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HEMATOPOIESIS
From Developmental Stem Cell Biology, The John P. Robarts Research Institute, London, ON, Canada; Department of
Microbiology and Immunology, University of Western Ontario, London, ON,
Canada; Second Research Department, Central Technology
Laboratory, Asahi Chemical Industry, Fuji, Shizuoka, Japan.
Delta-mediated Notch signaling controls cell fate decisions during
invertebrate and murine development. However, in the human, functional
roles for Delta have yet to be described. This study reports the
characterization of Delta-1 and Delta-4 in the human. Human
Delta-4 was found to be expressed in a wide range of adult and fetal
tissues, including sites of hematopoiesis. Subsets of immature
hematopoietic cells, along with stromal and endothelial cells that
support hematopoiesis, were shown to express Notch and both Delta-1 and
Delta-4. Soluble forms of human Delta-1 (hDelta-1) and hDelta-4
proteins were able to augment the proliferation of primitive human
hematopoietic progenitors in vitro. Intravenous transplantation of
treated cultures into immune-deficient mice revealed that hDelta-1 is
capable of expanding pluripotent human hematopoietic repopulating cells
detected in vivo. This study provides the first evidence for a role of
Delta ligands as a mitogenic regulator of primitive hematopoietic cells
in the human.
(Blood. 2001;97:1960-1967) The Notch signaling pathway has been well conserved
throughout evolution and has been shown to play a crucial role in
embryonic development and cell fate determination in a wide range of
organisms.1,2 Activation of the Notch pathway is thought
to be mediated by interactions of bordering cells via cell-to-cell
contact of the Notch receptor and its membrane-associated
ligands.3 Notch receptors represent a single-pass
transmembrane protein composed of a large extracellular domain
containing 36 epidermal growth factor (EGF)-like repeats in tandem and
3 cysteine-rich Notch/LIN-12 repeats2,4 and an
intracellular domain consisting of 6 tandem cdc10/ankyrin repeats, a
glutamine-rich domain (opa), and a PEST sequence critical to downstream
signaling.5 Similar to the Notch receptor, putative Notch
ligands are transmembrane proteins possessing EGF repeats in the
extracellular domain, in addition to a unique cysteine-rich N-terminal
region referred to as the Delta:Serrate:LAG2 (DSL) domain.6 The DSL domain of Notch ligands interacts stably
with EGF repeats 11 and 12 of the Notch receptor in a calcium ion
(Ca++)-dependent manner, leading to Notch
activation.7,8 In mammals, 4 Notch genes have been
described (Notch-1, Notch-2, Notch-3, and Notch-4),2,9
which interact with Notch ligands, such as Delta.8
Mammalian forms of Delta have recently been characterized and include
Delta-1, Delta-like-1, and Delta-like-3.10 Using genetic
and biochemical approaches, Artavanis-Tsakonas's group has previously
shown that Delta ligand can be proteolytically processed by Kuzbanian,
a member of the ADAM family of metalloproteases,11 thereby
allowing a diffusible form of Delta to be secreted.
A potential role for Notch-mediated signals in hematopoiesis was
introduced by the identification of a mutant form of the human
homologue of the Notch-1 receptor expressed within a subset of human
T-cell leukemias.12 Leukemic blasts were shown to contain a translocation between hNotch-1 and T-cell receptor In the prevailing model of Notch signal transduction, activation of the
pathway leads to transcriptional suppression of lineage-specific genes
that cause inhibition of differentiation, resulting in the maintenance
of cells in an uncommitted state.1,15,17-19 This role of
Notch activation has led to the hypothesis that Notch ligands may be
capable of modulating tissue-specific stem cell function.20,21 Here, we demonstrate that in addition to
the Notch receptor, both human Delta-1 (hDelta-1) and hDelta-4, along with the Delta-processing metalloprotease Kuzbanian, are expressed in
purified subsets of immature human hematopoietic cells and cells
composing their putative micro-environments. Soluble forms of hDelta-1
and hDelta-4 protein were produced to investigate the functional role
of these ligands in the regulation of human hematopoietic progenitor
proliferation and differentiation. We show that Delta ligands are
capable of expanding human clonogenic progenitors and pluripotent human
repopulating cells detected in vivo. Our results indicate that Delta
ligands represent hematopoietic growth factors capable of regulating
primitive human hematopoietic cells. On the basis of these studies, we
suggest that Delta ligands may provide a novel approach for enhanced ex
vivo expansion and retroviral-mediated gene transfer of human
repopulating cells.
Cloning and expression of human Delta ligands and
Notch receptors
Purification and isolation of primary human cells
Ex vivo culture of purified primitive hematopoietic cells Isolated CD34+CD38 Lin
populations were seeded in serum-free liquid cultures previously
designed and shown to sustain human repopulating stem
cells.24 Serum-free cultures contained 9500 BIT media
(Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 104 M  -mercaptoethanol, 2 mM L-glutamine (Gibco
BRL), and the following growth factors: 300 ng/mL rhu-Flt-3; 10 ng/mL
rhu-interleukin(IL)-3; 10 ng/mL rhu-IL-6, (R&D Systems, Thousand
Oaks, CA); 300 ng/mL rhu-stem cell factor (SCF); and 50 ng/mL
rhu-G-colony stimulating factor (CSF) (Amgen, CA). Cultured
subfractions were incubated with cytokine cocktail and the media
conditions listed above for indicated periods at 37°C and 5%
CO2 in the presence of recombinant IgG1 protein
(control treated) or 10 µg/mL hDelta-1-IgG1 or
hDelta-4-IgG1. Recombinant IgG1 proteins were
added to cultures treated with cytokine cocktail alone, in the absence
of hDelta ligands, to control for nonspecific effects of
IgG1 protein. Addition of recombinant IgG1
proteins had no effect when compared with conditions with hematopoietic
cytokines cocktail alone. Shorter-term cultures analyzed at 4 to 12 days were done independently of longer-term cultures analyzed at 15 to
25 days.
Assays for human hematopoietic progenitors Human clonogenic progenitor assays were performed by plating equal numbers of hDelta-1- or hDelta-4-treated or control-treated CD34+CD38Lin cells with the use
of Methocult H4434 (Stem Cell Technologies) containing 50 ng/mL
rhu-SCF, 10 ng/mL rhu-GM-CSF and rhu-IL-3, and 3 U/mL
rhu-erythropoietin. Differential colony counts were assessed following
incubation for 10 to 14 days at 37°C and 5% CO2 in a
humidified atmosphere as shown previously.27
Transplantation of ex vivo-cultured human hematopoietic cells into nonobese diabetic/severe combined immune-deficient mice We derived 8-week-old NOD/LtSz-scid/scid (nonobese diabetic/severe combined immune-deficient [NOD/SCID]) mice from breeding pairs originally obtained from Jackson Laboratories (Bar Harbor, ME) and maintained them under defined flora at the animal facility at the Robarts Research Institute at the University of Western Ontario. All animals were handled under sterile conditions and maintained in microisolators. Mice were sublethally irradiated at 355 cGy by means of a 137-Cs -irradiator prior to intravenous injection of cultured purified human cells. Mice were cotransplanted with 100 000 Lin+ irradiated (1500 rads) accessory
cells.28
Analysis of human stem cell engraftment Genomic DNA was extracted from cells harvested from the BM of the femurs, tibiae, and iliac crests of transplanted mice. For analysis, 1 to 2 µg DNA was digested with EcoRI, run on agarose gels, transferred, and probed on a Southern blot with the use of a human chromosome-17-specific -satellite probe (p17H8).27,29 Quantitation of levels of engraftment was performed by comparing the
2.7-kilobase band with standards of known mouse and human DNA mixtures;
this was done with a level of resolution equal to 0.05% human DNA. In
addition, BM cells from each mouse were stained with
fluorochrome-conjugated antibody specific to human CD45 (a pan-leukocyte marker) and CD38 (Becton Dickinson, Immunocytometry Systems [BDIS], San Jose, CA) and analyzed by flow cytometry by means
of a FACScalibur and Cell Quest software (BD). Murine BM cells from
engrafted animals were further stained with CD45 APC (Pharmingen
Canada) and gated to analyze human cells only in combination with either CD20 FITC and CD19 phycoerythrin (PE); CD33 FITC
(BDIS) and CD15 PE (Immunotech, Marseilles, France); or CD34 FITC and CD38 PE (BDIS) monoclonal antibodies for multilineage analysis.
Characterization of hDelta-4 The Delta family of Notch ligands was first described in Drosophila and has since been shown to be conserved in mammals.2,8 Using low-stringency hybridization and hDelta-1 cDNA as a probe to screen a human fetal lung library, we have isolated an additional member of the Delta family, termed human Delta-4. Using a different strategy for identification and isolation, Shutter et al30 have also reported the human Delta-4 sequence. This group has characterized the expression of the mouse homologue of Delta-4 in murine embryonic development and provided evidence for the role of murine Delta-4 in endothelial cell regulation. Figure 1A illustrates an alignment of the hDelta-4 amino acid sequence, comparing hDelta-1 (hdel1),8 Xenopus Delta-2 (xdel2),31 and murine Delta-3 (mdel3).32 Similar to that shown by Shutter et al,30 hDelta-4 encodes a transmembrane protein that has 8 EGF-like repeats and the DSL domain (Figure 1A). Although a second invertebrate Delta gene was reported in Xenopus as X-Delta-2,31 hDelta-4 appears to be unique among mammals and shares a low similarity (49.7%) with X-Delta-2, which is much lower than the homology of 76.2% shared by the hDelta-1 and X-Delta-1 orthologues. The intracellular domain of hDelta-4 is distinct and shares only low similarities with X-Delta-1 and X-Delta-2; these are 29.1% and 26.3%, respectively. On the basis of these comparisons, we suggest that hDelta-4 does not represent an orthologue of other mammalian Deltas.
The expression of hDelta-4 in human tissues was evaluated by hybridization to human mRNA Northern blots containing a variety of adult and fetal human tissues (Figure 1B). Variable levels of expression were detected in most adult human tissues, including sites of hematopoiesis such as BM and lymph nodes (Figure 1B). In early human development, hDelta-4 was ubiquitously expressed and was detected in all fetal tissues examined, including brain, lung, and kidney. In addition, hDelta-4 was highly expressed in the liver, which serves as a primary site of human fetal hematopoiesis (Figure 1B). Taken together, our results show that this previously uncharacterized form of human Delta is expressed in a variety of tissues composing both fetal and post-natal stages of human development. Both Notch and Delta ligands are expressed among human hematopoietic cells and cells composing the hematopoietic micro-environment Previous reports have shown that primitive human CD34+ blood cells express Notch-1 and Notch-2,14,33 suggesting that Notch signaling may play a role in modulating hematopoietic progenitors in the human. Activation of the Notch receptor expressed on hematopoietic cells has been postulated to be derived from Notch ligands expressed among cells composing the micro-environment of active hematopoiesis, such as BM stromal or human umbilical vein endothelial cells (HUVECs). Consistent with this notion, adult BM stromal cells and HUVECs were shown to express hDelta-1 and hDelta-4, whereas hDelta-3 was not detectable in either of these tissues (Figure 2A). In addition, Notch-1 and Notch-2 were shown to be expressed in both BM stromal cells and HUVECs (Figure 2A). On the basis of the expression of hDelta-1 and hDelta-4 in these tissues, we investigated whether Kuzbanian, the metalloprotease responsible for cleavage of Delta ligands,11 would also be expressed in human BM stromal cells and HUVECs. Human Kuzbanian was not expressed in adult stromal cells but could be detected in HUVECs (Figure 2A) and in stromal cells isolated from human fetal liver (data not shown).
To investigate the expression of Notch and the family of Delta
ligands in hematopoietic cells, highly purified subsets of myeloid, T-
and B-lymphoid cells were isolated from human hematopoietic tissue by
means of flow cytometric sorting (purity greater than 99%; data not
shown). In addition to mature subsets, rare primitive cells depleted of
lineage-committed cells (Lin Human Delta-1 and hDelta-4 proteins are capable of interacting with primitive human hematopoietic cells Previous studies examining the functional role of Notch ligands have used coculture or conditioned supernatants from cell lines transfected with cDNA expressing the Notch ligands.36,37 However, these studies are complicated by the potential presence of unknown factors resulting from the expression of the specific ligand in the transfected cell line. Accordingly, to examine the role of Delta in human hematopoiesis, soluble forms of hDelta-1 and hDelta-4 were produced in transfected cell lines and purified by affinity chromatography.22 Since the expression of hDelta-3 was not detected in human hematopoietic subsets or stromal cells, we focused our study on the functional role of hDelta-1 and hDelta-4 in human hematopoietic development. Purified hDelta-1 and hDelta-4, expressed as chimeric proteins of either human IgG1Fc or FLAG peptide sequences, were evaluated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and visualized by Coomassie blue staining (Figure 3A, i). The specificity of Delta-1 and Delta-4 proteins was verified by Western blotting with the use of anti-human IgG1 or FLAG antibodies (Figure 3A, ii). To determine whether soluble ligands of hDelta-1 and hDelta-4 were capable of interacting with human hematopoietic cells, full-term gestation umbilical cord blood (CB) mononuclear cells and primitive CB Lin fractions were examined. Cells were treated with
purified hDelta-1- and hDelta-4-FLAG chimeric proteins complexed to
anti-FLAG monoclonal antibody fluorochrome conjugates and then analyzed
by flow cytometry (Figure 3B). Soluble hDelta-1 was capable of binding
15% of mononuclear cells while cells with binding affinity to hDelta-4
were considerably higher, at approximately 60% (Figure 3B). Human
Delta-1 and hDelta-4 were capable of interacting with 10% and 20% of
primitive CB Lin hematopoietic cells, respectively
(Figure 3B, inset). The specificity of hDelta-1 and hDelta-4-FLAG
chimeric protein interactions with human hematopoietic cells was
verified by pretreating cells with hDelta-1- and hDelta-4-IgG
chimeric proteins (Figure 3B). Pretreatment with either hDelta-1- or
hDelta-4-IgG blocked the binding of the respective Delta-FLAG chimeric
ligand (Figure 3B). In addition, binding of both hDelta-1 and hDelta-4
could be abrogated by co-incubation of the ligand with 10 mM EDTA,
which prevents Ca++-mediated Delta-Notch interactions via
DSL-EGF domains (Figure 3B).7 Treatment with EDTA or the
IgG chimeric ligands did not affect the efficiency of binding of other
cell-surface-targeted fluorochrome-conjugated antibodies. Taken
together, these results provide evidence for the ability of purified
forms of soluble hDelta-1 and hDelta-4 to interact with subsets of
primary primitive human hematopoietic cells.
Human Delta-1 and hDelta-4 modulate the development of primitive human hematopoietic cells in vitro To investigate the role of human Delta ligands in human hematopoiesis, highly purified CD34+CD38Lin cells were
cultured in serum-free media containing hematopoietic cytokines25,38 and compared with cultures where soluble
forms of hDelta-1 or hDelta-4 were added. Recombinant IgG1
protein was added to cultures treated with cytokine (control
treated) in the absence of hDelta-1-IgG or hDelta-4-IgG to account
for any nonspecific effects of IgG1. Addition of
IgG1 had no effect in comparison with cells cultured with
hematopoietic cytokines alone (data not shown).
CD34+CD38Lin cells were
harvested at indicated times, and changes in total cell number,
primitive CD34+CD38 subsets, and
hematopoietic progenitors were compared (Figure 4). Treatment with hDelta-1 or hDelta-4
had no significant effect on total cell number (Figure 4A). However, in
contrast to the bulk culture of total cells, both hDelta-1 and hDelta-4
treatment were able to expand the subfraction of phenotypically
primitive CD34+CD38 cells (Figure 4B). Human
Delta-1 was able to expand the total number of
CD34+CD38 cells in both short- and long-term
cultures as compared with control-treated cells (Figure 4B), whereas
the proliferative effects of hDelta-4 was more pronounced after
extended culture periods beyond 15 days (Figure 4B).
Human hematopoietic progenitor function was examined by quantitative
analysis of the colony-forming unit (CFU) capacity of CD34+CD38 Effects of hDelta-1 and hDelta-4 on human hematopoietic cells capable of pluripotent repopulating ability in NOD/SCID mice (SRCs) To explore the potential effect of hDelta-1 and hDelta-4 in regulating primitive human repopulating cells, CD34+CD38Lin cells cultured
with or without soluble Delta ligands were transplanted into NOD/SCID
mice. This human-mouse xenotransplant model has been
established as a reliable measure of human hematopoietic repopulating
cell function and serves as a quantitative surrogate in vivo assay
amenable to limiting dilution analysis.24,39,40 To
quantitatively examine the role of hDelta ligands on SRCs, individual
wells seeded with 2500 highly purified
CD34+CD38Lin cells were treated
and transplanted after 4 and 6 days into NOD/SCID mice. The BM of
animals transplanted with cultured cells was analyzed 8 weeks
post-transplant by flow cytometry and Southern blot analysis to
determine the presence of human chimerism. A summary of the frequency
of human chimeric NOD/SCID mice transplanted with control-treated, hDelta-1-treated, or hDelta-4-treated cells is shown in Table 1. Primitive
CD34+CD38Lin cells cultured for
4 days in the presence of hDelta-1 resulted in an increase in the
number of human SRCs (frequency of 53%; n = 17), compared with
control-treated cultures containing hematopoietic cytokines (frequency
of 24%; n = 17) (Table 1). However, hDelta-1 had no effect on SRC
function after 6 days of culture (Table 1). Unlike hDelta-1-treated
cells, CD34+CD38Lin cells
cultured for 4 days in the presence hDelta-4 (n = 12) demonstrated the identical frequency of chimeric NOD/SCID mice as the group of mice
transplanted with control-treated cultures (Table 1). An additional 2 days of culture in the presence of hDelta-4 resulted in the loss of
repopulating function (Table 1).
Since addition of soluble hDelta-1 increased the frequency of
SRCs after 4 days, we examined whether hDelta-1 was capable of
expanding SRCs in these short-term cultures. Individual wells containing hDelta-1 were seeded with 2500 CD34+ CD38
A major impediment to successful expansion and gene transfer of
human repopulating cells has been the lack of defined conditions capable of inducing the proliferation of human hematopoietic stem cells.41 The ability of Notch signal activation to
maintain cells in a primitive state suggests that Notch ligands may
represent ideal candidates for the self-renewal and expansion of
hematopoietic stem cells.17 In the present study, the use
of soluble Notch ligands to expand human hematopoietic progenitors and
SRCs has important implications in the optimization of ex vivo culture conditions used for stem cell expansion and gene transfer procedures. Our study indicates that hDelta ligands are able to expand primitive CD34+CD38 Previous reports have shown that membrane-bound Delta ligands are
expressed by adult stromal or endothelial cells.30,51 Regulated expression of these ligands among stromal cells and endothelial cells is thought to create niches within the BM and to
activate Notch signals within adjacent hematopoietic cells that express
Notch receptors.17,21 In addition to the existence of
instructive signals provided by stromal or endothelial cells alone, the
expression of Delta-1 and Delta-4 in hematopoietic cells reported here
suggests that Delta ligands may mediate activation of Notch in a
multidirectional manner among hematopoietic progenitors. This is
supported by the ability of chimeric forms of hDelta-1 and hDelta-4
ligands to competitively bind hematopoietic cells in the
absence of stromal support. In addition, differences in the binding
capacity to Delta-1 or Delta-4 ligands were demonstrated with the use
of primitive human CB Lin On the basis of the proliferative effects of soluble Deltas on human hematopoietic repopulating cells illustrated in this study, we suggest that other tissue-specific stem cells may share common embryonic programs in response to environmental stimuli and therefore may respond similarly to human Delta proteins. Therefore, our results provide a basis for investigating whether soluble Delta ligands represent regulators of recently identified neural or myogenic stem cells.52,53 Experiments attempting to elucidate the fundamental mechanisms involved in Notch receptor and Delta ligand interactions that permit the expansion and maintenance of tissue-specific stem cells derived from human neural, hematapoietic, or muscle tissue is currently ongoing in our laboratory.
Amgen, Thousand Oaks, CA, for cytokines; the staff of the labor and delivery departments of St. Joseph's Hospital and London Health Sciences, London, ON, Canada, and especially Marlene Watson and Jane Popma for providing cord blood specimens; and Drs Michael Underhill, Joseph Verdi, and David Kelvin for critically reviewing this manuscript.
Submitted September 6, 2000; accepted November 16, 2000.
Supported by a grant (MT-15063) and a scholarship award (MSH-35681) to M.B. from the Canadian Institutes of Health Research, Ottawa, ON, Canada.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mickie Bhatia, The John P. Robarts Research Institute, Developmental Stem Cell Biology, 100 Perth Dr, London, ON, N6A 5K8; e-mail: mbhatia{at}rri.on.ca.
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M. De Smedt, T. Taghon, I. Van de Walle, G. De Smet, G. Leclercq, and J. Plum Notch signaling induces cytoplasmic CD3{epsilon} expression in human differentiating NK cells Blood, October 1, 2007; 110(7): 2696 - 2703. [Abstract] [Full Text] [PDF]
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 E. Fung, S.-M. T. Tang, J. P. Canner, K. Morishige, J. F. Arboleda-Velasquez, A. A. Cardoso, N. Carlesso, J. C. Aster, and M. Aikawa Delta-Like 4 Induces Notch Signaling in Macrophages: Implications for Inflammation Circulation, June 12, 2007; 115(23): 2948 - 2956. [Abstract] [Full Text] [PDF]
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C. Delaney, B. Varnum-Finney, K. Aoyama, C. Brashem-Stein, and I. D. Bernstein Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells Blood, October 15, 2005; 106(8): 2693 - 2699. [Abstract] [Full Text] [PDF]
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S. M. Vercauteren and H. J. Sutherland Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo Blood, October 15, 2004; 104(8): 2315 - 2322. [Abstract] [Full Text] [PDF]
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T. Schroeder, H. Kohlhof, N. Rieber, and U. Just Notch Signaling Induces Multilineage Myeloid Differentiation and Up-Regulates PU.1 Expression J. Immunol., June 1, 2003; 170(11): 5538 - 5548. [Abstract] [Full Text] [PDF]
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B. Varnum-Finney, C. Brashem-Stein, and I. D. Bernstein Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability Blood, March 1, 2003; 101(5): 1784 - 1789. [Abstract] [Full Text] [PDF]
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S. Weijzen, M. P. Velders, A. G. Elmishad, P. E. Bacon, J. R. Panella, B. J. Nickoloff, L. Miele, and W. M. Kast The Notch Ligand Jagged-1 Is Able to Induce Maturation of Monocyte-Derived Human Dendritic Cells J. Immunol., October 15, 2002; 169(8): 4273 - 4278. [Abstract] [Full Text] [PDF]
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 J. M. Lee, K.-H. Lee, M. Weidner, B. A. Osborne, and S. D. Hayward Epstein-Barr virus EBNA2 blocks Nur77- mediated apoptosis PNAS, September 3, 2002; 99(18): 11878 - 11883. [Abstract] [Full Text] [PDF]
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M. Dorsch, G. Zheng, D. Yowe, P. Rao, Y. Wang, Q. Shen, C. Murphy, X. Xiong, Q. Shi, J.-C. Gutierrez-Ramos, et al. Ectopic expression of Delta4 impairs hematopoietic development and leads to lymphoproliferative disease Blood, August 28, 2002; 100(6): 2046 - 2055. [Abstract] [Full Text] [PDF]
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 A. C. Jaleco, H. Neves, E. Hooijberg, P. Gameiro, N. Clode, M. Haury, D. Henrique, and L. Parreira Differential Effects of Notch Ligands Delta-1 and Jagged-1 in Human Lymphoid Differentiation J. Exp. Med., October 1, 2001; 194(7): 991 - 1002. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
)Lin(