
372 Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc.
initiated with a constant number of input LT-HSC measured at
?62 competitive repopulating units (CRU) per well (see Figures
S2B–S2C for CRU assessment). The MAS was 3-fold higher
for Hoxb4-transduced cells than for controls (Figure 3B). Simi-
larly, the MAS varied between 2.8 (Vps72) and 4.9 (Trim27) for
all ten hits identified in our screen (Figure 3B). Except for Sfpi1
(p=0.18), all values reached statistical significance (see p values
in red in Figure 3C).
Impact of Candidate Genes on Cell Proliferation,
Death, and Differentiation In Vitro
There is growing evidence to suggest that HSC self-renewal
involves the active repression of a differentiation program, which
is coupled to cell division (Cellot and Sauvageau, 2007). In
support of this,werecently foundthatHoxb4-orNA10HD-trans-
duced cells, which actively undergo in vitro self-renewal divi-
sions, show evidence of differentiation arrest (Figure 4A, left
panels) (Cellot et al., 2007). We investigated whether our newly
identified hits behave similarly. To achieve this, we first analyzed
the cytological characteristics of transduced and sorted
CD150+CD48?Lin?cells aftera 7day in vitro culture period (prior
to their transplantation). In this context, cultures initiated with
controlvector-transduced cellscontained70% ±8%ofdifferen-
tiated cells. These included neutrophils, monocytes, and mast
cells (Figure 4A, arrows in upper-left panel with summary of
results in right panel). Conversely, cellular differentiation was
reduced in cultures initiated with HSCs transduced with most
of the candidates (Figure 4A, right panel). The increase in the
proportion of undifferentiated to differentiated cells was most
important for Vps72, Fos, Sox4, Klf10, Ski, Prdm16, Erdr1, and
Sfpi1 when compared to cultures initiated with control vector-
transduced HSCs (see blue bars exceeding shaded area, repre-
senting mean of vector plus two standard deviations). Note that
while some of these genes are as potent as Hoxb4 in this assay,
none exceeds NA10HD in keeping cells undifferentiated.
We also monitored transduced CD150+CD48?Lin?cells for
cell death and proliferation. Figure 4B shows that all factors
analyzed conferred a significant reduction in the proportion of
dead cells harvested at day 4 of culture. Surprisingly, besides
Hmgb1, there were no significant increases in total cell numbers
in these cultures (Figure 4C). In fact, some genes such as Hoxb4,
and possibly NA10HD, were associated with a reduction in total
cell counts (Figure 4C).
Transduced HSCs Differentiate In Vivo
The in vitro differentiation arrest displayed by Hoxb4- or
NA10HD-transduced HSCs is eventually reverted after their
transplantation in vivo (Cellot et al., 2007; Ohta et al., 2007).
factors) or not affect (e.g., in vivo under steady state conditions)
HSC differentiation. To determine whether the newly identified
regulators of HSC activity are similarly permissive to HSC differ-
entiation in vivo, we used four different approaches. First, we
evaluated the general health, spleen size, and bone phenotype
(white versus red) of each recipient. Except for the recipients of
Prdm16-transduced cells, which eventually developed spleno-
megaly and myeloproliferation with white femurs at 20 weeks
after transplantation (data not shown), none of the mice trans-
planted with cells expressing our nine other genes ever pre-
sented this or any other hematological phenotype. Second, we
performed cytological evaluation of bone marrow and spleen
derived from representative mice for each gene. Results from
these analyses were normal for all groups, except for the
Figure 4. In Vitro Differentiation, Survival,
and Proliferation Profiles of Cultured Cells
(A) Morphological analysis of cytological prepara-
tions of the starting HSC fraction overexpressing
most of identified primary hits at day 7 of culture.
Proportions of immature (blasts: black arrow in
middle-left insert) versus terminally differentiated
cells (neutrophils, monocytes and masts cells:
black arrows in upper-left insert) for respective
cultures are depicted in the right panel. A field
comprising 100 cells was examined per indepen-
dent experiment (n), and values are presented as
mean; n = 3, except for vector (n = 6) and Hoxb4,
Ski, Tcfec, Sfpi1, and Hmgb1 (n = 1). The shaded
area represents the mean of vector plus two stan-
dard deviations. Note that a vast majority of the
less significant hits described in Figure 2C are
included in this study (i.e., Tcfec, Hmgb1, Cnbp,
Xbp1, Hnrpdl, and Hdac1). BM, bone marrow.
the fraction of cell death in cultures initiated with
our validated hits and the less potent gene candi-
dates (Tcfec and Hmgb1) as well as empty vector,
Hoxb4, or NA10HD, serving as controls. Assess-
ment was performed at the day 4 time point of
culture. Values are presented as mean ±SD of two independent experiments (n), where n represents the analysis of 30,000 cells. * p % 0.05.
(C) Cell proliferation was assessed by flow cytometry-based cell counts (y axis) at day 4 of cultures. Analyses were performed for the same gene candidates as in
(B). Values are presented as mean ±SD of two independent experiments. * p % 0.05.
Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc. 373
Prdm16 cohort, which showed an excessof poorly differentiated
myeloid cells in their bone marrow, as previously determined by
others (Shing et al., 2007), and the Ski cohort, in which the
number of lymphocytes in the bone marrow was reduced (data
not shown). Besides recipients of Prdm16-transduced cells,
spleens were never infiltrated with myeloid cells, nor did they
include enhanced numbers of erythroblasts. To confirm this,
we devised a third approach consisting of flow cytometry anal-
ysis on donor-derived (CD45.1+) cells. The results, presented
in Figure 5A for the peripheral blood, bone marrow, and thymus
of a representative mouse (Trim27) and summarized in Figure 5B
for all groups, largely confirmed our cytological evaluation.
Indeed, except for recipients of Ski-transduced cells, which
showed a marked reduction in B lymphocytes in their peripheral
blood and marrow with a compensatory increase in other cell
types, most groups of mice either showed normal distributions
of various cell types or presented some minor variations. We
further extended this analysis by gating only on CD45.1+/GFP+
cells for genes in which this was possible and ended with the
same conclusions, with the exception that B cell differentiation
was not observed in Klf10-transduced cells (Figure S3A). Finally,
clonal analyses of recipients that were reconstituted with retro-
virally marked cells were performed on bone marrow (less than
5% T cells) and thymus (less than 5% non-T cells). A representa-
tive result is presented in Figure 5C for Trim27, showing that
identical clones contributed to the reconstitution of these two
tissues, thus reinforcing the finding that these transduced
HSCs remain competent in T cell differentiation although they
displayed enhanced reconstitution activity. This finding can be
extended to all other genes except for Ski, Prdm16, and Erdr1,
for which we cannot be certain that the same clone contributed
to thymic and bone marrow reconstitution (Figure S3B).
We also used quantitative RT-PCR assays to monitor overex-
pression levels of the different transgenes in CD150+CD48?Lin?,
CD150+CD48+Lin?, B, and myeloid cells isolated from bone
marrow to verify whether the in vivo reversal of the differentiation
arrest noted in vitro (as shown in Figure 4A) was associated with
loss of transgene expression. Results presented in Figure 5D
suggest that overexpression of Vps72, Fos, Sox4, Klf10, Ski,
Erdr1, and Sfpi1 does not interfere with in vivo differentiation
as several types of immature/differentiated cells still express
the transgene. However, in vivo extinction of the transgene
may have occurred for the three following genes: Smarcc1,
Trim27, and Prdm16.
Together, these results confirm that the majority of the Hoxb4-
like genes identified in our screen conferred enhanced HSC
activity without causing hematological diseases or profoundly
altering cell differentiation, while still overexpressing the trans-
genes at least until 20 weeks after transplantation. Prdm16
was a notable exception.
Evidence of Self-Renewal Divisions by Transduced
We next verified whether HSCs transduced with each of the
confirmed hits remained capable of symmetrical self-renewal
divisions in vitro. To address this, we performed clonal analysis
(i.e., proviral integration pattern) of hematopoietic tissues
derived from selected recipients that were highly reconstituted
(17%–83% CD45.1+cells) at 20 weeks after transplantation,
a time point deemed sufficient for inferring that reconstitution
is solely derived from the LT-HSC. For eight of the ten newly
identified Hoxb4-like genes, namely Smarcc1, Vps72, Trim27,
Sox4, Klf10, Ski, Prdm16, and Erdr1, we observed proviral
DNA in the vast majority of mice that were analyzed (i.e., 43/49;
Figure 6, two upper panels). Several different clones with long-
term reconstitution ability contributed to hematopoiesis among
different recipients from a given experiment, and from subse-
quent validation experiments. In several instances, we could
identify the same proviral integrations in the DNA from two
different mice reconstituted by cells derived from the same
culture, demonstrating that LT-HSC self-renewal has indeed
occurred in these cultures (see ‘‘a’’–‘‘g’’ in Figure 6). We also
performed this analysis with the less potent candidates and
found evidence for HSC self-renewal for Cnbp and Xbp1 (see
clones ‘‘h’’ and ‘‘i’’ in Figure 6). Together, these data indicate
that a significant proportion of HSCs engineered to overexpress
our validated hits remained capable of symmetrical self-renewal
divisions in vitro.
Evidence of a Non-Cell-Autonomous Activity
for Selected Genes
Surprisingly, we could not reveal any integrated provirus in the
majority of recipients transplanted with cells transduced with
the Hoxb4-like genes Fos and Sfpi1 (Figure 6, bottom panels).
This suggests that untransduced HSCs in these cultures have
favorably responded to some extrinsic factors. A detailed evalu-
ation of recipients from which these observations are derived is
provided in Table S4. Consistent with the presence of a non-cell-
autonomous effect, we observed that upon exposure to Fos or
to 26%–31% with 7 additional days of culture (Table S4,
compare %CD45.1 at day 0 [seventh column] to that at day 7
[eighth column]). The absence of Fos and Sfpi1 proviruses in
the hematopoietic system of long-term recipients is surprising
considering the high level of gene transfer to transplanted cells
also intrinsically interfere with HSC repopulation when overex-
pressed, leading to depletion of transduced HSCs. Similar
observations were found with Tcfec and Hmgb1, members of
the less potent category (data not shown).
To provide more-direct evidence of non-cell-autonomous
activities for Fos, Sfpi1, Tcfec, and Hmgb1, we transduced
non-viral-producing NIH 3T3 cells with each of these constructs
and used them as feeder cells replacing the viral producers
described in Figure 1B. Overexpression of each of these four
factors in NIH 3T3 cells was verified by quantitative RT-PCR,
with a relative fold difference above baseline values ranging
from 3-fold (Hmgb1) to 18,000-fold (Tcfec) (data not shown).
As per our experimental protocol, 1500 CD150+CD48?Lin?cells
were seeded on non-viral-producing cells and maintained for
7 days prior to their transplantation into irradiated hosts. Strik-
ingly, we found that three of the four genes, namely Fos, Tcfec,
and Hmgb1, conferred a similar impact on HSC activity whether
viral-producing or non-viral-producing cells were present
in the cultures (see Table 1). Interestingly, background-level
374 Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc.
Figure 5. In Vivo Differentiation Potential of HSCs Overexpressing Validated Hits
(A) In vivo differentiation potential along the lymphomyeloid lineages was assessed in long-term recipients (20 weeks after transplantation) of HSCs transduced
with Trim27, used as an example. Immunophenotypic analysis by flow cytometry was performed with specific antibodies against B, T, and myeloid cell surface
markers (B220, CD3, and CD11b, respectively) on the CD45.1+population of cells derived from the peripheral blood, bone marrow (BM), and thymus of these
mice (and on CD45.1+/GFP+cells in Figure S3A).
(B) Compilation of B-/T-lymphoid and myeloid cell percentages within the CD45.1+population (left panels) and of CD45.1+cell proportions in the B-lymphoid,
T-lymphoid, or myeloid populations (right panels) of blood, thymus, and BM tissues, as gathered in (A), for most of the primary hits. Values are presented as
mean ±SD. Mouse identification numbers are presented between brackets. Dashed lines indicate values obtained with control mice (vector) for the different
hematopoietic tissues. Note that multilineage reconstitution was observed in all of mice analyzed except for those transplanted with cells transduced with
Ski, which showed a block in B-lymphoid differentiation. The proportion of CD45.1+cells within the B-lymphoid or myeloid populations in the thymus was not
calculated in the right panels because these populations represent less than 5% of total cells in this tissue. Since weak donor-derived reconstitution levels
were observed in mice transplanted with control vector cells at day 7 of culture (i.e., 4% in Figure 3A), we used mice transplanted with control vector cells at
day 0 of culture in this case. Values pertaining to less statistically significant hits are also presented (Tcfec, Zfp472, Hmgb1, Cnbp, Xbp1, Hnrpdl, and Hdac1).
(C) Proviral integration pattern studies by Southern blot analysis performed on genomic DNA extracted from bone marrow (left panel) and thymus (right panel) of
three long-term recipients of Trim27-overexpressing HSCs. Each lane represents a specific mouse (ID number below). For a given animal, identical integrations
are found in both tissues, indicating a common precursor cell origin. Identical clones are also observed in two distinct recipients, retrospective molecular
evidence for a self-renewal division in vitro. Multipotentiality (BM and thymus) analyses in reconstituted tissues harvested from primary recipients of culture cells
overexpressing the various validated hits are shown in Figure S3B.
(D) Quantitative RT-PCR analysis of mRNA expression levels of nine of the ten newly identified Hoxb4-like gene transcripts. For technical reasons, Prdm16 was
excluded from the analysis. RNA was extracted from distinct bone marrow cell fractions isolated from long-term, highly reconstituted recipients of day 7 culture
cells (S, CD150+CD48?Lin?; P, CD150+CD48+Lin?; B, B220+; M, Mac+). Average DCt values (representative of expression levels) were determined with b-actin
serving as an endogenous control to normalize levels of target gene expression. Relative fold differences were determined using control cells as a reference cali-
brator for each candidate gene. Reactions were done in triplicate; black bars represent mean endogenous expression levels ±SEM of three independent wild-
type mice (Ctrl); red bars represent mean overexpression levels ±SEM of two transplanted mice (Trx) from two independent experiments. The identification
numbers of mice used in this assay are presented between brackets. Note that control black bars on each panel are set at a relative fold difference value of 1.
Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc. 375
reconstitution was found in recipients of cells kept on Sfpi1-
transduced NIH 3T3 cells, possibly indicating a more complex
cellular network involved with this gene.
In this study, we combined the power of expression profiling and
functional studies to uncover candidate factors that impact on
HSC activity. Our experimental procedure included an efficient
production of high-titer retroviruses, a sharp and discriminating
HSC gain-of-function signal optimized by an ex vivo culture
step together with a robust and reliable in vivo assay (see impor-
tance of culture step for signal-to-noise discrimination in
Figure S4). Using this strategy, we individually tested 104 prese-
lected candidates revealing 18 genes that conferred a clear
repopulation advantage to HSCs. Of these, ten displayed
a Hoxb4-like effect, thereby greatly extending the repertoire of
potential regulators of HSC activity. These factors, which are
highly and in some case preferentially expressed in HSCs, are
Figure 6. Clonal Analysis to Track HSC
Southern blot analyses of genomic DNA extracted
from the bone marrow (BM) of selected long-term
recipients (20 weeks after transplantation) of day 7
specific probe and systematically exposed for the
same period of time. Each well was equally
loaded, taking into consideration donor-derived
peripheral blood reconstitution level of recipients
as measured 16 weeks after transplantation (i.e.,
17%–83% CD45.1+cells). Top and bottom panels
show gene sets for which proviral DNA was either
detected or absent in the majority of recipients
analyzed, respectively. For most bottom panels,
the brightness and contrast was enhanced using
Photoshop to further verify the absence of inte-
grated proviral DNA, and a b-actin probe was
also used to confirm the presence of genomic
DNA. Clones that self-renewed during the 7 day
culture, prior to transplantation, are labeled ‘‘a’’
to ‘‘i’’ and identified in more than one recipient.
‘‘X’’indicates thatGFP expression wasnotdetect-
able for these constructs/clones. Note that panel
5, representing the Trim27 gene, is also included
in Figure 5C (left panel).
implicated in a diversity of processes,
such as chromatin modification (e.g.,
Smarcc1 and Vps72), stress response
(e.g., Fos), and gene transcription (e.g.,
Trim27 and Klf10) (see Table S4 and
legend for more details on these Hoxb4-
like factors). With this significant cohort
of genes impacting HSC activity, it was
possible to discern a subset of four
Hmgb1) that exerted their influence on
stem cell function through a non-cell-
autonomous phenomenon. Importantly,
the non-cell-autonomous activity of three of these four factors
was confirmed in gene transfer-free conditions.
Identified Hits Display Hoxb4-like HSC Activity
Similar to our previous results with Hoxb4-overexpressing
HSCs, nine of the ten hits identified in this screen also signifi-
cantly conferred increased HSC activity to levels above those
observed with input cells (genes in red in Figures 3B–3C).
Considering the ?50% average gene transfer level, not taken
into consideration in the evaluation of the mean activity of stem
cells (MAS) reported in Figure 3B, the impact of some of our
confirmed hits was likely underestimated. Interestingly, our
newly identified candidates also induced a maturation block
in vitro, which was reversed in vivo. Moreover, and similar to
Hoxb4, the majority of these genes failed to enhance cellular
proliferation in vitro.
The increase in HSC repopulation potential (i.e., activity)
observed with Hoxb4 overexpression involves a net expansion
(self-renewal) of these cells (Antonchuk et al., 2002). Although
376 Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc.
our validated candidates show Hoxb4-like effects and we could
document self-renewal divisions in vitro for several HSCs engi-
neered to express these genes (Figure 6), it is important to stress
that the formal proof for HSC expansion (enhancement in self-
renewal divisions) as the driving force behind these effects will
require intensive investigations that combine serial determina-
tions of HSC numbers at consecutive time points (e.g., DCRU
assay) with large-scale clonal analyses as previously reported
for the Hoxb4 gene (Antonchuk et al., 2002; Cellot et al., 2007).
Indeed, among several possibilities, enhancement in prolifera-
tive potential combined with in vitro HSC maintenance could
explain the Hoxb4-like effects observed with our validated hits.
Secondary transplantation assays performed with selected
candidates (Vps72, Klf10, Erdr1, and Fos, Table S5) argue
against, but do not refute, such a possibility.
The eventual identification of shared target genes between
these factors would further strengthen this argumentation. Of
interest, attempts to build transcriptional networks, or hubs,
among the hit genes have currently proven inconclusive, when
most of the respective mRNA expression levels were assessed
in HSCs freshly transduced with each of the candidate genes
Non-Cell-Autonomous Enhancement in HSC Activity
The non-cell-autonomous influence exerted by Fos, Tcfec, and
Hmgb1 was confirmed in cultures with NIH 3T3 support, con-
firming that gene transfer to sorted HSCs was not required for
these three genes. In line with these findings, the proportion of
GFP-positive cells was 0% in recipients of cells kept for 7 days
in NIH 3T3-containing cultures (see column four in Table 1). It
is also important to stress that these so-called ‘‘non-cell-auton-
omous’’ factors were identified in a context of overexpression,
which in some cases (i.e., Fos) was outstanding (e.g., 38,000-
fold above endogenous levels in some experiments, data not
shown), potentially resulting in toxicities to transduced HSCs. It
is therefore likely that several of these factors normally perform
cell-autonomous activity in steady-state hematopoiesis. Sfpi1
is a notable example for this (Rosenbauer et al., 2006).
The results obtained with Sfpi1 during the screen and several
additional experiments could not be confirmed in viral-free
cultures. Reasons for this remain unclear. Several possibilities
by some differentiated hematopoietic cells found in our culture
or by essential cofactors unique to GP+E-86 viral producer cells.
even essential, contribution by the feeder cells (in this case NIH
3T3) to the observed effects on HSC activity. While this level of
characterization is beyond the scope of our paper, this set of
experiments exposes a complexity, often underestimated, when
therefore be taken into consideration in future experiments.
Although the demonstration of the non-cell-autonomous
activity is relatively straightforward (e.g., three of the four factors
described above), the proof for cell-autonomous function (e.g.,
the scope of our experimental design. Thus, it is possible that
some of the eight other Hoxb4-like genes (Smarcc1, Vps72,
Trim27, Sox4, Klf10, Ski, Prdm16, and Erdr1) also or only display
Future of the Resource
Entries pertaining to candidate nuclear factors implicated in
stem cell activity are stored in an open-access database
(http://www.bioinfo.iric.ca/self-renewal/), as part of a building
block in creating an interactive resource for the scientific
community. Information thus far includes mRNA expression
profiles, together with specific probe and primer sets, blood
reconstitutionpatternsovertimewith possibilities ofdatareanal-
ysis, and the complete nuclear factor list with ranking criteria.
This database will eventually include similar data emerging
from ongoing gain- and loss-of-function screens from different
laboratories that will exploit this resource.
Thus far, nuclear factor candidates clustering in the upper-
most levels (eight to ten) of our classification harbor a 23%
primary hit rate, compared to 12% for those scoring lower (six
to seven). This suggests that while positive HSC regulators
tend to segregate in the top ranks, other downstream genes
listed in our database (scores 1–5, n = 585 candidates) also
include potential determinants of HSC self-renewal, with puta-
tive derivation of cross-regulation nodes as mentioned.
Our understanding of HSC self-renewal will mirror advance-
ments in the rapidly evolving field of cellular therapy. As pio-
neered with Hoxb4 (Krosl et al., 2003a), recombinant TAT-fusion
proteins could be envisioned and therapeutically tested on
human cells. Moreover, findings from this work will potentially
pave the way to identify the mediator(s) that link these non-
cell-autonomous candidates to enhanced HSC activity, thereby
bypassing the requirement for retroviral vectors in the goal of
expanding HSCs for clinical purposes.
were previously described (Antonchuk et al., 2001; Ohta et al., 2007). For all
Table 1. HSC Activity Is Enhanced Extrinsically
GP+E86 (virus) NIH 3T3 (no virus)a
vector62 ± 264 ± 10 ± 05 ± 2
Fos71 ± 9 31 ± 80 ± 035 ± 14
Sfpi137 ± 17 26 ± 120 ± 02 ± 1
Tcfec48 ± 21 22 ± 80 ± 0 13 ± 5
Donor-derived reconstitution levels (%CD45.1+) observed 16 weeks after
transplantation in mice transplanted with cells cocultured either with
virus-producing cells (GP+E-86) or similar cells not producing virus
(NIH 3T3), both overexpressing selected genes. Values are presented
as mean ±SEM of independent experiments (n) per candidate gene,
where n = 4,except for control vector, where n = 8.For each independent
experiment, a mean of three mice per gene was evaluated.
aMice were analyzed at 16 (experiments 1 and 2) and 8 (experiments
3 and 4) weeks after transplantation; %GFP, gene transfer assessed by
22 ± 1420 ± 100 ± 014 ± 5
Cell 137, 369–379, April 17, 2009 ª2009 Elsevier Inc. 377
candidate genes, single open reading frames (ORFs) were amplified by PCR
(see Table S2 and the Supplemental Experimental Procedures for details).
(C57Bl/6J-CD45.2 x C3H/HeJ) F1 recipient mice and (C57Bl/6J-CD45.1-
Pep3b x C3H/HeJ) F1 congenic donor mice were bred at a specific path-
ogen-free (SPF) animal facility at IRIC in Montreal.
CRU Assay on HSC-Enriched Populations
CRU assay and calculation were performed as described originally (Szilvassy
et al., 1990) with modifications as in Sauvageau et al. (1995). Recipients were
considered reconstituted (i.e., positive) when R1% of their peripheral blood
leukocytes were of donor (CD45.1+) origin at 18–20 weeks after transplanta-
tion. Two CRU assays were performed with CD150+CD48?Lin?cells and
one CRU assay for the CD150+CD48?Lin?cKit+Sca1+subpopulation.
Bone Marrow Cell Culture, Retroviral Infection, and Transplantation
Generation of retrovirus-producing GP+E-86 cells, or gene-overexpressing
NIH 3T3, were performed as previously described (Krosl et al., 2003b) and
seeded in a 96-well plate format, enabling production of a single ecotropic
pseudotyped retrovirus per well (for GP+E-86). See the Supplemental Exper-
imental Procedures for details.
Flow Cytometry Assessment of Donor-Derived Hematopoiesis
The contribution of donor cells to peripheral blood reconstitution was deter-
mined at regular intervals after transplantation in individual recipients. See
the Supplemental Experimental Procedures for details.
Analysis of Proliferation and Cell Death
Transduced cells harvested at day 4 of culture with trypsinization were
counted with BD Trucount?Tubes (BD Biosciences, San Jose, CA) according
to manufacturer’s guidelines, or stained with Alexa350-annexin V (Invitrogen
flow cytometry. Gates were set to exclude GP+E86 retroviral producers by
forward- and side-scatter criteria.
Resource Database and Web Application
The open-source postgreSQL relational database engine was used to store
the accumulated data, and the web application was built with the open-source
Webware application server framework. The application is designed to house
results from other ongoing screens from our lab as well as screens performed
elsewhere. As such, a sections-based authentication system has been imple-
mented in order to restrict access to sensitive or nonpublished results. We
expect this website to become an open-access resource for teams working
on deciphering the molecular basis for stem cell self-renewal.
The significance of differences was determined by a two-tailed Student’s
Supplemental Data include Supplemental Experimental Procedures, five
figures, and six tables and can be found with this article online at http://
The authors thank J. Krosl, R. Bisaillon and B.T. Wilhelm for technical
help, C. Charbonneau from IRIC imagery platform, D. Gagne ´ from IRIC flow
cytometry platform, and P. Chagnon and R. Lambert from IRIC genomic plat-
form for their expertise with quantitative RT-PCR. We also want to acknowl-
edge C. Perreault, M. Therrien, K.J. Hope, and R.K. Humphries for discussions
and critical comments about the manuscript. This work was supported by the
Canadian Institute of Health Research (CIHR) Team Grant in Hematopoietic
Stem Cell Self-Renewal: From Genes to Bedside (Grant number 154290,
2006-2011). G.S. holds a Canada Research Chair on molecular genetics of
stem cells; E.D. and S.C. are recipients of a CIHR studentship and Clinician
Scientist award, respectively; J.C. holds an American Society of Hematology
fellowship; M.S. holds a National Canadian Institute of Cancer studentship;
and S.B.T. is the recipient of National Health Medical Research Council and
a Royal Australian College of Physicians fellowships. IRIC is supported in
part by the Canadian Center of Excellence inCommercialization and Research
(CECR), the Canada Foundation for Innovation (CFI), and the Fonds de
Recherche en Sante ´ du Que ´bec (FRSQ).
Received: September 9, 2008
Revised: January 19, 2009
Accepted: March 16, 2009
Published: April 16, 2009
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Cell, Volume 137
A Functional Screen
to Identify Novel Effectors
of Hematopoietic Stem Cell Activity
Eric Deneault, Sonia Cellot, Amélie Faubert, Jean-Philippe Laverdure, Mélanie Fréchette, Jalila
Chagraoui, Nadine Mayotte, Martin Sauvageau, Stephen B. Ting, and Guy Sauvageau
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Template cDNA listed in Table S2 are from the following source: cDNA corresponding to BC
accession numbers come from ATCC, Manassas, VA, USA; cDNA corresponding to AK
accession numbers come from Riken DNABook, Japan. Amplicons were digested with
appropriate restriction enzymes [Not1, EcoR1 (Invitrogen, Burlington, ON, Canada) and/or
Mfe1 (New England Biolabs, Ipswich, MA, USA)], and subcloned into 1 of 3 modified
MSCV-PGK-GFP vectors (pKOF-1, -2 or -3, containing different reading frames) in
accordance with specific frameshift requirements upon subcloning. Confirmatory sequencing
of all cloned cDNA was performed using the ABI 3730 Genetic Analyser at the Institute for
Research in Immunology and Cancer (IRIC) genomics platform in Montreal (Dr. Pierre
Animals were housed in ventilated cages and provided with sterilized food and acidified water
under veterinary supervision. Experimental procedures were revised and approved by
University of Montreal animal ethics committee (Comité de Déontologie de
l’Expérimentation sur les Animaux de l’Université de Montréal).
Hematopoietic stem cell isolation
Bone marrow cells harvested from C57Bl/6J-CD45.1-Pep3b mice were stained with
allophycocyanin (APC)-conjugated primary antibodies recognizing differentiation specific
cell surface markers [Gr-1, B220 and Ter119 (BioLegend, San Diego, CA)]. Cells were
washed, pelleted, and stained with anti-APC magnetic MicroBeads according to manufacturer
guidelines (Miltenyi Biotec Inc, Auburn, CA, Order no 130-090-855). Depletion of the
lineage positive cells was achieved using the AUTO-MACS magnetic cell separator system
(Becton-Dickinson, San Jose, CA, USA). The lineage negative cell fraction was subsequently
stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD48 and phycoerythrin
(PE)-conjugated anti-CD150 antibodies (BioLegend, San Diego, CA). This step was followed
by purification of the PE-CD150+/FITC-CD48-/APC-Lin- stem cell enriched subpopulation by
flow cytometry using the FACSAria cell sorter (Becton-Dickinson, San Jose, CA, USA). Cell
populations used for Q-RT-PCR expression studies were additionally stained with PE-Cy7-
conjugated anti-cKit and PE-Cy5-conjugated anti-Sca1 antibodies (BioLegend, San Diego,
CA) after the lineage depletion step, and sorting gates set to isolate the PE-CD150+/PE-Cy5-
Sca1+/PE-Cy7-cKit+/FITC-CD48-/APC-Lin- cell fraction from bone marrow, or PE-Cy7-
Cell, Volume 137
CD150+/PE-Cy5-Sca1+/APC-Cy7-cKit+/FITC-CD48-/PE-Mac+/APC-Lin- cell fraction from
Bone marrow cells culture, retroviral infection and transplantation
Freshly sorted CD150+CD48-Lin- CD45.1+ bone marrow cells were plated at a density of
1,500 cells per well, and co-cultured for 5 days on these confluent and irradiated (1,500 cGy
of 137Cs gamma radiation) retroviral producers in the presence of Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 10 ng/mL
human interleukin-6 (IL-6), 6 ng/mL murine interleukin-3 (IL-3), 100 ng/mL murine stem cell
factor (SF), and 6 µg/ml polybrene, 10 µg/ml ciprofloxacin and 10-4M β-mercaptoethanol.
Upon this infection interval (day 0), contents from each well were harvested by trypsinization
and the retrieved cell volume was partitioned between cell culture and transplantation
requirements. For every candidate gene, 2 independent C57Bl/6J-CD45.2 recipient mice were
sublethally irradiated (800 cGy of 137Cs gamma radiation) and received 1/8 of the cell
suspension, along with 2x105 whole bone marrow competitor cells (CD45.2+). One half of the
cell suspension remained in culture for an additional 7 day period, at the end of which (day 7),
3 independent recipient mice were similarly transplanted with a graft equivalent to 1/8 of the
original cell input, for each condition. A small aliquot was drawn from day 4 cultures to
assess gene transfer by EGFP expression using flow cytometry.
Flow cytometry assessment of donor-derived hematopoiesis
A 50 µl blood sample obtained from tail vein puncture was incubated with excess ammonium
chloride (StemCell Technologies, Vancouver, BC, Canada) to favour lysis of erythrocytes.
The washed cell pellet was then stained with a primary PE-conjugated antibody recognizing
the CD45.1+ (donor-derived) leucocyte cell surface marker (BioLegend, San Diego, CA), as
described (see HSC isolation section above). Immunophenotype was determined by flow
cytometry (BD LSR II flow cytometer, BD Biosciences, San Jose, CA, USA) and data
analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Similarly, at 20 weeks
post-transplantation, cells isolated from blood, bone marrow and thymus were stained with a
cocktail of antibodies directed against various cell surface differentiation markers [APC-Cy7-
conjugated anti-B220, PE-Cy5-conjugated anti-CD11b and PE-Cy5.5-congugated anti-CD3ε
antibodies (BioLegend, San Diego, CA)] and analyzed by flow cytometry.
Southern blot analysis of genomic DNA
High-molecular-weight DNA from hematopoietic tissues of long-term (20 weeks) post-
transplantation recipients was isolated with DNAzol reagent (Invitrogen, Carlsbad, CA,
USA), as recommended by the manufacturer guidelines. Proviral integration patterns were
determined by Southern blot analysis of isolated DNA as previously described (Krosl et al.,
2003) using the EcoRI restriction enzyme, with a unique specific recognition sequence within
the integrated provirus DNA. 20 µg of the digested genomic DNA were separated by 1%
agarose gel electrophoresis and transferred to a Zeta-probe membrane (Bio-Rad, Mississauga,
ON, Canada). A 710 bp [32P]dCTP EGFP probe, digested from pEYFP-N1 (Clontech
Laboratories Inc., Palo Alto, CA, USA) with EcoRI/HindIII (Invitrogen, Burlington, ON,
Canada), was used to reveal the integration patterns, and a 1.8 kb [32P]dCTP β-actin probe
was used for loading control.
Western blot analysis
Protein expression of cloned cDNA was assessed in retroviral producing cell lines. Protein
extracts were obtained from transfected GP+E86 cells grown in 96 well plates by incubation
with a 30 µl volume of 1x Laemmli (1/60 β-mercaptoethanol) solution per well, followed by a
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10 min boiling step. Western blots analyses were performed as previously described (Krosl et
al., 2003). A mouse anti-FLAG primary antibody (BioLegend, San Diego, CA) was used to
reveal the presence of the candidate protein, followed by a goat horseradish peroxidase-
conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA,
Q-RT-PCR expression studies
For gene expression profile analyses, cells were harvested following 5 days of infection using
trypsinization, and individual well contents resubmitted to cell sorting (FACSAria cell sorter,
Becton-Dickinson, San Jose, CA, USA). Gates were set to positively select for GFP+ cells,
excluding GP+E86 retroviral producers by forward- and side-scatter criteria. Cells were
directly collected in Trizol solution to isolate total RNA, according to the manufacturer’s
protocol (Invitrogen, Burlington, ON, Canada). Reverse transcription of total RNA was
performed using the MMLV-reverse transcriptase (RT) and random hexamers according to
manufacturer’s guidelines (Invitrogen, Burlington, ON, Canada). Resulting cDNA was pre-
amplified using a TaqMan® PreAmp (Applied Biosystems, Foster City, CA, USA) algorithm
in which candidate genes specific oligonucleotides were added to the PreAmp Master mix
(final concentration of 50nM). PCR conditions for the pre-amplification reactions were as
follows: 95°C for 10 minutes, followed by 12 cycles of 95°C/15 sec and 60°C/4 min. The
ABI Gene Expression Assay was performed to measure gene expression levels using primer
and probe sets from Applied Biosystems. Q-RT-PCR reactions were done on a high-
throughput ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). Briefly, from a
given test sample, the Ct (threshold cycle) values for each gene were normalized to the
endogenous control gene TBP or β-actin (Applied Biosystems; ∆CT = Cttarget - Ctendogenous) and
compared to the mean ∆CT from control sample (calibrator) using the ∆∆Ct method (∆∆CT =
∆CtSample - ∆CtCalibrator). Q-RT-PCR cycling conditions were as follows: 2 minutes at 50°C and
10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 59°C. The
primer sequences and probes used in qPCR assays are presented in Table S6.
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Figure S1. Expression levels of confirmed hits in purified HSC
(A) Endogenous expression levels of newly identified Hoxb4-like genes in highly purified
HSC at 3 different time points during development, i.e., in CD150+CD48-Lin-cKit+Sca1+Mac+
fetal liver (FL) at 14.5 days post-coitum (dpc), and in CD150+CD48-Lin-cKit+Sca1+ bone
marrow (BM) cells at 3 and 4 weeks after birth. Results are presented as the Ct values of
tested genes divided by the corresponding Ct values of TATA Binding Protein (TBP), serving
as an endogenous control to normalize levels of target gene expression. Reactions were done
(B) Quantitative analysis on HSC vs total tissues with the same genes at the same time points
as in (A). Average ∆Ct values were determined in highly purified HSC [see (A)] and in total
tissue, with TBP serving as an endogenous control to normalize levels of target gene
expression. Relative fold differences were calculated using the total tissue as a reference
calibrator for each candidate gene. Reactions were done in triplicate for each independent
experiment (n). Values are presented as mean ±SD for Smarcc1, Fos, Sox4 and Ski (n=1) and
as mean ±SEM for other genes (n=2).
(C) Quantitative analysis of the newly identified Hoxb4-like gene-overexpression levels in
HSC-enriched subpopulation. RNA was extracted from CD150+CD48-Lin-cKit+Sca1+ bone
marrow cells that were beforehand co-cultured with retroviral producers for 5 days and sorted
for the GFP positive fraction (or from GFP+ retroviral producers for Sfpi1). Average ∆Ct
values were determined with β-actin serving as an endogenous control to normalize levels of
target gene expression. Reactions were done in triplicate for each independent experiment (n).
Values are presented as mean ±SD for Ski (n=1) and as mean ±SEM for Hoxb4 and Klf10
(n=2), Sfpi1 (n=3) and other genes (n=4); n/a=not available.
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Figure S2. Purity and activity of sorted HSC subpopulation
(A) Left panel shows representative flow cytometry sorting profile of the starting HSC-
enriched population used in the primary screen. Events are gated on the Lin-APC- cell fraction
of freshly sorted bone marrow cells, with CD150-PE positivity and CD48-FITC negativity
along the y-and x-axis, respectively. Percentage of Sca1-PECy5+c-Kit-PECy7+ cells within
the CD150+CD48-Lin- contingent is depicted in the right panel.
(B) In vivo competitive repopulation assay performed using CD45.1+CD150+CD48-Lin- cells
described in (A). Results are presented as cell numbers (x-axis) against the corresponding
percentage of negative mice (y-axis) per dose. Hematopoietic reconstitution at 20 weeks post-
transplantation was deemed negative if less than 1% of the recipient blood cells were derived
from the sorted graft. The mean of two representative assays is shown. The obtained average
CRU frequency was 1 in 24 cells (95% CI: 1/10 to 1/57). In that respect, ~62 CRU per well
(or 8 CRU* per mouse) were seeded at day -5 (see Figure 1B).
(C) Results from in vivo limit dilution assays in (B) are displayed as peripheral blood
reconstitution values in relation to calculated CRU numbers. The extrapolated percentage of
blood reconstitution derived from 8 freshly sorted CRU, as indicated, is ~12.5%. This value
mirrors the mean reconstitution level observed in control mice transplanted with an equivalent
CRU fraction (i.e., 8 CRU/mouse; see Figure 2A) in the same competitive context, after 5
days of retroviral infection (day 0 of the screen), and suggests HSC activity maintenance after
this short co-culture period; CRU=competitive repopulation unit
*Based on the CRU evaluation of the starting CD150+CD48-Lin- population, it was possible
to estimate at ~8 the maximal number of CRU transplanted per mouse at day 0: 1,500 cells x
1/24 CRU (at day -5) x 1/8 well per mouse = 8 CRU per mouse.
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Figure S3. In vivo differentiation of HSC transduced with newly identified
(A) Differentiation potential along the lympho-myeloid lineages in long-term recipients (20
weeks post-transplantation) of HSC transduced with selected candidates. Immunophenotypic
analysis by flow cytometry was performed using specific antibodies against B, T and myeloid
cell surface markers and gated on CD45.1+/GFP+ populations derived from the peripheral
blood, bone marrow and thymus of these mice. Technical difficulties inherent to GFP
expression in certain contexts preclude this type of analysis for the following genes: Smarcc1
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and Prdm16 in all tissues analysed, and Ski, Klf10, and Erdr1 in the thymus. Values represent
mean ±SD and the number of mice analyzed (n) per candidate gene was n=2, except for
vector: n=5; NA10HD and Cnbp: n=3; Trim27 and Klf10: n=1.
(B) Southern blot analysis showing the proviral DNA in the BM (upper panel) and in the
thymus (lower panel) of selected recipients that were highly reconstituted at 20 weeks post-
transplantation. Transduced HSC remain competent in T cell differentiation although they
displayed enhanced reconstitution activity for each gene, except for Ski, Prdm16 and Erdr1,
where we cannot be certain that the same clone contributed to thymic and bone marrow
reconstitution. Membranes were exposed for 3 days and DNA loading normalized based on
CD45.1 level in peripheral blood. Trim27 blots are also presented in Figure 5C; n/a=not
Figure S4. Importance of the ex vivo culture in the specificity of the
The background noise characteristic of day 0 (left panel) was abolished by the 7 day ex vivo
culture (right panel) when comparing test values (black: negatives, and red: positives lines)
with vector (yellow lines). This variability at day 0 is due to the short in vitro exposure period
(5 days), the survival benefits granted by viral producer cells, and discrepancies in
proliferative potential (output) of individual HSC, despite equivalent cell doses per well at
initiation of culture. In sharp contrast, a week in vitro culture period led to HSC exhaustion in
the absence of a positive maintenance/self-renewal factor. Relying only on reconstitution data
from day 0 cultures, the rate of false positive would dramatically increase to an estimated
19%, negating this screening method from large-scale applications.
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Figure S5. Different forms of Trim27 with different potential
(A) Two different forms of Trim27 have been tested in this study, i.e., one containing a frame-
shift error (truncated and used in primary screen; accession number BC085503; upper panel)
preserving intact only the RING, B-box and first Coiled-coil domains, and another full-length
form (accession number BC003219; bottom panel) containing in addition the second Coiled-
coil and the SPRY domains.
(B) Western blot analysis presenting the detection of the 2 FLAG-Trim27 proteins of
expected sizes; *=degradation product or unknown isoform
(C) Competitive repopulation assays reporting the differential reconstitution level of recipient
mice by HSC transduced with the different forms of Trim27. Note that the SPRY domain
within the full-length form of Trim27 seems to limit the potential of this gene in enhancement
of HSC activity. The left panel is a replicate from that presented in Figure 2C. Values are
presented as mean ±SEM. The number of independent experiments equals 4 for the left panel
and 2 for the right panel.
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Table S1. Scoring system used in candidate selection
Candidates were selected using microarray gene expression profiling from a leukemia stem
cell line (FLA2, G.S. et coll., in preparation) and expression profiles from enriched stem cell
populations (Bhattacharya et al., 2004; Georgantas et al., 2004; Ivanova et al., 2002; Phillips
et al., 2000; Ramalho-Santos et al., 2002; Shim et al., 2004; Shojaei et al., 2004; Terskikh et
al., 2001). Nuclear factors were selected and rated relative to their expression in different
reports/databases (i.e., high rank=high expression) according to the criteria listed in the text
(full details available upon request). Using this ranking system, genes with a score of 6 or
above were selected for functional studies (n=139), of which 104 have been tested in the
screen (genes followed by an *).
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Table S2. Subcloning strategy and protein expression of candidates
The accession numbers corresponding to each cDNA used as template for PCR amplification
are presented in addition to the sequence of forward and reverse primers used for subcloning.
The size of corresponding FLAG-proteins produced in retroviral producers were estimated
and determined by western blot using an anti-FLAG antibody; sequences underlined in
columns 3-4 represent restriction sites used for subcloning; *=estimated according to
http://www.bioinformatics.org/sms/prot_mw.html; +=same molecular weight as estimated
within a margin of error of 10 kDa; n/a=not applicable.
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Table S3. Summary of gene transfer and reconstitution data obtained
from primary screen and validation experiments
Gene transfer efficiencies, based on the proportion of GFP+ cells at day 4 (4 days after day 0
in Figure 1B), are presented in the 2nd column for each candidate tested in the primary screen.
Values in other columns represent mean percentages of peripheral blood reconstitution levels
by CD45.1+ cells in independent experiments (see also http://www.bioinfo.iric.ca/self-renewal
for more details and mice identification numbers used in the different experiments). For
validation experiments (i.e., experiments 2 to 5), only day 7 data are presented. Note that
several mice were eliminated at 12 or 16 weeks post-transplantation because they did not
meet our reconstitution level criteria for primary screen hit selection (see also Figure 2B).
Except for day 0 (n=2 mice) and vector control (n=9 mice), all values represent mean of 3
different mice; expt=experiment; w=weeks; inf. lev.=infection level.
Table S4. Summary of Hoxb4-like candidates on HSC activity and
clonality of reconstitution
Columns 3 to 6 present results for selected mice, which were analyzed at 20 weeks post-
transplantation. These include gene transfer efficiencies as proportion of GFP-positive cells in
cultures at day 4 (mean±SD, column 3), peripheral blood reconstitution by CD45.1+ HSC
(mean±SD, column 4), proportion of selected mice containing proviral DNA in their BM
(column 5), and the minimal number of independent clones contributing to reconstitution
(column 6). Results presented in column 6 show that, except for the 2 non-cell autonomous
candidates Fos and Sfpi1, several different clones (n=4-8 per gene) contributed to
reconstitution in recipient mice. In some cases (e.g., Smarcc1), we could track up to 5 clones
reconstituting a cohort of mice derived from a single well for an estimated recovery of above
50% of the input LT-HSC introduced in such cultures. This suggests the absence of clonal
dominance, typically associated with low frequency events such as insertional mutagenesis.
Columns 7 to 9 present peripheral blood reconstitution values (in %) for the entire cohort of
mice transplanted at day 0 (mean±SEM, column 7) and at day 7 (mean ±SEM, column 8). See
legend of Figure 2C for numbers of independent experiments. For each experiment, a mean of
2 mice per gene was evaluated at day 0 (column 7) and a mean of 3 mice at day 7 (column 8).
Column 9 provides an assessment of possible non-cell autonomous (NCA) activity for each of
Cell, Volume 137
the candidate gene. Below is a brief description of the known activity for these 10 genes.
Smarcc1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin
subfamily C member 1, or BRG1-associated factor 155) overexpression positively correlates
with tumour recurrence and dedifferentiation in prostate cancer (Heeboll et al., 2008). Vps72
(vacuolar protein sorting-associated protein 72 homolog) is a new subunit of the
TRRAP/TIP60 HAT complex and seems to play multiple roles in chromatin modification and
remodelling in cells (Cai et al., 2005). Fos, or proto-oncogene protein c-Fos, is a nuclear
phosphoprotein, which is thought to have an important role in signal transduction, cell
proliferation, differentiation and protection against stress-induced cell death in certain cell
types (Zhou et al., 2007). Trim27 (tripartite motif-containing protein 27) represses
transcriptional activation by basic helix-loop-helix (bHLH) transcription factors (Bloor et al.,
2005). Note that 2 versions (truncated and full-length) of this gene were tested (see Figure S5
for details). Sox4 (SRY-related HMG-box gene 4) is overexpressed in t(8;21)(q22;q22) acute
myelogenous leukaemia (Tonks et al., 2007). Klf10 (Krüppel-like factor 10) acts as an inducer
or repressor of gene transcription to enhance the TGFβ/Smad pathway, as well as other
signaling pathways, to regulate cell proliferation, differentiation, and apoptosis in skeletal
disease (osteopenia/osteoporosis), heart disease (hypertrophic cardiomyopathy), and cancer
(breast and prostate) (Subramaniam et al., 2007). Ski (Sloan-Kettering viral oncogene
homolog) seems to be involved in the blocking of differentiation in AML via inhibition of
RARalpha signaling (Ritter et al., 2006). Prdm16 (PR domain-containing protein 16) is
involved in leukemias with chromosomal rearrangements. Overexpression of the short
isoform, sPRDM16, in mouse bone marrow induced AML with full penetrance, but only in
the absence of p53. Overexpression also increased the pool of HSC in vivo, and in vitro
blocked myeloid differentiation and prolonged progenitor life span (Shing et al., 2007). Erdr1
(erythroid differentiation regulator 1) expression is observed in many normal mouse tissues,
yet in hematopoiesis is largely confined to CD34+ cells (Dormer et al., 2004). Finally, Sfpi1
(transcription factor PU.1 or SFFV proviral integration 1 protein) acts as a lymphoid-specific
enhancer and is a transcriptional activator that may be specifically involved in the
differentiation or activation of macrophages and B cells. DDR=donor-derived reconstitution;
Table S5. Secondary transplantation assay for selected genes
For secondary transplantation experiments, frozen-thawed bone marrow cells isolated from
primary animals (20 weeks after transplantation) were cultured for 7 days in feeder-free
growth factor-supplemented media to simulate the conditions utilized between day 0 to day 7
in the primary screen (see Figure 1B). ~5 x 106 input cells (~1/4 femur) cultured cells were
transplanted per myeloablated secondary recipient and blood reconstitution (%CD45.1)
analyzed at 4, 8 and 14 weeks after transplantation (only 14 week data are presented). Note
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that, except for primary recipients of vector control cells (values presented as mean ±SD),
reconstitution levels were between 50-72% for all primary mice used in these experiments.
Note also that while proviral DNA for Vps72, Klf10 and Erdr1 was easily detected in the bone
marrow of each of the primary recipients (5th column in and Figure 6) it was not found for Fos
(Figure 6, bottom panels) and for control vectors (data not shown). The results from Q-RT-
PCR experiments show relative expression levels of the indicated transgenes over their
corresponding normal endogenous levels (expressed as Fold exp. in 4th column) in purified
CD150+CD48-Lin- cells isolated at the time the primary mice were sacrificed. Interestingly for
Klf10, there was a correlation between reconstitution level in secondary recipients and
transgene expression in primary donors (compare results for mouse 2111: 6-fold increase in
Klf10 and 25% CD45.1+ cells in corresponding secondary recipient, to that of mouse #3293 in
which Klf10 levels are lower). Also note that the peripheral blood reconstitution in all four
secondary recipients analyzed was multi-lineage. Consistent with a non-cell autonomous
function for Fos, secondary recipients of cells isolated from mice #2081 and #3472 were
poorly reconstituted by donor cells. Together, these results suggest that enhanced self-renewal
of HSC, rather than transient expansion of (multipotent) progenitor cells, is responsible for the
observed effects of Vps72, Klf10 and Erdr1 on HSC activity described in this study.
Secondary transplantation experiments also further validate the non-cell autonomous function
of Fos within this experimental setup. Wks=weeks post-transplantation; n/a=not applicable;
Table S6. Primer sequences and Universal ProbeLibrary probe numbers
used in Q-RT-PCR assays
“Universal ProbeLibrary #” refers to the identification number for the “Taqman-like” short
probes used in these studies. Information about these probes are available online at
Cell, Volume 137
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