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The Journal of Immunology, 2001, 166: 3042-3051.
Copyright © 2001 by The American Association of Immunologists

Phenotypic Distinction and Functional Characterization of Pro-B Cells in Adult Mouse Bone Marrow1

Mariluz P. Mojica*, S. Scott Perry{dagger}, A. Elena Searles{ddagger}, Kojo S. J. Elenitoba-Johnson{dagger}, L. Jeanne Pierce§, Anne Wiesmann§, William B. Slayton and Gerald J. Spangrude{dagger},{ddagger},§

Departments of * Human Genetics, {dagger} Pathology, {ddagger} Medicine (Division of Hematology), § Oncological Sciences, and Pediatrics, University of Utah, Salt Lake City, UT 84132


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1negSca-1posc-KitlowLinneg. These cells were CD43posCD24pos on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Combinations of surface proteins expressed by cells at different stages of blood cell development have enabled the isolation and subsequent functional characterization of phenotypically defined hemopoietic progenitor cell populations. In the adult mouse, the bone marrow (BM)3 is the site of hemopoiesis where mature blood cell lineages are generated from self-renewing multipotent hemopoietic stem cells (HSC) (1). These HSC express low levels of Thy-1.1 and high levels of stem cell Ag-1 (Sca-1pos) and lack expression of lineage-associated markers (Linneg) (2). Phenotypically defined progenitor cell populations with restricted myeloid and/or lymphoid lineage potentials have recently been described (3, 4, 5, 6). Expression of CD45R has been used by a number of investigators to isolate and characterize lymphoid progenitors (7, 8), while stages of B lymphoid development before expression of CD45R are only beginning to be explored (4, 9, 10).

A goal in enriching for early B lymphoid progenitors is to separate these cells from both HSC and differentiated cells of the B and other blood cell lineages. The latter can be achieved by depleting BM of cells expressing CD45R and other lineage-associated markers. Segregating early lymphoid progenitor cells from HSC has been more challenging. Previous studies have shown that although the Thy-1.1lowSca-1posLinneg (Thy-1.1low) cell population is markedly enriched for HSC, it is functionally heterogeneous (11, 12). This heterogeneity, which is reflected in the ability of the cell population to mediate both short term and long term BM engraftment, can be dissected using separations based on cell cycle activity and activation state (13, 14). Although experiments designed to segregate progenitors for lymphoid lineages have shown that subsets of Thy-1.1low HSC engraft in the BM, the kinetics of B cell engraftment exhibit a 2-wk delay relative to whole BM transplants (15). This suggests that a lymphoid progenitor cell population that is present in normal BM is missing from the Thy-1.1low HSC population.

The c-Kit molecule has also been used extensively as a cell surface marker in the purification of HSCs (16, 17, 18). We recently found that the c-Kitpos subset of Sca-1posLinneg BM cells contains two populations of cells which differ with respect to expression of Thy-1.1. The majority of Thy-1.1low cells express c-Kit at high levels (c-Kithigh), while Sca-1posLinneg BM cells lacking the Thy-1.1 Ag (Thy-1.1neg cells) include c-Kitlow as well as c-Kithigh subsets. Transplant studies demonstrated that the Thy-1.1lowc-Kithigh subset mediated full hemopoietic engraftment of lethally irradiated recipient animals with prominent erythroid, myeloid and platelet reconstitution and delayed lymphoid engraftment (19). In contrast, the Thy-1.1neg subset failed to provide erythroid and platelet engraftment to transplant recipients, but mediated rapid lymphoid engraftment with a minor degree of myeloid recovery. In the absence of full hemopoietic recovery, transplant recipients of Thy-1.1neg cells survive only 25–30 days before death due to hemopoietic failure.

The current studies were initiated to resolve the lymphoid and myeloid potentials of Thy-1.1neg cells at a clonal level. Using a number of additional phenotypic markers, we demonstrate three separate progenitor populations within the Thy-1.1neg subset of Sca-1posLinneg BM cells. These include separate committed progenitors for lymphoid and myeloid (predominantly macrophage) lineages as well as a mixed lineage progenitor population. Interestingly, expression of the AA4.1 Ag defines the onset of Pax5 transcription and marks the loss of pro-T cell potential. Thus, these studies have defined a very early stage of mouse pro-B cell development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse strains

B6.PL and AKR mice were obtained from The Jackson Laboratory (Bar Harbor, ME), while C57BL/Ka, B6-Thy-1.1-Ly-5.1, and B6-Ly-1.1 mice were bred and maintained at the Animal Resource Center facility of the University of Utah. Mice used were 4–16 wk of age.

Cytokines and Abs

Steel factor (STL) and G-CSF were gifts from Gemini Science (San Diego, CA), a subsidiary of Kirin Brewery (Tokyo, Japan). Flt3 ligand (Flt3L) and IL-6 were kindly provided by Immunex (Seattle, WA). Recombinant human erythropoietin (Epo) was purchased from Ortho (Raritan, NJ). Recombinant murine IL-3 and IL-7 were purchased from PeproTech (Rocky Hill, NJ). The cytokines were used at the following concentrations: STL, 100 ng/ml; G-CSF, 10 ng/ml; Flt3L, 75 ng/ml; IL-6, 20 ng/ml; Epo, 5 U/ml; IL-3, 10 ng/ml; and IL-7, 10 ng/ml.

mAbs against CD8 (53-6.7), Mac1 (M1/70), erythrocytes (TER119), Gr-1 (RB6-8C5), CD3 (KT3-1.1), CD5 (53-7.3), CD2 (Rm2.2), mouse Ig (RAM-HB58), CD45R (RA3-6B2), Thy-1.1 (19XE5), c-Kit (ACK-4), early B cell (AA4.1), and IL-7R were purified from media of cultured hybridoma cell lines, while the mAb against CD19 was purchased from PharMingen (San Diego, CA). mAbs used for cell surface staining of CD45R, Thy-1.1, c-Kit, Gr-1, CD62L, AA4.1, and IL-7R were conjugated with biotin, PE, or FITC in our laboratory. Biotinylated Abs were secondarily stained with either PE-streptavidin (PE-SAv; Biomeda, Foster City, CA) or PE-Texas Red-SAv (Red613; Life Technologies, Grand Island, NY). In addition, PE-conjugated mAbs to Sca-1, Mac-1, and CD45R, biotinylated Abs to CD24 (M1/69) and CD43 (S7), and allophycocyanin-conjugated c-Kit Ab were purchased from PharMingen. The IL-7R clone used in these studies was a gift from Richard R. Hardy (Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA).

Preparation of BM cells and isolation of hemopoietic stem/progenitor cell populations

The procedure for the preparation of BM cells for sorting has been previously described (20). Briefly, BM cells were isolated from femurs and tibia of donor mice, and the RBCs were lysed in an ammonium chloride potassium solution. The cells were incubated in a lineage cocktail containing optimized concentrations of Abs to CD2, CD3, CD5, CD8, Mac-1, Gr-1, TER119, CD45R, and CD19. The CD45R Ab was not included in the lineage cocktail whenever CD45R expression was evaluated after lineage depletion. Lineage depletion was conducted by two successive incubations of the BM cells in sheep anti-rat Ig-coupled magnetic beads (Dynal, Oslo, Norway). The Linneg cells were stained with PE-Sca-1 and sorted using the FACSVantage (Becton Dickinson, San Jose, CA) set at enrichment mode and thresholding on PE emissions above background levels. Dead cells were excluded from all analyses and sorts by gating on forward scatter and PI staining. The sorted Linneg Sca-1pos cells were pelleted and stained with allophycocyanin-c-Kit and FITC-Thy-1.1 and resorted into Thy-1.1lowc-Kitpos and Thy-1.1negc-Kitpos subsets. In experiments in which the Thy-1.1negc-Kitpos subset was further fractionated, the appropriate biotin-conjugated Ab stain was added and visualized using Red613-SAv. All cell sorting steps were performed using the FACSVantage, and an aliquot of the sorted cell population was always taken for reanalysis.

Methylcellulose assays

The sorted cell populations were cultured in methylcellulose at a plating density of ~100 cells/35-mm culture dish. Each milliliter of culture medium contained {alpha}-MEM (Life Technologies), 1.2% methylcellulose (Shinetsu, Tokyo, Japan), 30% FCS (Life Technologies), 1% deionized BSA (Sigma, St. Louis, MO), and 0.1 mM 2-ME (Mallinckrodt, Chesterfield, MO) supplemented with the indicated cytokine combinations. Culture dishes were incubated at 37°C and infused with 5% CO2. The number of colonies was counted using an inverted microscope after 7 days of culture to determine the cloning efficiency of each sorted cell population. Four to six plates were scored for each group, and the results were expressed as percentage of the total cells plated. In addition, individual colonies were plucked between days 6 and12 of culture and analyzed for both cell surface staining and cell morphology. Two-thirds of the cells harvested from each colony were stained with Abs to CD45R and Gr-1 and analyzed by flow cytometry, while cytospins were prepared from the remaining cells. Cytospins were stained with May-Grunwald-Giemsa for morphological analysis.

Liquid cultures

Liquid cultures of sorted cell populations were conducted using {alpha}-MEM (Life Technologies) containing 10% FCS, 1 mM MEM sodium pyruvate solution (Life Technologies), 10 mM HEPES (pH 7.3), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 0.1 mM 2-ME (Mallinckrodt) and supplemented with the indicated cytokine combinations. Cells were either grown in bulk in 24-well plates or seeded at limiting dilution (one cell per well) in 96-well plates with or without stromal cell feeder layers as indicated. Culture plates were incubated at 37°C and infused with 5% CO2. The 2018 stromal cell line (a gift from Kateri Moore) was maintained at 31–33°C. Cells were prepared the day before coculture by seeding 10,000 cells/well in 24-well plates for bulk cultures and 1,000 cells/well in 96-well plates for the limited dilution (clonal) assays. The presence of a single cell per well in 96-well plates was confirmed whenever possible after overnight culture using an inverted microscope. Positive clones (wells) were scored by day 5 and harvested for analysis between days 6 and 14. For bulk cultures, representative wells were harvested for analysis between days 5 and 14.

Intrathymic T cell development assay

Sublethally irradiated B6 (4- to 6-wk-old females) mice were anesthetized and immobilized with rubber bands. The skin over the chest was incised to reveal the sternum, which was cut. The thymus was visualized within the thoracic cavity, and 3 µl of fluid containing the sorted cell population of interest was directly injected into the thymic tissue using a Hamilton syringe (Reno, NV). The chest was closed using stainless steel surgical clips. The cells to be transplanted were obtained from the B6-Thy-1.1-Ly-5.1 double-congenic strain and were sorted directly into a microfuge tube containing a known amount of Hanks’ 10% FCS so that each 1 µl of fluid contained a known number of cells. Graded doses of cells were injected into groups of animals (10 animals/group) in the presence of an excess of Linneg cells obtained from a second B6 congenic strain (B6-Ly-1.1), which served as a carrier and as an internal control to indicate successful injections. Three or 4 wk later, the recipient B6 mice were sacrificed, and thymic tissue was isolated for analysis by flow cytometry to identify thymic lobes containing progeny cells derived from the injected populations. Successful intrathymic transfers were identified by the presence of Ly-1.1pos cells, and positive thymic lobes were scored for the presence of Ly-5.1pos cells. Limiting dilution statistics were applied to the resulting data to derive the frequency of repopulating cells in the sorted population.

RT-PCR assay

Sorted cells were lysed using 500 µl of TRIzol (Life Technologies) with 20 µg of glycogen (Roche, Indianapolis, IN) added as a carrier. The TRIzol protocol for RNA isolation prescribed by the manufacturer was followed using half volumes. After isopropanol precipitation, the RNA pellet was washed twice in 70% ethanol and resuspended in 8 µl of diethylpyrocarbonate-treated water. The RNA samples were incubated with 1 µl of amplification grade DNase I (Life Technologies) and 1 µl of 10x DNase I buffer at room temperature for 15 min to eliminate any contaminating DNA. The reaction was stopped with the addition of 1 µl of 25 mM EDTA and heating at 72°C for 10 min. Water was added to bring the total volume of each reaction to 20 µl. Five to 10 µl from each total RNA sample was used for first-strand synthesis using random primers (Life Technologies) and Moloney murine leukemia virus reverse transcriptase (Life Technologies) following the protocol provided by the manufacturer.

Semiquantitative PCR was used to compare the expression of genes between sorted cell populations. All primer sequences used in this study have been previously described. To equalize for cDNA input, each sample was first amplified by PCR using GAPDH primers (21), and the amount of input cDNA was adjusted to provide equivalent signals. Subsequent PCR amplifications used the predetermined amount of cDNA with gene-specific primers for sterile Ig heavy chain transcript µo, Rag-2, E2A, Pax-5, and CD19 (7, 22, 23). PCR cycle parameters used for GAPDH, µo, and Rag-2 were described by Li et al. (22), while those for E2A, Pax-5, and CD19 were described by Bain et al. (23). Fifteen-microliter aliquots were withdrawn at cycles 24, 27, and 30 (GAPDH) or cycles 27, 30, and 33 (µo, Rag-2, E2A, Pax-5, and CD19) to assure that amplification was within the linear range. The PCR products were separated by 1% agarose gel electrophoresis. Quantitation was performed using the MultiAnalyst program (Bio-Rad, Hercules, CA). Individual bands were measured and normalized using the GAPDH signal for each sample. Comparison of gene expression between samples was achieved by comparing the normalized value for each sample to the value obtained for CD45Rpos cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Thy-1.1neg cell population contains three separate progenitor subsets

Linneg mouse BM cells were stained with Abs to Sca-1, c-Kit, and Thy-1.1 and sorted to recover the Thy-1.1low and Thy-1.1neg cell populations (Fig. 1Go). The Thy-1.1neg subset comprised ~0.05 ± 0.01% (mean ± SD; n = 8) of nucleated BM cells, a frequency very similar to that of the Thy-1.1low subset as previously reported (1, 2). Virtually all cells expressing low levels of Thy-1.1 were c-Kithigh, while the Thy-1.1neg population included cells expressing both low and high levels of c-Kit. Cells lacking c-Kit expression were not further characterized in these studies. Because of concerns regarding contamination of Thy-1.1neg cell preparations with Thy-1.1low HSC, reanalysis of sorted populations was always performed as shown in Fig. 1Go, and Thy-1.1neg populations containing any discernable contamination with cells expressing Thy-1.1 at a level 5- to 10-fold above background levels were not used for functional studies. Although our previous transplant studies demonstrated an inhibitory influence of allophycocyanin-conjugated c-Kit Abs on in vivo engraftment of Thy-1.1neg cells (19), direct comparisons of cloning efficiencies and lineage potentials of Thy-1.1neg cells isolated using biotin or allophycocyanin conjugates of anti-c-Kit Abs showed no differences in our in vitro studies.



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  		FIGURE 1. BM cells isolated from adult mice were lineage depleted and subsequently stained with mAbs to c-Kit, Sca-1, and Thy-1.1. Dead cells were excluded using propidium iodide staining and forward scatter gating. Sca-1pos cells were selected by gating (A) and analyzed for Thy-1.1 expression (B). Thy-1.1negSca-1posc-Kitpos Linneg (Thy-1.1neg) and Thy-1.1low Sca-1posc-Kitpos Linneg (Thy-1.1low) cells were isolated by cell sorting as described in Materials and Methods, and aliquots were taken for reanalysis (C and D).

 
Initial in vivo transplant studies demonstrated that the Thy-1.1neg cell population mediates rapid BM engraftment and contributes to both lymphoid and myeloid lineages (19). To determine whether the Thy-1.1neg cell subset consisted of multipotent progenitors or separate progenitor cells committed to either lymphoid or myeloid lineages, we conducted clonal progenitor cell assays. The sorted cells were cultured in methylcellulose or single-cell liquid cultures supplemented with different cytokine combinations, and individual colonies were analyzed after 6–12 days in culture by both flow cytometry and cell morphology. These analyses showed that the Thy-1.1neg cell subset contained three types of progenitors (Table IGo). Stimulation with a mixture of seven cytokines, as detailed in Table IGo, allowed differentiation of multiple hemopoietic lineages. Under these culture conditions, we observed colonies consisting solely of myeloid lineage cells (macrophages, primitive granulocytes, and erythroid cells), colonies consisting solely of lymphoid lineage cells (CD45RposGr-1neg), and mixed colonies containing both lineages. Lymphoid lineage colonies represented 33% of the total Thy-1.1neg colonies analyzed, while those consisting of myeloid lineage cells represented 54% (Table IGo). Mixed lineage colonies, containing CD45Rpos cells as well as myeloid cells, were observed at a frequency of 13%. Similar results were obtained in liquid cultures initiated from single cells, suggesting that the mixed lineage colonies were not the result of sampling error in the methylcellulose assay. When Thy-1.1low HSC were cultured under the same conditions, pure lymphoid colonies were not observed, and very few mixed lineage colonies containing CD45Rpos cells were scored (4%, or 2 colonies of 45 examined; Table IGo). Stimulation of Thy-1.1low HSC with lymphoid-specific cytokines (STL, IL-7, and Flt3L) resulted in a marked decrease in cloning efficiency (88 vs 581 colonies/1000 cells plated).


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  		Table I. Cloning efficiency and lineage potential of Thy-1.1low and Thy-1.1neg subsetsa

 
IL-3 has been reported to inhibit early B lymphoid differentiation (24, 25). Consistent with this observation, we observed that omission of IL-3 from the cytokine cocktail resulted in a 40–50% decrease in the cloning efficiency of Thy-1.1neg cells. Analysis of the colonies that grew in the absence of IL-3 demonstrated a decrease in the frequencies of pure myeloid and mixed colonies and a concomitant increase in the proportion, but not the absolute number, of pure lymphoid colonies. Comparison between S7F6EG stimulation with or without IL-3 showed that 33% of 148 ± 44 colonies were lymphoid in the presence of IL-3 (48.8 ± 14 colonies/1000), whereas 60% of 81 ± 29 colonies were lymphoid in the absence of IL-3 (48.6 ± 17 colonies/1000; Table IGo). Thy-1.1neg cells grown in methylcellulose cultures supplemented with cytokines permissive only for lymphoid differentiation (S7F) exhibited a 6-fold decrease in cloning efficiency compared with the more complex cytokine combination, and all colonies evaluated from these cultures contained only cells with lymphoid morphology and surface Ag expression. The presence of pure lymphoid colonies after stimulation of Thy-1.1neg cells with the complex cytokine mixture confirms that a committed lymphoid progenitor population is contained within the Thy-1.1neg cell subset, but not in the Thy-1.1low HSC population. These cells grow in response to the cytokine combination of S7F and are insensitive to the inhibitory effects of IL-3.

A number of early B lymphoid markers fractionate the Thy-1.1neg cell population into distinct subsets

To determine whether additional markers could potentially fractionate the Thy-1.1neg cell population into functionally distinct subsets, we isolated Thy-1.1neg cells and evaluated the expression of a number of cell surface markers known to be expressed during the early stages of lymphoid development. Representative FACS plots of the staining analyses are shown in Fig. 2Go. As shown in Fig. 2GoA, the AA4.1 mAb identifies a subset comprising 30–50% of the Thy-1.1neg cells that expresses low levels of c-Kit. Expression of IL-7R also separated the Thy-1.1neg population into two distinct clusters that correlated with c-Kit staining intensities. Cells that were IL-7Rpos invariably expressed low levels of c-Kit, while IL-7Rneg cells were both c-Kitlow and c-Kithigh (Fig. 2GoB). In contrast, CD62L/MEL14 expression was observed on both the c-Kitlow and the c-Kithigh subsets of Thy-1.1neg cells (Fig. 2GoC). Of the other cell surface Ags tested, CD4 staining revealed only a minor subpopulation of positive cells (10% of Thy-1.1neg cells, equally distributed between the c-Kitlow and c-Kithigh subsets, Fig. 2GoD), which was similar to the distribution of the Sca-2 Ag (data not shown). CD3 served as a negative control and was expressed by <1% of Thy-1.1neg cells (Fig. 2GoE). Since expression of AA4.1 and IL-7R have previously been associated with early lymphoid progenitors (4, 6, 7, 26), we focused on the c-Kitlow subset of cells for additional phenotypic and functional analysis.



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  		FIGURE 2. BM cells from young (5- to 6-wk-old) adult mice were lineage depleted using an Ab cocktail as described in Materials and Methods, substituting a CD19 monoclonal in place of CD45R. This population was subsequently stained with mAbs to Sca-1, c-Kit, and Thy-1.1 and sorted for Thy-1.1neg and Thy-1.1low cells as described in Fig. 1Go. Sorted cell populations were then stained with mAbs to early lymphoid markers for subsequent analysis by FACS. A–E, Analyses of Thy-1.1neg CD45Rneg cells. F, The c-Kitlow subset defined in plots A–E was selected by gating for analysis of CD45R expression on c-Kitlow cells. G and H, Expression of CD24 and CD43 by Thy-1.1low c-Kithigh HSC () compared with Thy-1.1neg c-Kitlow lymphoid progenitors ({square}). Plot I shows gating for CD45Rneg and CD45Rpos subsets of Thy-1.1neg c-Kitlow cells, which were defined using a control stain of lymph node lymphocytes () to establish a sorting gate for negative and positive cells.

 
A hierarchy of progenitors in the B lymphocyte developmental pathway has been described by Hardy and colleagues, who used cell surface Ag expression to define specific developmental stages (4, 7, 27). To better place the Thy-1.1negc-Kitlow progenitor population in the context of the previous studies, we evaluated CD45R, CD24, and CD43 expression by these cells. Unlike the primitive pro-B cell described by Hardy’s stage A, Thy-1.1negc-Kitlow cells largely lack expression of CD45R and express high levels of CD24 (Fig. 2Go, F and G). In common with Hardy’s stage A of development, Thy-1.1neg c-Kitlow cells express lower levels of CD43 relative to Thy-1.1low stem cells (Fig. 2GoH). Hardy and colleagues used expression of AA4.1 along with CD4, CD24, and CD43 to identify a CD45Rneg stage of B cell development preceding the A stage (7), but the isolation protocol used in those studies failed to segregate these cells away from erythroid lineage progenitors (4). Furthermore, functional assessment of B lineage precursors in adult mouse bone marrow established that most early B lineage progenitors express CD24, as detected using either the M1/69 or 30-F1 Ab (9). To summarize, Thy-1.1negc-Kitlow cells overlap with LinnegTdT+ cells as defined by Tudor et al. (9), but differ from fraction A0 as defined by Hardy and colleagues (7) in that most Thy-1.1negc-Kitlow cells lack CD4 and express high levels of CD24 (Fig. 2Go, D and G).

The lymphoid-committed progenitors in the Thy-1.1neg cell population are predominantly c-Kitlow

To address the developmental potential of the individual subsets of cells isolated in the Thy-1.1neg population, we used levels of c-Kit and AA4.1 expression as selection criteria to sort the Thy-1.1neg population into three subsets: c-KithighAA4.1neg, c-KitlowAA4.1neg, and c-KitlowAA4.1pos (Fig. 2GoA). Methylcellulose and liquid cultures were initiated with each c-Kit/AA4.1 subset to ascertain their cloning efficiencies and myeloid and/or lymphoid differentiation potential. These cultures were supplemented with cytokine combinations selected to either support proliferation and differentiation toward the lymphoid lineage (STL+ IL-7 + Flt3L, S7F), the myeloid lineage (IL-3 + IL-6 + Epo + G-CSF, 36EG), or both lymphoid and myeloid lineages (STL + IL-7 + Flt3L + IL-3 + IL-6 + Epo + G-CSF, S7F36EG). Evaluation of the cloning efficiencies of the subsets showed that c-KithighAA4.1neg cells cloned at a frequency of 20% in S7F36EG and gave rise to myeloid or mixed colonies, but not pure lymphoid colonies (Table IIGo). Cloning efficiency dropped to 5% when the cells were stimulated under lymphoid conditions. In contrast, the two c-Kitlow populations resolved by AA4.1 staining showed equivalent colony growth in either lymphoid-specific or complex cytokine conditions, suggesting that the majority of clonogenic c-Kitlow cells are lymphoid committed (Table IIGo). This interpretation was strengthened by flow cytometric and cytospin analysis of individual colonies grown in S7F36EG. Of 99 separate c-KitlowAA4.1pos and c-KitlowAA4.1neg colonies analyzed, 88 (89%) contained only lymphoid lineage cells despite the presence of myeloid-promoting cytokines in the cultures. Mixed lineage colonies were most frequent in the c-KithighAA4.1neg subset, where they represented 15% of the total colonies. These progenitors were also identified in both the AA4.1pos and AA4.1neg subsets of c-Kitlow cells, with their frequency being higher in the AA4.1neg subset (13% of total colonies, compared with 6% among the AA4.1pos cells). Similar results were obtained in single-cell liquid cultures, suggesting that mixed lineage colonies were not due to inadvertant mixing of adjacent single-lineage colonies. Cytospin analysis confirmed the presence of myeloid cells as detected by flow cytometry. A limited variety of myeloid cells were observed. Neutrophil morphology was limited to very primitive-appearing cells with crescent nuclei. In contrast, mature-appearing macrophages were consistently observed in cytospin preparations. Purely myeloid colonies arising from Thy-1.1negc-Kitlow cells were very rare (2% of total colonies), and colonies containing erythroid lineage cells based on benzidine staining of hemoglobin were never observed in the myeloid or mixed colonies derived from Thy-1.1negc-Kitlow cells. These results confirm previous observations that pro-B cells are characterized by a low level of c-Kit expression (9, 28), and extend these findings by showing that these early pro-B cells can be completely separated from progenitors for nonlymphoid hemopoietic lineages using the Thy-1.1neg selection protocol.


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  		Table II. Cloning efficiency and lineage potential of Thy-1.1neg subsets defined by c-Kit and AA4.1a

 
Mixed lineage colonies generated by Thy-1.1negc-Kithigh cells maintained both macrophage and lymphoid progeny over the life of the cultures. In contrast, the mixed lineage colonies detected in cultures initiated with Thy-1.1negc-Kitlow cells (either AA4.1pos or AA4.1neg) were transient, as they could only be detected on day 6 or 7 of culture. A comparison of the growth kinetics of mixed lineage colonies in the c-KitlowAA4.1pos and c-KitlowAA4.1neg cell subsets is shown in Fig. 3GoA. The c-KitlowAA4.1neg subset did not produce visible colonies until day 7 in culture. At this time, an equivalent number of pure lymphoid and mixed colonies was observed. On subsequent days, only pure lymphoid colonies were detected. A similar pattern was observed when c-KitlowAA4.1pos cells were cultured, except that colonies appeared 1 day earlier, and pure lymphoid colonies always outnumbered mixed colonies. Thus, the mixed lineage potential of the Thy-1.1neg c-Kitlow subsets of cells was limited, and detection of the myeloid progeny was transient.



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  		FIGURE 3. A, Observed kinetics of lineage outcomes of colonies plucked from sorted Thy-1.1neg c-Kitlow AA4.1 subsets after 6–9 days of culture in methylcellulose supplemented with S7F36EG. {square} and {blacksquare}, Colonies from Thy-1.1neg c-KitlowAA4.1neg subset; {circ} and •, colonies from Thy-1.1neg c-KitlowAA4.1pos subset. {square} and {circ}, Lymphoid lineage colonies; {blacksquare} and •, mixed lineage colonies. B, Observed cell surface staining pattern of sorted Thy-1.1neg c-KitlowAA4.1neg cells after 5–8 days of culture in medium supplemented with S7F36EG. Most of the Thy-1.1neg c-Kitlow AA4.1neg cells became AA4.1pos and CD45Rpos by day 5 and retained expression of c-Kit. By day 8, virtually all cells were CD45RposGr-1neg.

 
The kinetics of colony evolution shown in Fig. 3GoA suggest that the c-KitlowAA4.1neg and the c-KitlowAA4.1pos subsets form a lineage, with c-KitlowAA4.1neg cells differentiating to generate the more lineage-restricted c-KitlowAA4.1pos cells. To address this hypothesis, Thy-1.1neg c-KitlowAA4.1neg cells were grown as replicate bulk cultures in the presence of S7F36EG in 24-well plates. Representative wells were harvested over a period of 8 days, and the cells were stained with mAbs to AA4.1, CD45R, c-Kit, and Gr-1. As shown in Fig. 3GoB, most of the cells isolated in the Thy-1.1neg c-KitlowAA4.1neg population become AA4.1pos and CD45Rpos after 5 days in culture. These cultures included rare cells expressing the myeloid lineage marker (Gr-1) on day 5 (data not shown) and day 6, but became predominantly CD45Rpos by day 8. These results, although not at a clonal level, recapitulate the kinetics of CD45R and Gr-1 cell surface expression observed in clonal colonies derived from the c-KitlowAA4.1neg cells (Fig. 3GoA). This experiment also provides further evidence that the c-KitlowAA4.1neg and the c-KitlowAA4.1pos subsets form a lineage.

It is interesting to note that virtually all cells in these cultures coexpressed AA4.1, CD45R and c-Kit at all time points (Fig. 3GoB and data not shown). In contrast, parallel cultures initiated with Thy-1.1neg c-Kithigh cells included a heterogeneous pattern of c-Kit expression and few CD45Rpos c-Kitpos cells (data not shown). This result underscores the multilineage potential of the Thy-1.1neg c-Kithigh subset in contrast to the predominantly lymphoid-committed Thy-1.1neg c-Kitlow subsets.

As shown in Fig. 2GoF, the majority of cells defined by the phenotype Thy-1.1neg c-KitlowAA4.1pos do not express CD45R, and our clonal assays indicate that this cell subset consists almost exclusively of lymphoid-committed progenitors (Table IIGo and Fig. 3GoA). However, the efficiency of magnetic bead depletion may not be absolute, particularly for cells expressing low levels of the target Ag. In addition, the cloning efficiency of Thy-1.1neg c-KitlowAA4.1pos cells (~5%) was equivalent to the low frequency of CD45Rpos cells contained in the Thy-1.1neg c-KitlowAA4.1pos subset (Table IIGo). To ascertain that lymphoid lineage potential is indeed contained within the CD45Rneg fraction of the c-KitlowAA4.1pos cells, we replaced anti-CD45R with anti-CD19 for magnetic lineage depletion and sorted these Linneg cells for CD45Rneg and CD45Rpos fractions within the Thy-1.1neg c-KitlowAA4.1pos cell subset (Fig. 2GoI). The cloning efficiencies of both CD45R fractions were comparable (5.8% for CD45Rneg, 4.9% for CD45Rpos), and 96% of colonies assayed (85 of 88) from the CD45Rneg c-Kitlow AA4.1pos cell subset were lymphoid in lineage (data not shown). This result suggests that the Thy-1.1neg c-Kitlow subset represents a developmental stage at which surface expression of CD45R is just beginning to be induced.

To investigate the influence of stromal cell monolayers on the cloning efficiency of the Thy-1.1neg c-Kitlow cell subsets, we established liquid cultures in the presence or the absence of cloned stromal cell lines. Single cells isolated from the AA4.1neg and AA4.1pos subsets of Thy-1.1neg c-Kitlow cells by automatic cell deposition FACS sorting were seeded into microtiter wells containing cytokines alone (S7F36EG or S7F) or in the presence of S7F plus three different stromal lines reported to support B lymphoid development (Fig. 4GoA). Any of three different bone marrow stromal cell lines (S17 (29), AC6–2.1 (30), and 2018 (31)) in the presence of exogenous S7F enhanced the cloning efficiency of AA4.1pos cells by about 2-fold, while the AA4.1neg subset cultured with S7F and the 2018 stromal line exhibited only a slight enhancement in cloning efficiency compared with S7F stimulation in the absence of stromal cells (Fig. 4GoA). Individual clones were harvested and evaluated for expression of surface Ags by flow cytometry. On days 11 and 12 of culture, single cells cocultured with 2018 in the presence of S7F had expanded to colonies ranging from 104 to 3 x 104 cells and expressed CD45R, CD24, and BP1 uniformly and CD43 at variable levels. Surface IgM (sIgM) expression was observed on very few cells (data not shown). Bulk cultures established from 5 x 102 AA4.1neg or AA4.1pos cells in S7F with or without 2018 cells expanded to 350- to 900-fold (60–70% viable) in 7 days and 1200-fold (30% viable) in 14 days, demonstrating the extensive proliferative potential of these cells. After 14 days in culture, sIgM expression could be detected on a small subset of cells growing either in cytokines alone or on 2018 cells in the presence of S7F (Fig. 4GoB). However, only about 10% of the cells in the sIgMneg population expressed cytoplasmic IgM on day 11, supporting the interpretation that most of the expanding cells in the cultures represent pro-B cells. This result is also consistent with recent studies suggesting that high levels of IL-7 lead to expansion of cIgMneg pro-B cells, and that selective regulation of IL-7 dose-response thresholds leads to preferential outgrowth of pre-B cells expressing productively rearranged IgM in association with the surrogate light chains and signaling components of the pre-B cell receptor (32). These results demonstrate high cloning efficiency and proliferative potential of the Thy-1.1neg c-Kitlow cell subsets.



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  		FIGURE 4. A, Cloning efficiencies of sorted c-Kit AA4.1 subsets of the Thy-1.1neg cell population grown in medium supplemented with different combinations of cytokines (S7F36EG and S7F) or cocultured with different stromal cell lines (AC6, S17, and 2018) supplemented with S7F. AC6 and S17 cultures were performed once, while all others represent mean results from three separate experiments ± SEM. In the case of AA4.1neg cells, the error in measurement of cloning efficiency was too small to plot. B, Flow cytometric analysis of bulk cultures of 500 AA4.1+ cells grown in medium supplemented with S7F (upper panels) or cocultured with 2018 stromal cells supplemented with S7F (lower panels) for 7 or 14 days. Among viable cells, 86% of cells derived from both cytokine-driven and stromal cell cocultures express CD45R on day 7, and this increased to 99% by day 14. The percentage of CD45Rpos cells expressing sIgM is indicated. On day 11, 10% of cells cultured with 2018 and S7F were positive for cytoplasmic IgM (data not shown).

 
The T cell potential of the Thy-1.1neg cell population is predominantly found in the c-KitlowAA4.1neg subset

To assess whether the lymphoid-committed Thy-1.1neg subsets have committed to the B lymphocyte lineage or, alternatively, are analogous to the previously reported common lymphoid progenitor (6), T cell progenitor cell assays were performed. Limiting dilution analysis was performed by injecting graded numbers of cells (0–85 c-KitlowAA4.1neg cells and 0–192 c-KitlowAA4.1pos cells) into thymic lobes of sublethally irradiated mice and scoring the lobes as positive or negative for T cell development 15–25 days later. To control for the efficiency of thymic injections, we coinjected the sorted cell subset of interest (isolated from B6 congenic mice carrying the Thy-1.1, Ly-1.2, and Ly-5.1 alleles) along with a saturating dose of Linneg cells derived from a second B6-congenic strain (Thy-1.2, Ly-1.1, Ly-5.2) into B6 mice (Thy-1.2, Ly-1.2, Ly-5.2). Successful intrathymic transfers were identified by the presence of Ly-1.1pos cells, and these thymic lobes were scored for the presence of Ly-5.1pos cells. This analysis showed that the frequency of T cell progenitors within the c-KitlowAA4.1neg and c-KitlowAA4.1pos cell subsets was 1 in 160 and <1 in 620, respectively (Fig. 5Go). This result supports the interpretation that expression of AA4.1 coincides with B lineage commitment, since a frequency of 1 in 620 could be explained by contamination with populations of cells outside our sorting gates. The low frequency of T cell progenitors in the c-KitlowAA4.1neg population suggests that common lymphoid progenitors can account for only a subset of these cells.



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  		FIGURE 5. Analysis of T cell progenitor frequency in the Thy-1.1neg c-KitlowAA4.1neg ({blacksquare}) and AA4.1pos ({square}) populations. Cell populations isolated from Ly-5.1 donor mice were injected into the thymic lobes of sublethally irradiated B6 animals along with Linneg BM derived from Ly-1.1 congenic animals, as described in Materials and Methods. After 12–20 days, animals were sacrificed, and thymic lobes were analyzed for the presence of Ly-1.1+ cells to indicate successful injections. Lobes were scored as positive when a clone of Ly-5.1+ cells >2% of the total thymic population was detected. By limiting dilution analysis, the AA4.1neg subset contained 1 thymic progenitor/160 cells (r = 0.991), while the AA4.1pos population contained <1 thymic progenitor/620 cells.

 
Expression of B lineage-associated genes in the Thy-1.1neg c-Kitlow lymphoid-committed progenitor subsets

The results of the in vitro clonal assays and the intrathymic injections suggest that the majority of c-KitlowAA4.1pos cells are committed to the B lymphoid lineage at a developmental stage coincident with up-regulation of CD45R expression. Commitment to the B lymphoid lineage occurs before Ig heavy chain rearrangement (4) and is associated with transcriptional activation of this locus, resulting in the expression of germline µ transcripts (µo) (7) as well as the expression of a number of genes that are required for Ig gene rearrangements (recombinase-activating gene (Rag)-1 and Rag-2) (22). Transcription of µo has been shown to reflect the accessibility and competence of the µ region for Ig gene rearrangements (7, 33). In addition, recent studies have established the importance of a number of transcription factors in lymphoid development (34). Three of these proteins, products of the Pax5, E2A, and EBF genes, are essential in both B cell lineage commitment and B cell development (23, 35, 36, 37). To better establish the stage of development represented by the two AA4.1 subsets of Thy-1.1neg c-Kitlow cells, we performed gene expression analysis by semiquantitative RT-PCR. We determined the expression of µo, Rag-2, E2A, Pax5, and CD19 in the various Thy-1.1neg cell populations compared with CD45Rpos BM cells (Fig. 6Go). Rag-2 expression was detected at equivalent levels in all samples. In contrast, µo transcripts were highly expressed by Thy-1.1neg c-Kitlow cells and by the two AA4.1 subsets relative to the total population of CD45R+ cells. Since the target sequence for the 5' oligonucleotide used to prime this amplification is deleted upon IgH D-J rearrangement (7, 33), this result demonstrates that both AA4.1 subsets include cells with at least one transcriptionally active allele of the IgH gene in germline configuration. Fig. 6Go also provides further evidence supporting the conclusion that AA4.1neg cells are the precursors of AA4.1pos cells. Although each population equally expressed the E2A gene, both Pax5 and CD19 were up-regulated in AA4.1pos cells relative to AA4.1neg cells. This observation is consistent with transfection experiments that indicate induction of Pax5 by E2A and of CD19 by Pax5 (38, 39, 40). Thus, the Thy1.1neg c-KitlowAA4.1neg stage of development corresponds to the aberrant B cell progenitors in Pax5-deficient mice that can be lineage redirected using IL-7 and other cytokines (36). Together with our functional data, these molecular results strongly support the conclusion that the Thy-1.1neg c-Kitlow subsets include cells at the earliest stages of B cell development in the mouse.



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  		FIGURE 6. RT-PCR analysis of early B lineage genes expressed by the subsets of cells described in these studies. Samples were adjusted based on GAPDH signal and amplified as described in Materials and Methods. Aliquots of PCR products were withdrawn at cycles 24, 27, and 30 (GAPDH) or cycles 27, 30, and 33 (µo, Rag-2, E2A, Pax-5, and CD19) and separated by 1% agarose gel electrophoresis for detection by ethidium bromide staining. The bar graphs depict expression levels relative to that detected in CD45Rpos BM cells, as assessed by densitometry scanning of the original gel. A, Thy-1.1neg; B, Thy-1.1neg c-Kitlow; C, Thy-1.1neg c-KitlowAA4.1neg; D, Thy-1.1neg c-KitlowAA4.1pos; E, CD45Rpos BM cells. Thus, B represents the sum of C and D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transplant studies have previously shown that the Thy-1.1neg population is enriched for committed lymphoid progenitors that can readily engraft in the recipient BM (19). Here we demonstrate that the Thy-1.1neg cell population contains three progenitor populations, including separate committed progenitors for both lymphoid and myeloid lineages as well as a mixed lineage progenitor. Compared with the Thy-1.1low HSC population, Thy-1.1neg cells have a more restricted pattern of myeloid lineage potential because most colonies contained macrophages, while mature neutrophils with segmented nuclei were rare. Moreover, the Thy-1.1neg cell population could be further segregated based on the level of c-Kit and AA4.1 expression. Using these Ags, we delineated two functionally distinct populations: c-KithighAA4.1neg cells, which are predominantly committed myeloid progenitors, and c-KitlowAA4.1neg/pos cells, which are predominantly lymphoid-committed progenitors (Table IIGo). Thy-1.1neg c-Kitlow cells largely share the CD45Rneg phenotype of the fraction A0 cells isolated by Allman et al. (4). However, in contrast to fraction A0, Thy-1.1neg c-Kitlow cells are CD24pos, CD4neg and do not include erythroid lineage progenitors. Tudor and colleagues have shown that the majority of cells comprising fraction A have a low cloning efficiency and thus probably do not represent a major intermediate in B cell development (9). The similarity in phenotype, frequency, cloning efficiency, and differentiation potential of Thy-1.1neg c-Kitlow cells compared with the Linneg c-KitlowIL-7R{alpha}posFlk-2posCD34neg cells described by Tudor and colleagues suggests that the two isolation protocols define a similar population of cells. However, the selection protocol described in the present studies may provide a better separation of lymphoid and myeloid progenitors, since up to 30% of clones grown on S17 stromal cells by Tudor and colleagues were of myeloid lineage. It is likely that the selection against Thy-1.1 expression in the present studies accounts for the increased resolution of B lymphoid progenitors from multipotent stem cells and myeloid progenitors.

Recently, a common lymphoid progenitor with the phenotype Thy-1.1negSca-1low c-KitlowLinnegIL7Rpos has been reported (6). This cell population possesses rapid and prominent lymphoid-restricted potential with limited or no self-renewal activity. Since most of the cells in the Thy-1.1neg c-Kitlow subset express IL-7R (Fig. 2GoB), this population of cells overlaps almost entirely with the common lymphoid progenitor of Kondo and colleagues. In contrast to the findings of that group, we demonstrate that T lineage potential is low among Thy-1.1neg c-KitlowAA4.1neg cells and is absent from the Thy-1.1neg c-KitlowAA4.1pos subset (Fig. 5Go). One obvious difference between the two sets of experiments is the short-term culture in S7F used by Kondo and colleagues to prove that the clonal progeny of single cells could differentiate into both the T and B lineages. A similar observation was reported by Jacobsen and colleagues, who showed that c-KithighSca-1posLinneg HSC could be cultured for up to 2 weeks in IL-7 and Flt3L and that the cultures, when transplanted i.v., reconstituted T and B, but not myeloid, lineages (41). Older studies by Phillips and colleagues reported similar findings using long term bone marrow cultures transplanted into immunodeficient recipient mice (42). These results are also consistent with studies using animals mutated at the Pax5 locus, since pro-B cells from these animals could be cultured long term in IL-7 while retaining T lineage potential (43). Taken with the results shown in Fig. 5Go, these observations indicate that common lymphoid progenitors will default to the B lineage unless specific stimulation with cytokines precedes thymic engraftment. This is consistent with an inductive mechanism for the T lineage, rather than a permissive or stochastic developmental pathway.

Kondo and colleagues proposed two pathways of development for conventional B lymphocytes. One of these branches early from the HSCs, has the potential to develop in the T or B lineage but has no myeloid potential, and expresses IL-7R. The other pathway branches later, has potential to develop as B lineage and a limited array of myeloid cells, and lacks the IL-7R. Although the Thy-1.1neg cell populations reported here as well as that reported by Kondo and colleagues are capable of rapid BM reconstitution, giving rise to predominantly lymphoid lineage cells, the Thy-1.1neg c-Kithigh cell subset as isolated in these studies retains some myeloid differentiation potential. Thus, we suggest that the mixed lineage progenitor described within the Thy-1.1neg c-Kithigh cell subset (Table IIGo) may represent the second branch of B cell progenitors postulated by Kondo and colleagues, while the Thy-1.1neg c-KitlowAA4.1neg cell population described here may include the common lymphoid progenitors described by Kondo and colleagues. It is not clear to what extent each of these mutually exclusive intermediates contributes to the Thy-1.1neg c-KitlowAA4.1pos cell subset and subsequent B lineage development (Fig. 7Go).



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  		FIGURE 7. The developmental relationships between the lymphoid progenitors described in this study and other identified intermediates in early lymphoid development. The HSC has been previously characterized as Thy-1.1low and negative for a variety of lineage Ags, including CD45R, but positive for c-Kit, Sca-1, CD24, and CD43. Although stem cells in yolk sac and fetal liver express AA4.1, adult BM stem cells do not (44 ). We have not established to what extent the pro-B cells described in this study are progeny of the common lymphoid progenitors (CLP) described by Kondo et al. (6 ) or a bipotent B/macrophage progenitor as described by numerous investigators. The T and B lineage potential of the pro-B transitional stage was derived from the frequencies of each activity (1 T progenitor/160 cells vs 1 B progenitor/20 cells). The CLP may be represented by the 10% of T lineage progenitors in the pro-B transitional stage described here, since the phenotypes of these two populations largely overlap.

 
Our analysis of T cell potential in the Thy-1.1neg cell population shows that these cells can differentiate in the thymus after intrathymic injection (Fig. 5Go). Most of the T cell potential is contained in the Thy-1.1neg c-KitlowAA4.1neg cell subset. However, the frequency of cells in this subset that engraft intrathymically is 32-fold lower than the frequency of T cell progenitors found when Thy-1.1low HSC are injected (2). This suggests that Thy-1.1neg c-Kitlow cells may predominantly be committed to the B cell lineage and that T cell progenitors may largely lie outside this subset of cells. Alternatively, the low frequency of T cell clones observed relative to Thy-1.1low HSC may represent a difference in proliferative expansion before the onset of TCR rearrangements. T cell clones derived from progenitors at later stages of development would appear earlier and would be smaller and may not be observed if they fail positive or negative selection before becoming large enough to be detected in this assay. Most studies indicate that the rate of emigration of progenitor cells into the thymus is quite low (45). Several lines of evidence suggest that it is a committed progenitor rather than a pluripotent HSC that seeds the thymus to initiate T cell development (46, 47, 48). We therefore expect to find a BM progenitor with sufficient proliferative activity to maintain production of T lineage cells in the thymus in the absence of large scale replenishment from marrow-derived sources. The sizes of thymic clones should reflect the amount of proliferation they can undergo before rearrangement of TCR genes and subsequent selection. Additional experiments to localize T cell progenitors immediately outside the HSC compartment will be addressed in the immediate future.

A number of investigators have characterized early B cell development by isolating and characterizing early progenitors based on CD45R expression. The earliest of these progenitors is thought to lack CD24 expression (7, 49, 50) and to up-regulate this marker as differentiation proceeds. The Thy-1.1neg c-Kitlow cell population isolated in these studies has strong hallmarks of being a transitional cell between the HSC and the progenitors for lymphoid lineages. Like the HSC, the Thy-1.1neg c-Kitlow progenitor is CD45Rneg and expresses CD24, with the CD24 expression being slightly higher compared with HSC (Fig. 2GoG). In addition, Thy-1.1neg c-Kitlow cells will proliferate in response to the lymphoid-selective cytokine combination of S7F, exhibiting a cloning efficiency of 4–8% under these conditions (Table IIGo and Fig. 4GoA). We observed that cytokine-driven bulk cultures of Thy-1.1neg c-KitlowAA4.1pos cells, in the presence or the absence of stromal cells, supported lymphoid differentiation up to the CD45RposIgMneg B cell stage, but were inefficient at supporting further differentiation (Fig. 4GoB). Modulation of IL-7 concentrations in these cultures may be necessary to select the rare cells that successfully complete Ig gene rearrangement (32, 51).

Recent studies have established the importance of a number of transcription factors in lymphoid development (52). Three of these proteins, products of the Pax5, E2A, and EBF genes, are specifically associated with commitment to the B cell lineage (53, 54, 55). Targeted mutation of E2A or EBF results in a block in B cell development at a stage before Ig gene rearrangements, while Pax5-deficient mice initiate D-JH rearrangement but arrest before V-DJH rearrangements (37). Interestingly, Pax-5 deficiency leads to the growth of pro-B cells at a stage of development that allows lineage reprogramming even after extensive culture in the presence of IL-7 (36). Thus, cultured Pax5-deficient cells that are phenotypically identical with native pro-B cells and have rearranged their Ig D-JH gene segments can subsequently be induced to differentiate as T lineage lymphocytes or even as macrophages, neutrophils, osteoclasts, dendritic cells, and NK cells (43). This lineage reprogramming requires withdrawal of IL-7 and substitution of other lineage-inducing cytokines or microenvironments, suggestive of a deterministic regulation of differentiation at this stage of development. Our results suggest that Thy-1.1neg c-KitlowAA4.1neg cells represent a normal counterpart to the pseudo-pro-B cell population found in Pax5-deficient animals, and that the developmental stage at which Pax5 expression is normally up-regulated during B cell development is associated with up-regulation of AA4.1 (Fig. 6Go). Enrichment of lymphoid progenitors within the Thy-1.1neg cell population using c-Kit and AA4.1 as additional phenotypic markers is further supported by the marked increase in one of the earliest markers of B lineage commitment, µo, that was observed in the lymphoid progenitor subsets compared with either the whole Thy-1.1neg cell population or the more mature CD45Rpos cells (Fig. 6Go). Furthermore, the onset of Pax-5 and CD19 transcription at the AA4.1pos stage of development corresponds to very recent activation of these genes, since the cell population does not yet express surface CD19 protein (data not shown).

The role of cytokines in supporting cell survival and promoting cell proliferation of HSCs has been the focus of several studies over the past few years (56). In addition, there is considerable debate about whether cytokines can directly influence lineage commitment decisions of progenitor cells (57, 58). Our data suggest that specific cytokine combinations can predictably influence the lineage outcomes of sorted Thy-1.1neg cells. The absence of IL-3 in the cytokine combination led to a marked decrease in myeloid lineage colonies, while the combination of S7F specifically supported lymphoid lineage colonies (Table IGo). In both cases, however, there was a decrease in cloning efficiency and no marked increase in the number of lymphoid colonies analyzed compared with the complex cytokine combination (S7F36EG). These observations led to the identification of separate committed progenitor cells for lymphoid and myeloid lineages contained within the Thy-1.1neg cell population (Table IIGo). The results also imply that the combination of S7F selectively supports the proliferation and differentiation toward the lymphoid lineage of lymphoid-committed progenitor cells, but is not sufficient to influence lineage commitment decisions of mixed lineage progenitor cells. Similar results have been reported for the role of IL-7 in lineage commitment decisions of bipotent lymphoid-myeloid progenitor cells from mouse fetal liver (59). The failure of apoptosis-inhibiting molecules to support continuing differentiation of B lineage cells strongly supports a selective role for extrinsic signals in lymphoid development (60).

Finally, it should be noted that erythroid and megakaryocyte lineage cells were rarely observed in the methylcellulose cultures of Thy-1.1neg cells and failed to recover in transplant studies. This observation that the Thy-1.1neg cell subset gives rise to lymphocytes, macrophages, and granulocytes, but not erythrocytes and megakaryocytes, provides evidence for the early separation of the myeloid-lymphoid progenitors from the erythroid-megakaryocyte progenitors. Several other laboratories have reported similar findings recently (3, 61, 62, 63). This is in contrast to the conventional view of the hierarchy of hemopoietic differentiation, where the lymphoid progenitors are usually depicted as separate from progenitors of the myeloid, megakaryocyte, and erythroid lineages early in the differentiation process. Cell isolation studies such as those reported here will be instrumental in defining the lineage relationships at early stages of hemopoietic development that until this point have remained elusive.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (RO1HL56857 and P50DK49219), the Lymphoma Foundation, and the Blood and Marrow Transplant Program at the University of Utah. The Flow Cytometry and Irradiation core facilities of the Huntsman Cancer Institute, supported by National Cancer Institute Cancer Center Support Grant P30CA42014, were used for these studies. Back

2 Address correspondence and reprint requests to Dr. Gerald J. Spangrude, Department of Oncological Sciences, University of Utah, 50 North Medical Drive, Room 5C334, Salt Lake City, UT 84132. Back

3 Abbreviations used in this paper: BM, bone marrow; HSC, hemopoietic stem cells; STL, Steel factor; Flt3L, Flt3 ligand; Epo, erythropoietin; Linneg, bone marrow cells depleted of cells expressing any of a panel of lineage-specific Ags; Thy-1.1low, Sca-1+c-Kit+LinnegThy-1.1low BM cells; Thy-1.1neg, Sca-1+c-Kit+LinnegThy-1.1neg BM cells; PI, propidium iodide; SAv, streptavidin; Rag, recombinase-activating gene. Back

Received for publication August 28, 2000. Accepted for publication December 18, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morrison, S. J., N. Uchida, I. L. Weissman. 1995. The biology of hematopoietic stem cells. Annu. Rev. Cell. Dev. Biol. 11:35.[Medline]
  2. Spangrude, G. J., S. Heimfeld, I. L. Weissman. 1988. Purification and characterization of mouse hematopoietic stem cells. Science 241:58.[Abstract/Free Full Text]
  3. Akashi, K., D. Traver, T. Miyamoto, I. L. Weissman. 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404:193.[Medline]
  4. Allman, D., J. Li, R. R. Hardy. 1999. Commitment to the B lymphoid lineage occurs before DH-JH recombination. J. Exp. Med. 189:735.[Abstract/Free Full Text]
  5. Payne, K. J., K. L. Medina, P. W. Kincade. 1999. Loss of c-kit accompanies B-lineage commitment and acquisition of CD45R by most murine B-lymphocyte precursors. Blood 94:713.[Abstract/Free Full Text]
  6. Kondo, M., I. L. Weissman, K. Akashi. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91:661.[Medline]
  7. Li, Y. S., R. Wasserman, K. Hayakawa, R. R. Hardy. 1996. Identification of the earliest B lineage stage in mouse bone marrow. Immunity 5:527.[Medline]
  8. Rolink, A., E. ten Boekel, F. Melchers, D. T. Fearon, I. Krop, J. Andersson. 1996. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J. Exp. Med. 183:187.[Abstract/Free Full Text]
  9. Tudor, K. S., K. J. Payne, Y. Yamashita, P. W. Kincade. 2000. Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events. Immunity 12:335.[Medline]
  10. Lu, L., D. G. Osmond. 1997. Apoptosis during B lymphopoiesis in mouse bone marrow. J. Immunol. 158:5136.[Abstract]
  11. Spangrude, G. J., G. R. Johnson. 1990. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 87:7433.[Abstract/Free Full Text]
  12. Uchida, N., L. Jerabek, I. L. Weissman. 1996. Searching for hematopoietic stem cells: II. The heterogeneity of Thy-1.1lowLin-/lowSca-1+ mouse hematopoietic stem cells separated by counterflow centrifugal elutriation. Exp. Hematol. 24:649.[Medline]
  13. Li, C. L., G. R. Johnson. 1992. Rhodamine123 reveals heterogeneity within murine Lin-, Sca-1+ hemopoietic stem cells. J. Exp. Med. 175:1443.[Abstract/Free Full Text]
  14. 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.[Abstract/Free Full Text]
  15. Nibley, W. E., G. J. Spangrude. 1998. Primitive stem cells alone mediate rapid marrow recovery and multilineage engraftment after transplantation. Bone Marrow Transplant. 21:345.[Medline]
  16. Osawa, M., K. Nakamura, N. Nishi, N. Takahasi, Y. Tokuomoto, H. Inoue, H. Nakauchi. 1996. In vivo self-renewal of c-Kit+ Sca-1+ Linlow/- hemopoietic stem cells. J. Immunol. 156:3207.[Abstract]
  17. Okada, S., H. Nakauchi, K. Nagayoshi, S. Nishikawa, Y. Miura, T. Suda. 1991. Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule. Blood 78:1706.[Abstract/Free Full Text]
  18. Orlic, D., R. Fischer, S. Nishikawa, A. W. Nienhuis, D. M. Bodine. 1993. Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 82:762.[Abstract/Free Full Text]
  19. Searles, A. E., S. J. Pohlmann, L. J. Pierce, S. S. Perry, W. B. Slayton, M. P. Mojica, G. J. Spangrude. 2000. Rapid, B lymphoid-restricted engraftment mediated by a primitive bone marrow subpopulation. J. Immunol. 165:67.[Abstract/Free Full Text]
  20. Spangrude, G. J., D. M. Brooks, D. B. 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.[Abstract/Free Full Text]
  21. Burn, T. C., M. S. Petrovick, S. Hohaus, B. J. Rollins, D. G. Tenen. 1994. Monocyte chemoattractant protein-1 gene is expressed in activated neutrophils and retinoic acid-induced human myeloid cell lines. Blood 84:2776.[Abstract/Free Full Text]
  22. Li, Y. S., K. Hayakawa, R. R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178:951.[Abstract/Free Full Text]
  23. Bain, G., E. C. Maandag, D. J. Izon, D. Amsen, A. M. Kruisbeek, B. C. Weintraub, I. Krop, M. S. Schlissel, A. J. Feeney, M. van Roon. 1994. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79:885.[Medline]
  24. Hirayama, F., S. C. Clark, M. Ogawa. 1994. Negative regulation of early B lymphopoiesis by interleukin 3 and interleukin 1{alpha}. Proc. Natl. Acad. Sci. USA 91:469.[Abstract/Free Full Text]
  25. Ball, T. C., F. Hirayama, M. Ogawa. 1996. Modulation of early B lymphopoiesis by interleukin-3. Exp. Hematol. 24:1225.[Medline]
  26. McKearn, J. P., C. Baum, J. M. Davie. 1984. Cell surface antigens expressed by subsets of pre-B cells and B cells. J. Immunol. 132:332.[Abstract]
  27. Hayakawa, K., Y. S. Li, R. Wasserman, S. Sauder, S. Shinton, R. R. Hardy. 1997. B lymphocyte developmental lineages. Ann. NY Acad. Sci. 815:15.[Medline]
  28. Rolink, A., D. Haasner, S. Nishikawa, F. Melchers. 1993. Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice. Blood 81:2290.[Abstract/Free Full Text]
  29. Henderson, A. J., A. Johnson, K. Dorshkind. 1990. Functional characterization of two stromal cell lines that support B lymphopoiesis. J. Immunol. 145:423.[Abstract]
  30. Whitlock, C. A., G. F. Tidmarsh, C. Müller-Sieburg, I. L. Weissman. 1987. Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule. Cell 48:1009.[Medline]
  31. Wineman, J., K. Moore, I. Lemischka, C. Muller Sieburg. 1996. Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells. Blood 87:4082.[Abstract/Free Full Text]
  32. Marshall, A. J., H. E. Fleming, G. E. Wu, C. J. Paige. 1998. Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression. J. Immunol. 161:6038.[Abstract/Free Full Text]
  33. Schlissel, M. S., L. M. Corcoran, D. Baltimore. 1991. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J. Exp. Med. 173:711.[Abstract/Free Full Text]
  34. Carsetti, R.. 2000. The development of B cells in the bone marrow is controlled by the balance between cell-autonomous mechanisms and signals from the microenvironment. J. Exp. Med. 191:5.[Free Full Text]
  35. Bain, G., E. C. Robanus Maandag, H. P. te Riele, A. J. Feeney, A. Sheehy, M. Schlissel, S. A. Shinton, R. R. Hardy, C. Murre. 1997. Both E12 and E47 allow commitment to the B cell lineage. Immunity 6:145.[Medline]
  36. Nutt, S. L., B. Heavey, A. G. Rolink, M. Busslinger. 1999. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401:556.[Medline]
  37. Nutt, S. L., P. Urbanek, A. Rolink, M. Busslinger. 1997. Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev. 11:476.[Abstract/Free Full Text]
  38. Kozmik, Z., S. Wang, P. Dorfler, B. Adams, M. Busslinger. 1992. The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. Mol. Cell. Biol. 12:2662.[Abstract/Free Full Text]
  39. Kee, B. L., C. Murre. 1998. Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12. J. Exp. Med. 188:699.[Abstract/Free Full Text]
  40. Nutt, S. L., A. M. Morrison, P. Dorfler, A. Rolink, M. Busslinger. 1998. Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments. EMBO J. 17:2319.[Medline]
  41. Borge, O. J., J. Adolfsson, S. Jacobsen. 1999. Lymphoid-restricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3 ligands. Blood 94:3781.[Abstract/Free Full Text]
  42. Fulop, G. M., R. A. Phillips. 1989. Use of scid mice to identify and quantitate lymphoid-restricted stem cells in long-term bone marrow cultures. Blood 74:1537.[Abstract/Free Full Text]
  43. Rolink, A. G., S. L. Nutt, F. Melchers, M. Busslinger. 1999. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401:603.[Medline]
  44. 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.[Medline]
  45. Scollay, R., J. Smith, V. Stauffer. 1986. Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus. Immunol. Rev. 91:129.[Medline]
  46. Rodewald, H. R., K. Kretzschmar, S. Takeda, C. Hohl, M. Dessing. 1994. Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization. EMBO J. 13:4229.[Medline]
  47. Aurrand-Lions, M., F. Galland, H. Bazin, V. M. Zakharyev, B. A. Imhof, P. Naquet. 1996. Vanin-1, a novel GPI-linked perivascular molecule involved in thymus homing. Immunity 5:391.[Medline]
  48. Kawamoto, H., K. Ohmura, Y. Katsura. 1998. Presence of progenitors restricted to T, B, or myeloid lineage, but absence of multipotent stem cells, in the murine fetal thymus. J. Immunol. 161:3799.[Abstract/Free Full Text]
  49. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.[Abstract/Free Full Text]
  50. Hunte, B. E., M. Capone, A. Zlotnik, D. Rennick, T. A. Moore. 1998. Acquisition of CD24 expression by Lin-CD43+B220lowckithi cells coincides with commitment to the B cell lineage. Eur. J. Immunol. 28:3850.[Medline]
  51. Ray, R. J., A. Stoddart, J. L. Pennycook, H. O. Huner, C. Furlonger, G. E. Wu, C. J. Paige. 1998. Stromal cell-independent maturation of IL-7-responsive pro-B cells. J. Immunol. 160:5886.[Abstract/Free Full Text]
  52. Georgopoulos, K., M. Bigby, J. H. Wang, A. Molnar, P. Wu, S. Winandy, A. Sharpe. 1994. The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143.[Medline]
  53. Urbanek, P., Z. Q. Wang, I. Fetka, E. F. Wagner, M. Busslinger. 1994. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79:901.[Medline]
  54. Zhuang, Y., P. Soriano, H. Weintraub. 1994. The helix-loop-helix gene E2A is required for B cell formation. Cell 79:875.[Medline]
  55. Lin, H., R. Grosschedl. 1995. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376:263.[Medline]
  56. Veiby, O. P., A. A. Mikhail, H. R. Snodgrass. 1997. Growth factors and hematopoietic stem cells. Hematol. Oncol. Clin. North Am. 11:1173.[Medline]
  57. Metcalf, D.. 1998. Lineage commitment and maturation in hematopoietic cells: the case for extrinsic regulation. Blood 92:345.[Free Full Text]
  58. Enver, T., C. M. Heyworth, T. M. Dexter. 1998. Do stem cells play dice?. Blood 92:348.[Free Full Text]
  59. Kee, B. L., C. J. Paige. 1996. In vitro tracking of IL-7 responsiveness and gene expression during commitment of bipotent B-cell/macrophage progenitors. Curr. Biol. 6:1159.[Medline]
  60. Tarlinton, D. M., L. M. Corcoran, A. Strasser. 1997. Continued differentiation during B lymphopoiesis requires signals in addition to cell survival. Int. Immunol. 9:1481.[Abstract/Free Full Text]
  61. Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara, S. Muramatsu, R. M. Steinman. 1993. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci. USA 90:3038.[Abstract/Free Full Text]
  62. McDonald, T. P., P. S. Sullivan. 1993. Megakaryocytic and erythrocytic cell lines share a common precursor cell. Exp. Hematol. 21:1316.[Medline]
  63. Choi, K., M. Kennedy, A. Kazarov, J. C. Papadimitriou, G. Keller. 1998. A common precursor for hematopoietic and endothelial cells. Development 125:725.[Abstract]



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