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First published online September 14, 2006
Stem Cells Vol. 25 No. 1 January 2007, pp. 203 -210
doi:10.1634/stemcells.2005-0303; www.StemCells.com
© 2007 AlphaMed Press

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THE STEM CELL NICHE

Notch Signaling Induces Apoptosis in Primary Human CD34+ Hematopoietic Progenitor Cells

Nicholas Chadwicka, Maria Cristina Nostrob, Martin Barona, Rachel Mottrama, Gerard Bradyb, Anne-Marie Bucklea

aFaculty of Life Sciences, University of Manchester, Manchester, United Kingdom;
bEpistem Ltd., Manchester, United Kingdom

Key Words. Notch • Hematopoiesis • Stem cell

Correspondence: Anne-Marie Buckle, Ph.D., Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom. Telephone: 0161-200-4214; Fax: 0161-236-0409; e-mail: a.buckle{at}manchester.ac.uk

Received July 6, 2005; accepted for publication September 8, 2006.
First published online in STEM CELLS EXPRESS   September 14, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Notch signaling regulates diverse cell fate decisions during development and is reported to promote murine hematopoietic stem cell (HSC) self-renewal. The purpose of this study was to define the functional consequences of activating the Notch signaling pathway on self-renewal in human HSCs. Subsets of human umbilical cord blood CD34+ cells were retrovirally transduced with the constitutively active human Notch 1 intracellular domain (N1ICD). N1ICD-transduced cells proliferated to a lesser extent in vitro than cells transduced with vector alone, and this was accompanied by a reduction in the percentage and absolute number of CD34+ cell populations, including CD34+Thy+Lin– HSCs. Ectopic N1ICD expression inhibited cell cycle kinetics concurrent with an upregulation of p21 mRNA expression and induced apoptosis. Transduction of cells with HES-1, a known transcriptional target of Notch signaling and a mediator of Notch function, had no effect on HSC proliferation, indicating that the mechanism of the Notch-induced effect is HES-1-independent. The results of this study show that activation of the Notch signaling pathway has an inhibitory effect on the proliferation and survival of human hematopoietic CD34+ cells populations. These findings have important implications for strategies aimed at promoting self-renewal of human HSCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Signaling through the Notch pathway plays an important role in determining cell fate decisions and maintaining progenitor cell populations. Active Notch signaling is achieved by the interaction of Notch cell surface glycoprotein receptors (Notch 1–4 in humans) with their ligands (Delta 1, 2, and 4 and Jagged 1 and 2), resulting in the release of the Notch intracellular domain (NICD) [1]. In the nucleus, NICD forms a complex with RBP-J{kappa} [2] (also called CBF1) and transcriptional coactivators of the Mastermind family [3] that stimulates the transcription of downstream target genes such as the basic helix-loop-helix genes HES-1 [4], HERP1, and HERP2 [5], which are thought to form transcriptional repression complexes [6]. Notch activation leads to transcriptional suppression of lineage-specific genes, inhibiting differentiation in response to inductive signals and leaving some progenitor cells uncommitted but competent to adopt alternative cell fates. The Notch signaling pathway is complex and involves modulation by a number of proteins such as Deltex, Suppressor of Deltex, and Numb [7].

In the hematopoietic system, the expression of Notch receptors and their ligands has been widely reported [8–13], and Notch signaling has been shown to influence differentiation at several stages of development. Ectopic expression of NICD in mouse bone marrow cells leads to extrathymic T cell development and a block in B cell development [14], whereas targeted knockout of Notch 1 blocks T cell development [15]. Effects on lymphoid development have also been shown in vitro using human CD34+ cells cultured with Notch ligand-expressing mouse stromal S17 cells to show Notch-induced promotion of T cell development at the expense of B cell differentiation [16]. Interestingly, this effect was only seen using Delta 1-expressing stroma and not Jagged 1-expressing stroma, indicating that some of functional effects of Notch signaling can be mediated by distinct Notch ligands.

Many studies have investigated the role of Notch in hematopoietic stem cell (HSC) self-renewal [17, 18]. A study using retroviral transduction of mouse bone marrow stem cells showed that Notch 1 intracellular domain (N1ICD) could effectively immortalize HSCs in vitro [17, 18]. The cytokine-dependent cell line generated in this study was shown to be pluripotent using both in vivo and in vitro assays. Stier et al. also used retroviral transduction of mouse Sca+Lin– bone marrow cells to demonstrate the increased long-term colony-forming potential of N1ICD-transduced cells and also the promotion of lymphoid versus myeloid differentiation of these cells [19]. Varnum-Finney et al. have reported the expansion of mouse hematopoietic progenitor cells in response to exogenous Jagged 1 stimulation [10] and Jagged 1 has also been shown to increase the colony-forming potential of fetal hematopoietic progenitors [20]. In addition to Jagged 1, activation of endogenous Notch receptors by Jagged 2 and Delta 1 has also been reported to expand mouse hematopoietic progenitor cell populations in vitro [21–23]. Most recently, a study in mice has shown that bone marrow osteoblasts express the Notch ligand Jagged 1 and promote stem cell self-renewal via Notch receptors expressed on adjacent HSCs [24]. In contrast with these findings, Dorsch et al. have shown that ectopic expression of Delta 4 leads to a reduction of hematopoietic progenitors in mice [25].

In humans, Carlesso et al. used retroviral expression of N1ICD in umbilical cord blood CD34+ cells to show a decrease in differentiation and an increase in colony-forming potential associated with Notch signaling [26]. In contrast, De Smedt et al. studied the effect of N1ICD on the differentiation of human CD34+ cord blood cells into dendritic cells and noted that transfection of CD34+ with N1ICD [27] resulted in a 10-fold decrease in absolute numbers of CD34+ cells compared with vector alone over a 2-week culture period. In a recent study [28], ectopic expression of constitutively active Notch 1 and Notch 4 was shown to inhibit the proliferation of CD34+ cord blood cells in short term cultures while increasing their long-term colony forming potential.

In humans, in vitro exposure of CD34+CD38–Lin– HSCs to soluble Jagged 1 has been investigated [12]. Although no effect of Jagged 1 was seen in vitro, subsequent in vivo analysis using a non-obese diabetic/severe combined immunodeficient (NOD-SCID) reconstitution assay showed an expansion of this cell population in response to Jagged 1 stimulation. Jagged 1 has also been shown to promote the expansion of CD34+ cells but not the CD34-Lin– population of primitive stem cells, and this may be due in part to the differential expression of Notch receptors on these distinct subpopulations [13]. In contrast, Walker et al. showed a decrease in proliferation of CD34+ cells exposed to Jagged 1 as assessed by colony formation on feeder cell layers [29]. Ohishi et al. have shown enhanced in vitro expansion and NOD-SCID mouse repopulating ability of human CD34+CD38– cells following exposure to Delta 1 [30]. From these studies, there is now good evidence that activation of Notch signaling, either by activation of endogenous Notch receptors using exogenous Notch ligands or by ectopic expression of NICD, promotes HSC self-renewal in mice. In humans, however, studies that have analyzed activation of the Notch signaling pathway have given rise to contrasting results.

In this study, we have analyzed the functional effect of Notch signaling on human hematopoietic progenitor cells (including the CD34+Lin–Thy+ HSC compartment), by ectopic expression of constitutively active Notch 1. We show that ectopic expression of N1ICD leads to cell cycle arrest and apoptosis in CD34+ cell populations in a mechanism that may be mediated by the upregulation of p21 and BCL2L1.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Vectors and Retrovirus Construction
cDNA encoding human N1ICD and membrane-tethered N1{Delta} E was ligated into the BamHI and XhoI sites of the green fluorescent protein (GFP) bicistronic retroviral vector pMX [31] (a kind gift from T. Kitamura, Tokyo, Japan). Likewise, cDNA encoding human HES-1 (a kind gift from M. Caudy, Weill Medical College of Cornell University, New York, NY) was ligated into the BamHI site of pMX. Recombinant vectors were grown in Stbl2 Escherichia coli (Invitrogen, Paisley, U.K., http://www.invitrogen.com) and purified vector DNA used to transfect the Phoenix amphotropic packaging cell line using a calcium phosphate transfection kit (Sigma-Aldrich, Poole, U.K., http://www.sigmaaldrich.com) according to the manufacturers' protocols. Viral supernatants were harvested at 48 and 72 hours and stored at –80°C. Empty pMX vector was used to make the vector-alone virus, a negative control throughout this study.

Isolation of CD34+ Progenitor Cells
Umbilical cord blood samples were obtained with approval from the Bolton Healthcare Trust Ethical Committee. Mononuclear cells were isolated from umbilical cord blood by Ficoll-Paque density centrifugation and CD34+ cells enriched using a direct CD34 microbead magnetic separation kit with MidiMacs columns (Miltenyi Biotec, Bisley, U.K., http://www.miltenyibiotec.com). Enriched cells were then stained with a fluorescein isothiocyanate-conjugated lineage antibody cocktail consisting of anti-CD2 (cloneRPA-2.10), CD14 (clone M5E2), CD15 (clone H198), CD16 (clone 3G8), and CD19 (clone HIB19), together with anti-CD34 allophycoerythrin conjugate (clone 581) and anti-Thy phycoerythrin conjugate (clone 5E10). All antibodies were purchased from BD Pharmingen (Oxford, U.K., http://www.bdbiosciences.com/pharmingen). CD34+Lin– or CD34+Lin–Thy+ populations were then sorted using a FACSVantage flow cytometer (Becton, Dickinson and Company, Oxford, U.K., http://www.bd.com).

Retroviral Transductions
Up to 105 CD34+ cells incubated overnight in serum-free expansion medium (SFEM) (Stem Cell Technologies, Meylan, France, http://www.stemcell.com) overnight with 50 ng/ml each stem cell factor (SCF), Flt3 ligand (Flt3-L), and thrombopoietin (Tpo). This cocktail of cytokines has been shown to be optimal for stem cell proliferation, enabling cells to be retrovirally transduced [32]. All cytokines were purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). Cells were then washed and resuspended in retroviral supernatant containing SCF, Flt3-L and Tpo, and transferred to a retronectin (Cambrex Bioscience, Wokingham, U.K.)-coated 24-well tissue culture plate. Virus and cells were centrifuged at 1,000g for 1 hour at room temperature and then incubated for an additional 2 hours at 33°C. Retroviral supernatant was replaced with SFEM containing SCF, Flt3-L, and Tpo, and cells were incubated for an additional 48 hours at 37°C. Cells were then removed from tissue culture plates using Cell Dissociation Buffer (Invitrogen).

Analysis of Cell Cycle and Apoptosis
For cell cycle analysis, cells were incubated for 45 minutes in 10 µM Hoechst 33342 and propidium iodide (Sigma-Aldrich) at 37°C and analyzed directly by flow cytometry. Cells were sorted on the basis of GFP expression. For apoptosis analysis, cells were incubated with APC-conjugated Annexin V (Caltag, Burlingame, CA, http://www.caltag.com) for 20 minutes at room temperature and analyzed directly with propidium iodide. For the determination of mitochondrial membrane potential, cells were incubated with 20 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide for 30 minutes at 37°C and analyzed directly by flow cytometry.

S17 Stromal Cell Cocultures
The mouse stromal S17 cell line (originally developed by Collins and Dorshkind [33]) was grown in MEM-{alpha} medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum. Monolayer cultures were prepared in 96-well tissue culture plates 1 week prior to seeding with primary cells.

Transduced GFP+ or untransduced GFP– cells were seeded onto S17-WT stroma at up to 1,000 cells per well. In some experiments, 50 ng/ml each of SCF, Flt3-L, and Tpo was added. Fresh medium was added after 7 days, and after 14 days, total cells were removed and analyzed by flow cytometry. Known numbers of FlowCheck beads (Beckman Coulter, High Wycombe, U.K., http://www.beckmancoulter.com) were added to each sample to quantify the absolute number of cells analyzed from each sample. During analysis of fluorescence-activated cell sorting (FACS) data, forward- and side-scatter gates were used to exclude stromal cells from analysis.

Poly(A) Polymerase Chain Reaction of cDNA and Subsequent Gene-Specific Polymerase Chain Reaction
Poly(A) polymerase chain reaction (PCR) was used to globally amplify cDNA derived from RNA isolated from limited numbers of FACS-sorted transduced CD34+ cells using methodology derived from Brady and Iscove [34]. Approximately 103 transduced cells were sorted on the basis of GFP expression and total RNA isolated using Total RNA Isolation Reagent (ABgene, Epsom, U.K., http://www.abgene.com) according to the manufacturers' instructions. Pellets of RNA were then used for cDNA synthesis and global amplification to generate cDNA products representative of the starting mRNA pool. In brief, reverse transcription was performed using AMV reverse transcriptase (Roche Diagnostics, Lewes, U.K., http://www.roche-applied-science.com) and an oligo(dT) primer (Sigma-Aldrich). dATPs were then ligated to the 3' cDNA ends using deoxyterminal transferase (Roche). In this manner, total cDNA could be globally amplified by 50 cycles of PCR using Taq polymerase (Roche) and an oligo(dT) primer. cDNA was then diluted 1:100 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR or 1:10 for all other gene-specific PCRs.

For gene-specific amplification, duplicate 10-µl PCRs were performed using RedTaq PCR reagent (ABgene) and 300 nM each primer (Sigma-Aldrich). Primers were designed against the 3' untranslated regions of mRNA transcripts within 300 base pairs of the poly(A) tail sequence (Table 1). For real-time PCR, triplicate 25-µl PCRs were assembled using the TaqMan Core PCR kit (Eurogentec, Southampton, U.K., http://www.eurgentec.be), according to the manufacturer's instructions, including primers and SybrGreen (for Deltex, p21, and BCL2L1) or TaqMan primers and probes (for GAPDH and HES-1). Forty cycles of amplification performed on an ABI 7300 Sequence Detector (Applied BioSystems, Warrington, U.K., http://www.appliedbiosystems.com). Relative gene expression levels were calculated based on CT values using the 2–{Delta} {Delta} CT formula.


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  		Table 1. Polymerase chain reaction primer details

 
Luciferase Assay
HEK 293T cells were transiently transfected with combinations of an RBP-J{kappa}-luciferase reporter construct containing a HES-1 promoter (Ga981–6; a kind gift from Ursula Just, GSF-National Research Center for Environment and Health, Munich, Germany), pMX N1ICD, and pMX HES-1 using Fugene6 (Roche). Luciferase activity was measured after 48 hours by incubation of cleared cell lysates with luciferin (Sigma-Aldrich) and analysis using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany, http://www.bertholdtech.com/ww/en/pub/home.cfm).

Statistical Analysis
Statistical analyses were performed using Student's t test, and all error bars represent the standard error of the mean.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Ectopic Expression of N1ICD in CD34+ Cells Prevents Expansion
To investigate the role of Notch 1 signaling in primary human CD34+ subsets, FACS-purified CD34+Lin–, CD34+Lin–Thy+, and CD34+Lin–Thy– cell populations were transduced with either bicistronic GFP-expressing retrovirus or a retrovirus containing N1ICD and GFP. After 48 hours, GFP+-transduced and GFP–-untransduced cells were FACS sorted and seeded onto a mouse S17 stromal cell monolayer in the presence of SCF, Flt3-L, and Tpo (a cytokine cocktail previously shown to promote stem cell proliferation [32]). After 2 weeks of coculture, total cells were harvested and analyzed by flow cytometry. A schematic diagram showing examples of FACS-purified CD34+ cell populations and transduced cells is shown in Figure 1A. During analysis, known numbers of fluorescent beads were added to each sample to allow absolute numbers of cells to be calculated. Survival and expansion of transduced cells was analyzed by determining the numbers of GFP+ cells recovered at the end of the experiment (day 17).


Figure 1
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  		Figure 1. Transduction of primary CD34+Lin– cells and CD34+Lin–Thy+ cells with N1ICD inhibits proliferation in long-term stromal cocultures. (A): Schematic diagram showing the experimental protocol used to isolate and transduce human cord blood CD34+ cell populations. CD34+Lin–, CD34+Lin–Thy+ and CD34+Lin–Thy– cells were purified by flow cytometry and incubated overnight in stem cell factor (SCF), Flt3 ligand (Flt3-L), and thrombopoietin (Tpo) to induce cell cycle progression. Cells were then transduced with vector alone or N1ICD retrovirus and sorted on the basis of GFP expression after 48 hours. GFP+ or GFP– cells were then seeded onto S17 stromal monolayers in the presence or absence of cytokines for 14 days. Cells were then harvested and stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry. (B): FACS plots of GFP+-transduced or GFP–-untransduced cells harvested from 14-day S17 stromal cell cocultures as outlined in (A). CD34+Lin–Thy+ (B, A–C) and CD34+Lin–Thy– (B, D–F) subsets of cells were isolated from cord blood and transduced with vector alone or N1ICD retrovirus. GFP+-transduced or GFP–-untransduced cells were seeded onto S17 stroma in the presence of SCF, Flt3-L, and Tpo. After 14 days, total cells were removed from stromal cocultures, stained for CD34, and analyzed by flow cytometry. Stromal cells were excluded from the analysis using forward- and side-scatter gating, and 5 x 103 events were analyzed to generate each FACS density plot. The numbers given below each plot represent the percentages of cells in each quadrant, and the numbers shown in boldface represent the percentage of CD34+ cells in the GFP+ or GFP– cell populations. The 1%–2% of GFP+ cells present in plots C and F presumably represent contaminants in the GFP-based FACS sort or transduced cells that were not GFP+ at the time of sorting but that subsequently expressed this marker. The plots displayed in this figure correspond with the CD34+Lin–Thy+ and CD34+Lin–Thy– data presented in Table 2. (C): Vector-alone-transduced or N1ICD-transduced CD34+Lin– cells were stained with Annexin V after 72 hours of culture to measure apoptosis. The percentage of GFP+ or GFP– cells staining for Annexin V was calculated for n = 9 samples. *, p < .001 for mock GFP+ versus N1ICD GFP+. (D): Measurement of loss of mitochondrial membrane potential (cells negatively stained for DiOC6) on vector-alone-transduced or N1ICD-transduced CD34+Lin– cord blood cell samples (n = 3). Abbreviations: DiOC6, dihexyloxacarbocyanine iodide; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; N1ICD, Notch 1 intracellular domain.

 


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  		Table 2. Fold expansion of transduced cells seeded onto S17 stromal monolayers for 14 days

 
From the data in Table 2 it is apparent that, in all but one experiment, a clear decrease in the expansion of N1ICD-transduced cells was seen compared with vector-alone-transduced cells, showing that ectopic expression of N1ICD has a deleterious effect on the expansion of human cord blood CD34+ cell populations.

From these data, it could be argued that N1ICD-transduced cells were entering a quiescent state and therefore not proliferating to the same extent as vector-alone-transduced cells. However, the effect of N1ICD in the absence of exogenous cytokines is shown in Table 3. Where cytokines were not added to the stromal cocultures, fewer N1ICD-transduced cells were recovered than were seeded, indicating that N1ICD may induce cell death rather than quiescence in CD34+Lin– cells.


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  		Table 3. Effect of cytokines on fold expansion of transduced CD34+Lin–GFP+ cells seeded onto S17 stromal monolayers for 14 days

 
Expression of N1ICD Decreases the Percentage of CD34+ Cells
Cells recovered from CD34+Lin–Thy+- and CD34+Lin–Thy–-transduced cell cocultures at day 17 were stained for CD34 and FACS plots of these recovered cells are shown in Figure 1B. From these plots, it can be seen that the majority of cells in the N1ICD cultures that retained GFP expression are CD34-negative, with few cells remaining as CD34+ (Fig. 1B, plots B and E), whereas CD34-expressing cells persisted to a greater extent in GFP+ vector-alone-transduced cells (Fig. 1B, plots A and D) and untransduced cells (Fig. 1B, plots C and F). These results were reproduced on a second cord blood sample (Table 2) and show that ectopic expression of N1ICD reduces the numbers of CD34+ cells recovered and that the remaining N1ICD+ are CD34–.

Expression of N1ICD Induces Apoptosis in CD34+Lin– Cells
To determine whether NICD-transduced cells undergo apoptosis, transduced CD34+Lin– cells were stained with Annexin V and the percentage of positive cells was determined in the GFP+ and GFP– populations. As shown in Figure 1C, NICD-transduced cells undergo a significantly higher level of apoptosis compared with mock-transduced cells or untransduced cells from the same culture. We have demonstrated a similar effect of N1ICD in TF-1 cells (a human CD34+ erythroleukemic cell line model of progenitor cells [N. Chadwick, C. Fennessy, M.C. Nostro, M. Baron, R. Mottram, G. Brady, A. Buckle, manuscript submitted for publication]). To further investigate the effect of ectopic N1ICD on CD34+ cells, N1ICD-transduced cells were stained with Hoechst 33342 dye and analyzed by flow cytometry 48 hours after transduction (supplemental online Fig. 1). The percentage of vector-alone-transduced cells in the S/G2/M phase of the cell cycle (mean, 31.6%; SD, 0.53) was significantly higher than those of N1ICD-transduced cells (mean, 28.2%; SD, 0.88; p < .01).

We also analyzed the mitochondrial membrane potential (MMP) of these cells using the mitochondrial dye DiOC6 and showed that N1ICD expression leads to a loss of MMP (Fig. 1E), an early event the apoptotic pathway. Our results with primary cells show that N1ICD induces apoptosis in primary human CD34+ cells, resulting in a decrease in the numbers of cells recovered from long-term stromal coculture assays.

Notch Signaling in CD34+Lin– and CD34+Lin–Thy+ Cells
To investigate the targets of Notch signaling primary CD34+ cells, RNA was isolated from GFP+ sorted cells 48 hours post-transduction. RNA was reverse transcribed, and the resulting cDNA was globally amplified to produce a pool of cDNA for gene-specific PCR analysis. This methodology has been shown to produce cDNA representative of the starting mRNA population [35]. Real-time PCR was performed for HES-1 and Deltex, known downstream transcriptional targets of Notch. Deltex has been shown to be upregulated in response to Notch signaling during thymocyte development [36]. As can be seen in Figure 2, an upregulation of HES-1 and Deltex occurred in response to N1ICD expression in both CD34+Lin– and CD34+Lin–Thy+ cells These data confirm the presence of functionally active Notch in N1ICD-transduced cells and identify transcriptional targets of Notch signaling in the context of both CD34+Lin– cells and CD34+Lin–Thy+ cells.


Figure 2
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  		Figure 2. Notch 1 intracellular domain (N1ICD) upregulates the expression of known downstream targets of active Notch signaling. GFP+ vector alone or N1ICD-transduced CD34+Lin– cells (n = 5), or CD34+Lin–Thy+ (n = 3) were sorted at 48 hours post-transduction and RNA generated. Poly(A) polymerase chain reaction (PCR) was used to generate a pool of globally amplified cDNA and duplicate gene-specific real-time PCR used to analyze the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, together with the known downstream targets of Notch signaling, HES-1, and Deltex to confirm the presence of active Notch in N1ICD-transduced cells.

 
HES-1 Does Not Affect the Proliferation of CD34+Lin– Cells
Because HES-1 is a known transcriptional target of Notch signaling, and we have shown N1ICD-induced upregulation of HES-1 in primary CD34+ cell populations, we sought to determine whether the observed N1ICD-induced apoptosis of primary CD34+Lin– cells could also be induced by HES-1. In mice, transduction of CD34low/–c-Kit+Sca-1+Lin– cells with HES-1 leads to a preservation of HSC phenotype, as shown by an increase in cells with a side-population phenotype and increased engraftment in recipient mice [37]. It could therefore be the case that HES-1, in contrast to N1ICD, promotes the expansion of human cord blood CD34+Lin– cell populations.

The HES-1 vector was shown to be functional using an RBP-J{kappa} luciferase assay, in which HES-1 inhibited Notch-induced activation of a HES-1 promoter (Fig. 3A), as has been shown previously [38]. Following transduction of primary cord blood CD34+Lin– cells, GFP+-transduced cells were sorted by flow cytometry and seeded onto S17 stroma, and after 14 days, cells were harvested and analyzed by flow cytometry as described above. As shown in Figure 3B, no difference in cell numbers was seen between cells transduced with vector alone and cells transduced with HES-1. Moreover, the percentage of GFP+ HES-1-transduced cells remained high in stromal cocultures (unlike N1ICD-transduced cultures, which lost GFP expression; Fig. 3C). HES-1 did not appear to have any effect on the proliferation of CD34+ progenitor cells because the percentage HES-1-transduced CD34+ cells was the same as vector-alone-transduced CD34+ cells. Representative FACS plots are shown in Figure 3D.


Figure 3
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  		Figure 3. Transduction of CD34+Lin– cells with HES-1 does not effect proliferation. (A): The activity of HES-1 was determined by its ability to inhibit N1ICD-induced transcription of a HES-1 promoter in a luciferase reporter assay using human embryonic kidney 293T cells. Results are given as fold increase over background and represent mean values of triplicate transfections. (B): CD34+Lin– cells were transduced with vector alone, pMX N1ICN, or pMX HES-1 and cultured with S17 stromal cells as described in Figure 1. Numbers of harvested GFP+ cells (relative to vector-alone-transduced cells) are shown. (C): Loss of GFP+ expression was measured by analysis of the percentage of GFP+ cells harvested from stromal cocultures. Lower levels of GFP expression presumably reflect a survival advantage of GFP– cells compared with GFP+ (N1ICD+) cells. (D): Representative fluorescence-activated cell sorting plots showing CD34+ expression on harvested cord blood cells following transduction with vector alone or HES-1. Abbreviations: GFP, green fluorescent protein; N1ICD, Notch 1 intracellular domain.

 
N1ICD Upregulates p21 and BCL2L1 in CD34+ Cells
Since the mechanism of N1ICD-induced cell cycle inhibition was not mediated by HES-1, we investigated other possible downstream mediators. PCR for a range of cell cycle genes was performed. These included the CDKIs p21, p27, and p53, which have previously been shown, individually or in combination, to mediate Notch-induced cell cycle arrest in other cell types [39–41]. Of these cell cycle genes, only p21 was shown to be significantly upregulated in response to N1ICD expression using real-time PCR (Fig. 4A), indicating that in primary human hematopoietic CD34+ cells, p21 may be the transcriptional target of N1ICD regulating cell cycle arrest. p21 was also upregulated by the membrane-tethered N1{Delta} E; however, this effect did not reach significance. Treatment of N1{Delta} E-trans-duced cells with a {gamma}-secretase inhibitor (GSI) inhibited the upregulation of p21, providing further evidence of Notch-induced p21 upregulation. Following Annexin V staining, the observed upregulation of p21 was found to correlate with the level of apoptosis in CD34+Lin– cells transduced with N1{Delta}E in the presence or absence of GSIs (Fig. 4B). We also performed PCR for genes related to apoptosis (Bcl-2, BCL2L1, BAK1, and PUMA). Of these genes, only BCL2L1 was consistently upregulated by Notch in three cord blood samples. Although this upregulation was not statistically significant, it was inhibited by treatment with GSIs (Fig. 4C). Two isoforms of BCL2L1 exist, an antiapoptotic long isoform (Bcl-xL) and a proapoptotic short isoform (Bcl-xS) [42, 43]. The global cDNA amplification method used in this study to analyze gene expression in small numbers of rare CD34+ subpopulations of cells did not enable us to distinguish between these two isoforms, but it is more likely that Notch induced apoptosis occurs via the upregulation of Bcl-xS rather than Bcl-xL. These findings demonstrate that the mechanism of Notch-induced cell cycle inhibition and apoptosis of human hematopoietic progenitor cells observed in this study and others [27, 28] involves the transcriptional upregulation of the cell cycle inhibitor p21 and BCL2L1.


Figure 4
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  		Figure 4. N1ICD upregulates p21 and BCL2L1 in CD34+Lin– cells undergoing apoptosis. (A): Real-time polymerase chain reaction (PCR) was used to determine the relative expression of p21 mRNA in vector-alone-transduced or N1ICD-transduced CD34+Lin– cells (n = 8), as well as N1{Delta} E-transduced CD34+Lin– cells treated with DMSO (0.1%) or GSI IX (10 µM) (n = 3). *, p < .05 for mock GFP+ versus N1ICD GFP+. (B): Apoptosis assay (Annexin V) performed with the same cells used for p21 real-time PCR. *, p < .001 for mock GFP+ versus N1ICD GFP+ and mock GFP+ versus N1{Delta} E (DMSO) GFP+. **, p < .05 for N1{Delta} E (DMSO) versus GFP+ N1{Delta} E (GSI IX) GFP+. (C): Real-time PCR for BCL2L1 using cDNA from CD34+Lin– cord blood cells (n = 3) transduced with vector alone, N1ICD, N1{Delta} E treated with DMSO (0.1%), or GSI IX (10 µM). Abbreviations: DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; GSI, {gamma}-secretase inhibitor; N1ICD, Notch 1 intracellular domain.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
This study demonstrates that constitutive activation of the Notch signaling pathway via ectopic expression of N1ICD in primary human CD34+ cell populations induces apoptosis. Moreover, of those cells that were recovered, a lower percentage of N1ICD-transduced cells were CD34+ compared with vector-alone-transduced cells. This effect was not due to toxicity of N1ICD since several cell lines did not undergo cell cycle inhibition in response to transduction with N1ICD (data not shown). It is possible that different levels of Notch signaling have different effects in terms of cell survival and proliferation. A recent report by Dallas et al. showed that exposure of mouse lymphoid progenitor cells to different densities of Delta ligand resulted in different differentiation patterns [44]. It may be the case that low levels of Notch signaling results in stem cell self-renewal, whereas higher levels of Notch signaling resulting from the retroviral transductions described here results in apoptosis. In our experiments, however, when transgene expression was analyzed in terms of GFP expression, no difference in cell survival was found between GFP-high and GFP-low populations, suggesting that the ability of N1ICD-transduced cells to survive was not necessarily related to the level of Notch signaling.

The main finding of this study, that Notch 1 prevents the expansion of human hematopoietic stem cells, is novel in that we have performed these experiments on the primitive Thy+ subset of CD34+Lin– population. Both N1ICD and N1{Delta} E were found to induce loss of mitochondrial membrane potential and apoptosis in CD34+Lin– cells. The dramatic effect of N1ICD is in agreement with data of De Smedt et al. [27], who showed reduced proliferation of CD34+Lin– cells transduced with NICD, together with a reduction in the percentage of CD34+ cells in stromal cocultures. We now show that this is also the case for the more primitive CD34+Lin–Thy+ population of HSCs. In addition, a recent study by Vercauteren and Sutherland [28] has shown that transduction of lineage-depleted cord blood cells with constitutively active Notch 1 or Notch 4 results in reduced cell numbers recovered from liquid cultures compared with vector-alone-transduced cells. The latter study described a decrease in colony forming ability of NICD-transduced cells, consistent with an inhibition of progenitor cell proliferation. However, when the effect of NICD on long-term self-renewal (over 4 weeks) was investigated using a long-term culture initiating cell (LTC-IC) assay and a NOD-SCID reconstitution assay, NICD was shown to promote the maintenance of hematopoietic progenitor cells. Although Notch expression has been detected in CD34 populations by PCR, the levels of functional extracellular Notch protein expressed on the cell surface of CD34 populations is not known, and it is possible that only a subfraction of the cells have functional cell surface Notch receptors. It may be the case that inappropriate expression of NICD in human cells that do not normally express Notch 1 leads to apoptosis. In contrast, ectopic expression of NICD in a population of cells that already express endogenous Notch may increase levels of appropriate Notch signaling and promote stem cell activity as determined by long-term assays. Additional studies are required to characterize those cells that survive short-term culture as identified by LTC-IC and NOD-SCID reconstitution assays.

N1ICD-transduced CD34+Thy+Lin– HSCs and CD34+Lin– progenitors were shown to upregulate the expression of the known targets of Notch transcription, Hes-1 and Deltex, demonstrating the presence of functionally active Notch 1 signaling in these cells. Indeed, this is the first time to our knowledge that active Notch signaling has been demonstrated in human CD34+Lin–Thy+ HSCs. However, we found that, unlike N1ICD, HES-1 had no effect on the proliferation of progenitor CD34+ cells, indicating that the mechanism of N1ICD-induced cell cycle inhibition is not mediated by HES-1. Two recent reports have described an increased reconstitution potential of hematopoietic progenitor cells transduced with HES-1. Shojaei et al. [45] has demonstrated this effect using human HSCs, whereas Yu et al. [46] have shown that the level of HES-1 is increased in quiescent human CD34+ cells and that transduction of mouse HSCs with HES-1 increased their reconstitution potential into secondary recipients. Although we found that HES-1 had no effect on HSC proliferation in short-term culture, it is possible that HES-1 increased the long-term self-renewal of these cells. The findings that HES-1 promotes long-term HSC self-renewal whereas Notch inhibits self-renewal may appear contradictory since HES-1 is a direct transcriptional target of Notch in HSCs as we have shown in this study. However, Notch may inhibit cell cycle kinetics via other mechanisms, leading to a situation where the balance of "self-renewal" (e.g., HES-1) versus "cell cycle inhibition" targets of Notch signaling determines the ability of HSCs to self-renew.

To determine HES-1-independent mechanisms underlying N1ICD-induced cell cycle inhibition in primary CD34+ cells, the expression of several cell cycle genes was analyzed, and p21 mRNA levels were found to be upregulated in N1ICD-transduced cells. p21 has previously been shown to be a direct transcriptional target of Notch/RBP-J{kappa} in keratinocytes [41], where activating transcription of this CDKI gene resulted in cell cycle arrest. Interestingly, Yu et al. also showed that p21 is upregulated by HES-1 in human CD34+ cells [46]. It may be that in our experiments, Notch upregulated p21 via HES-1, although if p21 upregulation was the only mechanism promoting cell cycle inhibition and apoptosis, this does not explain why HES-1 did not generate the same effect as Notch in our hands.

Interestingly, GSI treatment did not inhibit basal levels of p21 expression, as determined in GFP– untransduced cells incubated with GSI. One reason for this could be that endogenous levels of Notch signaling are low or absent in these cells and therefore any inhibition of Notch signaling has a minimal effect on p21 expression. In support of this, data from our laboratory show that only a minority of CD34+Lin– and CD34+Lin–Thy+ cells express Notch 1 at the cell surface.

This study shows for the first time that ectopic Notch induces apoptosis in primary human CD34+ cells and that this effect is concurrent with p21 upregulation, providing a possible mechanism for N1ICD-induced apoptosis in these cells. p21 has been reported to play a role in stem cell quiescence and self-renewal [47], and the level of p21 expression may influence whether a stem cell proliferates or undergoes cell cycle arrest. Thus, Notch-induced upregulation of p21 may represent a protective mechanism for inhibiting the proliferation of HSCs that have inappropriate signaling through this potentially oncogenic protein.

Notch signaling has also been shown to adversely affect the viability of several other diverse cell types in mechanisms involving the upregulation of p21, p27, and p53. In this study, no upregulation of p27 or p53 mRNA expression was observed in N1ICD-transduced primary CD34+ cells; however, it remains possible that Notch induces changes at the protein level that induce apoptosis.

The finding that BCL2L1 is upregulated by Notch in CD34+Lin– cells raises the possibility that the short isoform (Bcl-xS) is responsible for the apoptotic effect of Notch in these cells. More work is required to determine the isoform of BCL2L1 present in these cells and whether expression of this isoform can induce apoptosis in CD34+ cells.

This study is the first to demonstrate the effect of Notch signaling on the CD34+Lin–Thy+ stem cell population. The context-specific functional outcome of Notch signaling has been reported in various studies, showing Notch-induced cell cycle arrest in some systems [39–41, 48] (N. Chadwick, C. Fennessy, M.C. Nostro, M. Baron, R. Mottram, G. Brady, A. Buckle, manuscript submitted for publication) and Notch-associated proliferation in others [49, 50]. The results of our study show that the role of Notch in mediating HSC activity in the context of human cells may be different from that reported in the mouse. Stem cell fate decisions are influenced by a complex combination of factors. These include cytokine signaling, adhesion to bone marrow stroma, and activation of Notch signaling pathways. Recently, it has become apparent that the Wnt signaling pathway also plays a role in HSC self-renewal [51]. Studies in Drosophila have identified crosstalk between Notch and Wnt signaling pathways [52], and it is likely that these pathways combine to influence stem cell fate decisions in the hematopoietic system. With the generation of reagents to study these signaling pathways, it should be possible to investigate the role of Notch signaling in the context of other signaling pathways to understand more fully the functional effects of Notch and thereby manipulate HSCs for purposes such as ex vivo expansion.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Bolton General Hospital Maternity Unit staff for kindly collecting cord blood samples. We also thank T. Kitamura for the retroviral vector pMX and Spiros Artavanis-Tsakonas and Kenji Matsuno for Notch and Deltex constructs. We also thank P. Hurley and E. Grimes for help with real-time PCR and Eiji Hara for help and advice concerning cell cycle analysis. This work was funded by the Leukemia Research Fund.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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