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a Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine and Jonsson Comprehensive Cancer Center, Los Angeles, California, USA;
b Department of Psychiatry and Biobehavioral Science, UCLA Neuropsychiatric Institute, Los Angeles, California, USA;
c Department of Biological Chemistry, Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California, USA.
Key Words. Jagged1 • Notch • Hematopoiesis • Stem cell factor • CD34+ cells
Dr. Judith C. Gasson, Jonsson Comprehensive Cancer Center, 8-684 Factor, Box 951781, UCLA, Los Angeles, California 90095-1781, USA.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lineage commitment or self-renewal of a quiescent stem cell may result from intrinsic stochastic processes, after which the presence of appropriate cytokines allows the survival and/or proliferation of particular daughter cells. On the other hand, HSC may be instructed to adopt a particular fate by the binding of specific extracellular ligands. Current models include a role for both intrinsic and extrinsic processes in governing stem cell proliferation and maturation [1, 2].
In recent years, numerous soluble growth factors have been characterized as the molecular effectors of hematopoietic maturation. The effects of colony-stimulating factors, known for their ability to stimulate the proliferation and differentiation of committed progenitors, can be synergistically enhanced by interleukins (IL) such as IL-1, IL-4, and IL-6, as well as stem cell factor (SCF) and Flt3 ligand [5]. The latter are also known for their ability to enhance stem cell survival [5], but cell-cell interactions between HSC and the bone marrow stroma are also necessary [3, 4]. In vivo, stem cells adhere tightly to bone marrow stroma and, in vitro, stromal cell contact is required for stem cell maintenance in long-term bone marrow cultures [4]. A candidate molecule that may be involved in such interactions is the Notch receptor. Notch is thought to play a central role in development by regulating numerous types of cell fate decisions [6]. While in some instances the Notch signal may be instructive [7, 8], more commonly it has been shown to modulate responses to cell type-specification cues [6, 9], often rendering progenitors resistant to differentiation [10-19].
Members of the Notch receptor family and their ligands encode cell surface proteins whose extracellular domains include a series of epidermal growth factor (EGF)-like repeats that have been highly conserved throughout evolution and are widely expressed early in development [6, 9, 20]. First identified in Drosophila and C. elegans, Notch homologs have now been found in vertebrate species [9, 21] and four Notch isoforms (Notch1-4) have been isolated from mammals [20]. The Notch ligands fall into two classes based on the prototype Serrate and Delta ligands first identified in Drosophila. In mammals, two Delta-like molecules (Delta1 and Delta3) [22, 23] and two Serrate-like molecules (Jagged1 and Jagged2) have been identified [13, 14, 24-26]. Little is currently known about the specificity of the various Notch receptors for each ligand [27], although it has been shown that Jagged1 and Jagged2 can activate Notch1 in C2C12 myoblasts [13, 14].
A role for the Notch receptor and its ligands in hematopoiesis is emerging. Notch family members have been found in many blood cell types, including developing T cells [28, 29], B cells [30], and myeloblasts [10, 12]. Notch has also been identified in both murine [31] and human [32] hematopoietic precursors, including the immature subset that lacks expression of antigens associated with commitment to individual lineages [31]. To complement these observations, several investigators have looked for Notch ligands in bone marrow stroma, which provides the microenvironment necessary for the early development of all hematopoietic lineages. Jagged1 has now been identified in both human and murine bone marrow stroma [11, 31, 33].
Recently, roles for Notch signaling in lymphopoiesis and myelopoiesis have been identified. Activated forms of Notch expressed in the thymus of transgenic mice have been shown to affect commitment to the CD4, CD8 [7], and 
ß, 
[8] lineages. Signaling through the Notch receptor has been shown to inhibit granulocytic differentiation of the murine myeloblast 32D cell line [10-12] and to promote the in vitro survival of primitive precursor populations from normal murine bone marrow [31] and murine fetal liver [33]. We hypothesize that interactions between human Notch receptors and their ligands may influence the complex array of soluble and matrix-bound signals that regulate lineage commitment and maturation of human hematopoietic cells. To test this hypothesis, CD34+ cells were co-cultured in the presence or absence of recombinant cytokines on feeder layers that either did or did not express the Notch ligand, Jagged1. Our results demonstrate that Notch signaling can act as a modulator of the SCF signal, and that in the absence of recombinant growth factors, the Jagged1-Notch pathway acts to preserve CD34+ cells in an immature state. These results support a role for Notch signaling in the bone marrow microenvironment and the earliest events in stem cell commitment.
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Materials and Methods |
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To detect Jagged1 protein in cell lysates, 105 cells per lane in reducing sample buffer were fractionated on a 7.5% polyacrylamide mini-gel (BioRad; Hercules, CA). Separated proteins were transferred to Hybond nitrocellulose (Amersham; Arlington Heights, IL). Membranes were blocked overnight at 4°C with 5% nonfat milk in Tris-buffered saline containing 1% Tween, prior to incubation for 2 h at room temperature with a 1:2,000 dilution of J59 rabbit antiserum. Immunoreactivity was detected using a biotinylated donkey anti-rabbit IgG antibody (Amersham), horseradish peroxidase-conjugated streptavidin (Amersham), and enhanced chemiluminescence Western blot reagents (Amersham) according to the manufacturer's instructions. Films (ECL Hyperfilm, Amersham) were scanned and reproduced for publication using Photoshop (Adobe Systems).
RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Primary human stromal cultures were established from a normal donor. mRNA was harvested from 7 x 106 cells using Fast Track kit poly A+ mRNA isolation (Invitrogen; Carlsbad, CA). This mRNA was used as a template for subsequent RT-PCR reactions. Degenerate PCR primers based on homologies between rat and Drosophila amino acid sequences were used to amplify a 660-nucleotide (nt) cDNA fragment of the human Jagged EGF repeat region (amino acids 409-629). The purified product was then random prime-labeled (Prime-It II Kit, Stratagene; San Diego, CA) with 32P-dCTP (Amersham) for use as a cDNA library screen probe.
Library Screening
A cDNA library was prepared using mRNA from normal human stroma, which was oligo dT-primed and then directionally cloned into EcoRI-XhoI-digested 
ZapII phage (Stratagene). The 660-nt EGF repeat PCR product was used as a probe to screen this library. The screen resulted in the independent isolation of two human Jagged1 clones, each approximately 4,000 nt in length, spanning from approximately 1,500 nt upstream of the gene's 5' end to the poly A+ tail at the 3' end.
Isolation of CD34+ Cells
After obtaining informed consent, human bone marrow samples from breast cancer patients, normal donors, or autologous bone marrow donors were placed in preservative-free heparin. The marrow was diluted 1:3 in phosphate-buffered saline (PBS), and mononuclear cells were isolated by gradient centrifugation on Ficoll-Paque (Pharmacia Biotech), followed by two washes with PBS. CD34+ cells were purified using Miltenyi Mini Macs separation columns and a CD34 isolation kit according to the manufacturer's instructions (Miltenyi Biotech; Auburn, CA). Typically, cell preparations were 95% CD34+, as shown by FACS analysis.
Flow Cytometric Staining of CD34+ Cells for Notch1
Freshly prepared CD34+ cells were washed in PBS, gently fixed in 0.5% paraformaldehyde for 20 min at room temperature, then permeabilized in 70% EtOH for 10 min at 4°C. After two PBS washes, cells were resuspended in PBS pH 7.2 containing 5 mM EDTA and 0.5% bovine serum albumin (BSA) (Sigma; St. Louis, MO). Cells were allowed to rehydrate for 30 min at 37°C. Cells (106) were then blocked with normal human IgG (Miltenyi Biotech) for 10 min at room temperature and stained for 30 min with 0.4 µg of affinity-purified 93-4 rabbit antiserum [15] to the intracellular domain of rat Notch1 or with 0.4 µg of normal rabbit IgG (Sigma). Cells were then stained for 15 min with a 1:100 dilution of human-absorbed flourescein isothiocyanate-conjugated goat anti-rabbit IgG (Caltag; Burlingame, CA). Cells were washed and analyzed immediately on a FACScan flow cytometer using Cell Quest software (Becton-Dickinson; Mountain View, CA).
Transfection and Viral Transduction of Feeder Layers
The C2C12 mouse myoblast cell line (ATCC #1772-CRL) was transfected with full-length rat Jagged1 cDNA sequence [13] cloned in the mammalian expression vector, pEF1-BOS [34], along with the neomycin resistance gene to allow for selection of stable Jagged1-expressing cell lines. Stable lines were selected with 0.4 mg/ml Geneticin (G418, Life Technologies; Grand Island, NY), as previously described [15]. The full-length rat Jagged1 sequence tagged with HA [13] was cloned into the retroviral expression vector, 
E SRMSVtKNeo (generously provided by Owen Witte, UCLA). The construct was used to make ecotropic retrovirus, which was used to transduce the S17 murine stromal cell line (established by the laboratory of Dr. Kenneth Dorshkind, UCLA), and the NIH 3T3 murine embryo cell line (ATTC #1658-CRL). Nearly confluent cell cultures were incubated at 37°C in 5% CO2 for 2 h (S17) or overnight (NIH 3T3) in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (Omega Scientific; Tarzana, CA), 10 µg/ml hexadimethrine bromide (Sigma), and the viral supernatant. The cells were then washed twice with PBS and incubated in fresh medium for two days before being selected and maintained on 0.2 mg/ml (S17) or 0.3 mg/ml (NIH 3T3) Geneticin (G418, Life Technologies).
Co-Culture and Harvest of CD34+ Progenitors with or without Jagged1
Feeder layers (S17, C2C12, or NIH-3T3 cells) expressing either Jagged1 or vector sequence were irradiated with 2,000 rads (60Co or 137Cs). The resulting cell monolayers were 70%-100% confluent within 24 h, at which time CD34+ cells were added. The co-cultures were maintained in Iscove's modified Dulbecco's medium (IMDM), 20% FBS (Atlanta Biological; Norcross, GA), 50 ng/ml gentamicin, and 0.4 mM added L-glutamine. In some experiments the culture medium was supplemented with either a four factor cocktail consisting of IL-3 (50 ng/ml), IL-6 (50 ng/ml), SCF (50 ng/ml), and G-CSF (50 ng/ml), or with SCF alone (50 ng/ml) (generously provided by Amgen; Thousand Oaks, CA).
After three to eight days, hematopoietic cells were harvested. Culture medium and unattached cells were removed from each culture and placed in a fresh culture dish. The remaining cells were washed one to two times with PBS to harvest more tightly bound hematopoietic cells. Versene (0.02% EDTA, Irvine Scientific; Santa Ana, CA) was added to the remaining monolayer of cells, and after 5-15 min, the cultures were disrupted by vigorous pipetting and scraping. All cell supensions were pooled and pelleted. Cell pellets were resuspended in growth medium and added to the culture dishes with the original cells. After a 2-h incubation (5% CO2, 37°C to allow feeder cells to attach to dishes), the suspended cells were removed and plated in colony, 3H-thymidine, or long-term culture assays.
Colony Assays
After zero, three, five, or eight days in culture, CD34+ cells were harvested and plated in triplicate in a semisolid medium (0.3% bacto-agar in IMDM, 20% FBS, 50 ng/ml gentamicin, 100 U/ml penicillin, 0.1 mg/ml streptomycin) supplemented with 4 U/ml erythropoietin (Epoetin alpha, Epogen) and 50 ng/ml each of IL-3, IL-6, and SCF (generously provided by Amgen). Cultures were maintained at 37°C in 5% CO2 and enumerated after 14 to 20 days of culture.
Long-Term Cultures
Cells harvested from the co-culture assay were plated on a confluent layer of irradated (2,000 rads) normal human stroma (passage 4). Either 50, 130, or 250 cells from the co-culture were added to each well of stroma (96-well plates). These plates were maintained in IMDM supplemented with 30% FBS, 1% deionized bovine serum albumin (BSA) [35], 2 mM L-glutamine, 50 µg/ml gentamicin, 10–5 M ß-mercaptoethanol, and 10–6 M hydrocortisone (Sigma). Once a week, half of the medium was carefully removed from each well, and an equal volume of fresh medium was added.
After four weeks in culture, the cells from these wells were evaluated for cobblestone area-forming cells or harvested as follows. Half of the medium was carefully removed from the wells, and cells were then removed from the remaining medium by vigorous pipetting and scraping of the wells. Harvested cells were plated in colony assays, as described, using the entire contents of each well in a final colony assay volume of 0.5 ml.
3H-thymidine Incorporation
Co-cultures were plated (as previously described), and on the day indicated, either 3 µCi 3H-thymidine (New England Nuclear; Boston, MA), 3 µCi 3H-thymidine and 800-fold excess dTTP (Life Technologies), or 3 µCi 3H-thymidine and 800-fold excess dCTP (Life Technologies) was added to triplicate co-culture wells. After 22 h, cells were harvested as described above. Resulting hematopoietic cells were lysed with 0.5% Triton X-100, and lysates were collected on glass fiber filters using a vacuum manifold (V&P Scientific; San Diego, CA). After three washes with PBS, two washes with 95% EtOH and 5-10 min of air drying, the filters were placed in scintillation cocktail (Beckman Ready Safe, Beckman Scientific; Fullerton, CA) and counted in a Beckman LS1800 liquid scintillation counter. Unlabeled dTTP typically competed away 90% of the 3H-thymidine incorporation; addition of dCTP showed no effect.
Propidium Iodide Staining of Cells for Viability and Cell Cycle Analysis
Hematopoietic cells were harvested from five-day co-cultures, as described. Cells were then resuspended at either 106/ml in hypotonic DNA staining buffer [36] for cell cycle analysis or resuspended at 3 x 105/ml in PBS containing 1 µg/ml propidium iodide (PI), 0.2% BSA, and 0.1% sodium azide (Sigma) for viability analysis. Hypotonic DNA staining buffer consisted of 1 mg/ml sodium citrate, 0.3% Triton-X100, 100 ng/ml PI, and 20 µg/ml Rnase A (Sigma). Cells were analyzed on a FACScan flow cytometer using Cell Quest and Modefit software (Becton-Dickinson). At least 5,000 cells per sample were analyzed for viability, and 10,000 cells per sample were analyzed for cell cycle progression.
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Results |
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In the Absence of Recombinant Cytokines, Jagged1 Inhibits CD34+ Cell Proliferation
Hematopoietic growth factors stimulate proliferation of progenitors, which is tightly linked to differentiation in normal cells. To test the effect of Notch on these processes, we established an in vitro co-culture system in the absence of recombinant cytokines. The availability of stromal and fibrobast cell lines with low or undetectable expression of Jagged1 made it possible to evaluate the effect of this Notch ligand on CD34+ hematopoietic precursors derived from human adult bone marrow. Vector alone or full-length Jagged1 was transduced into C2C12, NIH 3T3, and S17 cell lines, all of which have low or undetectable levels of endogenous Jagged1 expression. Robust expression of Jagged1 in the transduced populations was confirmed by Western analysis ( Fig. 1B). The presence of Notch1 on purified human CD34+ cells was confirmed using FACS analysis ( Fig. 2).
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The Presence of Cytokines IL-3, IL-6, SCF, and G-CSF Overrides the Inhibitory Effect of Jagged1
The next series of experiments was designed to provide CD34+ cells with a potent signal to both proliferate and mature, in an effort to determine whether the effect of Jagged1 could be overcome. Previous work in vitro has shown that the addition of IL-3, IL-6, SCF, and G-CSF stimulates both optimal expansion and maturation of myeloid elements [37]. To determine the interaction of the Notch signaling pathway with a potent stimulus for the maturation of myeloid elements in vitro, CD34+ cells were cultured for one week in the presence of these four factors on control C2C12 feeder layers or on C2C12 feeders engineered to express recombinant Jagged1. Two independent experiments under these conditions showed that Jagged1 exerted no reproducible effect on either the overall expansion of hematopoietic cells or the numbers of erythroid and myeloid colony-forming cells (data not shown).
Jagged1 Increases Erythroid Progenitors in the Presence of SCF
It is possible that Notch signaling cannot alter the strong maturation stimulus provided by the IL-3, IL-6, SCF, and G-CSF cytokine combination. To test this idea, a third series of co-culture experiments was performed exactly as described above, except that only one factor, recombinant SCF, was added to the co-culture medium. SCF has been shown to act as a survival factor and to play an important role in erythropoiesis [5, 38, 39]. Hematopoietic progenitors were harvested on days 3 through 8, and colony-forming cells were enumerated in semisolid media.
Seven independent co-culture experiments were performed in the presence of SCF alone for seven to eight days using either C2C12 cells (n = 5) or 3T3 cells (n = 2) as feeders. The presence of Jagged1 increased erythroid colony formation approximately twofold ( Table 1; p < 0.03) compared to controls, regardless of the cell type used in the feeder layer. The increase in erythroid colonies in the presence of Jagged1 suggests that Notch cooperates with SCF to enhance the survival of erythroid progenitors. In contrast, no differences in myeloid colonies were observed in the presence of SCF after seven to eight days of co-culture.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies demonstrate, for the first time, a physiologic role of endogenous Notch in the modulation of human hematopoietic progenitor cell proliferation and maturation. Consistent effects of Jagged1 were observed in the absence of a strong proliferative signal from exogenously added growth factors. A clear decrease in myeloid colonies was closely mirrored and supported by a reduction in total cell numbers, 3H-thymidine incorporation and entry into S phase. Cells co-cultured on Jagged1-expressing feeder layers showed no significant loss in viability when compared to controls. Conversely, early precursors, as measured by the generation of colony-forming cells after long-term culture, were increased in the presence of Jagged1. The ability of Notch signaling to preserve CD34+ cells in an immature state is consistent with studies showing that Notch signaling can inhibit the differentiation of 32D cells [10-12] and the differentiation of murine myoblasts [13-19].
In contrast, when CD34+ cells were cultured in the presence of a strong signal to proliferate and mature (IL-3, IL-6, SCF, G-CSF), the presence of Jagged1 in the feeder layer did not significantly alter either the fold expansion or the types of colonies formed. These results suggest that Notch signaling induced by Jagged1 cannot override the response of relatively mature progenitors to a strong proliferative signal. In an attempt to reproduce a microenvironment in which an interaction between Notch signaling and a cytokine signal might be observed, we performed co-cultures in which only recombinant SCF was added. SCF enhances the viability of hematopoietic precursors, but on its own, does not drive proliferation and/or differentiation [5]. We observed a twofold increase in erythroid colonies derived from co-cultures supplemented with SCF alone, suggesting that Notch signaling enhances the survival of erythroid progenitors in the presence of SCF. The Notch-induced modulation of the SCF signal is consistent with the Notch-induced modulation of the G-CSF signal observed in 32D cells. G-CSF ordinarily induces cell cycle arrest and terminal differentiation in 32D cells, but activated forms of Notch1 and receptor engagement by Jagged1 allow 32D myeloid progenitors to survive in the presence of G-CSF [10-12].
The enhanced survival of erythroid progenitors observed in our Jagged1 co-cultures supplements the recent results of Varnum-Finney et al. [31] and Jones et al. [33], which suggest that Jagged1-Notch signaling in hematopoietic cells promotes the survival of a primitive precursor. Both groups used co-culture systems similar to the one used here, except that they employed murine rather than human hematopoietic precursors, which they derived either from adult bone marrow [31] or from fetal aorto gonadal mesonephros and fetal liver [33]. Neither group used more than a single growth factor combination. In one case, a cocktail consisting of IL-6, IL-11, SCF, and the Flt-3 ligand was used [31], and in the other, precursor cells were exposed to SCF and IL-7 [33]. These cytokines in all likelihood enhance precursor survival in vitro but do not deliver a strong erythroid or myeloid differentiation stimulus. Both groups observed increases in immature high proliferative potential colony-forming cells (HPP-CFC) and, in one case, increases in more mature, committed CFU-C in the presence of Jagged1 [31].
In the co-cultures described here, which were supplemented with SCF alone, we saw an increase in erythroid but not myeloid progenitors, suggesting that Notch signaling was active in a lineage-committed cell. Varnum-Finney et al. and Jones et al. observed that Notch signaling enhanced the survival of a more primitive cell type, the HPP-CFC, or possibly an HPP-CFC precursor. We also observed the maintenance of a primitive colony-forming cell, which we derived from long-term culture. Taken together, these results suggest that Notch signaling can act at different stages in hematopoietic differentiation, including both pluripotent stem cells and lineage-committed progenitors.
The Notch-induced modulation of the SCF signal suggests that signaling downstream of the Notch receptor may interact with signaling downstream of the tyrosine kinase SCF receptor. SCF is required to prime progenitors for signaling through the erythropoietin receptor [38, 39]. Through interaction with its receptor, SCF activates the erythropoietin receptor by tyrosine phosphorylation [39], which, in turn, induces proliferation and maturation of CFU-E [41]. It is unclear how the intracellular components of Notch signaling pathways might interact with this process.
Taken together, our experiments support a role for Notch in the earliest events of hematopoiesis and stem cell commitment. In the absence of recombinant growth factors, the Jagged1-Notch pathway acts to preserve cells in an immature state and to prevent their proliferation. However, once an appropriate stimulus is present, our experiments with SCF suggest that Notch signaling can enhance the survival of a more committed progenitor. Thus, it is likely that at many stages of hematopoiesis, just as in other developmental systems, Notch signaling alters the response of undifferentiated cells to the signals required for cell type specification.
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Acknowledgments |
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This work was supported in part by NIH grants R01 CA40163 and PO1 CA32737 (J.C.G.), and the UCLA Training Program in Biotechnology #5T32 GM08375 (L.W.). The UCLA Jonsson Comprehensive Cancer Center Flow Cytometry Core Laboratory and the Statistical and Epidemiology Service are both supported by the NIH Cancer Center support grant CA16042.
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Footnotes |
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ß versus T cell lineage decision. Cell 1997;88:833-843.[Medline]
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