By
From the * Department of Gene Therapy and Molecular Virology, The John P. Robarts Research
Institute, and the Department of Microbiology and Immunology, University of Western Ontario,
London, Ontario N6A 5K8, Canada; and the Program in Cancer/Blood and the Program in
Developmental Biology, Research Institute, Hospital for Sick Children, and the Department of
Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X8, Canada
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Abstract |
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The identification of molecules that regulate human hematopoietic stem cells has focused
mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies
in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs)
may play a critical role in the specification of hematopoietic tissue from the mesodermal germ
layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified
primitive human hematopoietic cells from adult and neonatal sources. Populations of rare
CD34+CD38Lin
stem cells were isolated from human hematopoietic tissue and were found
to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell
surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor
, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34+CD38
phenotype and repopulation capacity. In
contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34+
CD38
Lin
cells, whereas at higher concentrations BMP-4 extended the length of time that
repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem
cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a
new pathway for controlling human stem cell development and a powerful model system for
studying the biological mechanism of BMP action using primary human cells.
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Introduction |
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Knowledge of specific factors that regulate adult murine
and human blood stem cells is poor and has been
mainly limited to cytokines (1). Recent work using developmental systems has shown that mesodermal precursors
can be induced to differentiate into hematopoietic cells,
thereby giving rise to the earliest blood stem cells (4).
For example, cells purified from murine tissue that differentiate from the ventral mesoderm, such as the aorta-
gonad-mesonephros and fetal liver, have been isolated and shown to contain stem cells capable of multilineage hematopoietic repopulation (10, 11). These circulating stem
cells seed the embryonic rudiments of blood-forming tissue
to establish hematopoiesis. Analysis using developmental
models of lower organisms have identified soluble and/or
paracrine growth factors (GFs)1 that induce hematopoietic
tissue from ventral mesoderm (12, 13). The majority of
these factors are members of the TGF- superfamily of secreted polypeptide GFs (14, 15).
TGF- itself is a potent inhibitor of cell cycle progression of murine blood stem cells and primitive human long-term culture initiating cells (LTC-ICs) detected using in
vitro assays (16, 17). Treatment of quiescent LTC-ICs with
neutralizing antibody against TGF-
can induce cell cycle
entry (18). Moreover, TGF-
is also secreted by immature
human hematopoietic cells, suggesting that a TGF-
autoregulatory loop is an important component of regulation in
these primitive cells (19, 20). However, TGF-
is the prototype of a large family of cytokines that includes the TGF-
s, activins, inhibins, and bone morphogenetic proteins (BMPs) (21). This family exerts a wide range of biological
responses such as cell growth, apoptosis, and differentiation
in a variety of cell types, including potent responses in patterning during embryonic development (22). One member
of the BMP subfamily, BMP-4, has been shown to be a
potent ventralizing factor and can induce hematopoietic
tissue in Xenopus and differentiation of mouse embryonic
stem cells into hematopoietic lineages (23). BMP-4 and the
transcription factor GATA-2 can function in two adjacent
germ layers, mesoderm and ectoderm, respectively, to participate in blood cell formation during embryogenesis (9, 13). This observation suggests that this subfamily of BMPs
may play a role in the development of primitive hematopoiesis. In humans, BMPs are expressed in adult human
bone marrow (BM) and are essential in bone remodeling
and growth (24). However, the ability of BMPs to continue to play a role in regulating blood stem cells once the
tissue has committed to the hematopoietic lineage, or to
play any direct role in adult and neonatal human stem cells,
is unknown.
The TGF- family of molecules signal through two
serine kinase receptors and a family of intracellular signal
transducers termed SMADs (25, 26). Soluble TGF-
and
related molecules induce formation of heterodimeric complexes of type II and type I transmembrane kinase receptors
(27). Within this complex, the type II kinase transphosphorylates the type I receptor, which transmits downstream signals to receptor-related SMADs, thereby specifying the nature of the biological response to ligand. Phosphorylation of
receptor-regulated SMADs induces association in the cytoplasm with a common mediator of SMADs, called SMAD-4
(28, 29). This heteromeric complex then moves into the
nucleus to regulate gene expression (26). Introduction of homologous SMAD proteins from human or mouse into frog
embryos mimics the effects of TGF-
activation in the induction of mesodermal specification, illustrating the phylogenetic conservation of these molecules (25, 30). Moreover, recent evidence in humans indicates that disregulation of
the SMAD molecules can affect normal growth, leading to
neoplastic hematopoiesis (31).
Most of the studies aimed at understanding the potential
biological role of BMPs in hematopoietic tissue have relied
almost exclusively on model systems involving lower organisms and in vitro systems (32, 33). Therefore, there is a
great need for a more biologically relevant model system to
determine the role and function of BMPs in human hematopoietic development. In this study, to ascertain whether
BMPs are capable of regulating primitive blood cells, we
have used a highly purified fraction of human hematopoietic tissue enriched for human repopulating stem cells (34,
35). These human repopulating cells can be assayed by transplantation into nonobese diabetic (NOD)/SCID mice
(36, 37). This repopulating cell, termed SCID-repopulating
cell (SRC), is capable of extensive proliferation and multilineage engraftment (34, 36). Cell purification and retroviral gene-marking studies demonstrated that the SRCs are
the most primitive cell type detected in the human stem
cell hierarchy and are biologically distinct from most cells
detected using in vitro assays, including colony-forming cells (CFCs) and LTC-ICs (34, 35, 38). Here, we report
that BMP receptors activin-like kinase (ALK)-3 and -6 together with the signal transducers SMAD-1, -4, and -5 are
expressed by highly purified CD34+CD38Lin
cells isolated from various human hematopoietic tissues. The addition of human BMP-2, -4, and -7 into previously designed
serum-free ex vivo cultures (38) resulted in alterations in
the proliferation, differentiation, and number of clonogenic
progenitors within the CD34+CD38
Lin
population.
Among these ligands, BMP-4 had a regulatory function distinct from BMP-2 and -7, and was capable of acting
on rare repopulating SRCs. These data demonstrate that
BMPs modulate the developmental program of human
stem cells and provide a novel model system to further understand the mechanism of BMP action within primary human cells.
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Materials and Methods |
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Human Cells.
Samples of human cord blood (CB) were obtained from placental and umbilical tissues and diluted (1:3) in IMDM (GIBCO BRL). The mononuclear cells were collected by centrifugation on Ficoll-paque (Amersham Pharmacia Biotech).Cell Purification.
CD34+CD38Reverse Transcription PCR Analysis.
Purified cells were collected after sorting in 500-µl tubes, and mRNA was extracted from 1,000 cells for each PCR reaction using a purification kit (Amersham Pharmacia Biotech). The mRNA was reverse transcribed into cDNA by standard methods using Superscript II (GIBCO BRL) as the reverse transcriptional enzyme. PCR was performed for the detection of transcripts using a Perkin-Elmer 9700 cycler with the indicated specific primers for 40 cycles. Primer sequences used for transcript detection for SMADs and ALK receptor were as follows: SMAD-1F, 5'-CGAATGCCTTAGTGACAG-3', and SMAD-1R, 5'-GAGGTGAACCCATTTGAG-3'; SMAD-4F, 5'-AGGTGAAGGTGATGTTTG-3', and SMAD-4R, 5'-GCTATTCCACCTACTGAT-3'; SMAD-5F, 5'-TGTTGGTGGAGAGGTGTA-3', and SMAD-5R, 5'-AGATATGGGGTTCAGAGG-3'; ALK-3F, 5'-ACCATCGGAGGAGAAACT-3', and ALK-3R, 5'-CTGCTGCGCTCATTTATC-3'; ALK-6F, 5'-AAGTTACGCCCCTCATTC-3', and ALK-6R, 5'-TGATGTCTTTTGCTCTGC-3'.Clonogenic Progenitor Assays.
Human clonogenic progenitors were assayed under standard conditions as shown previously, which included the addition of 10% 5637 conditioned medium as a source of cytokines (38). In brief, 100-500 purified cells were plated in methylcellulose cultures aliquoted in 1-ml vol in 35-mm suspension culture dishes and incubated at 37°C. After 10-14 d, clonogenic progenitors were scored according to standard criteria (38).Liquid Suspension Cultures.
CD34+CD38Transplantation of Purified Cells into NOD/SCID Mice.
Cells were transplanted by tail vein injection into sublethally irradiated NOD/LtSz-scid/scid (NOD/SCID) mice (375-cGy 137Cs) according to our standard protocol (36, 39). In all cases, cells were cotransplanted with irradiated nonrepopulating CD34Analysis of Human Cell Engraftment.
High molecular weight DNA was isolated from the BM of transplanted mice, and the percentage of human cells was determined by probing with a human chromosome 17-specificStatistical Analysis.
The data were analyzed by the unpaired, two-tailed Student's t test assuming a Gaussian distribution (parametric test) using Prism® software, version 2.0 (GraphPad). ![]() |
Results |
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TGF- receptors have previously been shown to be expressed on
highly purified primitive hematopoietic populations in
which soluble TGF-
is capable of regulating proliferation
and progenitor cell content (19, 20). Our previous studies
demonstrated that highly purified SRCs derived from both
human CB and BM were found in the fraction of CD34+
CD38
Lin
cells (34, 35, 38). The type I BMP receptors
ALK-3 and ALK-6 are capable of binding BMPs in the
absence of type II receptors, and it has been suggested
that these receptors may be capable of ligand selection and
may be specific for BMPs (15, 40). Reverse transcription
(RT)-PCR analysis demonstrated that both ALK-3 and -6 are expressed in primitive CD34+CD38
Lin
cells isolated
from human CB and BM tissue (Fig. 1 A). Detection of
ALK-3 and -6 expression in BM samples was more difficult
compared with CB-derived primitive populations at similar
RT-PCR conditions, suggesting lower expression in BM
versus CB. SMAD-1 and -5 are restricted for BMP signaling, whereas SMAD-4 is a shared mediator of TGF-
signaling, and acts as a common partner with pathway-specific SMADs (25, 26). Purified CD34+CD38
Lin
cells isolated
from human CB, BM, mobilized peripheral blood (M-PB),
and human BM-derived stroma express SMAD-1 and -5 transcripts (Fig. 1 B). SMAD-4 expression was found in all
sources of primitive cells, but was more easily detected in
BM (n = 2) than in CB samples (n = 4) and was barely detectable in M-PB (n = 2). In summary, transducers of the
BMP signaling pathway are expressed in primitive subfractions of both embryonic and adult human hematopoietic
tissue, suggesting that candidate human stem cell populations have the capability of responding to BMPs.
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The proliferative response
of CD34+CD38Lin
cells in response to BMP treatment
was determined by comparing the number of cells obtained
after 3 d of culture in the presence or absence of BMPs to
the number originally seeded at day 0 (Fig. 2 A). Cells were seeded in ex vivo culture conditions with serum-free media
(control SF, which we had previously designed to allow for
the expansion of primitive human blood cells [38]) and
were compared with cultures in which TGF-
, BMP-2,
-4, -7, or 5% serum was added (Fig. 2 A). The addition of
TGF-
inhibited the proliferative response seen in control
SF conditions by 80% but did maintain the number of cells
initially incubated, whereas TGF-
neutralizing antibody had no additional effect on total cell number compared
with control conditions (Fig. 2 A). Consistent with effects
on cell growth, treatment with TGF-
maintained the
number of progenitors capable of producing CFCs after 3 d
of culture, whereas TGF-
antibody had no effect on CFC
capacity (Fig. 2 B). BMP-2 and -7 had a modest effect on
cell growth at concentrations of 5 ng/ml; however, at higher concentrations (50 ng/ml) both BMP-2 and -7 treatment inhibited cell proliferation similar to TGF-
.
The inhibitory growth effect of BMP-2 and -7 is consistent
with decreased CFC content of treated cultures. BMP-2
and -7 decreased the CFC capacity of CD34+CD38
Lin
cells in a dose-responsive manner; however, at 5 ng/ml
BMP-7 was less effective than BMP-2 (Fig. 2 B). In the
cases of TGF-
, BMP-2, and BMP-7, changes in total
cell number correlated with changes in CFC content but
did not selectively alter the specific type of progenitor
detected, demonstrating that BMPs do not affect lineage
commitment. This suggests that BMP-2 and -7 are capable of modulating proliferation of primitive CD34+CD38
Lin
cells in a manner that does not alter the developmental
program and differentiation capacity of primitive cell populations. Addition of 5% serum caused a massive proliferative
response (Fig. 2 A) along with a dramatic decrease in the
number of cells capable of producing progenitors, indicative
of differentiation induction (Fig. 2 B).
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Treatment of CD34+CD38Lin
cells with BMP-4 invoked a unique response compared with TGF-
, BMP-2,
and BMP-7. The addition of BMP-4 at 5 ng/ml inhibited
the cell growth of CD34+CD38
Lin
cells, in contrast to
the effects observed using BMP-2 and -7 at similar concentrations (Fig. 2 A). Increasing BMP-4 concentrations to 50 ng/ml was toxic to CD34+CD38
Lin
cells (data not
shown). Concentrations of 25 ng/ml of BMP-4 did not alter cell viability and were capable of inducing an increase in
cell number over control SF conditions (Fig. 2 A). The
ability of CD34+CD38
Lin
cells to produce CFCs in response to BMP-4 was also dose dependent. Although 5 ng/ml
of BMP-4 inhibited proliferation of CD34+CD38
Lin
cells, CFC content was only slightly decreased compared
with control, resulting in increased frequency. CD34+
CD38
Lin
cells treated with 25 ng/ml of BMP-4 dramatically expanded CFCs compared with control SF conditions (Fig. 2 B). These data indicate that members of the
TGF-
family, including those in the BMP subfamily, are
capable of modulating proliferation and differentiation of primitive human blood cells and that the effects are specific to the dose and subtype of BMP ligand.
To determine whether BMP treatment affected the differentiation program of primitive human CD34+CD38Lin
cells, cultures were analyzed by
flow cytometry for changes in CD34 and CD38 expression
(Fig. 3). Similar to that shown previously (38), control SF
cultures induced modest differentiation of CD34+CD38
Lin
cells into CD34+CD38+ cells (Fig. 3 A), whereas the
addition of 5% serum induced a differentiation response as
demonstrated by the acquisition of CD38 and loss of CD34
expression. Both TGF-
and TGF-
neutralizing antibody
had little effect on the CD34+CD38
phenotype and remained similar to control cultures (Fig. 3 A).
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Members of the BMP subfamily caused changes in
CD34+CD38 phenotype compared with control SF conditions (Fig. 3 A). At concentrations of 5 ng/ml, both
BMP-2 and -7 had a potent differentiation effect on
CD34+CD38
Lin
cells as demonstrated by the acquisition of CD38; however, the population remained relatively
immature since the cells were all CD34 positive. At higher
concentrations of 50 ng/ml, both BMP-2 and -7 maintained equivalent numbers of CD34+CD38
cells compared with control SF conditions (Fig. 3 B). Phenotypic analysis of CD34+CD38
Lin
cells after treatment with
BMP-4 was distinct from that observed for BMP-2 and -7. At low doses, BMP-4 induced a complete differentiation of
CD34+CD38
cells into CD34+CD38+ cells, whereas at
higher doses two populations developed: one that had differentiated and one that maintained a primitive phenotype (Fig. 3 B). To further investigate the unique response of
CD34+CD38
Lin
cells to BMP-4, phenotypic analysis
was extended to 6 d of ex vivo culture (Fig. 4). Parallel cultures of CD34+CD38
Lin
cells incubated in control SF
conditions for 6 d differentiated into CD34+CD38+ cells,
similar to the differentiation response seen when low concentrations of 5 ng/ml of BMP-4 were added (Fig. 4). In
contrast, the high concentration of BMP-4 treatment resulted in the maintenance of a significant proportion of
primitive CD34+CD38
cells. These changes in the phenotype of CD34+CD38
Lin
cells in response to BMP
treatment illustrate that these factors are capable of modulating the developmental program of primitive subsets of
CD34+ cells. Consistent with effects on proliferation and
CFC capacity, BMP-4 is capable of inducing distinct dose-dependent effects on the differentiation program of human
CD34+CD38
Lin
cells compared with other members of
the TGF-
superfamily tested.
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Using the SRC assay, we have previously demonstrated that serum-free ex vivo cultures were
the only conditions capable of maintaining pluripotent repopulating cells for as long as 4 d before transplantation
(38). However, even under these conditions, no SRCs
were present after 9 d although CFCs and CD34+ cells expanded enormously during this time period. These previous results indicated that even these optimized cultures had
limited ability to expand or even maintain SRCs. Accordingly, we have assessed the effect of BMP treatment on
SRC maintenance and expansion. Wells were seeded initially with 700-1,000 CD34+CD38Lin
cells in control
SF conditions representing 1 or 2 SRCs. BMPs were added
as indicated, and the entire contents of each well were then
transplanted into NOD/SCID mice at 2, 4, and 6 d. Human cell engraftment was determined 8 wk after transplant,
and results are summarized in Table I.
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The frequency of mice engrafted after transplantation of
cells cultured for 2 d in ex vivo cultures was similar in all
treatment groups, with the exception of cultures containing 5% serum, which were unable to sustain SRCs after 2 and 4 d (Table I). The addition of serum caused the majority of CD34+CD38 cells to acquire CD38 and/or lose
CD34 cell surface expression after 4 d (Fig. 2). This differentiation shift suggests that the SRCs have most likely undergone differentiation into more mature cells that no
longer repopulate. However, since primitive CD34+CD38
cells were present in serum-containing cultures, the loss of SRCs is most likely associated with maturation that occurred independent of CD38 acquisition. Similar results
have also been obtained in a separate study examining the
loss of SRCs during culture for retroviral transduction, and
demonstrate that there is dissociation between phenotype
and function as a consequence of downregulation of CD38
in differentiated cells after culture in serum (our unpublished data). All mice transplanted with purified cells
cultured for 4 d in control SF conditions were repopulated
(100%). However, cultures containing ligands at concentrations having inhibitory effects on cell growth and
CFC capacity, such as TGF-
, 50 ng/ml of BMP-2 and -7, and 5 ng/ml of BMP-4 (Fig. 2, A and B), all resulted in a
decrease in the frequency of engrafted animals, suggesting a
loss of SRCs during culture (Table I). In contrast, but consistent with mitogenic responses detected by cell growth
and CFC capacity, the addition of TGF-
neutralizing antibody, 5 ng/ml of BMP-2 and -7, or 25 ng/ml of BMP-4
allowed for the maintenance of SRCs after 4 d of culture as
indicated by 100% engraftment frequency in transplanted animals.
Since all mice transplanted with control SF cultures at 4 d
of culture were also engrafted, it was difficult to determine whether the addition of BMPs was affecting human repopulating cells. By using similar techniques of limiting dilution analysis employed in our previous studies (34, 38), we
compared cultures treated with BMPs or TGF- to assess
effects on the number of SRCs at day 4; no significant differences in the frequency of SRCs were found (data not
shown). To determine whether BMPs were capable of affecting the survival of SRCs, cultures were extended for up
to 6 d. After 6 d of ex vivo culture, SRCs could not be detected under SF conditions. In contrast, one out of four
cultures containing TGF-
neutralizing antibody or 5 ng/ml
of BMP-2 contained repopulating cells. The most dramatic
effect was seen in cultures containing 25 ng/ml of BMP-4,
where as few as 700 CD34+CD38
Lin
cells cultured under these conditions for 6 d were capable of engrafting 5 out of 6 mice (83%; Table I). The percentage of human
chimerism in the BM of all positive mice shown in Table I
ranged between 0.1 and 1%. These results are consistent
with our previous studies in which transplantation of one
SRC enriched in purified CD34+CD38
cells at limiting
dose allowed for similar levels of human engraftment (34,
38). Further extension of ex vivo cultures containing 25 ng/ml of BMP-4 to 8 d resulted in the loss of SRCs (data
not shown). These data demonstrate the novel role of
BMPs in regulating the repopulating function of primitive
human hematopoietic cells and demonstrate that BMP-4
acts as a survival factor for candidate human stem cells.
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Discussion |
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The development of serum-free ex vivo culture systems
together within xenogenic transplant systems that are capable of detecting human repopulating cells has provided a
method for the further identification of factors regulating
the developmental program of candidate human stem cells
(SRCs [38]). In this report, we provide the first evidence
for the role of BMPs in the modulation of the developmental program of primitive subfractions of CD34+CD38
Lin
cells that contain SRCs. Treatment with BMP-2, -4, and -7 resulted in dose-dependent effects on the growth,
differentiation, and repopulating function of CD34+
CD38
Lin
cells. Evidence of the regulatory role of BMPs
was best demonstrated by BMP-4 treatment. At low concentrations of BMP-4, rapid differentiation was seen as well
as loss of SRCs. At high doses, some differentiation occurred, but a significant proportion of primitive CD34+
CD38
Lin
cells remained and more importantly SRCs
were present. BMP-4 was capable of extending the duration of stem cell activity in culture for an additional 2 d in
comparison with all other previously optimized conditions
using cytokines believed to act on primitive blood cells.
Thus, BMP-4 is acting as a survival factor, preserving stem
cell function under conditions that normally lead to stem cell loss.
The expression of pathway-restricted SMAD-1 and -5, together with expression of BMP receptors ALK-3 and -6, suggest that the mechanism of BMP action is due to the
specific activation of the BMP pathway that is distinct from
TGF- signaling. In addition, BMP-2 and -4 normally induce similar cellular responses, and therefore the differential
effects of BMP-2 compared with BMP-4 on primitive hematopoietic tissue shown here are unique, and remain to
be tested in other species and tissue types using similar in
vivo model systems for primary tissue. This unique response of human blood stem cells to BMP-4 ligand may be
due to a previously unreported receptor and/or inhibitory
molecule mediating BMP-4 signals that is expressed by this
population of rare blood cells. Alternatively, the divergent
effect of BMP-2 and -4 may not be at the receptor level
and may be due to the synergistic effects of BMP-4 with other cytokines used in this ex vivo culture system, which
do not have overlapping effects on BMP-2-specific pathways. The differential response of BMP-2 and -4 may not
diverge at the level of intercellular signaling in individual
cells, but could be due to an intrinsic heterogeneity of
BMP receptor expression within the cells that comprise this
population. At day 3 and 6 of ex vivo culture, BMP-4 was
capable of inducing a differentiation response shown by the acquisition of CD38, but also of maintaining the primitive
phenotype of a potentially distinct subset of CD34+CD38
cells. These results are most easily interpreted by the existence of two differentially responsive populations that are
heterogeneous at the level of BMP binding proteins. Evidence from other gene transfer and cell purification studies
has already suggested that CD34+CD38
Lin
cells are heterogeneous and that there is a hierarchy of stem cells
within this cell fraction (35). To address this possibility, it
would be necessary to develop flow cytometric methods to
detect subpopulations within CD34+CD38
Lin
fractions
using fluorochrome-conjugated BMP ligands or antibodies to BMP receptors. Reagents to perform these experiments
are currently being developed.
The results reported here, together with studies using other developmental systems, underscore the role of BMP-4 in primitive hematopoietic tissue and demonstrate that BMPs continue to regulate blood development well after tissue specification (13, 41). Thus, an entirely new avenue remains to be explored to identify this new biological mechanism of stem cell regulation. Moreover, ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications, including gene therapy, tumor cell purging, and stem/progenitor cell expansion (42), and therefore the identification of this novel class of stem cell regulatory molecules opens the way to developing these clinical applications. Since current ex vivo culture systems for human blood cells are limited in their ability to maintain stem cells in vitro (38, 42), the ability to extend the period in which repopulating cells can be maintained in culture with the addition of factors such as BMP-4 represents a significant advance in these systems. Based on the novel role of this family of molecules in the regulation of primitive hematopoietic tissue, this study establishes the foundation for the use of these and other mesodermal regulators in the manipulation of human stem cells in a clinical setting.
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Footnotes |
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Address correspondence to Mickie Bhatia, The John P. Robarts Research Institute, Gene Therapy and Molecular Virology, 100 Perth Dr., London, Ontario N6A 5K8, Canada. Phone: 519-663-5777 ext. 4166; Fax: 519-663-3789; E-mail: mbhatia{at}rri.on.ca
Received for publication 20 November 1998 and in revised form 20 January 1999.
D. Bonnet's present address is Coriell Institute for Medical Research, Camden, NJ 08103.We thank Amgen, Inc. for cytokines, Vicki Rosen and Steve Neben at Genetics Institute for BMP-2 and BMP-4, respectively, Kuber Sampath at Creative Biomolecules for BMP-7, and L. McWhirter and M. Watson for providing CB specimens.
Supported by grants to M. Bhatia from the Medical Research Council of Canada (MRC) and Bayer Inc. Research Fund; to J.E. Dick from the MRC, the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society, the Bayer/Red Cross Research Fund, the Canadian Genetic Diseases Network of the National Centers of Excellence, and an MRC Scientist award; and by postdoctoral fellowships to D. Bonnet from the Human Frontier Science Organization Program and the French Cancer Research Association.
Abbreviations used in this paper ALK, activin-like kinase; BM, bone marrow; BMP, bone morphogenetic protein; CB, cord blood; CFC, colony-forming cell; GF, growth factor; Lin, lineage; LTC-IC, long-term culture initiating cell; M-PB, mobilized peripheral blood; NOD, nonobese diabetic; RT, reverse transcription; SRC, SCID-repopulating cell.
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