TY - JOUR
AU - Levy, Yves
AB - Abstract Notch and its ligands regulate multiple cell fate decisions. However, several questions on the timing, durability, and reversibility of Notch signaling effects on human hematopoietic precursors are still unresolved. Here, we used recombinant Delta ligands to deliver temporally and dose-controlled signals to human immature cord blood CD34+CD38low cells at clonal cell levels. Notch activation increased the frequency of multipotent progenitors, skewed the T and natural killer (NK) cell potential of CD34+CD38low clones in a dose- and ligand-dependent manner, and inhibited the differentiation of B cell clones. Low doses of ligands were sufficient for significantly increasing the frequency of NK cell precursors, whereas higher doses were required for increasing the frequency of T-cell clones. Interestingly, we demonstrate that temporary Notch activation prevents the subsequent differentiation of CD34+CD38low cells beyond a pro-B CD79a+CD19− stage characterized as a common lymphoid progenitor (CLP). Moreover, the lymphoid potential of this pro-B/CLP was skewed toward NK cell potential while the B cell precursor frequency was dramatically reduced. These results indicate critical timing and quantitative aspects of Notch/Delta interactions, imprinting the potential of CD34+CD38low hematopoietic progenitors. These results may have implications both in physiology and for cell manipulation because they demonstrate a tight regulation of the fate of human progenitors by Notch signaling. STEM CELLS 2009;27:1676–1685 Disclosure of potential conflicts of interest is found at the end of this article. B lymphocytes, CD34, Human cord blood, Lymphopoiesis, Multipotential differentiation, NK cells, Notch, Stem cell plasticity Introduction Notch receptors and their corresponding ligands constitute a family of evolutionarily conserved transmembrane proteins that regulate cellular differentiation in a variety of tissue types throughout the life of multicellular organisms [1]. Four receptors (Notch-1 to Notch-4) and five ligands (Delta-1, Delta-3 and Delta-4 and Jagged-1 and Jagged-2) [2] have been described in mammals. Ligand–receptor interactions lead to the cleavage of Notch and to the release of Notch intracellular domain (NICD), which translocates to the nucleus and associates with the transcriptional repressor core binding factor 1 (also called CSL, suppressor of Hairless, or Lag-1). Binding of NICD to CSL induces the dislocation of corepressors, such as Mint and Nrarp, and recruitment of coactivators, such as Mastermind, consequently stimulating the transcription of Notch target genes such as hairy and enhancer of split 1 (HES-1). Implications of Notch signaling during hematopoiesis development are widely studied, in particular in the cell fate decisions of multipotent progenitors. Gain- and loss-of-function studies have established the crucial role of Notch signaling during determination of T- versus B-cell differentiation [3–5]. Several studies confirmed that the Notch pathway enhances T-cell differentiation to the detriment of B lymphopoiesis [2, 6–10]. These studies provided evidence that Notch signaling is crucial for both thymus-dependent and thymus-independent T-cell development [11, 12]. These data suggest that Notch, in particular Notch-1, is sufficient for T-cell commitment and negatively modulates B-cell development. Delta-1 (DL1) and DL4 have been shown to be implicated in the promotion of early T-cell differentiation [13, 14]. The different cellular expression of Notch receptors and their ligands suggests that each ligand could carry a different specific function [4, 15–17]. Moreover, a dose-dependent effect of Notch ligands has been demonstrated, indicating an important role for ligand density in the differential promotion of cell-fate outcomes [18, 19]. Several questions on the timing, durability, and reversibility of Notch signaling's effects on hematopoietic precursors are still unresolved [20]. These questions remain difficult to address in the setting of human primitive progenitors. Evaluation of the biological potential of human primitive progenitors is challenging because a unique assay suitable for the evaluation of all potential fates does not exist. Several studies have established the role of Notch in human hematopoiesis using global primitive progenitor populations transduced with Notch intracytoplasmic domain [21], cocultured with purified Notch ligands immobilized [22] or expressed in murine stromal cell lines [23, 24]. In the present study, we evaluated the effects of Notch signaling on the fate of CD34+CD38low human primitive progenitors using a system in which cells were exposed to the recombinant Notch ligands DL1 and DL4 and then replated in culture conditions suitable for the evaluation of their lymphoid and macrophage potential [25]. We found that Notch/Delta interactions increased the frequency of T, natural (NK), and multipotent NK/T/B clones while inhibiting the development of monopotent B-cell clones from CD34+CD38low progenitors. These effects were modulated by the dose and type of ligand. Low and high doses were required, respectively, for increasing the NK- and T-cell potential of primitive progenitors. Moreover, DL4 was more efficient than DL1 at increasing the NK-cell potential of CD34+CD38low progenitors. We also show that Notch inhibited B-cell differentiation at a pro-B CD79+CD19−CD10+ stage, previously described as a pro-B/common lymphoid progenitor (CLP) [26]. Moreover, the lymphoid potential of this pro-B/CLP was skewed towards NK-cell differentiation whereas the B-cell precursor frequency was dramatically reduced. Materials and Methods Cell Preparation and Cell Lines Human cord blood was collected with informed consent from the mother. First, mononuclear cells were subjected to a standard CD34 immunomagnetic bead separation (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com), as previously described [26]. Second, bead-separated CD34+ cells were stained using anti-CD34-phycoerythrin (PE) and anti-CD38-fluorescein isothiocyanate (FITC) monoclonal antibodies, and the CD34+38low population was sorted by the EpicElite cell sorter (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Murine stromal MS-5 cells were passaged weekly in α-minimal essential medium (MEM) (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 10% fetal calf serum (FCS) (Life Technologies, Cergy-Pontoise, France, http://www.lifetech.com). OP9 cells expressing human DL1 (kindly provided by C. Zúñiga-Pflücker) were maintained in α-MEM supplemented with 20% FCS (HyClone, Perbio, France, http://www.hyclone.com) and passaged twice a week. All cell lines were incubated at 37°C in air atmosphere saturated with humidity and 5% CO2. CD34+38low Progenitor Exposure to Notch Ligands CD34+38low sorted cells were exposed for 6 days in 48-wells plates (Falcon, Becton Dickinson, Sunnyvale, CA, http://www.bd.com) precoated with either polyvalent Ig or human Notch ligand DL1 or DL4 (5-20 μg/ml). A noncompetitive γ-secretase inhibitor (GSI), compound E (Alexis Biochemicals Corporation, San Diego, CA, http://www.axxora.com), was added, or not, at a concentration of 250 nM every 4 days during the culture period [54]. Cells were cultured in α-MEM with glutamax supplemented with 20% FCS (HCC-6150; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) and 1% Penicillin-Streptomycin (PS) in the presence of 20 ng/ml human interleukin (hIL)-7, 50 ng/ml human FLT3 ligand (hFLT3L), 50 ng/ml human stem cell factor (hSCF), 20 ng/ml human thrombopoietin, and 10 ng/ml hIL-3 (all purchased from AbCys s.a., Paris, France, http://www.abcysonline.com). Purified human Notch ligand DL4 and DL1 consisted of the extracellular domain of DL4 or DL1 fused to the Fc portion of human Ig, produced as previously described [22] (both kindly provided by Asahi Kasei Corporation, Japan). Cell Cycle Analysis Analysis of the cell cycle was done by standard labeling of nuclei with propidium iodide, and by pulse (4 hours) incorporation of bromodeoxyuridine (BrdU, 10 μM) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). BrdU was revealed by labeling with an anti-BrdU antibody directly coupled to fluorescein (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Cells were acquired using a FACScalibur flow cytometer and results were analyzed with CellQuest software (Becton, Dickinson and Company). Single-Cell Culture Experiments Single CD34+38low cells were sorted using the automatic device of the EpicElite cell sorter into one well of a 96-well U-bottom plate (Falcon, Becton Dickinson) precoated with 5, 10, or 20 μg/ml of Ig, DL1, or DL4 and under cytokine conditions as described above. At day 6 of the culture, growing wells with >25 cells assessed by visual examination were selected and wells were harvested and seeded under the two following conditions. B-cell/NK-cell/macrophage conditions: cells were cocultured with a confluent layer of MS-5 cells in RPMI 1640 (Gibco-BRL) with 10% human AB serum (Institut Jacques Boy, Reims, France, http://www.biotechjboy.com) and 5% pretested FCS (HCC-6150; Stem Cell Technologies) in the presence of 10 ng/ml hIL-2, 1 ng/ml hIL-15, and 50 ng/ml hSCF. T-cell conditions: cells were cocultured with a layer of OP9-DL1 cells pretreated with mitomycin C (7 μg/ml) in reconstituted α-MEM supplemented with 20% FCS (HyClone), 1% PS, 5 ng/ml hFLT3L, and 2 ng/ml hIL-7. Sorting of the Pro-B CLP/Macrophage Population Sorted CD34+38low cells were exposed for 6 days to either DL1, DL4, or Ig (20 μg/ml) and then replated in B-cell/NK-cell/macrophage conditions. After 14 days, the pro-B/CLP population was sorted on the basis of CD10 expression and negativity for the lineage markers CD1a, CD3, CD4, CD5, CD8, CD14, CD15, CD19, and CD56. The sorted fraction intracellularly expressed CD79α antigen and was negative for CD19 surface expression, as described elsewhere [26]. To study their NK- and B-cell potential, these precursors were plated at 5, 10, 25, or 50 cells/well using a sorter under B-/NK-cell conditions for 15 days. Cell Phenotyping by Flow Cytometry The following monoclonal mouse Ig antibodies to human antigens were used: CD1a-PE, CD3ε-FITC, CD5-PC-5, CD10-PE, CD14-FITC, CD19-PE or PE-Cy5, CD34-PE, CD56-PE-Cy5, CD56-PE, CD79α-PE, and PE-CY5 (all from Beckman Coulter). The intracellular expression levels of CD79α and CD3ε were determined first by fixing cells with a 1% paraformaldehyde solution and, after a wash, using a 0.1% saponin solution for 30 minutes. Analysis was performed on a FACSCalibur (Becton Dickinson) using CellQuest software. Molecular Analysis of Transcript Expression Cells (2-3 × 104) were lysed in trizol (Invitrogen, Life Technologies, Carlsbad, California, http://www.invitrogen.com) and total RNA was extracted by the chloroform method. RNA was then reverse transcribed using oligo-dT and the Superscript kit (Invitrogen, Life Technologies) according to the manufacturer's instructions. Primers used for S14, HES, GATA-3, p-Tα, and Pax-5 reverse transcription-polymerase chain reaction (RT-PCR) were described previously [25]. Expression of each target gene was normalized using the endogenous gene S14. PCRs were performed in a 50-μl volume containing 200 μM of each deoxynucleotide triphosphate, 1 μM of each primer, 1.5 mM MgCl2, and 0.3U DNA polymerase (Invitrogen). Amplifications were done in a GeneAmp PCR System 2700 thermocycler (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). DNA Preparation and PCR Analysis of Ig Gene Rearrangements Sorted CD10+lin− DNA was prepared as previously described [26]. Amplification of incomplete DJH rearrangements was performed using a 3′ consensus JH primer and a mixture of consensus 5′ primers for each of the seven main DH gene families used as previously reported [26]. Statistical Analysis Comparisons were done using a paired Student's t-test on data for bulk conditions and using a χ2 test for clonal conditions. B- and NK-cell frequencies were calculated as the reciprocal of the concentration of cells that resulted in ≤37% negative wells using Poisson statistics and the weighted mean method. Results Dose-Dependent Inhibition of the Proliferation of Cord Blood CD34+CD38low Cells Exposed to Notch Ligands Sorted CD34+CD38low cord blood human primitive progenitors were exposed to various doses of human DL1 or DL4 Notch ligands or to Ig control in the presence of cytokines. As shown in Figure 1A, at day 6, CD34+CD38low cells expressed higher levels of HES-1, PT-α, and GATA-3 transcripts in the presence of 20 μg/ml of DL4 than with the lower doses of DL4 and with DL1. No induction of Pax-5 expression, a gene associated with B-cell differentiation, was noted. These results show that Notch ligand exposure activates the Notch pathway and a T-cell molecular program, as previously described [25]. Next, we evaluated the effects of Notch priming on CD34+CD38low progenitor proliferation. As shown in Figure 1B, at day 6, cell counts showed a significant and dose-dependent inhibition of the proliferation of CD34+CD38low cells cultured on either DL1 or DL4, compared with control conditions. Exposure to DL4, used at 10 μg/ml and 20 μg/ml, led, respectively, to 72% ± 2.8% and 80.6% ± 2.9% inhibition of cell proliferation, compared with control conditions (n = 4; p = .02 and p = .002; mean ± standard error of the mean [SEM]). DL1 exposure also significantly inhibited cell proliferation but only at the 20-μg/ml dose (69.3% ± 4.3% inhibition, compared with control, n = 4; p = .004). Effects of DL4 on cell proliferation were confirmed by cycle analysis using BrdU incorporation. As shown in Figure 1C, in a representative experiment, 24% ± 4% (mean ± SEM) (n = 4) of CD34+CD38low cells exposed to Ig, but only 4% ± 1% (n = 4) of those exposed to DL4, incorporated BrdU during a 4-hour pulse. Measurement of apoptosis at day 6 using annexin V staining did not show significant differences among the different culture conditions (6.9%, 7.8%, and 8.8% in for Ig, DL1, and DL4, respectively, mean of three experiments, data not shown). These results demonstrate that the induction of Notch signaling in CD34+CD38low cells by DL1 and DL4 decreased cell proliferation in a dose-dependent manner. This effect was more pronounced when cells were exposed to DL4. 1 Open in new tabDownload slide Gene expression and proliferation of CD34+CD38low progenitors exposed to Notch ligands DL1 or DL4. (A): RT-PCR for expression of S14, HES-1, T-cell (pTα, GATA3), and B-cell (PAX-5) associated transcripts in CD34+38low cells after 6 days of culture in the presence of 5 μg/ml or 20 μg/ml of either Ig, DL1, or DL4. Total cord blood mononuclear cells were used as a control. (B): Expansion indices of CD34+38low progenitors at day 6 of the culture in the presence of various concentrations of Ig (□), DL1 (), or DL4 (▾). All results are mean ± standard error of three or four independent experiments between Notch ligand and Ig control conditions (*, p < .05; **, p < .01). Expansion indices were calculated as: number of living cells/number of cells plated at day 0. (C): CD34+CD38low cells incubated on DL4 and Ig for 5 days were pulsed with BrdU for 4 hours and labeled with anti-BrdU-FITC and CD34-PC5. Percentages refer to the proportion of CD34+ cells that had incorporated BrdU during the 4 hours. One representative experiment of four with similar results is shown. Abbreviations: BrdU, bromodeoxyuridine; DL, Delta; FITC, fluorescein isothiocyanate; PE, phycoerythrin; RT-PCR, reverse transcription-polymerase chain reaction. 1 Open in new tabDownload slide Gene expression and proliferation of CD34+CD38low progenitors exposed to Notch ligands DL1 or DL4. (A): RT-PCR for expression of S14, HES-1, T-cell (pTα, GATA3), and B-cell (PAX-5) associated transcripts in CD34+38low cells after 6 days of culture in the presence of 5 μg/ml or 20 μg/ml of either Ig, DL1, or DL4. Total cord blood mononuclear cells were used as a control. (B): Expansion indices of CD34+38low progenitors at day 6 of the culture in the presence of various concentrations of Ig (□), DL1 (), or DL4 (▾). All results are mean ± standard error of three or four independent experiments between Notch ligand and Ig control conditions (*, p < .05; **, p < .01). Expansion indices were calculated as: number of living cells/number of cells plated at day 0. (C): CD34+CD38low cells incubated on DL4 and Ig for 5 days were pulsed with BrdU for 4 hours and labeled with anti-BrdU-FITC and CD34-PC5. Percentages refer to the proportion of CD34+ cells that had incorporated BrdU during the 4 hours. One representative experiment of four with similar results is shown. Abbreviations: BrdU, bromodeoxyuridine; DL, Delta; FITC, fluorescein isothiocyanate; PE, phycoerythrin; RT-PCR, reverse transcription-polymerase chain reaction. Priming of CD34+CD38low Cells by Notch Ligand Increases the Frequency of T- and NK-Cell Potential at the Single-Cell Level In order to evaluate the effects of DL1 and DL4 ligands on the potential of primitive CD34+CD38low precursors, single cells were sorted and cultured on wells precoated with different doses of ligands (5 μg/ml and 20 μg/ml). At day 6, wells containing >25 cells under microscopic visual evaluation (proliferating clones) were harvested and seeded in culture conditions suitable for either NK-/macrophage/B- or T-cell differentiation (Fig. 2A). First, we confirmed that cells cultured at a clonal level in ligand conditions, but not in control wells, expressed HES and GATA-3 transcripts at day 6 (not shown). Over a total of five experiments, we were able to transfer into secondary cultures 332 of 1,644 (20%), 224 of 1,176 (20.5%), and 237 of 1,644 (14.5%) clones generated in control Ig, DL1, and DL4 conditions, respectively. The number of proliferating clones generated under DL4 conditions was significantly lower (p = .001) than the number generated under control conditions. This result corroborates the inhibitory effect observed with DL4 under bulk conditions whereas no significant effect was observed with DL1. The single-cell analysis showed that CD34+CD38low cell exposure to Notch ligands resulted in a greater frequency of T- and NK-cell precursors (Fig. 2B). A 130% higher number of T-cell clones was obtained when cells were exposed to 20 μg/ml of either DL1 or DL4 (p = .016 and p = 0.019 under DL1 and DL4 conditions, respectively), whereas no difference in T-cell potential was noted when progenitors were exposed to 5 μg/ml of both ligands. However, exposure to this lower dose increased the NK-cell potential by up to 50% (p = .014 and p = .006 for DL1 and DL4, respectively). The number was up to 100%–300% higher at higher doses of ligands (p < .035). Interestingly, DL4 was more efficient than DL1 in priming NK-cell potential (Fig. 2B). Moreover Notch signaling decreased macrophage potential (Fig. 2B). 2 Open in new tabDownload slide Analysis of the frequency of lymphoid and myeloid precursors generated from single Notch/Delta primed CD34+38low cells. (A): Single CD34+38low cells were sorted and initially cultured on 96-well plates coated with various concentrations of Ig, DL1, or DL4 (5-20 μg/ml). At day 6, the progeny of each of the outgrowing cells was replated, half under OP9-DL1 T-cell differentiation conditions and half under B-cell/NK-cell/macrophage differentiation conditions to determine the frequency of lineage-committed clones. Representative phenotypic analysis of CD19+ B cell, CD14+ macrophage, CD56+ NK cell, and intracellular CD3ε/CD5+ (iCD3ε/CD5+) precursor T clones are shown. (B): Percentage change from control conditions in the frequency of iCD3ε+CD5+ precursor T, CD56+ NK, and CD14+ macrophage clones generated from single CD34+38low cells primed in the presence of 5 μg/ml or 20 μg/ml of DL1 (gray bars) or DL4 (black bars) ligand and then transferred to appropriate culture conditions. (C): Frequency of clones with single (B, T, and NK), bipotent (B/NK and T/NK), or multipotent (NK/T/B) potentials generated from single CD34+CD38low cells primed with Ig (white bars) or 20 μg/ml of DL4 (black bars) and then transferred to appropriate culture conditions. Abbreviations: DL, Delta; NK, natural killer. 2 Open in new tabDownload slide Analysis of the frequency of lymphoid and myeloid precursors generated from single Notch/Delta primed CD34+38low cells. (A): Single CD34+38low cells were sorted and initially cultured on 96-well plates coated with various concentrations of Ig, DL1, or DL4 (5-20 μg/ml). At day 6, the progeny of each of the outgrowing cells was replated, half under OP9-DL1 T-cell differentiation conditions and half under B-cell/NK-cell/macrophage differentiation conditions to determine the frequency of lineage-committed clones. Representative phenotypic analysis of CD19+ B cell, CD14+ macrophage, CD56+ NK cell, and intracellular CD3ε/CD5+ (iCD3ε/CD5+) precursor T clones are shown. (B): Percentage change from control conditions in the frequency of iCD3ε+CD5+ precursor T, CD56+ NK, and CD14+ macrophage clones generated from single CD34+38low cells primed in the presence of 5 μg/ml or 20 μg/ml of DL1 (gray bars) or DL4 (black bars) ligand and then transferred to appropriate culture conditions. (C): Frequency of clones with single (B, T, and NK), bipotent (B/NK and T/NK), or multipotent (NK/T/B) potentials generated from single CD34+CD38low cells primed with Ig (white bars) or 20 μg/ml of DL4 (black bars) and then transferred to appropriate culture conditions. Abbreviations: DL, Delta; NK, natural killer. The CD34+CD38low population is heterogeneous and contains multipotent (T/B/NK), bipotent (T/NK or B/NK), and monopotent (T, B, or NK) clones [27]. Our system allowed us to analyze, after transfer into appropriate culture systems, the potential of daughter cells arising from the progeny of a single cell exposed for 6 days to DL4. Thus, we investigated whether Notch signaling affected the frequency of these different categories of clones. We found that Notch priming (20 μg/ml of DL4) led to a greater frequency of monopotent T- and NK-cell clones, whereas the development of monopotent B-cell clones was completely abrogated (Fig. 2C). Moreover, the numbers of tripotent NK+/T+/B+ and bipotent T+/NK+ and B+/NK+ clones were greater, suggesting that Notch priming affected the potential of multipotent progenitors. Exposure of CD34+CD38low Cells to Increasing Doses of DL1 and DL4 Inhibits the Generation of B Cells at a Pro-B/CLP Stage We have previously shown that CD34+CD38low cells cocultured with MS-5 stromal cells differentiate into pro-B CD79α+CD19− cells able to give rise in vitro to B cells, NK cells, and macrophages, representing a pro-B/CLP stage [26]. Thus, we investigated whether Notch priming could influence this early stage of lymphoid development. CD34+CD38low cells were exposed for 6 days to Notch ligands and then replated in coculture conditions with MS-5 murine stromal cells. In accordance with single-cell experiments, we found that exposure of CD34+CD38low cells to 20 μg/ml of DL1 and DL4 inhibited, by 51.1% ± 11% and 70.6% ± 12%, respectively, the generation of CD79α+CD19+ B cells (p = .05 and p = .006 for DL1 and DL4, respectively, mean ± SEM) (Fig. 3A, 3D). Only a trend to a lesser generation of B cells was noted at lower doses of ligands (p = .3 at 10 μg/ml for both ligands) (Fig. 3A, 3D). As show in Figure 3B, in bulk culture conditions, the generation of CD56+ NK cells and CD14+ macrophages was not affected by pre-exposure of CD34+CD38low cells to Notch ligands. Strikingly, as shown in Figure 3A and 3C, we found that Notch ligand treatment did not affect the generation of CD79α+CD19− pro-B cells (Fig. 3C). These results demonstrate that the priming of CD34+CD38low cells by Notch ligand blocks the transition of pro-B cells toward more mature CD19+ B cells. 3 Open in new tabDownload slide Phenotypic analysis of B-cell differentiation from CD34+CD38low progenitors exposed to DL1 or DL4. (A): Phenotypic analysis of pro-B (CD79α+CD19−) and pre-B (CD79α+CD19+) cells generated from CD34+38low cells primed in the presence of various doses (5 μg/ml, 10 μg/ml, or 20 μg/ml) of either control Ig, DL1, or DL4 for 6 days and then transferred to secondary cultures on MS-5 stromal cells for 21 days. (B): Phenotypic analysis of NK cells (CD56+) and macrophages (CD14+) generated from CD34+38low cells cultured as in (A). (C): Percentages of CD79α+CD19− pro-B cells generated at day 21 of secondary cultures from CD34+38low cells primed by control Ig (white bars), DL1 (gray bars), or DL4 (black bars) at 5 μg/ml, 10 μg/ml, or 20 μg/ml. (D): Percentages of CD79α+CD19+ pre-B cells were assessed as in (B). All results are mean ± standard error of three independent experiments (*, p < .05; **, p < .01). Abbreviations: DL, Delta; NK, natural killer. 3 Open in new tabDownload slide Phenotypic analysis of B-cell differentiation from CD34+CD38low progenitors exposed to DL1 or DL4. (A): Phenotypic analysis of pro-B (CD79α+CD19−) and pre-B (CD79α+CD19+) cells generated from CD34+38low cells primed in the presence of various doses (5 μg/ml, 10 μg/ml, or 20 μg/ml) of either control Ig, DL1, or DL4 for 6 days and then transferred to secondary cultures on MS-5 stromal cells for 21 days. (B): Phenotypic analysis of NK cells (CD56+) and macrophages (CD14+) generated from CD34+38low cells cultured as in (A). (C): Percentages of CD79α+CD19− pro-B cells generated at day 21 of secondary cultures from CD34+38low cells primed by control Ig (white bars), DL1 (gray bars), or DL4 (black bars) at 5 μg/ml, 10 μg/ml, or 20 μg/ml. (D): Percentages of CD79α+CD19+ pre-B cells were assessed as in (B). All results are mean ± standard error of three independent experiments (*, p < .05; **, p < .01). Abbreviations: DL, Delta; NK, natural killer. A 48-Hour Notch Signaling Is Sufficient to Inhibit the Proliferation and B-Cell Differentiation of CD34+CD38low Cells Next, we wanted to determine the minimal duration of Notch signal able to affect the B-cell potential of hematopoietic progenitors. Notch signaling was inhibited using a γ-secretase inhibitor, GSI, that was added at different time points of a 6-day culture of CD34+CD38low cells exposed to either DL1, DL4, or Ig control (20 μg/ml) (Fig. 4A). Then, at day 6, treated cells were transferred to MS-5 stromal cells in order to evaluate their B-cell potential. 4 Open in new tabDownload slide Kinetics of Notch signaling effect on B-cell differentiation. (A): Sorted CD34+38low CB progenitors were incubated for 6 days in the presence of 20 μg/ml of the Notch ligands DL1 and DL4 and in the presence of GSI added either at 0, 24, 48, 72, 96, or 110 hours following the initiation of the culture period. At day 6, progenitors were collected and replated under either B-cell/NK-cell/macrophage or T-cell culture conditions. Control conditions are represented by sorted CD34+38low CB progenitors incubated for 6 days in the presence of 20 μg/ml of Ig plus either GSI or an equivalent amount of DMSO (GSI vehicle) added at H0. (B): Expansion indices of Ig- (white bars), DL1- (gray bars), or DL4- (black bars) primed CD34+38low cells after 6 days of culture. In Ig conditions, cells were cultured throughout the culture period in the presence of GSI added at day 0 (Ig + GSI) or DMSO (Ig + DMSO). Under Notch ligand conditions, GSI was added at different times throughout the culture period as indicated. All results are mean ± standard error of three independent experiments (*, p < .05). Mean expansion indices were calculated as: number of living cells/number of cells plated at day 0. (C): Time-dependent inhibitory effect of Notch signaling on B-cell differentiation. The figure shows the percentage inhibition of the differentiation of CD79α+CD19+ B cells generated from CD34+CD38low progenitors cultured for 6 days in the presence of 20 μg/ml of DL4 and GSI added either at 48 hours or 96 hours and then replated on MS-5 stromal cells. Results are mean ± standard error of three independent experiments (*, p < .05). The mean inhibitory effect of B-cell differentiation was calculated as: number of B cells in DL4 and GSI conditions/number of B cells in Ig conditions. Abbreviations: CB, cord blood; DL, Delta; DMSO, dimethylsulfoxide; GSI, γ-secretase inhibitor; NK, natural killer. 4 Open in new tabDownload slide Kinetics of Notch signaling effect on B-cell differentiation. (A): Sorted CD34+38low CB progenitors were incubated for 6 days in the presence of 20 μg/ml of the Notch ligands DL1 and DL4 and in the presence of GSI added either at 0, 24, 48, 72, 96, or 110 hours following the initiation of the culture period. At day 6, progenitors were collected and replated under either B-cell/NK-cell/macrophage or T-cell culture conditions. Control conditions are represented by sorted CD34+38low CB progenitors incubated for 6 days in the presence of 20 μg/ml of Ig plus either GSI or an equivalent amount of DMSO (GSI vehicle) added at H0. (B): Expansion indices of Ig- (white bars), DL1- (gray bars), or DL4- (black bars) primed CD34+38low cells after 6 days of culture. In Ig conditions, cells were cultured throughout the culture period in the presence of GSI added at day 0 (Ig + GSI) or DMSO (Ig + DMSO). Under Notch ligand conditions, GSI was added at different times throughout the culture period as indicated. All results are mean ± standard error of three independent experiments (*, p < .05). Mean expansion indices were calculated as: number of living cells/number of cells plated at day 0. (C): Time-dependent inhibitory effect of Notch signaling on B-cell differentiation. The figure shows the percentage inhibition of the differentiation of CD79α+CD19+ B cells generated from CD34+CD38low progenitors cultured for 6 days in the presence of 20 μg/ml of DL4 and GSI added either at 48 hours or 96 hours and then replated on MS-5 stromal cells. Results are mean ± standard error of three independent experiments (*, p < .05). The mean inhibitory effect of B-cell differentiation was calculated as: number of B cells in DL4 and GSI conditions/number of B cells in Ig conditions. Abbreviations: CB, cord blood; DL, Delta; DMSO, dimethylsulfoxide; GSI, γ-secretase inhibitor; NK, natural killer. When Notch signaling was abrogated throughout the culture period by adding GSI at day 0, no inhibition of cell proliferation was noted, compared with control conditions (Fig. 4B). When GSI was added either at day 1 or day 2 of culture, 24 or 48 hours of Notch signaling was sufficient to significantly inhibit the rate of cell proliferation under DL4 and DL1 conditions, respectively (Fig. 4B). CD79+CD19+ B-cell differentiation was significantly lower following 48 hours of Notch signaling (Fig. 4C). Results also showed a correlation between the duration of Notch signaling and the inhibition of the generation of CD79+CD19+ cells under DL4 conditions (Fig. 4C). This inhibition was up to 32.8% ± 12% (p = .05) and 82% ± 9% (p = .003) when GSI was added at day 2 and day 4, respectively (n = 3). According to the absence of inhibitory effects of Notch/Delta interactions in the generation of CD79a+CD19− pro-B/CLPs (Fig. 3B), the addition of GSI at different time points of the culture period did not change the generation of those cells (not shown). These results demonstrate a time-dependent effect of Notch/Delta interactions in the inhibition of B-cell lineage differentiation beyond the pro-B/CLP stage. Notch Signaling Directs the Fate of CD79α+CD19−CD10+ Pro-B/CLPs The above results showed that priming of CD34+CD38low cells with Notch ligands blocked the subsequent differentiation of these cells toward the B-cell lineage at a pro-B CD79α+CD19− stage. Next, we investigated whether Notch priming of primitive progenitors may have an impact on the biological potential of their pro-B/CLP progeny. After a 6-day priming of CD34+CD38low progenitors under control or Notch ligand conditions, those cells were transferred to MS-5 stromal cells (Fig. 5). Because CD79α is an intracellular antigen, pro-B cells generated at day 14 were sorted on the basis of a CD10+lin− phenotype. As shown in Figure 5B, and previously described elsewhere [26], these cells were CD79α+ and had initiated a DJH rearrangement of the Ig locus. Moreover, we found that whatever the priming conditions (i.e., Ig, DL1, or DL4), these cells gave rise to CD19+ B cells, CD56+ NK cells, CD14+ macrophages, and CD5+CD1a+ precursor T cells (Fig. 5C). However, priming with DL4 or DL1 of hematopoietic progenitors modified the fate of those cells by inhibiting the generation of CD19+ B cells and CD14+ macrophages and increasing the generation of NK cells. Finally, the generation of T-cell precursors was not significantly affected. In order to confirm the B- and NK-cell potential alterations of pro-B/CLPs generated from CD34+CD38low progenitors exposed to DL4, we precisely evaluated the frequency of B- and NK-cell precursors by limiting dilution analysis (Fig. 6). Results show that exposure of CD34+CD38low cells to DL4 alters the frequency of progenitors generated from pro-B/CLPs, increasing the NK-cell potential (1 in 16 versus 1 in 38 for pro-B CLPs generated from CD34+CD38low cells exposed to control Ig) and decreasing the B-cell potential (1 in 714 versus 1 in 48 generated under control Ig conditions). Together, these results show that Notch signaling imprinted the potential of subsequent CLP progeny from CD34+CD38low hematopoietic progenitors. Moreover, Notch signaling irreversibly altered the generation of CD19+ B cells and CD14+ macrophages and increased the differentiation of pro-B/CLPs toward the NK-cell lineage. 5 Open in new tabDownload slide Differentiation potential of pro-B/CLPs generated from CD34+CD38low progenitors primed with DL1, DL4, or control. (A): Sorted CD34+38low progenitors were pretreated for 6 days with 20 μg/ml of either DL1, DL4, or control Ig and then transferred to B-cell differentiation conditions and cultured for 2 weeks. At that time, generated CD10+lin− pro-B/CLP cells were sorted and replated under T-cell or B-cell/NK-cell/macrophage conditions for 35 days or 21 days, respectively. (B): CD10+lin− sorted cells expressed intracellular CD79α antigen and had initiated DJH gene rearrangements similar to CD19+ more mature cells as assessed by PCR DNA analysis. Blin-1, a pre-B cell line was used as a positive control. (C): Phenotypic analysis of the generation of CD19+ B cells, CD56+ NK cells, CD14+ macrophages, and CD5+CD1a+ precursor T cells from CD10+CD79α+lin− cells generated from CD34+CD38low progenitors primed with Ig, DL1, or DL4. One representative experiment of two with similar results is shown. Abbreviations: CLP, common lymphoid progenitor; DL, Delta; NK, natural killer; PCR, polymerase chain reaction. 5 Open in new tabDownload slide Differentiation potential of pro-B/CLPs generated from CD34+CD38low progenitors primed with DL1, DL4, or control. (A): Sorted CD34+38low progenitors were pretreated for 6 days with 20 μg/ml of either DL1, DL4, or control Ig and then transferred to B-cell differentiation conditions and cultured for 2 weeks. At that time, generated CD10+lin− pro-B/CLP cells were sorted and replated under T-cell or B-cell/NK-cell/macrophage conditions for 35 days or 21 days, respectively. (B): CD10+lin− sorted cells expressed intracellular CD79α antigen and had initiated DJH gene rearrangements similar to CD19+ more mature cells as assessed by PCR DNA analysis. Blin-1, a pre-B cell line was used as a positive control. (C): Phenotypic analysis of the generation of CD19+ B cells, CD56+ NK cells, CD14+ macrophages, and CD5+CD1a+ precursor T cells from CD10+CD79α+lin− cells generated from CD34+CD38low progenitors primed with Ig, DL1, or DL4. One representative experiment of two with similar results is shown. Abbreviations: CLP, common lymphoid progenitor; DL, Delta; NK, natural killer; PCR, polymerase chain reaction. 6 Open in new tabDownload slide Limiting dilution analysis of B- and NK-cell potential from CD10+CD79α+lin− progenitors. CD10+lin− pro-B/CLPs generated from CD34+CD38− cells (see Fig. 5A) exposed to either DL4 (▴) or Ig (□) culture conditions were sorted and replated under B-/NK-cell conditions at 5, 10, 25, and 50 cells/well. The frequencies of CD56+ (A) and CD19+ (B) progenitors were evaluated at day 14 of the culture (see Material and Methods). Abbreviations: CLP, common lymphoid progenitor; DL, Delta; NK, natural killer. 6 Open in new tabDownload slide Limiting dilution analysis of B- and NK-cell potential from CD10+CD79α+lin− progenitors. CD10+lin− pro-B/CLPs generated from CD34+CD38− cells (see Fig. 5A) exposed to either DL4 (▴) or Ig (□) culture conditions were sorted and replated under B-/NK-cell conditions at 5, 10, 25, and 50 cells/well. The frequencies of CD56+ (A) and CD19+ (B) progenitors were evaluated at day 14 of the culture (see Material and Methods). Abbreviations: CLP, common lymphoid progenitor; DL, Delta; NK, natural killer. Discussion In this study, we investigated the subsequent fate of human hematopoietic progenitors primed with Notch ligands in a culture system allowing the modulation of the dose and the time line of Notch/Delta signaling. Analyses at a clonal level demonstrated that Notch activation increased the frequency of multipotent progenitors and skewed the T- and NK-cell potential of CD34+CD38low clones in a dose- and ligand-dependent manner. Low doses of ligands were sufficient for significantly increasing the frequency of NK-cell precursors, whereas higher doses were required for increasing the frequency of T-cell clones. Moreover, Notch signaling prevented the differentiation of B-cell clones. Interestingly, we were able to determine that Notch blocks B-cell lineage differentiation at a pro-B CD79α+CD19− stage, previously described as a pro-B/CLP population [26]. We demonstrate here that a transitory exposure of CD34+CD38low progenitors to Notch ligand altered the fate of these pro-B/CLP progeny. In fact, these cells exhibited greater NK-cell potential whereas their capacity to differentiate into CD19+ B cells or CD14+ macrophages was lower. The observation that single CD34+CD38low cells exposed to DL1 or DL4 maintained diverse lymphoid and macrophage potential suggests the lack of any kind of definitive lock of their differentiation response despite prolonged activation of the Notch pathway. Theoretically, this observation suggests at least two possible and nonexclusive responses of CD34+CD38low cells to Notch ligands: first, intrinsic reversibility of the early specification process of CD34+CD38low cells, and second, the ability of some of these clones to delay specification as described in fetal mice liver hematopoietic precursors [28]. Finally, the fact that distinct differentiation responses remained at the single-cell level after switching cells from a Notch/Delta activation culture system to another system proves the plasticity of some of these clones. We found that the effects of Notch signaling on the T- and NK-cell potential of primitive progenitors was dose and ligand dependent. Several models have shown that various intensities of Notch signaling may induce different levels of expression of target genes in a single cell, leading to different cell fate outcomes [29]. These observations are fully in accordance with several reports showing that a critical threshold of Notch signaling is required for inducing different cell fate outcomes [19, 30]. Finally, single-cell experiments offer the advantage of limiting, in contrast to bulk cultures, cell–cell interactions, which may modulate responses to Notch activation [31]. We, and others, have already reported that activation of Notch in immature progenitors favors differentiation toward CD56+ NK cells [25, 32, 33]. However, the precise conditions of Notch activation required for NK induction remain debatable because it has been shown that transduction of NICD into human thymic lymphomyeloid precursors inhibits their NK-cell potential [34]. Similarly, partial inhibition of the Notch signaling pathway with intermediate doses of GSI allows the generation of NK cells [21]. Experiments in mice [35] and rat [36] also strongly suggest that inhibition of Notch signaling is responsible for directing lymphoid progenitors into NK cells. Our results demonstrate that a 1-week activation of the Notch/Delta pathway increased, by up to 250%, the frequency of NK cells. In accordance with our results, recent studies performed in mice demonstrate that Pax5−/− pro-B cells could not differentiate efficiently to NK cells unless they had received a transient Notch signal [37, 38]. Furthermore, we observed here that signals delivered by DL4 enhanced NK-cell commitment more efficiently than those delivered by DL1, a feature of NK-cell differentiation that has not been explored in previous studies performed exclusively with DL1 ligand. This result is in accordance with our previous study showing that 2-4 days of priming of CD34+ cells with DL4 induced high levels of HES-1 and ID2, a key transcription factor of NK-cell specification [25]. Thus, our results demonstrate that transient Notch/DL4 interaction is an inducer of human progenitor differentiation toward the NK-cell lineage. There are several lines of evidence that some NK and T cells are closely related and arise from common T/NK progenitors [39]. T- and NK-cell potentials are also closely linked and dependent on Notch signaling [21, 33, 40, 41]. We found that the percentage of multipotent and bipotent clones with NK- and T-cell potentials was greater among the progeny of single cells that had experienced Notch signaling. Globally, our results confirm, at the single-cell level, the close developmental behavior toward the T-/NK-cell lineage of pluripotent human precursors primed with Notch. In contrast to T-/NK-cell differentiation, the distinct response to Notch signaling of early progenitors is evidence of the early separation of the T- and B-cell lineages. The B-cell lineage specification also depends on the activity of the E2A, EBF, and PAX-5 transcriptions factors. PAX-5 is a master factor that activates the expression of CD19 and locks B-cell specification [42]. We found here, as previously reported, that Notch signaling inhibited the generation of CD19+ B cells from CD34+CD38low immature progenitors. However, for the first time, to our knowledge, we show that Notch/Delta interaction did not prevent the first steps of human B-cell differentiation to pro-B CD79α+CD19− cells. As previously described [26] and shown here, those cells are characterized by initiation of the rearrangement of the DJH genes of the Ig locus, the expression of EBF, but not PAX-5, and multipotentiality [26]. Likely, our results suggest, as recently demonstrated in vitro for prethymic precursors [28], that, in our system, Notch prevents B cell lineage specification from pro-B cells by blocking induction of PAX-5 expression by these cells. Other studies have shown that Notch may also antagonize EBF activity [43, 44] or trigger E2A stabilization [45]. We do not favor this latter effect because multipotent pro-B CD79a+CD19− cells generated from immature CD34+ progenitors primed with Notch ligands maintained T- and NK-cell potentials. Mice studies have shown that PAX-5-deficient pro-B cells can efficiently differentiate into T cells when exposed to Notch/Delta signaling [46, 47]. Along the same line, conditional deletion of PAX-5 allowed mature B cells to dedifferentiate into functional T cells [48], showing the plasticity of lymphoid progenitors. On the other hand, specific coupling between Notch and its ligands may also impair B-cell differentiation. Specific expression of the activated form of Notch-2 in murine hematopoietic progenitors blocks the maturation of conventional B cells at the pre-B stage [49]. Analysis of the differentiation potential of pro-B/CLPs revealed that Notch signaling in primitive hematopoietic progenitors represses the subsequent B-cell and macrophage differentiation of CLP/macrophage progenitors. However, the fate of these cells was clearly directed toward the NK-cell lineage. In contrast, a transient Notch signal in primitive hematopoietic precursors was not sufficient to increase the generation of T-cell progenitors from pro-B/CLPs. This confirms reports in mice showing that continuous Notch signaling is required for the induction, survival, and/or maintenance of T-cell specification [28]. These results suggest that Notch signaling exhibits a durable and delayed effect on the differentiation and survival capabilities of pro-B/CLP cells. Study of the biological potential of human primitive progenitors is challenging, and single-cell experiments are required for the precise analysis of the impact of environmental factors on lineage specification. Using this approach, globally, our results revealed complex behavior of uncommitted hematolymphoid progenitors following Notch activation. Instead of a binary choice toward either T- or B-cell specification, some progenitors maintained pluripotency following 1 week of Notch/Delta interaction, demonstrating the degree of plasticity of these cells. Single-cell experiments allowed us to determine the T-cell, B-cell, NK-cell, and macrophage potentials of CD34+CD38low progenitors. We confirmed that the CD34+CD38low population is heterogeneous and consists not only of multipotential cells but also of more restricted progenitors. For example, we found that around 6%–7% of clones generated under Ig and DL4 culture conditions exhibited bipotent T-cell/macrophage potential (data not shown). These results are in accordance with recent studies in mice showing that some early T progenitors retain the ability to generate macrophages even if they lose their B-cell potential [50, 51]. These results argue against the classical model of hematopoiesis, based on first-level segregation between myeloid and lymphoid potential. We have identified an early pro-B stage of B-cell differentiation that was not inhibited by Notch/Delta interaction, indicating that some uncommitted progenitors maintain their capacity to undergo the first stages of B-cell differentiation despite Notch/Delta interactions. Initial education of primitive hematopoietic progenitors in the presence of Notch ligands may durably imprint the fate of a pro-B/CLP population generated several weeks later. For the T-cell part, Notch signaling increased the T-cell potential of primitive cells, the majority of which are characterized by combined NK-cell potential. The strongest inducer of T-/NK-cell differentiation was high-density DL4 ligand. Likely, other signal or other conditions not yet identified are needed for the generation of fully differentiated monopotent T-cell precursors from primitive human progenitors. Thus, our results may have physiological implications because they demonstrate that tight regulation of Notch signaling may be achieved in vivo by the type of cell, the density of ligands, and possibly by specific Notch/Delta couples in the microenvironment of hematopoietic progenitors, or in anatomic regions such as observed within the thymus [16]. Acknowledgements This work was supported by grants from INSERM, the Juvenile Diabetes Research Foundation, and the Association Française contre les Myopathies through the Lymphoid Network. 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Copyright © 2009 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Notch Increases T/NK Potential of Human Hematopoietic Progenitors and Inhibits B Cell Differentiation at a Pro-B Stage
JF - Stem Cells
DO - 10.1002/stem.94
DA - 2009-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/notch-increases-t-nk-potential-of-human-hematopoietic-progenitors-and-dVMJlLuumf
SP - 1676
EP - 1685
VL - 27
IS - 7
DP - DeepDyve
ER -