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THE STEM CELL NICHE

Cell Cycle Quiescence of Early Lymphoid Progenitors in Adult Bone Marrow

^aImmunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA;
^bDepartment of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

Key Words. Adult stem cells • B lymphopoiesis • Fetal stem cells • G₀ • Hematopoiesis • Hematopoietic cell transplantation Hematopoietic progenitor cells • Hematopoietic stem cell

Correspondence: Paul W. Kincade, Ph.D., 825 NE 13th Street, Oklahoma City, Oklahoma 73104, USA. Telephone: 405-271-7905; Fax: 405-271-8568; e-mail: kincade{at}omrf.ouhsc.edu

Received April 12, 2006; accepted for publication August 15, 2006.
First published online in STEM CELLS EXPRESS   August 24, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Lymphocyte production in bone marrow (BM) requires substantial cell division, but the relationship between largely quiescent stem cells and dividing lymphoid progenitors is poorly understood. Therefore, the proliferation and cell cycle status of murine hematopoietic progenitors that have just initiated the lymphoid differentiation program represented the focus of this study. Continuous bromo-2'-deoxyuridine (BrdU) incorporation and DNA/RNA analysis by flow cytometry revealed that a surprisingly large fraction of RAG-1⁺c-kit^hi early lymphoid progenitors (ELPs) and RAG-1⁺c-kit^lo pro-lymphocytes (Pro-Ls) in adult BM were in cell cycle quiescence. In contrast, their counterparts in 14-day fetal liver actively proliferated. Indeed, the growth fraction (cells in G₁-S-G₂-M phases) of fetal ELPs was on average 80% versus only 30% for adult ELPs. After 5-fluorouracil treatment, as many as 60% of the adult ELP-enriched population was in G₁-S-G₂-M and 34% incorporated BrdU in 6 hours. Transcripts for Bcl-2, p21Cip1/Waf1, and p27 Kip1 cell cycle regulatory genes correlated inversely well with proliferative activity. Interestingly, adult lymphoid progenitors in rebound had the high potential for B lymphopoiesis in culture typical of their fetal counterparts. Thus, lymphocyte production is sustained during adult life by quiescent primitive progenitors that divide intermittently. Some, but not all, aspects of the fetal differentiation program are reacquired after chemotherapy.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Development and replenishment of the immune system require differentiation from rare hematopoietic stem cells (HSCs). This dynamic process can be viewed as a gradual progression from very primitive hematopoietic progenitors with multiple lineage potentials through more restricted progenitors [1], and it is tightly linked to proliferation. There has been substantial progress in understanding how pre-B cells progressively expand, complete immunoglobulin (Ig) gene rearrangement, and give rise to functional B cells [2]. In contrast, methods have only recently been developed to study cells that have just initiated the lymphoid differentiation program [3], and little information is available about their proliferation.

A strain of heterozygous RAG-1/green fluorescent protein (GFP) knockin mice are used to isolate the earliest known lymphoid progenitors from adult bone marrow (BM) and fetal liver [4, 5]. This is possible because GFP fluorescence corresponds to the presence of RAG-1 transcripts in cells that are primitive in terms of transcription factors, surface markers, and time required to differentiate into lymphoid cells. These Lin^–c-Kit^HiSca-1⁺CD27⁺Flk-2⁺RAG-1⁺ early lymphoid progenitors (ELPs) in adult BM have tremendous potential for generating all lymphoid cell lineages and likely give rise to Lin^– c-Kit^LoSca-1^±CD27⁺Flk-2⁺RAG-1⁺ pro-lymphocytes (Pro-Ls). Similarly, the RAG-1-expressing cells in embryos can be resolved into a series of differentiation stages beginning with c-Kit^HiSca-1⁺GFP^lo and culminating in c-Kit^lo/–GFP^hi subsets [5]. The cell cycle status of primitive lymphoid progenitors represented the main focus of this study.

HSCs in a state of prolonged cell cycle quiescence have been proposed to support hematopoiesis through clonal succession. That is, one or a small number of HSC clones give rise to mature blood cells as needed, and the remaining HSCs are inactive and do not contribute to hematopoiesis until the proliferative capacity of the cycling HSC clone is exhausted [6–8]. To address this issue, the proliferation of HSCs in adult mice was analyzed in vivo by means of bromo-2'-deoxyuridine (BrdU) incorporation kinetics [9, 10]. By the end of 6 months of continuous BrdU administration, 99% of HSCs had incorporated BrdU during DNA synthesis and, although 75% of HSCs are quiescent in phase G₀ at any one time, all HSCs are recruited intermittently into the cell cycle so that 99% of them divide on average every 57 days. Whether cycling HSCs contribute directly to cells entering the lymphopoietic program has not been directly addressed.

The growth, differentiation, and survival of HSCs are regulated by a number of cytokines and chemokines and by the relative basal expression level of cyclins, cyclin-dependent kinases (cdks), and cdk-inhibitors (cdkis) [8]. Whereas stem cell factor (SCF), Flt3-ligand, thrombopoietin, interleukin (IL)-3, and IL-6 promote the growth of human HSCs in vitro [11], transforming growth factor-ß (TGF-ß1) and MCP-1 (monocyte chemoattractant protein-1) induce cell cycle arrest of HSCs [12] and primitive hematopoietic progenitor-enriched fractions [13, 14], respectively. Similar information is emerging about extracellular cues that regulate the earliest stages of lymphopoiesis [15].

HSC cyclins are negatively regulated by cdkis [10]. Among the cdkis, p21 is highly expressed in the quiescent HSC-like fraction of BM cells. Moreover, HSCs in the G₀ phase are reduced and the total number of HSCs increased in p21^–/– mice [16], and survival in p21^–/– mice treated with the myelotoxic agent 5-fluorouracil (5-FU) is much lower than in littermate controls. These results indicate that p21 is a key molecule that restricts HSC entry into cell cycle, thereby imposing limits on their pool size and preventing their exhaustion. On the other hand, cdki p27 seems to govern the expansion of progenitor cell populations [17–19].

Many transcription factors such as c-Myb, GATA-2, Gfi-1, Bmi-1, and those of the homeobox (Hox) family have been shown to be additional key players in the proliferation and differentiation of early BM progenitor cells. A recent study using Hoxb4-deficient mice demonstrated reduced proliferative capacity of BM and fetal liver HSCs without affecting differentiation or lineage choice [20]. HOXB4, like Notch, has been reported to induce or enhance the expression of c-myc, cyclin D2, cyclin D3, cyclin E, and E2F1 in murine HSCs [21], and modulation of their signaling in hematopoietic precursors could represent an interesting approach to improve cell-based therapy [22]. Key to the long-term success of these strategies will be a molecular understanding of the population dynamics in vivo of HSCs and their more-committed downstream progenitors [23].

B lymphopoiesis has been depicted as a unidirectional process in which developing cells transit through successive differentiation stages in an irreversible, synchronous manner. Recently, some studies have examined this view by quantification of specific BM precursor B-cell populations [24] or by combining kinetic analysis of developing B-cell subsets in the BM with mathematical modeling [2, 25]. Asynchronous differentiation models explain BM-labeling kinetics and predict reflux between the pre- and immature B-cell pools. Additionally, studies in normal, gene-deleted, transgenic, and mutant mice have shown that the apoptotic index and apoptotic rate are maximal during the pro-/pre-B-cell transition and among immature B lymphocytes in BM [26].

We have now studied the proliferation status of mouse lymphoid progenitors that are recently derived from HSCs. The results indicate that ELPs in fetal liver proliferate much faster than their counterparts in adult BM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of key cell cycle mediators identified several that could account for the fetal/adult disparity. A BrdU-labeling pattern for ELPs and Pro-Ls in adult BM suggests that cells in both compartments are cycling asynchronously and intermittently. Furthermore, adult progenitors acquire some, but not all, characteristics of fetal cells during rebound from chemotherapy. Although primitive lymphoid progenitors sustain replenishment of the immune system throughout adult life, they exist in two kinetic states and only a minority is in cell cycle at any one time.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Mice and Cell Suspensions
RAG-1/GFP knockin mice have been described [4, 27] Wild-type (WT) C57BL/6 strain mice were purchased from The Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Heterozygous F₁ RAG-1/GFP mice were generated at the OMRF (Oklahoma Medical Research Foundation) Laboratory Animal Research Facility (Oklahoma City, OK) by mating homozygous male RAG-1/GFP knockin mice with WT female mice. Fetal liver cells were obtained at embryonic days 13–16 (E13–E16). Adult BM cells were purified from 3- to 5-month-old heterozygous RAG-1/GFP knockin mice.

BrdU Treatment of Mice and Cell Cycle Analyses
Groups of 35–55 mice for adult BM studies were given an initial intraperitoneal injection of BrdU (100 µg/100 µl of phosphate-buffered saline [PBS]), while groups of three pregnant mice for fetal studies were given BrdU intravenously at zero time. This was to establish a satisfactory concentration of label and a valid starting time. In each case, BrdU was then administered continuously in drinking water (0.8 mg/ml) for the duration of the experiment. At defined time points, adult or fetal lymphoid progenitors were purified from treated mice by sorting pooled BM or fetal liver samples, respectively, followed by intracellular staining with monoclonal antibody (mAb) to BrdU (BrdU flow kit; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Phycoerythrin (PE)-labeled, rather than fluorescein isothiocyanate (FITC)-labeled, anti-BrdU mAb was used to visualize BrdU in RAG-1/GFP⁺ progenitors. The cells were analyzed on a FACScan (BD Biosciences), using Cell Quest and WinMDI 2.8 software programs. Representative analyses are shown in supplemental Figure 1.

Flow Cytometry and Cell Sorting
Adult BM was flushed from femurs, tibias, and humeri with 3% fetal calf serum (FCS) PBS. Cells were enriched by incubation with antibodies to lineage markers Gr1 and CD11b/Mac1 for myeloid cells, CD19 and CD45R/B220 for B-lineage cells, and TER-119 for erythroid cells, followed by negative selection using the Bio-Mag cell separation system (Qiagen Inc., Valencia, CA, http://www1.qiagen.com). These partially lineage-depleted cells were further blocked with anti-FcR and stained with allophycocyanin-anti-c-kit antibody and with biotin-anti-lineage markers (Gr-1, Mac-1, CD3, CD8a, CD19, CD45R, DX-5, and TER-119) combined with streptavidin (Sav)-R613. CD11b/Mac1 was not included in the lineage-depletion protocol after 5-FU treatment. Sorting on MoFlo (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) was performed on the basis of Lin^–GFP⁺c-kit^hi (ELP) and Lin^–GFP⁺c-kit^lo (Pro-L). Sca-1 was used as an additional gating parameter for lymphoid progenitors in our previous studies but is expressed at artificially high levels on cells in rebound BM. Fetal livers (E13–E16) were minced, and the suspensions were subjected to depletion of TER-119⁺ cells prior to two-steps cell sorting. In the first step, cells were sorted into GFP^–, GFP^lo, and GFP^hi. Background autofluorescence was discriminated from authentic GFP by collecting data in two fluorescence channels without compensation [5]. An illustration of this method is provided as supplemental online Figure 2. Sorted cells were incubated with anti-FcR before staining with APC-anti-c-kit and bio-Sav-R613-anti-Sca-1 or PE-anti-Sca-1 antibodies and subjected to a second round of sorting. Hematopoietic progenitors were fractionated according to Sca-1 and c-kit expression, as previously described [5].

Treatment with 5-FU
Adult mice were given a single intraperitoneal injection with 5-FU (150 mg/kg of body weight) in PBS. BM was recovered at the times indicated.

Cell Cycle Fractionation with Hoechst and Pyronin Y
A combination of Hoechst 33342 (Hst) and pyronin Y (PY) was used for the differential staining of cellular RNA and DNA, as described elsewhere [28]. Briefly, lymphoid progenitors were fixed in 70% ethanol overnight, resuspended in a solution of 2 µg/ml Hst (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and 4 µg/ml PY (Polysciences, Inc., Warrington, PA, http://www.polysciences.com), and measured by flow cytometry on a MoFlo equipped with UV laser (DakoCytomation). Because RNA staining with PY yields a continuous histogram without demarcation between positive and negative cells [29], an arbitrary analysis window comprising approximately 15% of fetal liver cells displaying minimal PY staining was used to designate G₀ fraction in all experiments.

Real-Time PCR Analysis of Cell Cycle Gene Expression
Lymphoid progenitors were sorted at high purity. Sequences of cell cycle candidate genes were obtained from the UCSC (University of California at Santa Cruz) genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway), and specific forward and reverse oligonucleotide primers were designed using Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (supplemental online Table 1). The real-time PCR amplification mixture contained template cDNA, 2x SYBR Green Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) and 2 µM each primer mix. Reactions were 10 minutes at 95°C followed of 40 cycles of 95°C for 15 seconds and 60 seconds at 60°C in an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The relative gene expression was calculated using ß-actin cDNA as an endogenous control [relative gene expression = 2^{(Ct ctrl – Ct gene)} x 10³].

Telomerase Activity Detection
Telomerase activity was measured quantitatively in 1 x 10³ lymphoid progenitors by one-step real-time reaction (Express Biotech International, Frederick, MD, http://www.expressbiotech.com). Briefly, the active telomerase from lysed cells added a varied number of telomeric repeats onto the 3'-end of a substrate oligonucleotide. The extension products were amplified by PCR and then detected by measuring the increase in fluorescence by binding of SYBR green to double-stranded DNA. A standard curve was performed to calculate activity using an oligonucleotide with a sequence identical to telomere primers.

Stromal Cell Cocultures
Sorted lymphoid progenitors were cocultured for up to 3 weeks with delta-like-1 and GFP retrovirally transduced OP9 stromal cells (OP9-DL1 and OP9-GFP, kindly provided by Dr. J.C. Zúñiga-Pflücker, University of Toronto, Ontario, Canada), as previously described [30] with modifications. During the first week, the -minimal essential medium 10% FCS contained 5 x 10^–5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, plus 2 ng/ml SCF, 5 ng/ml Flt3-L, and 2 ng/ml IL-7. During the second and third weeks of coculture, the IL-7 concentration was increased to 5 ng/ml.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
ELPs and Pro-Ls in Adult BM Are Each Heterogeneous with Respect to Cell Cycle Kinetics, and a Significant Fraction Are Quiescent
BM cells that lack all markers of differentiated blood cells were resolved into primitive and more mature progenitors according to levels of c-kit and RAG-1 in RAG-1/GFP knockin mice. Stem cells, multipotent progenitors, and ELPs are in the c-kit^hi fraction of lineage-marker negative (Lin^–) BM, with one of the distinguishing characteristics of ELPs being activation of the RAG-1 locus [3, 4]. The c-kit^lo fraction of Pro-L (including common lymphoid progenitor [CLP]) generate B- and natural killer (NK)-lineage cells more quickly in short-term cultures [3, 4, 31, 32].

Continuous administration of BrdU yielded biphasic labeling curves for both ELP and Pro-L populations (Fig. 1A), which were simultaneously sorted for analyses (supplemental online Figs. 1 and 2). An initial steep rise in the labeling index, which represented the DNA labeling of a fraction of rapidly cycling cells, was soon followed by a second slower rise, indicating the gradual entry of labeled cells into a considerably larger cell fraction [33]. Extrapolating from the 36-hour interval to the time necessary to reach complete (100%) labeling yielded an apparent average cell cycle time of 11.9 days for the ELP population as a whole and 11.4 days for the Pro-L population, both cell cycle times long in comparison with that of the Lin^– c-kit^hi GFP^– fraction of BM (5 days). Consistent with these findings, an analysis of DNA/RNA cell content revealed that a majority of ELPs and Pro-Ls are in a G₀ state at any given time (Fig. 1B). Taken together, these results indicate that ELPs and Pro-Ls in adult BM consist of a mixture of cells representing two kinetic states, a minor set of cycling cells and a major set of quiescent cells which represented approximately two thirds of the total populations of ELPs and Pro-Ls. The slow linear BrdU labeling of the second subset of cells indicates that these ELPs and Pro-Ls do not remain permanently in a dormant G₀ state but are slowly turning over. Periodically, G₀ cells are triggered to enter cell cycle, after which some or all of their labeled progeny may revert to the G₀ state.

View larger version (24K): [in this window] [in a new window]   Figure 1. Most lymphoid progenitors in adult bone marrow are in a state of cell cycle quiescence. (A): Adult bone marrow ELP, Pro-L, and a cohort Lin–GFP–c-kithi population were purified from bone marrow pooled from at least 35 BrdU-treated mice, followed by intracellular staining for BrdU. Each point on the curves represents a single measurement for one pool. The numbers of mice used per time point were 35 for 6 hours, 55 for 24 hours, 52 for 36 hours, 55 for 72 hours, 50 for 120 hours, and 50 for 156 hours. Linear regression by least squares analysis was used to fit two sets of lines for each cell type. (B): ELP, Pro-L, and Lin–GFP–c-kithi cells were stained for Hoechst 33342 and Pyronin Y. The diagram at the side illustrates how actively dividing cells can be resolved on the basis of RNA/DNA staining into G1 or S+G2+M fractions. Cell frequencies in G0 or each cell cycle phase are shown. The data are derived from and representative of two different experiments that were conducted independently from that shown in (A). Abbreviations: BrdU, bromo-2'-deoxyuridine; ELP, early lymphoid progenitor; GFP, green fluorescent protein; Pro-L, pro-lymphocyte.

View larger version (20K): [in this window] [in a new window]   Figure 2. RAG-1lo and RAG-1hi subsets of lymphoid progenitors are not homogeneous with respect to proliferative activity. (A): GFP+ ELPs and GFP+ Pro-Ls were sorted from adult bone marrow of mice after 5 days of BrdU treatment. The two subsets were then resolved according to GFP density (left panels), and the incorporation of BrdU was analyzed for each population by means of intracellular staining (right panels). (B): Average percentages for BrdU labeling of RAG-1/GFPlo and RAG-1/GFPhi cells across all intervals are shown. **p = .0076. Abbreviations: BrdU, bromo-2'-deoxyuridine; ELP, early lymphoid progenitor; GFP, green fluorescent protein; Pro-L, pro-lymphocyte.

View larger version (29K): [in this window] [in a new window]   Figure 3. The entire lymphopoietic series in fetal liver is rapidly dividing. (A): Cell cycle status was determined by flow cytometry of Hoechst 33342- and Pyronin Y-stained ELP GFPloc-kithi, Pro-L GFPloc-kitlo, and GFP–c-kithi cells sorted from E14 fetal liver. (B): Pregnant mice (E13–E16) were given BrdU intravenously, and in drinking water for 6 hours. The lympho-hematopoietic progenitors were sorted and stained with monoclonal antibody to BrdU. The subsets are arranged according to probable degree of maturity, beginning with the stem cell-rich fraction on the left and CD19+-expressing cells at the far right. Results are representative of two independent experiments. Abbreviations: BrdU, bromo-2'-deoxyuridine; E, embryonic day; ELP, early lymphoid progenitor; GFP, green fluorescent protein; Pro-L, pro-lymphocyte.

These data suggest that apparent proliferation rates of fetal lymphoid progenitors are substantially higher than those of adult progenitors. Indeed, a 30-fold longer interval was needed to achieve the equivalent degree of labeling with adult cells. Furthermore, two-parameter flow cytometry revealed that the growth fraction of fetal progenitors is correspondingly high with an average of only 20% in the G₀ phase of the cell cycle at any one time (Fig. 3A). We conclude that few of these cells are in a state of cell cycle quiescence prior to birth.

Adult Lymphoid Progenitors Enter Rapid Cycle During Rebound from 5-FU Treatment
Fetal, but not adult, stem cells express Flk-2, Mac-1, and CD34 [5, 36, 37]. It is interesting that stem cells regenerating in the BM of adult mice after chemotherapy are CD11b/Mac-1⁺ and CD34⁺ [38, 39]. There are also fetal/adult differences with respect to the markers displayed on lymphoid progenitors, and we wondered whether proliferative characteristics of fetal cells could be also induced on those within BM. RAG-1/GFP mice were treated with a single injection of 5-FU and then examined for up to 8 days, resulting in a transient depletion in lymphoid progenitors. RAG-1/GFP⁺ cells could be identified among the Lin^– fraction of 5-FU-treated BM, and numbers of these cells recovered dramatically from day 4 after 5-FU treatment, when rebound from chemotherapy is said to begin (Fig. 4A) [38].

View larger version (29K): [in this window] [in a new window]   Figure 4. Transient depletion of lymphoid progenitors along with loss of c-kit expression after 5-FU chemotherapy. Mice were treated with a single dose of 5-FU, and early lymphoid progenitors and pro-lymphocytes were sorted after the indicated intervals. (A): Absolute numbers of each cell type recovered from four bones are given. (B): Temporal changes in the expression of c-kit on Lin–GFP+ cells after treatment is shown. The top left panel shows the results of staining with an isotype control antibody. Abbreviations: 5-FU, 5-fluorouracil; GFP, green fluorescent protein.

To assess proliferation, the animals were injected with and then fed BrdU for 6 hours before analysis. Approximately one-third of recovering adult progenitors incorporated the label (Fig. 5A), and the percentage of progenitors in the inactive G₀ fraction was also greatly reduced (Fig. 5B). It is noteworthy that BrdU incorporation indices were lower than the cell cycle activity determined by Pyronin Y/Hoechst staining. The latter method may be more sensitive, especially when only 6 hours are allowed for tissue equilibration and uptake of BrdU. Telomerase is important for maintaining stem cell chromosome integrity, and we evaluated this enzyme in ELPs. Telomerase activity in fetal cells was fourfold higher than in adult cells and markedly upregulated in progenitors recovered during marrow rebound (Fig. 5C).

View larger version (30K): [in this window] [in a new window]   Figure 5. Lymphoid progenitors rebounding from chemotherapy are, like fetal progenitors, in active cell cycle. After 90 hours of 5-FU treatment, mice were given BrdU intraperitoneally, and in drinking water for 6 hours. ABM was recovered at 96 hours, and the purified 5-FU Lin–GFP+c-kit+ lymphoid progenitors and control ELPs were stained with anti-BrdU to examine proliferation rates (A) or were stained for Hoechst 33342 and Pyronin Y (B). In parallel, BrdU was given to pregnant mice for 6 hours and FL E14 ELPs were analyzed. Telomerase activity was measured quantitatively in FL E14 ELP, ABM ELP, and ABM 5-FU Lin–GFP+c-kit+ cells by real-time PCR (C). The graph shows median values (amol/µl) with bars representing standard errors. *p = .025. Abbreviations: 5-FU, 5-fluorouracil; ABM, adult bone marrow; BrdU, bromo-2'-deoxyuridine; E, embryonic day; ELP, early lymphoid progenitor; FL, fetal liver; GFP, green fluorescent protein.

View larger version (23K): [in this window] [in a new window]   Figure 6. Changes in cell cycle-regulated gene expression during rebound from chemotherapy correspond to the fetal pattern in early lymphoid progenitors. Lymphoid progenitors from FL E14, ABM, and 5-FU-treated ABM were sorted, and transcripts for cell cycle regulators were detected by cDNA-based real-time amplification. The data are expressed relative to levels of ß-actin cDNA. Abbreviations: 5-FU, 5-fluorouracil; ABM, adult bone marrow; BrdU, bromo-2'-deoxyuridine; E, embryonic day; ELP, early lymphoid progenitor; FL, fetal liver; GFP, green fluorescent protein.

Rebounding Adult Lymphoid Progenitors Are Not Harmed by Chemotherapy and Have Some Functional Properties Typical of Their Fetal Counterparts
Stromal cell cocultures were used to compare the differentiation potential of fetal, adult, and rebound lymphoid progenitors (Fig. 7). All three populations produced B220/CD45R⁺ CD19⁺ GFP⁺ CD11b^– B-lineage lymphocytes in 3-week cultures on OP-9 stromal cells (Fig. 7A), whereas T-lineage cells were made when the same cells received a Notch signal by culture on OP9-DL1 stromal cells (Fig. 7B). In the latter circumstance, recovering lymphoid progenitors had a tendency to make T-cell receptor (TCR)-/⁺ rather than TCR-ß⁺ cells. Yields of lymphocytes were extremely dependent on the source of the progenitors (Fig. 7C). Approximately 2 logs more B-lineage cells were produced from fetal or rebound progenitors on OP-9 as compared with those freshly isolated from normal adult marrow. The situation was quite different with respect to T-lineage cell differentiation, in which only fetal progenitors yielded high numbers of T cells within 3 weeks of culture on OP9-DL1.

View larger version (37K): [in this window] [in a new window]   Figure 7. Lymphoid progenitors in rebound from chemotherapy resemble their fetal counterparts with respect to B-, but not T-, differentiation potential. Lymphoid progenitors from FL E14 mice, nontreated adult mice, or 4-day 5-FU-treated mice were cultured for 3 weeks on OP9-GFP or OP9-DL1 stromal cells. (A): B-lineage GFP+ CD19+ B220+ cells were stained after harvesting from OP9-GFP cocultures. Plots show the populations gated on CD45R/B220. (B): T-lineage TCR-ß+ and TCR-+ cells were assessed in OP9-DL1 cocultures. (C): The yields per input progenitor for each lineage are depicted in the time-course graphs. Abbreviations: 5-FU, 5-fluorouracil; ABM, adult bone marrow; E, embryonic day; ELP, early lymphoid progenitor; FL, fetal liver; GFP, green fluorescent protein; Pro-L, pro-lymphocyte; TCR, T-cell receptor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Environmental cues in a specialized BM niche may determine what fraction of stem cells gives rise to proliferating lymphoid progenitors, and a subset of those replenishes the immune system. The population dynamics of T lymphocyte-lineage cells in the thymus have been extensively studied, and many aspects of B-lymphocyte formation have been similarly investigated [2, 40–44]. However, no comparable information was available about the most primitive lymphoid progenitors, and our understanding of fetal/adult differences in lymphopoiesis was incomplete. Here, we have exploited Rag-1/GFP knockin mice [4] and BrdU incorporation by DNA-synthesizing cells to explore those important issues. Although more mitotically active than stem cells, the most primitive cells in the lymphoid series divide only once every 12 days. Changes that occur in ELPs during recovery from 5-FU treatment may reflect processes that are important during transplantation and chemotherapy.

ELPs begin the lymphoid differentiation program and express several lymphocyte-related genes at low levels but have greatly reduced myeloid potential relative to stem cells. They are part of the Lin^– Sca-1⁺ c-Kit^hi CD27⁺ Flk-2⁺ fraction of BM that has been variously referred to as multipotent progenitors (MPPs) or lymphocyte-primed MPPs [15, 37]. Although the RAG-1/GFP knockin model used here allows isolation of viable, functional ELPs, cells prepared this way may be very closely related to GFP^– ones expressing other lymphoid genes such as TdT [4, 45]. In any case, ELPs are potent progenitors for T-, B-, NK-, and plasmacytoid dendritic cell (pDC) lineages [4, 32, 46]. They likely give rise to Lin^– Sca-1⁺ c-kit^lo Pro-Ls that include most cells designated CLPs on the basis of IL-7R expression [31].

Neither of these two lymphocyte progenitor populations is homogeneous, and at any given time, they existed in two kinetic states. Additionally, both ELPs and Pro-Ls had a range of GFP levels, and this inversely corresponded to degrees of BrdU incorporation. That is, the GFP brightest progenitors labeled more slowly than the dim ones, making it tempting to propose an inverse relationship between activation of the RAG-1 locus and proliferation. There is a coordination of RAG-2 protein with the cell cycle, in parallel with fluctuations in the activity of cyclinA/CDK2 [47, 48]. RAG-2 preferentially accumulates in G₀/G₁, declines before cells enter S-phase, and remains low throughout the S-, G₂-, and M-phases. Although levels of RAG-1 show less fluctuation, it has been suggested that its E3 ligase activity may target cell cycle regulatory proteins [49]. Our experience with the RAG-1/GFP knockin mice indicates that endogenous RAG-1 is initially expressed in strict concordance with GFP levels [5], but decay of the GFP protein is thought to be slower than the RAG-1 protein [4, 50]. Regardless, initiation and progression of progenitor cells through the earliest stages of lymphopoiesis are unlikely to be synchronous.

Cells with low RNA content maintain a state of dormancy in G₀. As the cells enter G₁, they accumulate RNA mainly in the form of ribosomal RNA until reaching S-phase [28, 29]. Many long-term hematopoietic cell-initiating cultures in humans and stem cells in mice are present in the G₀ fraction of adult BM [16, 29, 39]. Cycling and other HSC behavior is thought to be regulated by coordination of environmental signals and intrinsic programs. The environmental cues may be provided by a niche composed of specialized cell populations located in unique sites. Arai and colleagues recently demonstrated that Lin^– Sca-1⁺ c-Kit^hi Tie-2⁺ BM in G₀ efflux dye and adhere to osteoblasts at the subendosteal bone surface. This and other recent studies are beginning to reveal signaling pathways, cytokines, and adhesion molecules that may regulate cell quiescence in the postulated stem cell niche [51–53].

Surprisingly, a substantial majority of primitive lymphoid progenitors were in G₀ and additional ones were in early G₁ (Fig. 1B). Although some of these cells are in proximity to the endosteal surface of the bone [54], that is not the case for a majority of the population and signals for quiescence might be delivered in other sites. That possibility accords with a recent report that quiescent HSCs can be found in tissues outside BM [55]. In any case, the slow population turnover for ELPs and Pro-Ls contrasts with the rapid proliferation of the large pro-B and pre-B cells that derive from them [2, 43, 44].

It is not known whether fetal lymphocyte progenitors are intrinsically different from those that arise as the hematopoietic system is replenished during adult life [1, 5]. BrdU labeling now shows that fetal lymphocyte progenitors divide considerably more rapidly than their adult counterparts, the rate of BrdU labeling gradually declining with gestational age (Fig. 3A). ELP and Pro-L populations in adult BM have low incidences, and their numbers remain constant without gross disturbance of the steady state. The primitive GFP^loc-kit^hiSca-1⁺ subset of fetal progenitors does not numerically increase in parallel with embryo size, whereas the more mature GFP^hic-kit^loSca-1^– fraction expands explosively from E15 [56]. Therefore, rapid differentiation, cell death, and/or export to other tissues must balance production of the earliest lymphopoietic cells.

Many previous studies have used 5-FU treatment to deplete actively proliferating marrow cells after which several characteristics of fetal stem cells are reactivated. Our analysis centered on day 4 after treatment because that is when c-kit⁺ RAG-1⁺ lymphoid progenitors began to emerge. This is consistent with previous descriptions of the recovery of HSCs [38, 57]. CD11b/Mac-1 and CD34 are present on fetal and rebound HSCs, but not those in BM of normal mature animals [36, 38]. Changes in marker expression might reflect either the recent regeneration of lympho-hematopoietic cells after chemotherapy or abnormalities in the marrow microenvironment. Although lymphoid progenitors did not appear to reacquire these two fetal markers during rebound, they were in active cell cycle and had at least one function characteristic of fetal cells.

Substantial information is available about proteins that govern progression through the cell cycle, and we sought explanations for fetal/adult/rebound disparities by extensive real-time RT-PCR analyses. Of particular interest were mediators that were dramatically elevated or depressed in fetal ELPs and rebound lymphoid progenitors as compared with those taken from adult marrow. These include cyclin D2, c-myc, Bcl-2, Ink4d/p19, Cip1Waf1/p21, Kip1/p27, HoxB4, and TGF-ß2R. Of that group, Bcl-2, Cip1Waf1/p21, and Kip1/p27 have been described as inhibitors of cell cycle progression [8, 16, 58] and thus match the pattern of quiescence we observed for adult ELPs. The last two of these have been implicated in cell cycle regulation in HSCs and multipotent progenitors, respectively [8]. Therefore, further investigation of these molecules might provide a mechanistic explanation for fetal/adult differences in proliferative activity.

Although ELPs are not stem cells, they can sustain lymphocyte production in the thymus for at least 6 weeks [4]. Furthermore, memory lymphocytes can expand and survive for long periods after participation in immune responses [59]. Telomerase is thought to be important for maintaining chromosome integrity in cells with such replicative potential, and its activity is known to be higher in fetal than adult life [60, 61]. Our results with ELPs correspond to that pattern, and it is interesting that telomerase activity increased during rebound from chemotherapy.

Some components of the immune system, especially CD4⁺ T lymphocytes, are slow to recover after the marrow ablation treatment used for chemotherapy and transplantation [62, 63]. However, we found no alteration in the T-lineage differentiation potential for post-5-FU treatment lymphoid progenitors. It could be that performance in the stromal cell coculture system we employed does not accurately reflect the ability to migrate to and colonize the thymus [30]. However, other culture assays for progenitors of NK and pDC also did not reveal an obvious influence of chemotherapy on differentiation potential (not shown). In striking contrast, formation of B-lineage lymphocytes from rebound progenitors was considerably greater than that from normal adult BM and equivalent to that observed with fetal cells.

As this manuscript was being completed, mitotic properties were described for hematopoietic cells defined with different criteria [64]. Some 50% of MPPs, a category that includes ELPs, were found to be in G₀. The MPP fraction also includes Lin^–c-Kit^HiRAG-1^– cells that we found to be 30% quiescent. Thus, RAG-1⁺ ELPs (67% in G₀) are less mitotically active than otherwise-similar cells. CLPs comprise most of the Pro-L fraction, and Passegue et al. reported that 74% were in G₀+G₁ [64], which is in general agreement with our finding of 85% for Pro-Ls.

Overall, this analysis provides important information about the earliest events in lymphopoiesis and raises a number of interesting questions. For example, do ELPs and stem cells share a common niche in BM and depend on the same signals to maintain quiescence? We also need to learn whether and how mitotic activity of lymphoid progenitors relates to their ability to differentiate and rapidly restore the immune system.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
We thank Drs. Michael Cancro and Linda Thompson for critical reading of this manuscript and suggestions. We also thank Robert Welner for valuable discussion, Jacob Bass and Diana Hamilton for expert sorting, Tara Khamphanthala for technical assistance, and Shelli Wasson for professional editorial assistance. This work was supported by Grants AI 20069 and AI 58162 from the National Institutes of Health. P.W.K. holds the William H. and Rita Bell Endowed Chair in Biomedical Research. This work was submitted in partial fulfillment of the requirements for the D.Sc. for R.P. at Universidad Nacional Autonoma de Mexico.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 

1. Baba Y, Pelayo R, Kincade PW. Relationships between hematopoietic stem cells and lymphocyte progenitors. Trends Immunol 2004;25:645–649.[CrossRef][Medline]

2. Mehr R, Shahaf G, Sah A et al. Asynchronous differentiation models explain bone marrow labeling kinetics and predict reflux between the pre- and immature B cell pools. Int Immunol 2003;15:301–312.[Abstract/Free Full Text]

3. Pelayo R, Welner RS, Nagai Y et al. Life before the pre-B cell receptor checkpoint: Specification and commitment of primitive lymphoid progenitors in adult bone marrow. Sem Immunol 2006;18:2–11.[CrossRef][Medline]

4. Igarashi H, Gregory SC, Yokota T et al. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 2002;17:117–130.[CrossRef][Medline]

5. Yokota T, Kouro T, Hirose J et al. Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression. Immunity 2003;19:365–375.[CrossRef][Medline]

6. Snodgrass R, Keller G. Clonal fluctuation within the haematopoietic system of mice reconstituted with retrovirus-infected stem cells. EMBO J 1987;6:3955–3960.[Medline]

7. Capel B, Hawley R, Covarrubias L et al. Clonal contributions of small numbers of retrovirally marked hematopoietic stem cells engrafted in unirradiated neonatal W/W^v mice. Proc Natl Acad Sci U S A 1989;86:4564–4568.[Abstract/Free Full Text]

8. Ezoe S, Matsumura I, Satoh Y et al. Cell cycle regulation in hematopoietic stem/progenitor cells. Cell Cycle 2004;3:314–318.[Medline]

9. Bradford GB, Williams B, Rossi R et al. Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment. Exp Hematol 1997;25:445–453.[Medline]

10. Cheshier SH, Morrison SJ, Liao X et al. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A 1999;96:3120–3125.[Abstract/Free Full Text]

11. Nakauchi H, Sudo K, Ema H. Quantitative assessment of the stem cell self-renewal capacity. Ann N Y Acad Sci 2001;938:18–24.[Medline]

12. Fan X, Valdimarsdottir G, Larsson J et al. Transient disruption of autocrine TGF-ß signaling leads to enhanced survival and proliferation potential in single primitive human hemopoietic progenitor cells. J Immunol 2002;168:755–762.[Abstract/Free Full Text]

13. Cashman JD, Eaves CJ, Sarris AH et al. MCP-1, not MIP-1, is the endogenous chemokine that cooperates with TGF-ß to inhibit the cycling of primitive normal but not leukemic (CML) progenitors in long-term human marrow cultures. Blood 1998;92:2338–2344.[Abstract/Free Full Text]

14. Cashman JD, Clark-Lewis I, Eaves AC et al. Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model. Blood 1999;94:3722–3729.[Abstract/Free Full Text]

15. Adolfsson J, Mansson R, Buza-Vidas N et al. Identification of Flt3⁺ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 2005;121:295–306.[CrossRef][Medline]

16. Cheng T, Rodrigues N, Shen H et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 2000;287:1804–1808.[Abstract/Free Full Text]

17. Taniguchi T, Endo H, Chikatsu N et al. Expression of p21^{Cip1/Waf1/Sdi1} and p27^Kip1 cyclin-dependent kinase inhibitors during human hematopoiesis. Blood 1999;93:4167–4178.[Abstract/Free Full Text]

18. Steinman R, Yaroslavskiy B, Goff JP et al. Cdk-inhibitors and exit from quiescence in primitive haematopoietic cell subsets. Br J Haematol 2004;124:358–365.[CrossRef][Medline]

19. Yaroslavskiy B, Watkins S, Donnenberg AD et al. Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells. Blood 1999;93:2907–2917.[Abstract/Free Full Text]

20. Brun AC, Bjornsson JM, Magnusson M et al. Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells. Blood 2004;103:4126–4133.

21. Satoh Y, Matsumura I, Tanaka H et al. Roles for c-Myc in self-renewal of hematopoietic stem cells. J Biol Chem 2004;279:24986–24993.[Abstract/Free Full Text]

22. Nakano T. Hematopoietic stem cells: Generation and manipulation. Trends Immunol 2003;24:589–594.[CrossRef][Medline]

23. Bruno L, Rocha B, Rolink A et al. Intra- and extra-thymic expression of the pre-T cell receptor alpha gene. Eur J Immunol 1995;25:1877–1882.[Medline]

24. Lu L, Smithson G, Kincade P et al. Two models of murine B lymphopoiesis: A correlation. Eur J Immunol 1998;28:1755–1761.[CrossRef][Medline]

25. Asquith B, Debacq C, Macallan DC et al. Lymphocyte kinetics: The interpretation of labelling data. Trends Immunol 2002;23:596–601.[CrossRef][Medline]

26. Lu L, Osmond DG. Apoptosis and its modulation during B lymphopoiesis in mouse bone marrow. Immunol Rev 2000;175:158–174.[CrossRef][Medline]

27. Kuwata N, Igarashi H, Ohmura T et al. Cutting edge: Absence of expression of RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent protein gene. J Immunol 1999;163:6355–6359.[Abstract/Free Full Text]

28. Darzynkiewica Z, Juan G, Srour EF. Differential staining of DNA and RNA. Current Protocols in Cytometry.Hoboken, NJ: John Wiley & Sons, Inc.,2005;7:3.1–7.3.16.

29. Gothot A, Pyatt R, McMahel J et al. Functional heterogeneity of human CD34(+) cells isolated in subcompartments of the G0/G1 phase of the cell cycle. Blood 1997;90:4384–4393.[Abstract/Free Full Text]

30. Huang J, Garrett KP, Pelayo R et al. Propensity of adult lymphoid progenitors to progress to DN2/3 stage thymocytes with Notch receptor ligation. J Immunol 2005;175:4858–4865.[Abstract/Free Full Text]

31. Kondo M, Weissman IL, Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 1997;91:661–672.[CrossRef][Medline]

32. Kouro T, Kumar V, Kincade PW. Relationships between early B- and NK-lineage lymphocyte precursors in bone marrow. Blood 2002;100:3672–3680.[Abstract/Free Full Text]

33. Osmond DG, Hales P. Methodology of lymphocyte kinetics. In: Herzenberg LA, ed. The Integrated Immune System. Weir's Handbook of Experimental Immunology.Cambridge: Blackwell Science, Inc.,1996;Vol IV:209.1–209.22.

34. Pelayo R, Welner R, Perry SS et al. Lymphoid progenitors and primary routes to becoming cells of the immune system. Curr Opin Immunol 2005;17:100–107.[CrossRef][Medline]

35. Kincade PW, Owen JJT, Igarashi H et al. Nature or Nurture? Steady state lymphocyte formation in adults does not recapitulate ontogeny. Immunol Rev 2002;187:116–125.[CrossRef][Medline]

36. Ogawa M. Changing phenotypes of hematopoietic stem cells. Exp Hematol 2002;30:3–6.[CrossRef][Medline]

37. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001;98:14541–14546.[Abstract/Free Full Text]

38. Randall TD, Weissman IL. Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood 1997;89:3596–3606.[Abstract/Free Full Text]

39. Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hematopoietic stem cells. Blood 1999;94:2548–2554.[Abstract/Free Full Text]

40. Porritt HE, Rumfelt LL, Tabrizifard S et al. Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages. Immunity 2004;20:735–745.[CrossRef][Medline]

41. Ceredig R, Rolink T. A positive look at double-negative thymocytes. Nat Rev Immunol 2002;2:888–897.[CrossRef][Medline]

42. Laurent J, Bosco N, Marche PN et al. New insights into the proliferation and differentiation of early mouse thymocytes. Int Immunol 2004;16:1069–1080.[Abstract/Free Full Text]

43. Rolink A, Karasuyama H, Haasner D et al. Two pathways of B-lymphocyte development in mouse bone marrow and the roles of surrogate L chain in this development. Immunol Rev 1994;137:185–201.[CrossRef][Medline]

44. Lu L, Osmond DG. Apoptosis during B lymphopoiesis in mouse bone marrow. J Immunol 1997;158:5136–5145.[Abstract]

45. Medina KL, Garrett KP, Thompson LF et al. Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen. Nature Immunol 2001;2:718–724.[CrossRef][Medline]

46. Pelayo R, Hirose J, Huang J et al. Derivation of two categories of plasmacytoid dendritic cells in murine bone marrow. Blood 2005;105:4407–4415.[Abstract/Free Full Text]

47. Lin WC, Desiderio S. Cell cycle regulation of V(D)J recombination-activating protein RAG-2. Proc Natl Acad Sci U S A 1994;91:2733–2737.[Abstract/Free Full Text]

48. Jiang H, Chang FC, Ross AE et al. Ubiquitylation of RAG-2 by Skp2-SCF links destruction of the V(D)J recombinase to the cell cycle. Mol Cell 2005;18:699–709.[CrossRef][Medline]

49. Sadofsky MJ. Recombination-activating gene proteins: More regulation, please. Immunol Rev 2004;200:83–89.[CrossRef][Medline]

50. Igarashi H, Kuwata N, Kiyota K et al. Localization of recombination activating gene 1/green fluorescent protein (RAG1/GFP) expression in secondary lymphoid organs after immunization with T-dependent antigens in rag1/gfp knockin mice. Blood 2001;97:2680–2687.[Abstract/Free Full Text]

51. Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149–161.[CrossRef][Medline]

52. Suda T, Arai F, Hirao A. Hematopoietic stem cells and their niche. Trends Immunol 2005;26:426–433.[CrossRef][Medline]

53. Li L, Xie T. Stem cell niche: Structure and function. Annu Rev Cell Dev Biol 2005;21:605–631.[CrossRef][Medline]

54. Hirose J, Kouro T, Igarashi H et al. A developing picture of lymphopoiesis in bone marrow. Immunol Rev 2002;189:28–40.[CrossRef][Medline]

55. Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109–1121.[CrossRef][Medline]

56. Yokota T, Tavian M, Hirose J et al. Tracing the first wave of lymphopoiesis in mice. Development 2006;133:2041–2051.[Abstract/Free Full Text]

57. Nishio N, Hisha H, Ogata H et al. Changes in markers, receptors and adhesion molecules expressed on murine hemopoietic stem cells after a single injection of 5-fluorouracil. STEM CELLS 1996;14:584–591.[Abstract]

58. O'Reilly LA, Harris AW, Tarlinton DM et al. Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo. J Immunol 1997;159:2301–2311.[Abstract/Free Full Text]

59. Lanzavecchia A, Sallusto F. Progressive differentiation and selection of the fittest in the immune response. Nat Rev Immunol 2002;2:982–987.[CrossRef][Medline]

60. Blasco MA, Funk W, Villeponteau B et al. Functional characterization and developmental regulation of mouse telomerase RNA. Science 1995;269:1267–1270.[Abstract/Free Full Text]

61. Blasco MA. Telomerase beyond telomeres. Nat Rev Cancer 2002;2:627–633.[CrossRef][Medline]

62. Fry TJ, Mackall CL. Immune reconstitution following hematopoietic progenitor cell transplantation: Challenges for the future. Bone Marrow Transplant 2005;35 (Suppl 1):S53–S57.[CrossRef][Medline]

63. Hakim FT, Memon SA, Cepeda R et al. Age-dependent incidence, time course, and consequences of thymic renewal in adults. J Clin Invest 2005;115:930–939.[CrossRef][Medline]

64. Passegue E, Wagers AJ, Giuriato S et al. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med 2005;202:1599–1611.[Abstract/Free Full Text]

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