Cell Cycle Quiescence of Early Lymphoid Progenitors in Adult Bone Marrow - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:58 |只看该作者 |倒序浏览 |打印

作者：Rosana Pelayoa, Kozo Miyazakia, Jiaxue Huanga, Karla P. Garretta, Dennis G. Osmondb, Paul W. Kincadea作者单位：aImmunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA;bDepartment of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada . c$ }; \9 `" b, ]

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      【摘要】
   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-kithi early lymphoid progenitors (ELPs) and RAG-1 c-kitlo 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. 1 ~% ?$ O% h/ T& u% N
      【关键词】 Adult stem cells B lymphopoiesis Fetal stem cells G Hematopoiesis Hematopoietic cell transplantation Hematopoietic progenitor cells Hematopoietic stem cell
   INTRODUCTION
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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 , 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 . The cell cycle status of primitive lymphoid progenitors represented the main focus of this study.6 i- y; X8 n7 a9 c) E" s
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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 . 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.9 z, j: g  S# Q8 l% U2 ]+ E

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) .; H' A4 F: H# S# n. B
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HSC cyclins are negatively regulated by cdkis .7 s+ e$ O7 s( m3 A* A

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 .3 W& q- s( [. H- ^4 z# F

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 .( j& c2 t6 y+ t+ R+ r

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." S! M: |2 ]! A: M; B
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MATERIALS AND METHODS! [9 v( ~1 B/ R  I& L& Y6 `6 T
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Mice and Cell Suspensions
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RAG-1/GFP knockin mice have been described  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¨C16 (E13¨CE16). 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¨C55 mice for adult BM studies were given an initial intraperitoneal injection of BrdU (100 µg/100 µl of phosphate-buffered saline ), 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 1.1 y4 t8 ]' ^" ^5 e; `$ j8 v$ u- C

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¨CGFP c-kithi (ELP) and Lin¨CGFP c-kitlo (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¨CE16) 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¨C, GFPlo, and GFPhi. Background autofluorescence was discriminated from authentic GFP by collecting data in two fluorescence channels without compensation .

Treatment with 5-FU4 `$ U) s8 L7 P0 B) B# F4 a
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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.
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Cell Cycle Fractionation with Hoechst and Pyronin Y
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A combination of Hoechst 33342 (Hst) and pyronin Y (PY) was used for the differential staining of cellular RNA and DNA, as described elsewhere , an arbitrary analysis window comprising approximately 15% of fetal liver cells displaying minimal PY staining was used to designate G0 fraction in all experiments.
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Real-Time PCR Analysis of Cell Cycle Gene Expression  I* ~* }/ ^: J5 m; ~. E

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¡ãC followed of 40 cycles of 95¡ãC for 15 seconds and 60 seconds at 60¡ãC in an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The relative gene expression was calculated using ß-actin cDNA as an endogenous control .

Telomerase Activity Detection9 \$ k& J: I2 K2 r1 P$ [& G

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.# S( E4 K! |1 l
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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. Z¨²ñiga-Pfl¨¹cker, University of Toronto, Ontario, Canada), as previously described  with modifications. During the first week, the -minimal essential medium 10% FCS contained 5 x 10¨C5 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.
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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-kithi fraction of lineage-marker negative (Lin¨C) BM, with one of the distinguishing characteristics of ELPs being activation of the RAG-1 locus .9 F3 |, g# O+ i  K

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 . 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 c-kithi GFP¨C 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.8 x% y; c4 _3 @2 ^% {4 s/ u4 O0 t
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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¨CGFP¨Cc-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¨CGFP¨Cc-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.( r7 S' q7 x! y& a# {" g

The designation ELP does not imply homogeneity . Rather, the most primitive lymphoid cells express TdT, RAG-1, a human µ transgene (in transgenic mice), or some combination of these characteristics. Also, levels of GFP vary among cells from RAG-1/GFP mice gated for any given set of cell surface markers. We now report that ELPs with the highest levels of RAG-1 incorporate BrdU at a substantially lower rate than ELPs with less RAG-1. This analysis was performed after 24, 36, 120, and 156 hours of continual treatment with BrdU (Fig. 2 and data not shown). The same parameter revealed similar heterogeneity among Pro-Ls, the cells showing the highest rate of BrdU uptake having relatively lower levels of RAG-1. Therefore, the kinetic data shown in Figure 1 represent average values for cell subsets that are not homogenous with respect to mitotic and RAG-1 gene activity. Variations in RAG-1 gene transcriptional activity could be inversely linked to proliferation such that Ig gene recombination can safely occur.

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.! N. H! ^: t, f
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The Lymphopoietic Series in Fetal Liver Is Rapidly Dividing& r. O- j5 V9 a$ C" H' K! l
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Many studies have revealed fetal/adult differences in lymphopoiesis, raising important questions about the properties of stem cells present at different developmental ages . An analysis of cell cycle status indicated that approximately 20% of E14 fetal liver c-kithi Sca-1  RAG-1/GFPlo ELPs were in the S G2 M phases of the cell cycle and an additional 58% were in G1 (Fig. 3A). Similarly, high values for the proportion of cycling cells were observed with all other lymphoid populations in the E14 fetal liver, including cells in the c-Kitlo Sca-1¨C RAG-1/GFPhi category, which are largely CD19 . These observations, consistent with relatively short cell cycle times, demonstrate a high growth fraction of proliferating early lymphoid cells in the fetal liver.
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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¨Cc-kithi cells sorted from E14 fetal liver. (B): Pregnant mice (E13¨CE16) 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.' {  z9 }8 U* S, p( r

We then injected pregnant mice with BrdU and performed an incorporation analysis of fetal liver subsets 6 hours later (Fig. 3B). Unlike the case with adult animals, very high labeling indices could be observed during this short interval. Indeed, at 13 days of gestation, more than 70% of all lymphoid cells incorporated BrdU in 6 hours. Thereafter, BrdU labeling indices for most subsets tended to decline with gestational age but were extremely high at all time points relative to adult BM progenitors. In fact, BrdU incorporation rates for adult ELPs were less than fetal ELPs even after 6 days of BrdU injection.& R5 {; R; D: X% A/ A# Z2 a
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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 .# Y. @4 \' }9 T% g" a

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¨CGFP  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.' W( J9 g, q. C- {2 O
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As previously reported, BM hematopoietic cells at early times post 5-FU treatment expressed only low levels of the c-kit receptor for SCF (Fig. 4B) . Therefore, in subsequent experiments conducted at 4 days after treatment, we did not use c-kit density to discriminate ELPs from Pro-Ls. A minority expressed detectable amounts of IL-7R (data not shown), and we assume that most progenitors at that time would be recently generated and thus equivalent to ELPs. Very small numbers of Lin¨C GFP  c-kit  cells displayed low levels of the CD11b myeloid marker after 5-FU treatment, but this was not comparable with ELPs or Pro-Ls present in fetal liver (supplemental online Fig. 3). Furthermore, the CD34 marker characteristic of fetal stem/progenitor cells was not re-expressed on adult lymphoid progenitors in rebound (supplemental online Fig. 3).
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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).
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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¨CGFP 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¨CGFP 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.

Mitotic activity was also assessed by measuring transcripts for key cell cycle mediators (Fig. 6). Transcripts for cyclin A2, cyclin D1, and CDK4 were much lower in adult than fetal progenitors, and the pattern was not reversed after chemotherapy. CDK2 and Gfi-1 were low in fetal cells and again unchanged by 5-FU treatment. In contrast, levels of cyclin D2, c-myc, Bcl-2, Ink4d/p19, Cip1Waf1/p21, Kip1/p27, HoxB4, and TGF-ß2R levels in lymphoid progenitors from rebound adult BM closely resembled those from fetal liver.9 @: R0 ?5 Q" k7 j$ T: W1 N

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.8 B3 j1 O0 `& _% p

Thus, some, but not all, aspects of the fetal differentiation program could be reacquired by adult lymphoid progenitors during rebound from chemotherapy. Levels of 8 of 13 cell cycle regulators paralleled re-entry into rapid division, but there was no corresponding re-expression of the fetal cell surface markers CD11b and CD34.0 C0 B1 w& ^% c/ h( ]+ B
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Rebounding Adult Lymphoid Progenitors Are Not Harmed by Chemotherapy and Have Some Functional Properties Typical of Their Fetal Counterparts
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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¨C 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.
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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.6 U' t& E: h, c! U: G$ q! o

Thus, chemotherapy by 5-FU did not alter the ability of adult lymphoid progenitors in rebound to generate T-lineage cells. Remarkably, B lymphopoiesis from rebound adult BM progenitors was as robust as that typical of fetal cells.

DISCUSSION$ X: G! @3 o  e* w( B

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  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.9 j  M1 U8 D; C* W! C
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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¨C Sca-1  c-Kithi CD27  Flk-2  fraction of BM that has been variously referred to as multipotent progenitors (MPPs) or lymphocyte-primed MPPs .4 _) o7 f8 H% [: d5 E9 X
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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 . Regardless, initiation and progression of progenitor cells through the earliest stages of lymphopoiesis are unlikely to be synchronous.; @& {+ T% D0 G( A( J9 D) I( R6 m

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 .8 M) N6 y. T5 n* e! \! l- N/ D

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 .

It is not known whether fetal lymphocyte progenitors are intrinsically different from those that arise as the hematopoietic system is replenished during adult life . Therefore, rapid differentiation, cell death, and/or export to other tissues must balance production of the earliest lymphopoietic cells.1 \) W% j5 U% c
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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 . 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.+ E, J& h3 x; T2 }

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 . Therefore, further investigation of these molecules might provide a mechanistic explanation for fetal/adult differences in proliferative activity.0 `6 J) t' t/ o; e) b3 j9 U7 e( Y
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Although ELPs are not stem cells, they can sustain lymphocyte production in the thymus for at least 6 weeks . Our results with ELPs correspond to that pattern, and it is interesting that telomerase activity increased during rebound from chemotherapy.
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Some components of the immune system, especially CD4  T lymphocytes, are slow to recover after the marrow ablation treatment used for chemotherapy and transplantation . 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 , which is in general agreement with our finding of 85% for Pro-Ls.4 ^/ h4 _9 E% E0 w

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." Q/ j& \/ l3 G. ]* Q
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS

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.
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