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*Stem Cells
The Journal of Immunology, 2006, 177: 201-208.
Copyright © 2006 by The American Association of Immunologists

Prolonged Cell Cycle Transit Is a Defining and Developmentally Conserved Hemopoietic Stem Cell Property1

Jens M. Nygren, David Bryder and Sten Eirik W. Jacobsen2

Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hemopoietic 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, 6, 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 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, 11, 12, 13, 14, 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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

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 µm). 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 µM) (Abbott Scandinavia) and incubated at 37°C for 30 min with 2.5 µg/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%).

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 (Sigma-Aldrich), 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 x 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 re-enter a transient state of relative quiescence and prolonged G1 transit.


Figure 1
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  		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.

 
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 (Sca-1+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 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.


Figure 2
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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+, {blacktriangleup}) and HSCs (Sca-1+c-kit+, {blacksquare}), 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.

 
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 long-term multilineage competitive repopulating activities of the two fractions by transplantation of limited numbers (100 cells per 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 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.


Figure 3
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  		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 ({square}) and S-G2-M ({blacksquare}) 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.

 
Ex vivo self-renewing adult HSCs remain predominantly in G1

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 self-renewal 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.


Figure 4
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  		FIGURE 4. Experimental design. Purified LSKCD34– HSCs cultured for 6 days (primary culture, 1°), under ex vivo conditions promoting HSC self-renewal 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.

 

Figure 5
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  		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 ({blacksquare}) or S-G2-M ({square}) LSKCD34– expansion equivalents and of the calculated (based on purity analysis) number of contaminating G1 cells among sorted S-G2-M cells ({blacktriangledown}). 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.

 
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 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).


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  		Table I. Reconstituting units in G1 and S-G2-M fractions of 1° and 2° ex vivo expansion culturesa

 
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, 11, 12, 13, 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).

Re-entry of ex vivo-expanded S-G2-M HSCs into G1 does not enhance their repopulating ability

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 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 re-entry 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.


Figure 6
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  		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 long-term 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.

 
Actively proliferating LT-HSCs sustain a prolonged cell cycle transit

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 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.


Figure 7
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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 ({square}) and PKH26high ({blacksquare}) 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.

 
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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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, 32, 33, 34, 35, 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 LT-HSCs 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 cell division but also subsequent cell divisions. Of interest, our data also suggest that ST-HSCs have a prolonged cell cycle transit relative to that of committed progenitors, although shorter than that of LT-HSCs. The prolonged cell cycle transit of LT-HSCs must primarily be due to a slower progression through the G1 phase, as we observed an accumulation of proliferating LT-HSCs specifically in G1. This is in agreement with G1 being the cell cycle phase that is most regulatable in time (37).

Previous studies have suggested that the expression of adhesion molecules and BM homing properties of HSCs fluctuate with cell cycle passage (12, 38). Although our studies support that cells in all phases of the cell cycle phase can repopulate lethally irradiated mice, and that the main explanation for enrichment of repopulating HSCs in G0-G1 is the prolonged G0-G1 transit of HSCs, it is important to emphasize that this does not exclude the possibility that HSCs in S-G2-M might have somewhat reduced repopulating ability, perhaps reflecting changes in adhesion molecule expression or function. However, when performing intrafemoral transplantations, we have observed the same differences in repopulating ability between S-G2-M and G0-G1 cells, as when transplanted i.v. (J. M. Nygren, unpublished observations).

Our studies point to the importance in evaluating LT-HSCs by functional (long-term competitive repopulation) rather than phenotypical (cell surface marker expression) methods. Consequently, the conclusions from our studies could only be reached by identifying and evaluating LT-HSCs through their function, because there is no reliable phenotype of LT-HSCs following in vitro and in vivo expansion (39, 40). Thus, previous studies implicating cell cycle-specific effects on LT-HSC engraftment (10, 12, 13, 14) as well as gene expression (41), should be re-evaluated in light of the fact that functionally defined LT-HSCs, due to a prolonged cell cycle transit, always are enriched in G1 relative to S-G2-M.

In conclusion, the prolonged transit through G0-G1 is not limited to HSCs in steady-state adult BM but is a defining and developmentally conserved stem cell property. We propose that this distinct regulation of cell cycle transit in HSCs (and potentially other somatic stem cells) could be a requirement for their self-renewal (42) and to avoid exhaustion of the LT-HSC compartment by limiting their proliferative capacity (43, 44). Thus, it will be of considerable importance to identify the unique regulatory mechanisms of HSC cell cycle transit during development as well as in steady-state adult hemopoiesis.


   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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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. Back

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: sten.jacobsen{at}med.lu.se Back

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. Back

Received for publication March 7, 2006. Accepted for publication April 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Spangrude, G. J., S. Heimfeld, I. L. Weissman. 1988. Purification and characterization of mouse hematopoietic stem cells. Science 241: 58-62. [Abstract/Free Full Text]
  2. Cumano, A., I. I. Godin. 2001. Pluripotent hematopoietic stem cell development during embryogenesis. Curr. Opin. Immunol. 13: 166-171. [Medline]
  3. Ikuta, K., I. L. Weissman. 1992. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc. Natl. Acad. Sci. USA 89: 1502-1506. [Abstract/Free Full Text]
  4. Pawliuk, R., C. Eaves, R. K. Humphries. 1996. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 88: 2852-2858. [Abstract/Free Full Text]
  5. Jordan, C. T., C. M. Astle, J. Zawadzki, K. Mackarehtschian, I. R. Lemischka, D. E. Harrison. 1995. Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation. Exp. Hematol. 23: 1011-1015. [Medline]
  6. Rebel, V., C. Miller, C. Eaves, P. Lansdorp. 1996. The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood 87: 3500-3507. [Abstract/Free Full Text]
  7. Rebel, V. I., C. L. Miller, G. R. Thornbury, W. H. Dragowska, C. J. Eaves, P. M. Lansdorp. 1996. A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp. Hematol. 24: 638-648. [Medline]
  8. Cheshier, S. H., S. J. Morrison, X. Liao, I. L. Weissman. 1999. In vivo proliferation and cell cycle kinetics of long-term self- renewing hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 96: 3120-3125. [Abstract/Free Full Text]
  9. Bradford, G. B., B. Williams, R. Rossi, I. Bertoncello. 1997. Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment. Exp. Hematol. 25: 445-453. [Medline]
  10. Habibian, H. K., S. O. Peters, C. C. Hsieh, J. Wuu, K. Vergilis, C. I. Grimaldi, J. Reilly, J. E. Carlson, A. E. Frimberger, F. M. Stewart, P. J. Quesenberry. 1998. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J. Exp. Med. 188: 393-398. [Abstract/Free Full Text]
  11. Fleming, W. H., E. J. Alpern, N. Uchida, K. Ikuta, G. J. Spangrude, I. L. Weissman. 1993. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J. Cell Biol. 122: 897-902. [Abstract/Free Full Text]
  12. Orschell-Traycoff, C. M., K. Hiatt, R. N. Dagher, S. Rice, M. C. Yoder, E. F. Srour. 2000. Homing and engraftment potential of Sca-1+lin – cells fractionated on the basis of adhesion molecule expression and position in cell cycle. Blood 96: 1380-1387. [Abstract/Free Full Text]
  13. Glimm, H., I. H. Oh, C. J. Eaves. 2000. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S-G2-M transit and do not reenter G0. Blood 96: 4185-4193. [Abstract/Free Full Text]
  14. Jetmore, A., P. A. Plett, X. Tong, F. M. Wolber, R. Breese, R. Abonour, C. M. Orschell-Traycoff, E. F. Srour. 2002. Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients. Blood 99: 1585-1593. [Abstract/Free Full Text]
  15. Quesenberry, P. J., G. A. Colvin, J. F. Lambert. 2002. The chiaroscuro stem cell: a unified stem cell theory. Blood 100: 4266-4271. [Abstract/Free Full Text]
  16. Passegue, E., C. H. Jamieson, L. E. Ailles, I. L. Weissman. 2003. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics?. Proc. Natl. Acad. Sci. USA 100: (Suppl. 1):11842-11849. [Abstract/Free Full Text]
  17. Szilvassy, S. J., T. E. Meyerrose, B. Grimes. 2000. Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells. Blood 95: 2829-2837. [Abstract/Free Full Text]
  18. Srour, E. F., R. Abonour, K. Cornetta, C. M. Traycoff. 1999. Ex vivo expansion of hematopoietic stem and progenitor cells: are we there yet?. J. Hematother. 8: 93-102. [Medline]
  19. Miller, C. L., C. J. Eaves. 1997. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc. Natl. Acad. Sci. USA 94: 13648-13653. [Abstract/Free Full Text]
  20. Bryder, D., S. E. Jacobsen. 2000. Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro. Blood 96: 1748-1755. [Abstract/Free Full Text]
  21. Yuan, R., C. M. Astle, J. Chen, D. E. Harrison. 2005. Genetic regulation of hematopoietic stem cell exhaustion during development and growth. Exp. Hematol. 33: 243-250. [Medline]
  22. Morrison, S. J., H. D. Hemmati, A. M. Wandycz, I. L. Weissman. 1995. The purification and characterization of fetal liver hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 92: 10302-10306. [Abstract/Free Full Text]
  23. Kubbutat, M. H., G. Key, M. Duchrow, C. Schluter, H. D. Flad, J. Gerdes. 1994. Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein). J. Clin. Pathol. 47: 524-528. [Abstract/Free Full Text]
  24. Osawa, M., K. Hanada, H. Hamada, H. Nakauchi. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273: 242-245. [Abstract]
  25. Ramsfjell, V., D. Bryder, H. Bjorgvinsdottir, S. Kornfalt, L. Nilsson, O. J. Borge, S. E. Jacobsen. 1999. Distinct requirements for optimal growth and in vitro expansion of human CD34+CD38 – bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells. Blood 94: 4093-4102. [Abstract/Free Full Text]
  26. Reddy, G. P., C. Y. Tiarks, L. Pang, J. Wuu, C. C. Hsieh, P. J. Quesenberry. 1997. Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells. Blood 90: 2293-2299. [Abstract/Free Full Text]
  27. Uchida, N., B. Dykstra, K. J. Lyons, F. Y. Leung, C. J. Eaves. 2003. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp. Hematol. 31: 1338-1347. [Medline]
  28. Harrison, D.. 1980. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 55: 77-81. [Abstract/Free Full Text]
  29. Yan, F., M. I. Collector, S. Tyszko, S. J. Sharkis. 2003. Using divisional history to measure hematopoietic stem cell self- renewal and differentiation. Exp. Hematol. 31: 56-64. [Medline]
  30. Lemischka, I. R., D. H. Raulet, R. C. Mulligan. 1986. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 45: 917-927. [Medline]
  31. Glimm, H., C. J. Eaves. 1999. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood 94: 2161-2168. [Abstract/Free Full Text]
  32. Ema, H., H. Takano, K. Sudo, H. Nakauchi. 2000. In vitro self-renewal division of hematopoietic stem cells. J. Exp. Med. 192: 1281-1288. [Abstract/Free Full Text]
  33. Moore, K. A., H. Ema, I. R. Lemischka. 1997. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood 89: 4337-4347. [Abstract/Free Full Text]
  34. Peters, S., E. Kittler, H. Ramshaw, P. Quesenberry. 1996. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL- 6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood 87: 30-37. [Abstract/Free Full Text]
  35. Fraser, C., S. Szilvassy, C. Eaves, R. Humphries. 1992. Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability. Proc. Natl. Acad. Sci. USA 89: 1968-1972. [Abstract/Free Full Text]
  36. Traycoff, C. M., K. Cornetta, M. C. Yoder, A. Davidson, E. F. Srour. 1996. Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential. Exp. Hematol. 24: 299-306. [Medline]
  37. Pardee, A. B.. 1989. G1 events and regulation of cell proliferation. Science 246: 603-608. [Abstract/Free Full Text]
  38. Becker, P. S., S. K. Nilsson, Z. Li, V. M. Berrios, M. S. Dooner, C. L. Cooper, C. C. Hsieh, P. J. Quesenberry. 1999. Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status. Exp. Hematol. 27: 533[Medline]
  39. Spangrude, G., D. Brooks, D. Tumas. 1995. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood 85: 1006-1016. [Abstract/Free Full Text]
  40. Sato, T., J. H. Laver, M. Ogawa. 1999. Reversible expression of CD34 by murine hematopoietic stem cells. Blood 94: 2548-2554. [Abstract/Free Full Text]
  41. Lambert, J.-F., M. Liu, G. A. Colvin, M. Dooner, C. I. McAuliffe, P. S. Becker, B. G. Forget, S. M. Weissman, P. J. Quesenberry. 2003. Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit. J. Exp. Med. 197: 1563-1572. [Abstract/Free Full Text]
  42. Hock, H., M. J. Hamblen, H. M. Rooke, J. W. Schindler, S. Saleque, Y. Fujiwara, S. H. Orkin. 2004. Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells. Nature 431: 1002-1007. [Medline]
  43. Allsopp, R. C., S. Cheshier, I. L. Weissman. 2001. Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells. J. Exp. Med. 193: 917-924. [Abstract/Free Full Text]
  44. Allsopp, R. C., G. B. Morin, R. DePinho, C. B. Harley, I. L. Weissman. 2003. Telomerase is required to slow telomere shortening and extend replicative lifespan of HSC during serial transplantation. Blood 27: 27

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