Originally published online as doi:10.1189/jlb.1003462 on January 23, 2004

Published online before print January 23, 2004

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(Journal of Leukocyte Biology. 2004;75:714-720.)
© 2004 by Society for Leukocyte Biology

Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay

Virág Vas^*, László Szilágyi, Katalin Pálóczi and Ferenc Uher^*,1

^* Stem Cell Biology, National Medical Center, Budapest, Hungary;
Department of Biochemistry, Loránd Eötvös University, Budapest, Hungary; and
Department of Immunology, Haematology, and Transfusiology, Semmelweis University, Budapest, Hungary

¹Correspondence: National Medical Center, Stem Cell Biology, Diószegi ut 64., Budapest, Hungary, H-1113. E-mail: uher{at}ohvi.hu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Stem cells reside in customized microenvironments (niches) that contribute to their unique ability to divide asymmetrically to give rise to self and to a daughter cell with distinct properties. Notch receptors and their ligands are highly conserved and have been shown to regulate cell-fate decisions in multiple developmental systems through local cell interactions. To assess whether Notch signaling may regulate hematopoiesis to maintain cells in an immature state, we examined the functional role of the recombinant, secreted form of the Notch ligand Jagged-1 during mouse hematopoietic stem cell (HSC) and progenitor cell proliferation and maturation. We found that ligand immobilization on stromal layer or on Sepharose-4B beads is required for the induction of self-renewing divisions of days 28–35 cobblestone area-forming cell. The free, soluble Jagged-1, however, has a dominant-negative effect on self-renewal in the stem-cell compartment. In contrast, free as well as immobilized Jagged-1 promotes growth factor-induced colony formation of committed hematopoietic progenitor cells. Therefore, we propose that differences in Jagged-1 presentation and developmental stage of the Notch receptor-bearing cells influence Notch ligand-binding results toward activation or inhibition of downstream signaling. Moreover, these results suggest potential clinical use of recombinant Notch ligands for expanding human HSC populations in vitro.

Key Words: cobblestone area-forming cell • mouse • Notch

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hematopoietic stem cells (HSCs) have the ability to renew themselves and to differentiate into mature blood cells of all lineages [^{1 2 3} ]. In the mouse, long-term, self-renewing HSCs make up 0.01–0.005% of bone marrow and can be isolated by their expression of undetectable levels of lineage markers, low levels of Thy-1.1, and high levels of c-kit and Sca-1 [⁴ ]. Although HSCs have been purified and characterized successfully, a fundamental question remains how their self-renewing growth is regulated. The Notch pathway is used widely, an evolutionarily conserved regulatory system that plays a central role in the fate decision of multipotent precursor cells. Notch often acts by inhibiting differentiation along a particular pathway while permitting or promoting self-renewal or differentiation along alternative pathways. In mammals, gene duplication has led to a diverse repertoire of Notch-related molecules. Four Notch receptor genes (Notch1, -2, -3, and -4) and five Notch ligand genes [Jagged-1 and -2; (-like)-1, -3, and -4] have been described [⁵ , ⁶ ]. Notch receptors and Notch ligands have been found on hematopoietic precursors as well as on marrow stromal cells [⁷ , ⁸ ]. After ligand binding, Notch transmembrane domains are processed proteolytically, and the released intracellular region moves to the cell nucleus, where it will act as a transcriptional activator after association with the DNA-binding protein CBF-1, Suppressor of Hairless, Lag-1 (CSL) [⁶ ].

Evidence for the importance of Notch signaling in hematopoiesis is plenty, albeit controversial. Retrovirus-mediated expression of activated Notch1 enhanced self-renewal and immortalized hematopoietic precursor cells in the context of appropriate cytokines, indicating Notch’s potential capacity for enhancing stem-cell self-renewal in nonmutant cells [⁹ ]. In contrast, multiple studies have reported only a modest increase in hematopoietic precursor cell numbers following culture with Notch ligand presented on cell surfaces or with engineered Notch ligands such as Delta-1, Jagged-1, or Jagged-2 in solution [^{10 11 12 13 14 15 16} ]. However, in many of those previous studies, hematopoietic precursors were cocultured with nonhematopoietic cells expressing cell-bound ligand. In those cases, ligand-presenting cells may have generated factors that interfered with the activity of the Notch ligand. In other studies, the engineered ligand was presented in solution, and in those cases, Notch activity may have been induced only partially. Recently, massive expansion of cytokine-stimulated, murine hematopoietic precursors was achieved by incubation with Delta-1 immobilized to the surface of plastic culture plates. Transplantation of these cultured cells resulted in a multiple log increase in the number of short-term bone marrow-reconstituting cells [¹⁷ ]. However, Jagged-1, not Delta-1, appears to be the major Notch ligand expressed by bone marrow stromal cells [¹¹ , ¹⁸ ].

In the present studies, we investigated the effect of soluble (monovalent) and immobilized (multivalent) forms of recombinant Jagged-1 protein on murine HSC and progenitor cell proliferation and fate decision. Our results show that ligand immobilization is required for the induction of self-renewing division of days 28–35 cobblestone area-forming cell (CAFC). The soluble ligand has a dominant-negative effect on self-renewal in the stem-cell compartment, presumably by sequestering the Notch receptor and preventing Notch interaction with the immobilized ligand. In contrast, soluble as well as immobilized Jagged-1 promotes cytokine-induced colony formation of committed progenitor cells. Thus, Jagged-1 is, in either form, a weak growth factor for hematopoietic progenitor cells.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
Male and female (C57Bl/6xDBA/2)F1 (BDF1) mice were obtained from the animal facility of the National Institute of Oncology (Budapest, Hungary) and were used at 6–8 weeks of age.

Preparation of bone marrow cells
Murine bone marrow was extracted from the femurs and tibias of the mice. After standard erythrocyte lysis, nucleated cells were placed into 25 cm² culture flasks (Costar, Cambridge, MA) and incubated overnight in minimum essential medium (MEM; Gibco, Grand Island, NY), supplemented with 20% fetal calf serum (FCS) and antibiotics (Gibco). Subsequently, nonadherent cells were harvested and stained with a panel of biotinylated lineage-specific antibodies (mouse-lineage panel, containing anti-CD3e, anti-CD45R/B220, anti-CD11b, anti-Ly-6G, and anti-TER-119; BD PharMingen, San Diego, CA) for 30 min at 4°C. Cells were washed twice and incubated with streptavidin-coated magnetic particles (BioSource, Camarillo, CA) for an additional 30 min at 4°C. The particle-free cell fraction was retrieved by exposure to a magnetic field (Polar Bear magnet, BioSource), and the magnetic depletion procedure was repeated on this fraction. Unstained cells were separated and collected as the lineage-negative (Lin⁻) fraction of bone marrow cells. This noncommitted cell population represents 0.2–0.6% of original, unfractionated bone marrow-nucleated cells. To ensure >98% purity, lin⁻ cells were labeled with Streptavidin–phycoerythrin (PE) and were reanalyzed by fluorescein-activated cell sorter.

Flow cytometry
Unfractionated or lin⁻ cells were resuspended in phosphate-buffered saline (PBS). Cells were stained with PE-conjugated monoclonal anti-Sca-1 (clone D7), fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (clone RAM34), and biotinylated anti-c-kit (clone 2B8) antibodies (BD PharMingen). c-kit-positive cells were detected with Streptavidin–PE or Extravidin–FITC (Sigma Chemical Co., St. Louis, MO). In all cases, stained cells were washed with PBS containing 12.5 µg/ml propidium iodide to gate for dead cells and were analyzed immediately on a FACScan flow cytometer using Cell Quest software (Becton Dickinson, Mountain View, CA).

Recombinant Jagged-1 protein–mono- and multivalent Notch ligands
To generate soluble Jagged-1 protein [sJG1^{extracellular domain (ECD)}], we used the pUSEamp vector with the insert of the cDNA encoding the ECD of rat Jagged-1 (Upstate Biotechnology, Lake Placid, NY). COS7 cells were transfected with the vector using the LipofectAmine^TM Plus reagent and the manufacturer’s protocol (Invitrogen Life Technologies, Rockville, MD). Briefly, COS7 cells to be transfected were cultured onto six-well plates in 2 ml Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS (Gibco) until the cells became confluent. A solution of LipofectAmine^TM Plus reagent–pUSEamp vector (1.5 µg/ml final concentration) was added for each well. After 3 h incubation, transfected cells were washed with serum-free DMEM and replaced with 2 ml medium supplemented with 10% FCS. Control cells were treated in an identical manner, but DNA was not added. Conditioned medium samples were collected and pooled after 3, 5, and 7 days. The sJG1^ECD was then absorbed on, and eluted from, a goat anti-rat Jagged-1 antibody (Sigma Chemical Co.)-coated Sepharose 4B column. The elution buffer was 3.5 M KSCN, and the peak dialyzed exhaustively against PBS (monovalent Notch ligand). A part of the affinity-purified sJG1^ECD protein was coupled to BrCN-activated Sepharose 4B as recommended by the manufacturer (Amersham Pharmacia Biotech, Uppsala, Sweden; multivalent Notch ligand).

Progenitor (colony-forming cell) assay
Quantification of the number of colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythroid (BFU-E), and CFU granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) was performed using a semisolid cytokine flow cytometry assay. Cells were plated in Iscove’s modified Dulbecco’s medium supplemented with 1% methylcellulose, 30% horse serum (Gibco), 10% WEHI-3B-conditioned medium as source of growth factors, 4 x 10⁻³ M/l L-glutamine, 2.5 x 10⁻⁴ M/l -thioglycerol (Gibco), 1% deionized bovine serum albumin (Sigma Chemical Co.), and antibiotics (Gibco). Cells were cultured in 35 mm petri dishes (Costar) at 37°C in 5% CO₂ in air. CFU-GM and BFU-E were counted on days 7 and 9 of culture in the same dish.

CAFC assay
In vitro determination of HSC and progenitor cell frequencies was performed by limiting dilution analysis of CAFC in microcultures, according to methods described previously [¹⁹ ] with some modifications. Bone marrow cells were extracted and purified as described above. The lin⁻ cells or whole bone marrow cells (unfractionated cells) were washed twice with CAFC medium [¹⁹ ] before the CAFC assay. For each sample, six to eight serial twofold dilutions of cells were prepared and plated in flat-bottom 96-well plates (Costar) over preestablished confluent layers of GY30 stromal cells (GY30, a Jagged-1⁺ murine cell line generously provided by Dr. Donna Rennick, DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, CA) [²⁰ ]. Twenty-four to 36 wells per dilution were plated for each group of cells and maintained at 33°C and 5% CO₂. Once a week, half of the medium (100 µl) was carefully removed from each well, and an equal volume of fresh medium was added. Wells were evaluated for cobblestone areas weekly from days 7–35. The proportion of negative wells at each dilution was used in a Poisson-based limiting dilution analysis calculation to determine the frequency of CAFC using the L-Calc software (Stem Cell Technologies, Vancouver, BC, Canada).

In vitro expansion of lin⁻ bone marrow cells
Stroma-free expansion cultures were performed as follows. Five thousand lin⁻ cells were cultured at 37°C in U-bottomed 96-well plates in 200 µl MEM medium supplemented with 10% FCS, antibiotics (Gibco), and cytokines (Sigma Chemical Co.). The final concentrations of cytokines were as follows: mouse stem cell factor (SCF), 100 ng/ml; Flk-2/Flt-3 ligand (FL), 100 ng/ml; and thrombopoietin (TPO), 50 ng/ml. After 7 or 14 days of culture, all cells were harvested by vigorous pipetting, washed in Hanks’ balanced saline solution, counted, and then used for further analysis. For 14-day cultures, after the first week, one-half of the medium was removed and replaced with fresh medium and growth factors.

Statistical analysis
Results are presented as the mean ± SE. Significance was determined using the two-tailed paired Student’s t-test.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Notch signaling modestly augments the cytokine-induced proliferation of committed hematopoietic progenitor cells
The effect of sJG1^ECD, a soluble form of recombinant Jagged-1 protein, on the colony formation of unfractionated bone marrow cells in semisolid medium is shown in Table 1 . CFU-GEMM, CFU-GM, and BFU-E formation of nucleated marrow cells in the presence of WEHI-3B-conditioned medium as a source of growth factors was slightly but significantly stimulated by sJG1^ECD in a dose-dependent manner. Furthermore, a similar stimulatory effect was observed in cultures incubated with sJG1^ECD-coated Sepharose-4B beads. When exogenous hematopoietic growth factors were not added to the cultures, no colony formation was observed in the presence of Notch ligands (data not shown). Thus, monovalent as well as multivalent Jagged-1 acted directly on committed hematopoietic progenitor cells and augmented their colony formation induced by various growth factors.

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Table 1. Effect of Notch Ligand-Dependent Activation on Colony-Forming Ability of Nucleated Bone Marrow Cells

Effect of sJG1 on the development of CAFCs
Unfractionated bone marrow cells were deposited in a limiting dilution setup in 96-well plates containing a pre-established confluent stromal cell layer, as described in Materials and Methods. It has been shown repeatedly that the time point of appearance of CAFCs in such long-term bone marrow cultures strongly correlates with the maturity of the hematopoietic cells. In the mouse system, committed progenitors form colonies after 7–14 days (hence, they are referred to as day-7–14 CAFC), whereas more primitive stem cells start proliferating only after 4–5 weeks (day-28–35 CAFC) [¹⁹ ]. sJG1^ECD had a biphasic effect in this assay system. Day-7–14 CAFC frequencies were increased, whereas day-28–35 CAFC frequencies were significantly decreased in the presence of high amounts (2–10 µg/ml) of sJG1^ECD. In contrast, low amounts (10–100 ng/ml) of sJG1^ECD enhanced the day-28–35 CAFC frequencies in the cultures. Day-21 CAFC frequencies were unaffected by sJG1^ECD (see an example in Fig . 1A 1B 1C ).

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Figure 1. CAFC frequencies of whole bone marrow cells in the presence or absence of sJG1ECD. Bone marrow cells were isolated as described in Materials and Methods and overlaid on 96-well plates in multiple dilutions for limiting dilution analysis of CAFC in the presence of 50 ng/ml and 5 µg/ml sJG1ECD. Wells were evaluated for cobblestone areas weekly from days 7 through 35. Frequencies of day-7 (A), day-21 (B), and day-35 (C) CAFCs were calculated by the L-Calc software (Stem Cell Technologies). Data are representative of five independent experiments.

To confirm the above data, we performed CAFC assays in which lin⁻ marrow cells were deposited in the plates under limiting dilution conditions. The lin⁻ (36% Sca-1⁺, 73% c-kit⁺, and 68% CD34⁺) cells are highly enriched for day-35 CAFC compared with unfractionated bone marrow cells (1:400–500 vs. 1:80,000–120,000, respectively; data not shown). Again, sJG1^ECD was able to inhibit as well as to stimulate day-35 CAFC formation in a dose-dependent manner (Fig. 2 ). However, when the pre-established stromal layers were only preincubated with a high amount (5 µg/ml) of sJG1^ECD and washed before the lin⁻ cells were seeded, a similar increase of day-35 CAFC frequencies was seen, such as in the presence of 50 ng/ml sJG1 (Fig. 3 ). These data suggest that a small amount of sJG1^ECD is able to bind to the pre-established stromal layer and together with the transmembrane form of Jagged-1 molecules expressed by the stromal cells, form multivalent Notch ligands, increasing day-35 CAFC frequencies. However, sJG1^ECD in excess inhibits multivalent ligand binding to Notch receptor-bearing HSCs.

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Figure 2. Day-35 CAFC frequencies of lin− bone marrow cells in the presence or absence of sJG1ECD. Lin− marrow cells were isolated as described in Materials and Methods and overlaid on 96-well plates in multiple dilutions for limiting dilution analysis of CAFC in the presence of 50 ng/ml and 5 µg/ml sJG1ECD, respectively. Wells were evaluated for cobblestone areas weekly from days 7 through 35. The L-Calc software (Stem Cell Technologies) was used to calculate frequencies of day-35 CAFC. Data are representative of three independent experiments.

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Figure 3. Day-35 CAFC frequencies of lin− bone marrow cell on stromal layer preincubated with sJG1ECD. Established stromal layers were preincubated with a high amount (5 µg/ml) of sJG1ECD and washed before the lin− cells were seeded. Wells were evaluated for cobblestone areas at day 35. The L-Calc software (Stem Cell Technologies) was used to calculate frequencies of day-35 CAFC. Data are representative of three independent experiments.

Multivalent Jagged-1 promotes the survival and expansion of HSCs in vitro
Next, we wished to assess whether stroma-free culture of lin⁻ marrow cells in the presence of multivalent Jagged-1 resulted in expansion of HSCs. To this end, lin⁻ marrow cells were cultured with recombinant SCF, FL, and TPO in the presence or absence of Notch ligands in U-bottomed 96-well plates. After 1 and 2 weeks, cultures were harvested, the cells counted, and the number of day-35 CAFC was determined. A summary of one typical experiment’s data is shown in Table 2 . After 2 weeks in expansion culture, the absolute number of cells was increased eight- to 14-fold in all cultures containing recombinant growth factors. However, the day-35 CAFC frequencies were dropped markedly at the same time, except in cultures containing growth factors and sJG1^ECD-coated Sepharose-4B beads. (Monovalent sJG1^ECD had no similar beneficial effect on the day-35 CAFC frequencies in parallel cultures.) Thus, the number of early hematopoietic cells was increased ten- to 20-fold in the presence of multivalent Jagged-1. This selective expansion of the day-35 CAFC subset was in full agreement with the expansion of Sca-1⁺ c-kit⁺ cells (data not shown) and strongly suggested the in vitro amplification of HSCs.

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Table 2. Effect of Jagged-1-Coated Beads on ex vivo Expansion of lin–Marrow Cells

sJG1 functions as an inhibitor of multivalent ligand-induced Notch signaling during stem-cell proliferation
Finally, based on preliminary experiments (data not shown), we have repeated the in vitro expansion experiments using an optimal (10,000/well) and suboptimal (2000/well) number of sJG1^ECD-coated beads in the presence or absence of different amounts of free sJG1^ECD. As anticipated, sJG1^ECD repressed the ability of sJG1^ECD-coated Sepharose-4B beads to induce self-renewal of day-35 CAFC in a dose-dependent manner (Table 3 ). Thus, we suggest that the sJG1 protein functions as an inhibitor of Notch/multivalent Jagged-1 signaling on day-35 CAFC, leading to a decrease in HSC self-renewal.

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Table 3. Effect of sJG1 and Insolubilized Jagged-1 on ex vivo Expansion of lin–Marrow Cells

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Most growth factors that act on HSCs in culture induce no or limited expansion or are unable to prevent differentiation [² ]. Thus, one of the most notable findings of our work is the induction of proliferation and the prevention of HSC differentiation by the multivalent Notch ligand sJG1^ECD-coated Sepharose-4B beads in lin⁻ bone marrow cell cultures supplemented with SCF, FL, and TPO. Our findings have shown that sJG1^ECD lacking the transmembrane and intracellular domains is inhibitory to Notch signaling in HSC expansion cultures. Recently, Varnum-Finney et al. [²¹ ] have reported that soluble mutants of the Notch ligand Delta are also capable of activating Notch on C2 myoblasts but only when they are immobilized onto a surface and mimic full-length ligands. In contrast, soluble forms of Delta act as repressors of CSL-dependent Notch-mediated activity, an observation consistent with our results. Conversely, secreted Delta/Serrate/Lag-2 ligands expressed in flies (Drosophila melanogaster) do not appear to activate Notch signaling but rather produce phenotypes reminiscent of losses in Notch signaling [²² , ²³ ]. Conversely, monovalent (soluble) as well as multivalent (insolubilized or transmemrane) Jagged-1 is able to modestly augment growth factor-induced proliferation of committed progenitor cells. Therefore, we suggest that Jagged-1 exists in three forms: as a transmembrane molecule, as an integral component of the extracellular matrix (ECM; based on our CAFC data), and as a soluble protein found in intercellular fluids. Each form has biologically significant functions of which some are shared and some are distinct (Fig. 4 ). Moreover, the different effects of Jagged-1 ligands may suggest that alternate Notch-signaling pathways mediate HSC self-renewal and augmentation of committed progenitor cell proliferation. Of course, this hypothesis needs further experimental support.

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Figure 4. Jagged-1 as multifunctional regulator of HSC fate decision. A possible mechanism based on our data. (A) In the CAFC system, some sJG1ECD is able to bind to the pre-established stromal layer (probably to the ECM). The insolubilized sJG1ECD acts synergistically with the transmembrane form of Jagged-1 molecules expressed by the stromal cells. Thus, they increase self-renewing divisions of HSCs together. sJG1ECD in excess, however, inhibits the interaction between multivalent ligands and Notch receptor-bearing HSCs. (B) In the ex vivo expansion system, sJG1ECD-coated beads are much stronger multivalent ligands than the Jagged-1+ stromal cells; however, sJG1ECD in excess is still able to reduce self-renewal.

We have not yet made any attempts to further optimize our expansion protocol, and it is conceivable that even more robust amplification can be obtained when culturing conditions are altered, or culture time is extended. In addition, whether the potent expansion activity is restricted to Jagged-1 remains to be seen, but considering the redundancy of the various Notch ligands, this may be unlikely. Moreover, other signals that increase proliferation of HSCs without genetic manipulation include Sonic Hedgehog (Shh) [²⁴ ], Wnt [²⁵ , ²⁶ ], and bone morphogenetic proteins (BMP)-2 and -4 [²⁷ ]. In the bone marrow, it is not clear if the three families of morphogens and the Notch system modulate one another’s expression or function. It will be, however, interesting to understand the relationships between these different signaling pathways during hematopoiesis. For example, Bhardwaj et al. [²⁴ ] have shown that Shh signals regulate the proliferation and differentiation of early human hematopoietic cells by modulating the morphogenic function of BMP-4. Additionally, Reya et al. [²⁵ ] have found that Notch1 receptors are up-regulated in response to Wnt signaling in HSCs. Components of these pathways have also been shown to promote proliferation of primitive cells in the brain [²⁸ ], in the gut [²⁹ ], and in the skin [³⁰ ], raising the possibility that the Notch system, with or without different morphogens, may be used as a general cue for self-renewal in adult stem cells from diverse tissues.

Our findings may have important implications for human hematopoietic cell transplantation. Induction of HSC growth by Notch signaling may allow in vitro expansion of a patient’s own or an allogeneic donor’s HSC without genetic manipulation and could provide an increased source of cells for future transplantation. Experiments are already in progress to test the in vivo reconstitution ability of our in vitro, expanded HSCs.

 
This work was supported by grants from the Hungarian Scientific Research Foundation (OTKA T037579) and Hungarian Ministry of Welfare (ETT 203/2001). We thank Prof. Dr. Susan R. Hollan for reading the manuscript and for helpful suggestions.

Received October 8, 2003; revised December 12, 2003; accepted December 15, 2003.

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