Notch Signaling Induces Apoptosis in Primary Human CD34 Hematopoietic Progenito - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Nicholas Chadwicka, Maria Cristina Nostrob, Martin Barona, Rachel Mottrama, Gerard Bradyb, Anne-Marie Bucklea作者单位：aFaculty of Life Sciences, University of Manchester, Manchester, United Kingdom;

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      【摘要】$ a% @5 _; s/ L, F0 |# x
   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¨C 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. 8 ?7 \% v: s4 B+ U) ?! n
      【关键词】 Notch Hematopoiesis Stem cell
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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¨C4 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) .
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In the hematopoietic system, the expression of Notch receptors and their ligands has been widely reported . 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.7 K. |+ Y" @) n+ q* E% r6 @# A2 C4 Y9 |

Many studies have investigated the role of Notch in hematopoietic stem cell (HSC) self-renewal .

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 , 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¨CLin¨C HSCs to soluble Jagged 1 has been investigated . 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¨CThy  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.
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MATERIALS AND METHODS7 k5 ~8 e( ?0 u) B+ b
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Vectors and Retrovirus Construction
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cDNA encoding human N1ICD and membrane-tethered N1 E was ligated into the BamHI and XhoI sites of the green fluorescent protein (GFP) bicistronic retroviral vector pMX  (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 ¨C80¡ãC. Empty pMX vector was used to make the vector-alone virus, a negative control throughout this study.
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Isolation of CD34  Progenitor Cells2 y9 }1 _+ c. H
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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¨C or CD34 Lin¨CThy  populations were then sorted using a FACSVantage flow cytometer (Becton, Dickinson and Company, Oxford, U.K., http://www.bd.com).) m: e" B" Q' V1 c. X$ S

Retroviral Transductions
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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 . 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¡ãC. Retroviral supernatant was replaced with SFEM containing SCF, Flt3-L, and Tpo, and cells were incubated for an additional 48 hours at 37¡ãC. Cells were then removed from tissue culture plates using Cell Dissociation Buffer (Invitrogen).; P: e) I. ?! a- d2 ~
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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¡ãC 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¡ãC and analyzed directly by flow cytometry.  s$ d5 s1 a: z3 B

S17 Stromal Cell Cocultures. j- c3 S, q% Q6 |* Q
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The mouse stromal S17 cell line (originally developed by Collins and Dorshkind ) was grown in MEM- 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.
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Transduced GFP  or untransduced GFP¨C 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
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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 . 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.2 }- _3 }* o  ]  I
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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¨C  CT formula.9 `9 x6 y1 I# J" f& [! t0 F7 S! R9 P
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Table 1. Polymerase chain reaction primer details

Luciferase Assay% z3 o' ~( W0 t, X

HEK 293T cells were transiently transfected with combinations of an RBP-J-luciferase reporter construct containing a HES-1 promoter (Ga981¨C6; 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).
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Statistical Analysis

Statistical analyses were performed using Student's t test, and all error bars represent the standard error of the mean.4 {, ]) p5 g, K2 C2 {, C7 K) M/ f

RESULTS
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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¨C, CD34 Lin¨CThy , and CD34 Lin¨CThy¨C cell populations were transduced with either bicistronic GFP-expressing retrovirus or a retrovirus containing N1ICD and GFP. After 48 hours, GFP -transduced and GFP¨C-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 ). 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).+ j4 H4 H* M6 l2 C+ a( t

Figure 1. Transduction of primary CD34 Lin¨C cells and CD34 Lin¨CThy  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¨C, CD34 Lin¨CThy  and CD34 Lin¨CThy¨C 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¨C 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¨C-untransduced cells harvested from 14-day S17 stromal cell cocultures as outlined in (A). CD34 Lin¨CThy  (B, A¨CC) and CD34 Lin¨CThy¨C (B, D¨CF) subsets of cells were isolated from cord blood and transduced with vector alone or N1ICD retrovirus. GFP -transduced or GFP¨C-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¨C cell populations. The 1%¨C2% 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¨CThy  and CD34 Lin¨CThy¨C data presented in Table 2. (C): Vector-alone-transduced or N1ICD-transduced CD34 Lin¨C cells were stained with Annexin V after 72 hours of culture to measure apoptosis. The percentage of GFP  or GFP¨C cells staining for Annexin V was calculated for n = 9 samples. *, p
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Table 2. Fold expansion of transduced cells seeded onto S17 stromal monolayers for 14 days
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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.# l3 h0 z- t+ D* V. P5 j' g. C( _
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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¨C cells.) n* ^* A5 u8 T% ^; p" P; a

Table 3. Effect of cytokines on fold expansion of transduced CD34 Lin¨CGFP  cells seeded onto S17 stromal monolayers for 14 days, X; \: f' W5 P0 T  V

Expression of N1ICD Decreases the Percentage of CD34  Cells  }) w2 d( O9 |  ^( P: C$ d" w
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Cells recovered from CD34 Lin¨CThy - and CD34 Lin¨CThy¨C-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¨C.

Expression of N1ICD Induces Apoptosis in CD34 Lin¨C Cells4 k; Q; z2 g, Q8 V" S
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To determine whether NICD-transduced cells undergo apoptosis, transduced CD34 Lin¨C cells were stained with Annexin V and the percentage of positive cells was determined in the GFP  and GFP¨C 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 ). 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
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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.8 _4 V, W+ ^4 O2 P. Y

Notch Signaling in CD34 Lin¨C and CD34 Lin¨CThy  Cells
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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 . As can be seen in Figure 2, an upregulation of HES-1 and Deltex occurred in response to N1ICD expression in both CD34 Lin¨C and CD34 Lin¨CThy  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¨C cells and CD34 Lin¨CThy  cells.& b& f+ j5 o  x( x/ @6 g
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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¨C cells (n = 5), or CD34 Lin¨CThy  (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.# U6 m! f$ o6 e" b9 ]

HES-1 Does Not Affect the Proliferation of CD34 Lin¨C Cells6 |& e0 o4 `) g2 G  Q3 `! v) y8 Q% W$ q
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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¨C cells could also be induced by HES-1. In mice, transduction of CD34low/¨Cc-Kit Sca-1 Lin¨C 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 . It could therefore be the case that HES-1, in contrast to N1ICD, promotes the expansion of human cord blood CD34 Lin¨C cell populations.3 e* z0 c& e" j0 J  u7 g  ]9 Z& o

The HES-1 vector was shown to be functional using an RBP-J luciferase assay, in which HES-1 inhibited Notch-induced activation of a HES-1 promoter (Fig. 3A), as has been shown previously . Following transduction of primary cord blood CD34 Lin¨C 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.4 ~- G6 w$ X/ |( ]7 x4 O

Figure 3. Transduction of CD34 Lin¨C 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¨C 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¨C 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.. @) s0 {0 f5 g* p# B

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  involves the transcriptional upregulation of the cell cycle inhibitor p21 and BCL2L1.% I. `0 o, k; B3 e
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Figure 4. N1ICD upregulates p21 and BCL2L1 in CD34 Lin¨C 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¨C cells (n = 8), as well as N1 E-transduced CD34 Lin¨C cells treated with DMSO (0.1%) or GSI IX (10 µM) (n = 3). *, p
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DISCUSSION

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 . 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.
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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¨C population. Both N1ICD and N1 E were found to induce loss of mitochondrial membrane potential and apoptosis in CD34 Lin¨C cells. The dramatic effect of N1ICD is in agreement with data of De Smedt et al.  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.0 K- J  B( _$ x8 i& A
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N1ICD-transduced CD34 Thy Lin¨C HSCs and CD34 Lin¨C 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¨CThy  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.  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 in keratinocytes . 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¨C 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¨C and CD34 Lin¨CThy  cells express Notch 1 at the cell surface.* s4 w! }# E% [( K( x6 X& R: r

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 , 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.* t: _& S; A6 Z6 b, ^

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.
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The finding that BCL2L1 is upregulated by Notch in CD34 Lin¨C 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.
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This study is the first to demonstrate the effect of Notch signaling on the CD34 Lin¨CThy  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 , 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.
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DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS2 K* a; K3 T# A% {, h7 O, c! b
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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.
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