Prolonged Cell Cycle Transit Is a Defining
and Developmentally Conserved Hemopoietic
Stem Cell Property
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J Immunol 2006; 177:201-208; ;
doi: 10.4049/jimmunol.177.1.201
http://www.jimmunol.org/content/177/1/201
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References
Jens M. Nygren, David Bryder and Sten Eirik W. Jacobsen
The Journal of Immunology
Prolonged Cell Cycle Transit Is a Defining and
Developmentally Conserved Hemopoietic Stem Cell Property1
Jens M. Nygren, David Bryder, and Sten Eirik W. Jacobsen2
H
emopoietic stem cells (HSCs)3 constitute a small population of cells defined by their unique ability to differentiate into all blood cell lineages as well as to generate
identical progeny with the same unrestricted hemopoietic potential
through self-renewal (1). The regulation of HSC fate decisions
remains largely unknown, and no unique characteristics of the
HSC cell cycle transit have been identified, which might be decisive for securing symmetrical and asymmetrical self-renewing
divisions.
Formation of definitive HSCs in the developing embryo occurs
in the dorsal aorta region around 10 days postcoitum (dpc) (2).
Embryonic blood cell production starts as these stem cells migrate
and populate the liver. The fetal liver microenvironment not only
supports blood formation but importantly also an exponential expansion of HSC numbers through self-renewing cell divisions (3,
4). This is reflected by the fact that fetal liver HSCs, despite being
largely similar to and the ancestors of adult HSCs, are much better
than their adult counterparts in repopulating the bone marrow
(BM) and reconstituting hemopoiesis following transplantation
into myeloablated hosts (5–7). Thus, the in vivo repopulating ability of highly proliferative fetal HSCs is superior to that of the more
quiescent and slowly cycling adult HSCs (8, 9). As much as this
Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell
Biology and Cell Therapy, Lund University, Lund, Sweden
Received for publication March 7, 2006. Accepted for publication April 21, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Swedish Research Council and the
Swedish Cancer Society. The Lund Stem Cell Center is supported by a Swedish
Center of Excellence grant in life sciences from the Swedish Foundation for Strategic
Research.
2
Address correspondence and reprint requests to Dr. Sten Eirik W. Jacobsen, Hematopoietic Stem Cell Laboratory, BMC B10, 221 84 Lund, Sweden. E-mail address:
[email protected]
3
Abbreviations used in this paper: HSC, hemopoietic stem cell; HPC, hemopoietic
progenitor cell; dpc, days postcoitum; BM, bone marrow; RU, reconstituting unit;
SCF, stem cell factor; TPO, thrombopoietin; LT-HSC, long-term hemopoietic stem
cell; ST-HSC, short-term hemopoietic stem cell.
Copyright © 2006 by The American Association of Immunologists, Inc.
might seem reasonable from a developmental point of view, it is
seemingly in conflict with several studies in which HSC function
(repopulating ability) has been suggested to be compromised while
transiting through the S-G2-M phases of the cell cycle (10 –15).
In steady-state adult BM, HSCs transit the cell cycle on average
as infrequently as once every 4 – 8 wk (8, 9) and are therefore
highly enriched in the G0 cell cycle phase (16). For this reason, it
has not been possible to establish whether the cell divisions of
adult HSCs when entering active cell cycle in steady state, occur
with similar kinetics to that of progenitor cells that have lost the
ability to self-renew. However, based on in vitro studies, it has
been proposed that the first cell division of HSCs is prolonged due
to the need to exit G0, and although complicated by most HSC
daughter cells in such cultures being progenitors rather than HSCs,
it has been assumed that subsequent ex vivo HSC divisions occur
with the same kinetics as downstream progenitors (10, 13, 17).
This combined with ex vivo expanding HSCs being highly enriched in G1 have led to the conclusion that HSCs are severely
compromised in their engraftment potential when transiting
S-G2-M phases of the cell cycle. If so, it could have important
clinical implications (15), because a reduced repopulating ability
of cycling HSCs could explain why extensive efforts to ex vivo
expand HSCs have in most cases failed (15, 18), and only in a few
cases resulted in marginal increases in HSC numbers (19, 20).
Herein, to better establish the cell cycle transit of HSCs actively
undergoing self-renewing divisions under physiological conditions, we investigated for the first time the cell cycle kinetics of
HSCs in the 14.5 dpc fetal liver, a stage when all HSCs were found
to divide within 48 h. We demonstrate that the average cell division of HSCs (10.6 h) is twice that of hemopoietic progenitors (5.6
h), due to a prolonged G1 transit and passage through a state of
relative G0 quiescence, resulting in also fetal HSCs being highly
enriched in G1. Furthermore, we demonstrate for the first time that
also ex vivo expanding adult HSCs sustain a prolonged G1 and cell
cycle transit when compared with hemopoietic progenitors, and
consequently the relative enrichment of HSCs in G0-G1 does at
least in part reflect the uniquely prolonged and developmentally
conserved cell cycle transit of HSCs.
0022-1767/06/$02.00
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Adult mouse hemopoietic stem cells (HSCs) are typically quiescent and enter and progress through the cell cycle rarely in
steady-state bone marrow, but their rate of proliferation can be dramatically enhanced on demand. We have studied the cell cycle
kinetics of HSCs in the developing fetal liver at a stage when they expand extensively. Despite that 100% of fetal liver HSCs divide
within a 48-h period, their average cell cycle transit time (10.6 h) is twice that of their downstream progenitors, translating into
a prolonged G1 transit and a period of relative quiescence (G0). In agreement with their prolonged G1 transit when compared with
hemopoietic progenitors, competitive transplantation experiments demonstrate that fetal HSCs are highly enriched in G1 but also
functional in S-G2-M. This observation combined with experimental data demonstrating that adult HSCs forced to expand ex vivo
also sustain a uniquely prolonged cell cycle and G1 transit, demonstrate at least in part why purified HSCs at any state of
development or condition are highly enriched in the G0-G1 phases of the cell cycle. We propose that a uniquely prolonged cell cycle
transit is a defining stem cell property, likely to be critical for their maintenance and self-renewal throughout development. The
Journal of Immunology, 2006, 177: 201–208.
202
Materials and Methods
Mice
Congenic C57BL/6 strains differing only at the CD45 locus were used in
all experiments. Experiments were approved by the ethical committee at
Lund University. All mice were given sterile food and autoclaved acidified
water, and housed under pathogen-free conditions in individually ventilated cages.
BrdU retention and cell cycle analysis
Pregnant mice were given an i.p. injection of BrdU (Sigma-Aldrich) in
0.9% saline (1 mg of BrdU per 6 g of body weight) and for 2– 48 h allowed
to freely drink water containing BrdU (1 mg/ml), before isolation of livers
from fetuses at 14.5 dpc for analysis. Evaluation of BrdU incorporation
as well as cell cycle analysis was done using a BrdU and a Ki67 and
7-aminoactinomycin D intracellular staining kit (BD Pharmingen) according to the manufacturer’s protocol.
Stem cell purification and cell cycle analysis
Competitive repopulation assays
Ex vivo-expanded BM HSCs (300 – 600 cells) or fresh fetal liver HSCs
(100 cells) from C57BL/6 mice (CD45.2) were transplanted into lethally
irradiated (925 rad) congenic recipients (CD45.1 or CD45.1/CD45.2) together with 200,000 unfractionated BM competitor cells (CD45.1), allowing quantification of reconstitution activity and ensuring survival of lethally irradiated mice. Hemopoietic donor cell reconstitution and lineage
distribution was evaluated in peripheral blood at different time points posttransplantation by FACS, as previously described (20). Total reconstitution
from 1000 control LSKCD34⫺ HSCs transplanted directly from the primary adult HSC culture or following the Hoechst 33342 staining did not
differ significantly (56.7 ⫾ 1.9 and 51.7 ⫾ 6.8%, respectively), suggesting
that staining procedures did not affect their viability or repopulating ability.
Calculation of reconstituting units (RUs) was performed based on a definition of 1 RU as the repopulating ability of 105 competitor BM cells, as
previously described (21).
Ex vivo expansion
Sorted HSCs cells were expanded ex vivo (20) using serum-free medium
X-Vivo 15 supplemented with 1% detoxified BSA, 1% penicillin/streptomycin, 1% L-glutamine (all from BioWhittaker), 1% 2-ME (SigmaAldrich), and cytokines (50 ng/ml each of stem cell factor (SCF; Immunex), Flt3 ligand (Immunex), and thrombopoietin (TPO; Amgen), and 20
ng/ml IL-3 (PeproTech)). Cell densities were never allowed to exceed
0.5 ⫻ 106 cells/ml.
Semisolid clonogenic progenitor cell assay
Cells were plated in duplicate in IMDM, supplemented with 20% FCS, 1%
penicillin/streptomycin, 1% L-glutamine, 1% 2-ME, methylcellulose
(StemCell Technologies), and cytokines (50 ng/ml each of SCF, Flt3 ligand, and TPO, 20 ng/ml IL-3, and 10 ng/ml granulocyte CSF (Amgen))
in 35-mm Petri dishes. Cultures were incubated at 37°C in a humidified
atmosphere with 5% CO2 in air for 7 days. Only colonies with ⬎50 cells
were scored.
Cell division tracking
Sorted HSCs cells were expanded ex vivo under conditions described
above and subsequently stained with the mitotic tracker dye PKH26
(Sigma-Aldrich) according to the manufacturer’s protocol and after 2 days
of additional expansion culture, sorted on a FACSDiva or FACSVantage to
fractionate the rapid (PKH26low, 35% of population with lowest PKH26
intensity) and slow (PKH26high, 20% of population with highest PKH26
intensity) proliferating cells. On average, 1,000 PKH26high cells or 3,000 –
5,000 PKH26low cells were transplanted together with 200,000 unfractionated BM cells, and hemopoietic reconstitution was evaluated as described
above. Note that, to compensate for the potentially higher number of asymmetric cell divisions in PKH26low compared with PKH26high cells, three to
five times as many PKH26low cells were transplanted into each recipient.
High-resolution cell division tracking using CFSE (Molecular Probes) was
conducted as previously described (20).
Statistics
All data are reported as means ⫾ SD or SEM. Statistical comparisons were
made using Student’s t test for unpaired samples and differences with p ⬍
0.05 were regarded as significant.
Results
Prolonged cell cycle transit of fetal liver HSCs during
physiological expansion
We investigated the cell cycle status and transition time of HSCs
in the fetal liver, at a time when HSC numbers expand extensively
in vivo (3, 4). Based on their rapid expansion, it was expected,
although not previously demonstrated, that 14.5 dpc fetal liver
HSCs would be actively dividing. Indeed, we found that 100% of
Sca-1⫹c-kit⫹ HSCs (22) as Sca-1⫺c-kit⫹ hemopoietic progenitor
cells (HPCs; Fig. 1A) had proliferated within 48 h, as demonstrated
through uptake of the thymidine analog BrdU (Fig. 1B). Although
the Sca-1⫹c-kit⫹ cell surface phenotype does not exclusively identify HSCs, all HSCs are Sca-1⫹. As the goal was to measure times
for complete BrdU incorporation within each population and because long-term HSCs (LT-HSCs) have the slowest division kinetics, impurities within the Sca-1⫹c-kit⫹ population would in fact
tend to underestimate differences in cell cycle transit times between LT-HSCs and progenitors. In light of the BrdU incorporation data, the distinct differences in cell cycle distribution between
HPCs and HSCs were striking (Fig. 1C). Whereas the majority of
fetal liver HPCs resided in S-G2-M (78%), the majority of fetal
liver HSCs were in G1 (51%), with just 34% in S-G2-M. Moreover,
whereas virtually no HPCs were found in G0, as much as 14% of
the HSC population appeared to be in a G0 state, as indicated by
undetectable Ki67 expression (23). Thus, despite extensive expansion and continuous proliferation, fetal liver HSCs appear to reenter a transient state of relative quiescence and prolonged G1
transit.
Distinct cell cycle transit times of fetal liver stem and
progenitor cells
To compare the cell cycle kinetics of fetal liver HSCs and HPCs,
pregnant females were treated with BrdU, and enriched HSC (Sca1⫹c-kit⫹) or HPC (Sca-1⫺c-kit⫹) populations of 14.5 dpc fetal
liver cells were analyzed for BrdU incorporation after 2– 48 h.
Within 2 h, as much as 42% of enriched HSCs and 74% of HPCs
( p ⬍ 0.05) had incorporated BrdU (Fig. 2A). After 9 h, 64% of
HSCs and virtually all (99%) HPCs had incorporated BrdU ( p ⬍
0.05), indicating that the HSC population in the fetal liver has a
much slower turnover rate than the HPCs. Plotting the log values
of undivided cells (that had not incorporated BrdU) over time
gives a linear regression curve (8) (Fig. 2B). Based on this, the
constant fraction of HSCs and HPCs entering the cell cycle in a
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Isolation of LSK and LSKCD34⫺ cells from the BM of ⬎10-wk-old
C57BL/6 mice was performed using FACS, as previously described (20).
Cells were sorted on a FACSVantage or FACSDiva (BD Biosciences).
Reanalysis of sorted cells reproducibly showed a high purity (⬎96%).
For isolation of LSKMac1low HSCs from fetal liver, C57BL/6 breeders
were put together in late afternoon and were checked for vaginal plugs the
following morning (designated 0.5 dpc). At 14.5 dpc, livers were dissected
from fetuses and single-cell suspensions were filtered through a nylon
mesh (70 ␮m). Cells were lineage depleted and stained with specific Abs
as previously described (20), using a mixture of lineage-specific Abs
against B220, CD3, CD8, Gr-1, Ter-119, and Abs against Mac-1, Sca-1,
and c-kit or isotype-matched control Abs (all from BD Pharmingen).
LSKMac1low cells were subsequently sorted on a FACSDiva with a purity
of ⬎96%.
To separate HSCs in G0-G1 and S-G2-M fractions, cells were diluted to
106 cells/ml in PBS supplemented with 5% FCS (BioWhittaker) and Verapamil (100 ␮M) (Abbott Scandinavia) and incubated at 37°C for 30 min
with 2.5 ␮g/ml Hoechst 33342 (Molecular Probes). Cells were kept in PBS
containing Verapamil at 4°C to prevent dye efflux and fractionated based
on cell cycle distribution using a FACSVantage or FACSDiva. As a purity
control of sorted S-G2-M cells, we in one experiment transplanted recipients with a number of G0-G1 cells corresponding to the impurity of the
S-G2-M sort (S-G2-M impurity was at 0.7%).
PROLONGED CELL CYCLE IS A DEFINING HSC PROPERTY
The Journal of Immunology
203
FIGURE 1. BrdU incorporation and cell cycle status of HPCs and HSCs
in fetal liver. A, Fetal liver HPCs (Sca-1⫺c-kit⫹) and HSCs (Sca-1⫹c-kit⫹)
from 14.5 dpc embryos were analyzed for (B) BrdU incorporation following 48-h in vivo treatment. Numbers represent mean percentages of BrdU⫹
cells from six fetuses in two experiments. C, Distribution in G0, G1, and
S-G2-M. A significant (p ⬍ 0.05) shift of HSCs to G0 and G1 compared
with HPCs was detected. Numbers represent mean percentages of cells in
each phase from eight fetuses representing two litters.
random fashion was calculated to be 8 and 18% per hour, respectively, and the 50% BrdU incorporation point for HSCs and HPCs
was 3.8 and 1.7 h, respectively. Although the HSC and HPC populations analyzed are heterogeneous and cycling asynchronously,
the average cell cycle time for each population can be calculated
by dividing the BrdU incorporation rate (0.08 and 0.18 for HSCs
and HPCs, respectively) with the fraction of proliferating cells (G1
⫹ S-G2-M ⫽ 0.85 and 0.99 for HSCs and HPCs, respectively; Fig.
1C). Thus the calculated mean cell cycle transit time of enriched
HSCs and HPCs in fetal liver was 10.6 and 5.6 h ( p ⬍ 0.05),
respectively. Although we used the same methods as in previous
studies of HSC kinetics in adult mice to calculate the above cell
cycle transit times (8), the calculated times might be unrealistically
short. As an almost complete (99%) BrdU labeling was observed
within 25 h for HSCs and within 11 h for HPCs (Fig. 2B), these
times might be more reasonable estimates for the generation times.
Transplantable fetal HSCs are enriched in G0-G1
The above studies of cell cycle distribution and BrdU incorporation in phenotypically defined fetal liver HSCs suggested that fetal
liver LT-HSCs, like their adult counterparts, would preferentially reside in G0-G1 of the cell cycle. To test this, we isolated G0-G1 and
S-G2-M fractions of Lin⫺Sca-1⫹c-kit⫹Mac-1low (LSKMac1low) fetal
liver cells (22), a population which is highly enriched for LT-HSCs
but predominately contains short-term HSCs (ST-HSCs) and multipotent progenitors (11) (Fig. 3A). We then evaluated the longterm multilineage competitive repopulating activities of the two
fractions by transplantation of limited numbers (100 cells per
FIGURE 3. Fetal liver HSCs are enriched in G0-G1. A and B, Purified
LSKMac1low HSCs from 14.5 dpc liver were separated into G0-G1 and
S-G2-M fractions (A) and transplanted (100 cells per mouse in competition
with 200,000 BM cells) into lethally irradiated congenic recipients (B).
Peripheral blood was investigated at 3, 8, 16, and 24 wk posttransplantation
for contribution of G0-G1 (䡺) and S-G2-M (f) HSCs to peripheral blood
reconstitution. Mean ⫾ SEM values from 9 to 14 mice at each time point
and group, from two independent experiments. Frequencies indicate fraction of transplanted mice positive for myeloid reconstitution 24 wk
posttransplantation.
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FIGURE 2. In vivo BrdU incorporation kinetics of fetal liver HPCs and
HSCs. A, Kinetics of BrdU incorporation in 14.5 dpc enriched fetal liver
HPCs (Sca-1⫺c-kit⫹, Œ) and HSCs (Sca-1⫹c-kit⫹, f), evaluated following
2– 48 h of BrdU treatment. Mean ⫾ SD percentages of BrdU-positive cells
at each time point represents 5–10 mice. B, The log values of BrdU-negative HPCs and HSCs plotted against time in hours. The lines were generated by using linear regression.
204
PROLONGED CELL CYCLE IS A DEFINING HSC PROPERTY
activity and provided as much as 4-fold higher reconstitution (Fig.
3B) when compared with S-G2-M cells. Thus, as LT-HSCs expand
in the fetal liver, they are preferentially distributed in G0-G1, as
would be predicted due to their uniquely protracted G0-G1 transit.
Ex vivo self-renewing adult HSCs remain predominantly in G1
mouse) into lethally irradiated congenic adult recipients. Although
long-term reconstitution ability was detected in both fractions,
G0-G1 cells were enriched for multilineage LT-HSC repopulating
FIGURE 5. ST- and LT-HSCs reside in
G1 and S-G2-M following primary ex vivo
expansion, but are enriched in G1. A, Cell cycle distribution of LSKCD34⫺ BM cells cultured under ex vivo self-renewing conditions
for 6 days. Fresh BM cells show distribution
in all cell cycle phases. Shown are representative profiles and mean percentages of two
experiments. B, Six-day ex vivo-expanded
LSKCD34⫺ cells analyzed for cell cycle distribution by Hoechst staining. Mean percentages of cells in G1 and S-G2-M from four
independent experiments. C, Purity analysis
of sorted G1 and S-G2-M cells. Mean ⫾ SEM
percentage purities from four experiments. D,
Ex vivo myeloid progenitor potential of
sorted G1 and S-G2-M cells from primary
LSKCD34⫺ expansion cultures. Mean ⫾ SD
values from three experiments. E, In vivo
competitive repopulating potential of
300 – 600 G1 (f) or S-G2-M (䡺) LSKCD34⫺
expansion equivalents and of the calculated
(based on purity analysis) number of contaminating G1 cells among sorted S-G2-M cells
(). Results represent mean ⫾ SEM values
from 25 to 26 mice in each group from four
independent experiments. F, Relative B cell,
T cell, and myeloid (M) reconstitution of G1
and S-G2-M HSCs at 16 wk.
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FIGURE 4. Experimental design. Purified LSKCD34⫺ HSCs cultured for
6 days (primary culture, 1°), under ex vivo conditions promoting HSC selfrenewal divisions are FACS purified into G0-G1 and S-G2-M fractions. Both
populations are evaluated for their hemopoietic potential by in vivo competitive repopulation of lethally irradiated congenic recipients and ex vivo by
myeloid colony-forming ability. Purified 1° G0-G1 and S-G2-M cells are also
cultured for an additional 6 days (secondary culture, 2°) before being reinvestigated for cell cycle distribution and repopulating activity.
Lin⫺Sca-1⫹c-kit⫹CD34⫺ (LSKCD34⫺) cells represent only
0.01% of total BM cells, but contain virtually all LT-HSCs in adult
mice (24). Defined conditions efficiently promoting in vitro selfrenewal of mouse LT-HSCs have been established (19, 20, 25).
However, because HSCs at best are maintained in such cultures,
HSC divisions (as in vivo) seem to be primarily asymmetric or
non-self-renewing, sustaining rather than expanding the number of
LT-HSCs, resulting in the vast majority of cells produced in such
cultures representing committed progenitors rather than HSCs. It is
well established that the first in vitro cell division of LT-HSCs in
mouse BM requires prolonged cytokine stimulation (26, 27) and
we have previously demonstrated that by 3 days of combined stimulation with the early acting cytokines SCF, Flt3 ligand, TPO, and
IL-3, all LSKCD34⫺ HSCs have undergone their first cell division
(20, 25). Thus, in the present studies, we used the same ex vivo
self-renewing conditions for 6 days (Fig. 4), to ensure that all
HSCs have divided at least once, and found that expanding cells in
these expansion cultures re-enter G1 but not G0 (Fig. 5A). Subsequently, using the viable DNA dye Hoechst 33342, ex vivo-expanded cells were sorted into highly purified G1 and S-G2-M populations (11) (Fig. 5, B and C) and evaluated for their progenitor
and HSC activities.
Whereas G1 and S-G2-M populations contained indistinguishable frequencies of myeloid progenitor cells (Fig. 5D), G1
cells were most highly enriched in LT-HSC repopulating activity (Fig. 5, E and F), as evaluated using an in vivo competitive
repopulation assay (28), where we found as much as a 10-fold
enrichment of RUs in the G1 compared with the S-G2-M
The Journal of Immunology
205
Table I. Reconstituting units in G1 and S-G2-M fractions of 1° and 2°
ex vivo expansion culturesa
Culture
G1 RU
S-G2-M RU
Fold
Difference
p Value
Primary
Secondary
1.36 ⫾ 0.29
0.62 ⫾ 0.26
0.14 ⫾ 0.04
0.05 ⫾ 0.01
9.7
12.4
⬍0.05
⬍0.05
a
Reconstituting units were calculated as described in Materials and Methods
based on 16-wk reconstitution data in Figs. 5 and 6.
Re-entry of ex vivo-expanded S-G2-M HSCs into G1 does not
enhance their repopulating ability
Actively proliferating LT-HSCs sustain a prolonged cell cycle
transit
Although our initial experiments clearly demonstrated that transplantable ex vivo self-renewing adult HSCs reside in G1 as well as
S-G2-M phases of the cell cycle, LT-HSCs capable of reconstituting lethally ablated recipients remained highly enriched in G1. This
To reconcile our inability to enhance the repopulating activity of
S-G2-M cells by promoting their transit to G1, with the predominant localization of HSCs in G1 during active proliferation, we
hypothesized that actively in vitro proliferating HSCs might have
FIGURE 6. Re-entry of ex vivo secondary
expanded S-G2-M HSCs into G1, does not enhance their reconstituting ability. LSKCD34⫺
cells were ex vivo expanded for 6 days before
being sorted into G1 and S-G2-M cells. Following an additional 6 days of 2° ex vivo expansion, 1° G1 and S-G2-M cultures were reinvestigated for cell cycle distribution (A),
colony-forming activity (B), short- and longterm competitive reconstitution (C), and relative contribution to the different blood cell lineages (D). Short- and long-term reconstitution
of 2° cultures of 1° S-G2-M cells was significantly lower than from 1° G1 cells (p ⬍ 0.05).
Results represent mean ⫾ SEM values from 25
to 26 mice in each group from four independent experiments.
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fraction (21) (Table I). Notably, there was less enrichment of
ST-HSCs from ex vivo-expanded LSKCD34⫺ cells; 3-wk engraftment levels were only 2-fold higher from the G1 compared
with the S-G2-M fraction (Fig. 5E).
In agreement with previous studies, detectable LT-HSC activity
was present in the S-G2-M population (11) (Fig. 5E). Because BM
HSCs in S-G2-M have been suggested to be severely compromised
in their repopulating ability (10 –14), we addressed whether the
ST- and LT-HSC activity of transplanted S-G2-M cells could be
due to contaminating G1 cells by transplanting a low number of G1
cells corresponding to the impurities of G1 cells in FACS-sorted
S-G2-M cells (Fig. 5C). Importantly, the marginal reconstituting
activity of this low number of contaminating G1 cells (Fig. 5E),
demonstrate that ex vivo-expanded adult LSKCD34⫺ cells residing in S-G2-M in fact contain ST-HSC and LT-HSCs, which can
reconstitute lethally irradiated recipients. In further support of this,
S-G2-M HSCs showed similar long-term contributions to B, T, and
myeloid lineage reconstitution as HSCs residing in G1 (Fig. 5F)
and were also able to self-renew, as demonstrated through their
ability to reconstitute secondary recipients (J. M. Nygren, unpublished data).
was in agreement with previous studies (12, 14, 15, 18) and has
been proposed to reflect that S-G2-M HSCs less efficiently than G1
HSCs home and/or engraft upon transplantation. Alternatively or
additionally, it could reflect a prolonged transit of ex vivo expanding LT-HSCs through G1, similar to that of physiologically expanding fetal liver HSCs. If so, this would unavoidably result in an
accumulation of HSCs in the G1 fraction of the cell cycle, when
compared with progenitors and ST-HSCs with a shorter G1 transit,
which therefore rather accumulate in S-G2-M.
If the reduced HSC repopulating ability in S-G2-M would reflect
a truly compromised HSC engrafting ability, it should be possible
to improve their reconstitution potential by promoting their reentry into G0-G1. Thus, G1 and S-G2-M fractions were purified
after 6 days of ex vivo expansion of LSKCD34⫺ cells, recultured
under identical conditions for an additional 6 days to promote additional HSC self-renewing cell divisions, and then investigated
for changes in HSC activity upon redistribution of cells into the
different phases of cell cycle (Fig. 4). Notably, G1 and S-G2-M
populations purified from primary (1°) expansion cultures showed
comparable cellular expansion in secondary (2°) cultures (J. M.
Nygren, unpublished data), displayed indistinguishable cell cycle
profiles after 6 days of 2° expansion (Fig. 6A), and as in primary
cultures contained similar frequencies of myeloid progenitors (Fig.
6B). However, despite the acquisition of indistinguishable cell cycle distribution profiles following 2° expansion, with most cells
now residing in G1 (Fig. 6A), competitive transplantation of cells
from the unfractionated 2° expansion cultures showed that the differences in ST-HSC and LT-HSC repopulating activities were retained (Fig. 6, C and D, and Table I). Thus, re-entry of S-G2-M
HSCs into G1 did not to affect their long-term repopulating ability.
206
PROLONGED CELL CYCLE IS A DEFINING HSC PROPERTY
a significantly longer cell cycle (G1) transit than committed progenitors, as demonstrated for fetal liver HSCs. To test this hypothesis, PKH26 was used to label HSCs that had already undergone at
least one division ex vivo, as indicated by decreased CFSE levels
(Fig. 7A), to test the timing of subsequent cell divisions. Because
PKH26 is distributed proportionally among daughter cells following mitosis (29), it allows high-resolution cell division tracking.
Following two additional days of ex vivo expansion, cells
that continued to divide with rapid (PKH26low) and slower
(PKH26high) kinetics could be distinguished (Fig. 7B). These
were FACS sorted to high purities (Fig. 7C) and transplanted in
competition with 200,000 BM cells into lethally irradiated recipients for evaluation of their total (Fig. 7D) and myeloid (Fig.
7E) long-term reconstitution potentials. Only the PKH26high
(slowly dividing) cells contained LT-HSC activity, whereas
PKH26low (rapidly dividing) cells provided only short-term
multilineage reconstitution, despite the fact that 3- to 5-fold
more PKH26low cells were transplanted. These findings are
compatible with ex vivo self-renewing adult LT-HSCs having
slower cell cycle kinetics, and therefore preferentially reside in
the G1 phase of the cell cycle.
Discussion
Whereas it was previously believed that only a very small fraction of mouse LT-HSCs cycle actively in steady-state adult BM
(30), more recent studies have suggested that all adult LT-HSCs
cycle within a period of 4 – 8 wk (8, 9). In the present studies,
we investigated the cell cycle kinetics of HSCs in the 14.5 dpc
fetal liver at a stage of normal development when HSCs expand
extensively (4). Our studies demonstrate that fetal liver HSCs,
although all dividing within as short a time as 48 h, have extensively prolonged cell cycle kinetics when compared with fetal HPCs. This striking difference in cell cycle dynamics reflects
that HSCs pass through a stage of relative quiescence
(Ki67low/neg) and prolonged G1, translating into a doubling in
the cell cycle transit time when compared with HPCs. Such G1
prolongation might reflect a specific G0 state entry of HSCs, a
notion supported by recent findings that long-term repopulating
HSCs in adult bone marrow are highly enriched in G0 compared
with G1 (16). Thus, although mechanisms must be in place to
dramatically enhance the speed of HSC expansion at this stage
of development, opposing mechanisms are obviously distinctly
prolonging the HSC cell cycle transit time.
Purified adult BM HSCs have been demonstrated to need considerably longer time to complete the first cell division ex vivo
when compared with more committed progenitors. However, subsequent ex vivo divisions have been assumed to have a G1 transit
time comparable to progenitors (13, 26, 27), but as current ex vivo
expansion conditions, dramatically expand cell numbers but only
maintain or at best slightly expand LT-HSCs over time (19, 20,
31–36), the vast majority of cells are committed progenitors rather
than LT-HSCs, complicating the interpretation of such experiments. Furthermore, our finding of considerable prolonged cell
cycle kinetics of fetal HSCs expanding under physiological conditions raised the possibility that also actively proliferating adult
HSC might have cell cycle kinetics distinct from those of adult
progenitor cells. Thus, herein we took a number of functional approaches, to directly establish the cell cycle kinetics of adult LTHSCs actively proliferating ex vivo (20). We were able to demonstrate that LT-HSCs, not only in steady-state adult BM (8, 9),
but unexpectedly also during active proliferation reside predominately in G1 of the cell cycle. We also found for the first time that
purified S-G2-M cells from ex vivo-expanded HSC cultures, upon
re-entering G1 in reculture, fail to regain repopulating activity and
that purified G1 cells, upon cell culture-induced S-G2-M re-entry,
did not lose significant HSC potential.
Our data would be best compatible with LT-HSCs having a
prolonged cell cycle transit, such that the primary sort of S-G2-M
cells mainly consisted of short-term stem/progenitor cells. To obtain further and direct support for this, a second line of experiments
was performed using advanced viable cell division tracking.
Through this approach, we obtained direct evidence that the cell
cycle transit time of ex vivo self-renewing LT-HSCs is prolonged
compared with their committed progeny, not only during the first
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FIGURE 7. Ex vivo self-renewing LT-HSCs have a prolonged cell cycle transit. A, Freshly isolated HSCs were stained with CFSE (0 h), cultured under
self-renewing conditions and, following 96 h, reinvestigated for CFSE intensity to demonstrate that all cells have divided at this time. B, Ex vivo-expanded
(96 h) LSK cells were stained with PKH26 and cultured for an additional 48 h before being reinvestigated for PKH26 intensity (144 h) and sorted into rapid
(PKH26low) and slow (PKH26high) proliferating fractions. C, Purity reanalysis of sorted PKH26low and PKH26high cells. D and E, Transplanted mice were
investigated 3, 8, and 17 wk following primary transplantation, as well as 8 wk following secondary (time point for secondary transplantation indicated
with 2° in the graphs) transplantation (26 wk) for contribution of PKH26low (䡺) and PKH26high (f) cells to total (D) and myeloid (E) peripheral blood
reconstitution. Total and myeloid absolute reconstitution was significantly (p ⬍ 0.05) higher from PKH26high than PKH26low cells at all investigated time
points. Results are mean ⫾ SEM values from seven mice in each group in one representative experiment of three.
The Journal of Immunology
Acknowledgments
We thank Lilian Wittman for expert technical assistance; the FACS Facility at Lund Strategic Research Center for Stem Cell Biology and Cell
Therapy for assistance with cell sorting; and Drs. Connie Eaves, Norman
Iscove, and Jennifer Antonchuk for helpful discussions and review of the
manuscript.
Disclosures
The authors have no financial conflict of interest.
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