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Expression of Stromal Cell-Derived Factor-1/Pre-B Cell Growth-Stimulating Factor Receptor, CXC Chemokine Receptor 4, on CD34⁺ Human Bone Marrow Cells Is a Phenotypic Alteration for Committed Lymphoid Progenitors¹

Takefumi Ishii^*, Masamichi Nishihara^*, Feng Ma, Yasuhiro Ebihara, Kohichiro Tsuji, Shigetaka Asano, Tatsutoshi Nakahata and Taira Maekawa^2,*

Departments of ^* Transfusion Medicine and Cell Therapy, Clinical Oncology, and Pathological Pharmacology, The Institute of Medical Science, University of Tokyo, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34⁺ bone marrow (BM) cells. Stringently FACS-sorted CD34⁺CXCR4⁺ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34⁺CXCR4^- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34⁺ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34⁺CXCR4⁺ cells are differentiated from CD34⁺CXCR4^- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34⁺ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cultures of hemopoietic cells have indicated the pivotal role of stromal cells in regulating hemopoiesis (1). Stromal cell-derived factor-1 (SDF-1)³ and SDF-1ß, cloned using a signal sequence-trapping strategy, were shown to be a member of the intercrine cytokine family, although their biological activities remained to be defined (2). Subsequently, pre-B cell growth-stimulating factor (PBSF), which supports the proliferation of murine pre-B cells, was cloned from the PA6 murine stromal cell line, and was found to be identical to SDF-1 by amino acid sequence analysis (3). SDF-1/PBSF is a member of the CXC group of chemokines, a large family of structurally related cytokines that has four conserved cysteines, the first two of which are separated by one amino acid.

It has been reported that SDF-1/PBSF is a natural ligand for the previously cloned orphan human chemokine receptor LESTR/fusin (4, 5). A cDNA that encodes a seven-transmembrane-spanning domain receptor was also isolated and was designated as a pre-B cell-derived chemokine receptor (6). Being the fourth cloned chemokine receptor, it was renamed CXC chemokine receptor 4 (CXCR4). The physiological roles of SDF-1/PBSF and CXCR4 were investigated by the use of mutant mice with a targeted disruption of these genes, showing that they are responsible for B lymphopoiesis. The mutant mice also had defects in vascularization of the gastrointestinal tract, cardiac ventricular septal formation, and neuronal development (7, 8, 9, 10).

It has been recently established that CXCR4 and the CC chemokine receptor, CCR5, act as coreceptors that are required for HIV to infect host cells (11). Coexpression of CXCR4 and the CD4 molecule on the surface of T cells is necessary for infection by syncytium-inducing, T-tropic HIV isolates (12). Anemia, neutropenia, lymphocytopenia, and thrombocytopenia are commonly found in AIDS patients (13). The growth of hemopoietic progenitors from HIV-1-infected individuals was found to be impaired (14), and CD34⁺ cells obtained from healthy volunteers have a reduced colony-forming capacity when exposed to HIV-1 (15). However, other investigators have reported that primitive CD34⁺ bone marrow (BM) cells are not susceptible to HIV-1 infection (16, 17), and that HIV-1 proviral DNA was rarely found in purified CD34⁺ BM cells from asymptomatic patients (18, 19). Therefore, the susceptibility of hemopoietic stem (or progenitor) cells to HIV-1 remains uncertain.

In an attempt to address these questions, we investigated whether CXCR4 is expressed on CD34⁺ BM cells obtained from healthy individuals, and examined their related functional characteristics in vitro. We found that highly purified CD34⁺CXCR4⁺ BM cells do not have the potential to form colonies by myeloid, erythroid, megakaryocytic, and mixed CFU (myeloid progenitors), while they can develop and differentiate into B and T lineage cells. In contrast, CD34⁺CXCR4^- BM cells generated colonies formed by these myeloid progenitors, and they also developed into both B and T lineage cells. We also observed that CD34⁺CXCR4⁺ cells differentiated from CD34⁺CXCR4^- cells, indicating that the latter are more primitive than the former. These results suggest that CD34⁺CXCR4⁺ BM cells are committed lymphoid progenitors in the early development of human lymphohemopoiesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells

BM cells were taken from the posterior iliac crest of healthy adult volunteers after obtaining informed consent according to institutional guidelines. Cells diluted with PBS were centrifuged over Ficoll-Metrizamide (density = 1.077 g/ml, Lymphoprep; Nyegaad, Oslo, Norway) for 30 min at 400 x g. Light-density mononuclear cells were harvested, washed twice in PBS supplemented with 5% FCS (HyClone, Logan, UT). Adherent cells were removed by incubation in plastic petri dishes (Costar, Cambridge, MA) at 37°C in a humidified atmosphere of 5% CO₂ in air.

Flow cytometry

Mononuclear nonadherent cells were suspended in PBS at a concentration of 5 x 10⁵ cells per tube for staining. Surface staining of BM mononuclear nonadherent cells was performed using the following mAbs: FITC-labeled anti-CD3 (Leu-4; Becton Dickinson, San Jose, CA), anti-CD4 (NU-TH/I; Nichirei, Tokyo, Japan), anti-CD8 (Leu-2a; Becton Dickinson), anti-CD10 (SS2/36; Dako, Glostrup, Denmark), anti-CD13 (WM47; Dako), anti-CD14 (Leu-M3; Becton Dickinson), anti-CD19 (CLB-CD19; Nichirei), anti-CD38 (AT13/5; Dako), anti-CD41a (HIP8; PharMingen, San Diego, CA), anti-c-kit (Nu-c-kit; Nichirei), anti-TdT (HT-6; Dako), PE-labeled anti-CXCR4 (12G5; PharMingen), PE-Cy5-labeled anti-CD34 (581; Immunotech, Marseille, France), anti-CD4 (13B8.2; Immunotech), purified anti-GM-CSFR -chain mAb (S-20; Santa Cruz Biotechnology, Santa Cruz, CA), anti-IL-3R -chain (S-12; Santa Cruz), anti-IL-7R -chain (R34.34; Immunotech), and biotin-labeled anti-G-CSFR (LMM741; PharMingen). In all experiments, the cells were first preincubated in PBS supplemented with 0.5% human -globulin (Sigma, St. Louis, MO) for 30 min at room temperature to block nonspecific binding. After washing with PBS, cells were stained with respective mAbs for 30 min on ice. To detect GM-CSFR, IL-3R, and IL-7R, cells were incubated with anti-mouse FITC-conjugated goat IgG (Dako) for 30 min on ice. For detection of G-CSFR, cells were incubated with FITC-conjugated streptavidin (Dako) for 30 min on ice. For staining of intracellular TdT, cells were prepared following fixation with 4% formaldehyde (Polysciencies, Warrington, PA) in PBS, and permeabilization with 0.1% saponin (Wako, Osaka, Japan) in PBS containing 1% FCS. Permeabilized cells were then incubated with FITC-conjugated anti-TdT or FITC-conjugated control mouse IgG1 (Becton Dickinson). Expression of cell surface Ags was determined by flow cytometry using a Cytoron Absolute (Ortho-Clinical Diagnostics, Tokyo, Japan) or FACSCalibur (Becton Dickinson, Mountain View, CA). Cells exhibiting low forward and low right angle scattering properties (lymphoid gate) were analyzed.

Cell sorting

Cell sorting was performed using FACSVantage (Becton Dickinson) equipped with 488-nm argon lasers, followed by FACS acquisition with a stringent gate, as previously described (20). In brief, BM cells were passed through a cell strainer (Falcon 2350; Becton Dickinson Labware, Lincolon Park, NJ) to remove cell aggregates. Cells were stained for cell sorting with PE-Cy5-labeled CD34 mAb and PE-labeled anti-CXCR4 mAb, as described above. A lymphocyte-sorting gate was established for both forward and side scatterings. Then gates displaying PE-Cy5 fluorescence (CD34) and PE fluorescence (CXCR4) were generated. Gates for CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells were stringently determined to obtain pure fractions. For negative controls, cells were stained with PE-Cy5-labeled mouse IgG1 (Immunotech), and PE-labeled mouse IgG2a were used. Using these gates, CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells were sorted. Immediately after being sorted, their fluorescence profiles were reanalyzed by a different flow cytometry (Cytoron Absolute). The viabilities of the sorted cells were determined by dye exclusion.

Cytokines

Stem cell factor (SCF) was kindly provided by Amgen Biologicals (Thousand Oaks, CA). IL-3 and erythropoietin (EPO) were generously provided by Kirin Brewery (Tokyo, Japan). G-CSF and IL-6 were kindly provided by Chugai Pharmaceutical (Tokyo, Japan) and Tosoh (Kanagawa, Japan), respectively. All cytokines were pure recombinant human molecules and were used at concentrations that induce optimal response in methylcellulose culture containing human hemopoietic cells. Concentrations used in cultures for myeloid progenitors except megakaryocytic progenitors were 100 ng/ml rhSCF, 100 ng/ml rhIL-6, 200 U/ml rhIL-3, 2 U/ml rhEPO, and 10 ng/ml rhG-CSF.

Clonal culture

Sorted CD34⁺CXCR4⁺ and CD34⁺CXCR4^- BM cells were incubated in methylcellulose culture in triplicate at concentrations of 300 cells/ml. One milliliter of culture including cells, -medium (Life Technologies, Grand Island, NY), 0.9% methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% FCS, 1% deionized fraction V BSA (Wako), 5 x 10^-5 M 2-ME (Wako), and the combinations of SCF, IL-6, IL-3, EPO, and G-CSF were plated in a 35-mm standard nontissue culture dish (Becton Dickinson) and incubated at 37°C in a humidified atmosphere flushed with 5% CO₂ in air. Serum-free methylcellulose culture contained components identical to those in serum-containing culture, except 1% pure BSA (Sigma), 300 µg/ml human transferrin (Sigma), 160 µg/ml soybean lecithin (Sigma), and 96 µg/ml cholesterol (Nacalai Tesque, Kyoto, Japan) replaced BSA and FCS. All cultures were scored on day 14 of culture according to criteria reported previously (21). To investigate megakaryocytic colony-forming capacity, FACS-sorted CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells were incubated in collagen-based cultures MegaCult-C (Stem Cell Technologies, British Columbia, Canada) that has been developed for optimal detection of CFU megakaryocytes (Mk). Each culture, performed in triplicate, contained 2500 cells, 1.1 mg/ml collagen, 1% BSA, 0.01 mg/ml bovine pancreatic insulin, 0.2 mg/ml human transferrin, 50 ng/ml recombinant human thrombopoietin, 10 ng/ml rhIL-6, and 10 ng/ml rhIL-3. After 12 days, whole cultures were stained with mouse anti-human GPIIb/IIIa mAb, and then colonies containing four or more Mk were classified into pure CFU-Mk (only containing Mk), mixed CFU-Mk (containing other lineages in addition to Mk), and others (non-Mk colonies).

RT-PCR

mRNAs were isolated from the sorted CD34⁺CXCR4⁺ and CD34⁺CXCR4^- BM cells under conditions using QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden), and cDNAs were synthesized from the same amount (0.5 µg) of each mRNA using T-primed First-Strand Kit (Amersham Pharmacia Biotech), according to the manufacturer’s instruction. The sequences of PCR primer for human G-CSFR were as follows: sense, 5'-ACCTGGGCACAGCTGGAGTGG-3'; antisense, 5'-CAGGCTGCTGTGAGCTGGGTC-3'. The PCR primer sets for human ß-actin were purchased from Clontech (Palo Alto, CA). All of PCR primers were intron spanning, and we confirmed that they could distinguish cDNA from contamination of genomic DNA. The annealing temperatures for G-CSFR and ß-actin were 60°C, respectively. The cDNAs were amplified with Taq polymerase (Ampli Taq; Perkin-Elmer, Foster City, CA) using GenAmp PCR system 2400 (Perkin-Elmer). Each PCR cycle consists of denaturation at 94°C for 30 s, annealing for 1 min, and extension at 72°C for 2 min. The PCR products were electrophoresed on 2% agarose gel for analysis. The PCR products were subcloned into pCR2.1 TA-cloning vector (Invitrogen, San Diego, CA), and sequences were confirmed using a Big Dye Terminator Cycle Sequencing Ready Reaction (Perkin-Elmer) and an ABI Prism 310 genetic analyzer (Perkin-Elmer), according to the manufacturer’s instruction.

B cell differentiation

The potential of CD34⁺CXCR4⁺ and CD34⁺CXCR4^- BM cells to differentiate into progenitor B cells was tested in the presence of murine stromal cells MS-5 (kindly provided by Dr. K. J. Mori, Niigata University, Niigata, Japan). Detailed methods for cultures of progenitor B cells have been described elsewhere (22). We have previously confirmed that human IgM was expressed on these cells after 6 wk of culture. Briefly, cultures were maintained in -medium supplemented with 10% FCS, 100 ng/ml SCF, 10 ng/ml G-CSF, and antibiotics. Cultures were incubated continuously at 37°C in a humidified atmosphere flushed with 5% CO₂ in air and fed each week by removing half of the medium and replacing it with fresh medium containing the cytokines listed above. After 4 wk of culture, cells were harvested using 0.05% trypsin (Life Technologies) plus 0.02% EDTA (Wako), and the number of variable cells per well was determined. The absolute number of CD10⁺CD19⁺ cells was calculated from the number of viable cells, and the percentage of positive cells was confirmed by two-color cytometry after 2, 3, and 4 wk of culture.

T cell differentiation

Thymus lobes were dissected from fetal nonobese diabetic-SCID mice at day 14.5 of gestation. To inoculate cells into the fetal thymus, hanging drops were maintained in Terasaki plates (Sumilon, Tokyo, Japan) by adding 25 µl of complete medium consisting of RPMI 1640 with 20% FCS in each well, supplemented with 5 x 10^-5 M 2-ME, nonessential amino acids, minimum amino acids, vitamins, and HEPES (Cosmo Bio, Tokyo, Japan) for 48 h at 37°C in a humidified atmosphere flushed with 7% CO₂ in air. Five thousand CD34⁺CXCR4⁺ and CD34⁺CXCR4^- BM cells were inoculated into the fetal thymus. After incubation for 48 h, the lobes were removed from the hanging drop, and put on the surface of a nuclepore filter (Corning, NY) above a sterile sponge (Pharmacia-Upjohn, Tokyo, Japan). In all experiments, lobes were treated with 1.35 mM 2-deoxy-guanosine (Nacalai Tesque) for 5 days at 37°C to deplete resident murine lymphocytes before reconstitution. After 5 wk of incubation, the cells were harvested to examine the generation of immature T cells with CD4⁺CD8⁺ phenotype, and mature T cells with CD3⁺CD4⁺ or CD3⁺CD8⁺ phenotype by flow cytometry, FACSCalibur (Becton Dickinson). As a negative control, we used hanging drops by complete medium without cells.

Emergence of CD34⁺CXCR4⁺ cells from CD34⁺CXCR4^- cells

To confirm the emergence of CD34⁺CXCR4⁺ cells from the CD34⁺CXCR4^- population, sorted CD34⁺CXCR4^- cells were cultured on the MS-5 stromal layer for 2 wk in the absence of cytokines. To remove mouse stromal cells, cell suspensions were incubated for 2 h in plastic petri dishes (Costar) containing -medium, supplemented with 10% FCS at 37°C in a humidified atmosphere of 5% CO₂ in air. Then nonadherent cells were analyzed by flow cytometry.

Statistical analysis

For statistical comparison in scoring the number of colonies, two-sided Student’s t test was applied.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sorting for CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells

CXCR4 was expressed on 39.7% ± 9.3% of CD34⁺ BM cells (mean ± SD, in five separate experiments). A lymphoid gate (R1) was established for both forward and side scatterings, as shown in Fig. 1A. Representative sorting gates for CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells are shown in Fig. 1C as R2 and R3, respectively. Fluorescent profiles of sorted CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells were reanalyzed using flow cytometry, Cytoron Absolute. As shown in Fig. 1, D and E, the purity of the sorted CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells was 97.4% and 99.1%, respectively. The purities exceeded 97%, in four separate experiments. More than 98% of the sorted cells were viable.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Sorting of CD34+CXCR4+ and CD34+CXCR4- cells. A, A lymphoid gate (R1) for BM cells established by forward and side scatterings was shown. B, A cytogram of BM cells stained with isotype control. C, Representative sorting gates for CD34+CXCR4+ cells (R2) and CD34+CXCR4- cells (R3). To verify the purity of each sorted cell population, cytograms of the two populations were reanalyzed using Cytoron Absolute immediately after sorting by FACSVantage. Representative cytograms of reanalysis of sorted CD34+CXCR4+ (D) and CD34+CXCR4- (E) cells were shown.

To examine hemopoietic colony-forming potentials of highly purified CD34⁺CXCR4⁺ and CD34⁺CXCR4^- BM cells, 300 cells were cultured in 1 ml of methylcellulose medium containing IL-3, IL-6, G-CSF, EPO, and SCF. As shown in Table I, the sorted CD34⁺CXCR4⁺ population had few colony-forming cells, while the CD34⁺CXCR4^- population contained cells able to generate colonies formed by CFU granulocyte, CFU granulocyte/macrophage, CFU macrophage, burst-forming units of erythroid, and CFU mixed lineage. Similar results were obtained in five separate experiments. Similar results were also obtained when cells were cultured with GM-CSF in addition to the above cytokine mixtures. Again, colonies formed by CFU macrophage were preferentially observed in the CD34⁺CXCR4^- population. Also in serum-free cultures, CFU granulocyte, CFU granulocyte/macrophage, CFU macrophage, burst-forming units of erythroid, and CFU mixed lineage were included in the CD34⁺CXCR4^- population, and no colonies were generated in the case of the CD34⁺CXCR4⁺ population (data not shown). As shown in Table II, nearly all colonies formed by CFU-Mk were developed from CD34⁺CXCR4^- cells. Similar findings were observed in four separate experiments.

We analyzed the expression of lineage or differentiation-specific Ags and cytokine receptors on CD34⁺ BM cells defined by mAbs for CD3, CD4, CD8, CD13, CD14, CD10, CD19, CD38, CD41a, c-kit, HLA-DR, IL-3R, G-CSFR, GM-CSFR, IL-7R, and TdT in comparison with staining profiles by mAb for CXCR4. As shown in Fig. 2, a few CD34⁺CXCR4⁺ cells expressed CD3 Ag, whereas CD34⁺CXCR4^- cells did not. A small number of CD34⁺CXCR4⁺ cells and CD34⁺CXCR4^- cells expressed CD4 Ag. CD8 Ag was not detected either in CD34⁺CXCR4⁺ or CD34⁺CXCR4^- cells. Concerning the B lineage markers, a considerable number of CD34⁺CXCR4⁺ cells simultaneously expressed CD10 Ag, while CD19 Ag was weakly positive on CD34⁺CXCR4⁺ cells and was not expressed on CD34⁺CXCR4^- cells. CD14 Ag for myeloid and monocyte/macrophage lineage was not observed on either CD34⁺CXCR4⁺ cells or CD34⁺CXCR4^- cells, whereas CD13 Ag was detected on CD34⁺CXCR4^- cells, but not on CD34⁺CXCR4⁺ cells. Almost all of the CD34⁺CXCR4⁺ cells expressed CD38 Ag, while a small number of cells that did not express CD38 Ag could be detected in the CD34⁺CXCR4^- fraction. Similar findings were observed in HLA-DR-negative cells, in which a small number of CD34⁺CXCR4^- cells were negative for HLA-DR Ag. The megakaryocytic lineage marker CD41a was expressed on CD34⁺CXCR4^- cells, but less so on CD34⁺CXCR4⁺ cells. c-kit expression on CD34⁺CXCR4^- cells was observed with various fluorescent intensities, c-kit^high, c-kit^low, and c-kit^-, while CD34⁺CXCR4⁺ cells did not express c-kit. Most CD34⁺CXCR4^- cells were found to be in the c-kit^low to c-kit^high fraction. Both CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells were low positive with GM-CSFR, but only CD34⁺CXCR4^- cells expressed IL-3R, albeit with a low fluorescent intensity. Although G-CSFR was barely detected by flow cytometry in either fraction, RT-PCR clearly showed the expression of mRNA of this receptor in CD34⁺CXCR4^- cells, but not in CD34⁺CXCR4⁺ cells (Fig. 3).

View larger version (53K): [in this window] [in a new window]   FIGURE 2. Three-color analysis of surface phenotype of CD34+CXCR4+ and CD34+CXCR4- cells. Surface expressions of various cell lineage Ags, including CD3, CD4, CD8 (for T lineage), CD10, CD19 (for B lineage), CD13, CD14, CD38 (for myeloid lineage), CD41a (for megakaryocytic lineage), HLA-DR, c-kit, GM-CSFR (-chain), IL-3R (-chain), and G-CSFR on CD34+CXCR4+ and CD34+CXCR4- cells, were examined. BM cells were stained with PE-Cy5-conjugated anti-CD34 mAb, PE-conjugated anti-CXCR4 mAb, and FITC-conjugated respective mAbs, as described in Materials and Methods, and then stringently gated CD34+ cells were analyzed using flow cytometry, Cytoron Absolute. Percentages of positive cells among total CD34+ cells are indicated in each quadrant set.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Expression of G-CSFR mRNA on CD34+CXCR4+ and CD34+CXCR4- cells. mRNAs from FACS-sorted CD34+CXCR4+ cells and CD34+CXCR4- cells were extracted, and cDNAs were synthesized as described in Materials and Methods. G-CSFR and ß-actin mRNAs were detected using RT-PCR (40 cycles), and the sequences of PCR products were confirmed. Size of the markers is indicated on the left.

View larger version (48K): [in this window] [in a new window]   FIGURE 4. Intracellular expression of TdT and surface expression of IL-7R on CD34+CXCR4+ and CD34+CXCR4- cells. Intracellular expression of TdT and surface expression of IL-7R on CD34+CXCR4+ and CD34+CXCR4- BM cells were investigated. BM cells were stained with PE-Cy5-conjugated anti-CD34 mAb, PE-conjugated anti-CXCR4 mAb, FITC-conjugated anti-TdT mAbs, or anti-IL-7R mAbs, as described in Materials and Methods, and then stringently gated CD34+ cells were analyzed. Cytograms of cells stained with isotype controls respective to each mAb are shown in the left column.

Next, we designed experiments to determine whether CD34⁺CXCR4⁺ and CD34⁺CXCR4^- BM cells could generate pro-B cells in vitro. Each cell fraction was cultured on MS-5 stromal cells in the presence of SCF and G-CSF. After 2, 3, and 4 wk of incubation, cells were harvested and expression of CD10 and CD19 Ag was analyzed by flow cytometry. In all three separate experiments, both CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells developed CD10⁺CD19⁺ pro-B cells. A representative cytogram after 4 wk of culture is shown in Fig. 5A. The absolute number of pro-B cells was calculated from the number of cells harvested and the percentage of cells with CD10⁺CD19⁺ phenotype. From 1 x 10³ CD34⁺CXCR4⁺ cells and 1 x 10³ CD34⁺CXCR4^- cells, 5.8 x 10³ ± 0.2 x 10³ pro-B cells, and 45.5 x 10³ ± 5 x 10³ (mean ± SD, in triplicate cultures) pro-B cells developed after 4 wk of culture, respectively. Three separate experiments showed similar results (Fig. 5B). A kinetic analysis for the emergence of CD10⁺19⁺ cells from the CD34⁺CXCR4⁺ and CD34⁺CXCR4^- populations was shown in Fig. 5C. B lineage cells have developed sooner from CD34⁺CXCR4⁺ cells than the CD34⁺CXCR4^- population.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Emergence of progenitor B cells from CD34+CXCR4+ and CD34+CXCR4- cells. Each of 1000 CD34+CXCR4+ and CD34+CXCR4- cells was cultured on the murine stromal cell line MS-5 in the presence of rhSCF and rhG-CSF. A, After 4 wk of culture, adherent cells were stained with PE-conjugated CD10 mAb and PE-Cy5-conjugated CD19 mAb, and then cells in lymphocyte gate were analyzed by flow cytometry. Cytograms of cells stained with isotype controls are shown on the left. B, Absolute number of CD10+CD19+ cells was calculated from the number of viable cells, and the percentage of positive cells was obtained by two-color cytometry. C, A kinetic analysis for the emergence of CD10+19+ cells from the CD34+CXCR4+ and CD34+CXCR4- populations was shown. Data are represented by mean ± SD from triplicate cultures.

Some investigators have reported that murine fetal thymus lobes isolated from both normal and scid/scid (SCID) mice were colonized by donor cells from either human BM or CB in vitro (23). Preliminary studies of fetal thymus organ culture using nonobese diabetic-SCID mice have shown that CD4 CD8 double-positive (DP) cells, CD4 single-positive (SP) cells, and CD8 SP cells are generated from CD34⁺ CB cells (F. Ma et al., unpublished observation). To determine whether sorted CD34⁺CXCR4⁺ cells or CD34⁺CXCR4^- cells can generate T cells, five thousand cells from each fraction were colonized per one lobe. After 5 wk of culture, the number of cells harvested was 1.9 x 10⁴ ± 0.5 x 10⁴ cells/lobe from CD34⁺CXCR4⁺ cells, and 3.5 x 10⁴ ± 0.3 x 10⁴ cells/lobe (mean ± SD) from CD34⁺CXCR4^- cells. Cells positive for T lineage markers developed from both CD34⁺CXCR4⁺ and CD34⁺CXCR4^- populations. CD4 SP cells, CD8 SP cells, and CD4CD8 DP cells were generated in the thymus inoculated with CD34⁺CXCR4^- cells. In the thymus inoculated with CD34⁺CXCR4⁺ cells, however, CD8 SP cells were not evident compared with those from CD34⁺CXCR4^- cells, whereas CD4 SP and CD4CD8 DP cells were identified (Fig. 6). Similar results were obtained in three separate experiments.

View larger version (46K): [in this window] [in a new window]   FIGURE 6. Generation of T cells from CD34+CXCR4+ and CD34+CXCR4- cells in fetal thymus organ culture. Harvested cells obtained from the fetal thymic lobes inoculated with CD34+CXCR4- cells (A) and CD34+CXCR4+ cells (B) after 5 wk of culture were stained with PE-conjugated anti-CD3 mAb, PE-Cy5-conjugated anti-CD4 mAb, and FITC-conjugated anti-CD8 mAb. Fluorescent profiles were analyzed by flow cytometry, FACSCalibur. Percentages of positive cells are indicated in each quadrant.

To investigate whether CD34⁺CXCR4⁺ cells develop from CD34⁺CXCR4^- cells during incubation, five thousand CD34⁺CXCR4^- cells were cultured on MS-5 stromal layer for 14 days in the absence of cytokines. Cultured cells were harvested, and again stained with CD34 and CXCR4 mAbs. Seventy-five percent of harvested cells in lymphocyte gate expressed CD34 Ag, and among these CD34⁺ cells, more than sixty percent expressed CXCR4 (Fig. 7).

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Emergence of CD34+CXCR4+ cells from CD34+CXCR4- cells. FACS-sorted CD34+CXCR4- cells were cultured on the murine stromal cell line, MS-5, for 2 wk, and then harvested cells were again stained by PE-Cy5-conjugated anti-CD34 mAb and PE-conjugated anti-CXCR4 mAb. A, Before culture, and B, after 2 wk of culture.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been reported that SDF-1 induces myelosuppression by activation of CXCR4 (24). To exclude the possibility that the loss of colony-forming capacity of CD34⁺CXCR4⁺ cells may have been caused by inhibitory signals of SDF-1 contained in FCS, the cells were cultured without serum. Results similar to those found in serum-containing cultures were obtained (data not shown). It is also unlikely that anti-CXCR4 mAb may activate CXCR4 resulting in myeloid suppression, because both CD34⁺CXCR4⁺ and CD34⁺CXCR4^-CB cells sorted using anti-CXCR4 mAb could similarly generate myeloid progenitors in response to CSFs (data not shown). Someone may argue that CD34⁺CXCR4⁺ cells consist of a noncycling G₀ population of stem cells. However, long-term culture-initiating cells were found only in the CD34⁺CXCR4^- population, but not in the CD34⁺CXCR4⁺ population (Ishii et al., unpublished observation).

The phenotype of CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells has been characterized extensively. As shown in Fig. 2, CD4 Ag was expressed in a small number of both CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells. Because some investigators have reported that CD34⁺CD4⁺ cells are multipotential progenitors, CD34⁺CXCR4^-CD4⁺ cells may be multipotential in nature (25, 26, 27, 28). With regard to B cell lineage markers, CD10 Ag was expressed both in CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells, while only CD34⁺CXCR4⁺ cells were positive for CD19 Ag. Therefore, CXCR4 may appear before CD19 Ag during early B cell development. Although CD10 Ag is thought to be expressed on more immature pro-B cells compared with CD19 Ag (Nishihara et al., unpublished observation), it remains to be determined whether CXCR4 is expressed earlier than CD10 during early B lymphopoiesis. With regard to myeloid lineage markers, CD13 Ag, expressed on CFU granulocyte/macrophage, granulocytes, and monocytes, was preferentially expressed on CD34⁺CXCR4^- cells, not on CD34⁺CXCR4⁺ cells, thereby supporting that myeloid progenitors are included in the CD34⁺CXCR4^- population. CD38 and HLA-DR Ags found on committed hemopoietic progenitors were expressed both in CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells. Because a small number of CD38^- and HLA-DR^- cells was found in the CD34⁺CXCR4^- population, and not in CD34⁺CXCR4⁺ cells, as shown in Fig. 2, uncommitted progenitors with CD34⁺CD38^- and CD34⁺HLA-DR^- phenotype (29, 30, 31) were negative for CXCR4. This result is consistent with the recent report by Weichold et al. that CXCR4 was not expressed on CD34⁺CD38^- cells (32). It has been reported that CD34⁺ cells with G-CSFR, GM-CSFR, or IL-3R expression have multilineage colony-forming capacity (33, 34). Primitive multilineage cells and committed progenitor cells are concentrated in the c-kit^low to c-kit^high fraction (35). These results are in agreement with our present findings, which indicate that CD34⁺CXCR4^- cells are c-kit^low to c-kit^high. Although analysis by flow cytometry did not show the expression of G-CSFR in either population, G-CSFR mRNA could be detected by RT-PCR in the CD34⁺CXCR4^- population, but not in the CD34⁺CXCR4⁺ population. The expression of G-CSFR mRNA restricted to CD34⁺CXCR4^- cells is consistent with the present results that CD34⁺CXCR4⁺ cells could not form myeloid colonies when stimulated by a cytokine mixture including G-CSF.

Megakaryocytic lineage marker CD41a expressed on CD34-positive CFU-Mk and late megakaryocyte progenitors (36, 37) was found only in CD34⁺CXCR4^- cells. In contrast to a recent report (38), we found that colonies formed by CFU-Mk developed only from CD34⁺CXCR4^- cells. It has been reported that CXCR4 was not expressed on CB CD34⁺ cells at the beginning of culture, while its expression was gradually enhanced as the megakaryocytes matured when stimulated by thrombopoietin and SCF (39).

In this repopulation assay using fetal thymus lobes, we have clearly demonstrated that both CD34⁺CXCR4⁺ and CD34⁺CXCR4^- cells give rise to T cells. Because a few CD34⁺CXCR4⁺ cells express CD3 Ag, as shown in Fig. 2, it raises the possibility that the low numbers of CD4⁺ T cells may be derived in part from the small numbers of CD34⁺CXCR4⁺CD3⁺ cells. Although CD8⁺ SP T cells did not develop from the thymus inoculated with CD34⁺CXCR4⁺ BM cells, 8% CD4⁺ SP T cells were derived from T cell progenitors as a consequence of typical thymocyte development, suggesting that the present culture system using fetal thymus lobes would show an early predominance of CD4⁺CD8⁺ cells that would progress to the CD8^- stage. It is also possible that mature T cells generated in the culture emigrate into the surrounding culture medium, which are usually not recovered for analysis. Further studies are definitely needed to investigate whether CD34⁺CXCR4⁺ cells only differentiate into a restricted population among the T cell repertoire (40).

A small number of cells in normal BM cells that can give rise to all blood cell types, and that can self renew are defined as hemopoietic stem cells (41). Along the path of differentiation, progenitors from hemopoietic stem cells gradually lose their self-renewal activity, and then start to include the irreversible restriction of one or more lineage commitments by sequential activation or inactivation of various genes including cytokine receptors (42, 43). It remains to be investigated whether B and T lymphocytes are derived only from committed lymphoid progenitors, or if they are derived directly from multipotential progenitors. It had been reported that expression of TdT on CD34⁺ BM cells may be the earliest phenotypic marker for lymphoid progenitors (44), and that IL-7R expression is a component of the differentiation program specifying the progressive development of lymphoid potential (45). Furthermore, it has been reported that CD34⁺Lin^-c-kit^-Thy-1^-CD10⁺ are a distinct population with a capacity to form B cells, T cells, NK cells, and dendritic cells in human BM, and that these cells do not form myeloid, erythroid, or megakaryocytic cells (46). This finding suggests the existence of committed lymphoid progenitors within the BM population. In a murine system, it was noted at a clonal level that a single Lin^-IL-7R⁺Thy-1^-ScaI^lowc-kit^low cell from adult BM had a lymphoid-restricted reconstitution capacity in vitro, and completely lacked myeloid colony-forming potentials (47). In the present study, we demonstrated that the CD34⁺CXCR4⁺ population could develop and differentiate into B and T lineage cells in vitro, but did not have the capacity to form colonies by myeloid progenitors. Results shown in Fig. 4 suggest that CXCR4 is expressed earlier than IL-7R and TdT. Therefore, it is probable that the expression of CXCR4 on CD34⁺ human BM cells is one of the earliest markers for lymphoid differentiation, and it is also conceivable that CD34⁺CXCR4⁺ BM cells are committed lymphoid progenitors that develop into both B and T lymphoid cells. Since our findings must be verified at a clonal level, further studies using a single CD34⁺CXCR4⁺ cell and a single CD34⁺CXCR4^- cell are mandatory.

Deletion of the CXCR4 gene is embryologically lethal in mice and produces various developmental defects (8, 9, 10). With regard to hemopoiesis, these groups reported that the number of B cell progenitors in the fetal liver of CXCR4^-/- mice was severely reduced. Being impaired in the BM, cells of myeloid lineage, including macrophages, granulocytes, monocytes, megakaryocytes, and erythrocytes, developed normally in the fetal liver of CXCR4^-/- mice (8). This suggests that the expression of CXCR4 is mandatory for the migration or homing of myeloid stem cells from the fetal liver to the BM environment during fetal development. Very recently, using SCID repopulating assay, Peled et al. have reported human CD38^-/lowCXCR4⁺ CB cells as stem cells endowed with migration and repopulation potential (48). It has been reported that CD34⁺ CB cells require a positive gradient of SDF-1/PBSF for their homing to the BM environment (49), and that an altered response to SDF-1/PBSF is associated with progenitor cell mobilization to the blood (50). It is likely that the requirement of the functional expression of CXCR4 for CB cells may differ from that for adult BM cells, because we have observed that both CD34⁺CXCR4⁺ and CD34⁺CXCR4^- CB cells could similarly generate myeloid progenitors. The dynamic alterations of the functional expression of CXCR4 on human hemopoietic stem/progenitor cells during fetal development, in the neonate, in the adult, and in mobilization of these cells to the blood yet remain to be determined. Understanding these processes during the homing and the engraftment, and in the steady state of hemopoiesis in more detail could be of value for stem cell transplantations in the clinical setting.

	Acknowledgments

 
We thank Y. Wada, S. Hosoda, and K. Ogami for their skillful technical assistance. We also thank Dr. P. Hughes for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

² Address correspondence and reprint requests to Dr. Taira Maekawa, Department of Transfusion Medicine and Cell Therapy, The Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address:

³ Abbreviations used in this paper: SDF, stromal cell-derived factor; BM, bone marrow; CB, umbilical cord blood; CXCR4, CXC chemokine receptor 4; DP, double positive; EPO, erythropoietin; Mk, megakaryocytes; PBSF, pre-B cell growth-stimulating factor; rh, recombinant human; SCF, stem cell factor; SP, single positive.

Received for publication April 19, 1999. Accepted for publication July 16, 1999.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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