Blood, 15 August 2000, Vol. 96, No. 4, pp. 1215-1222
PERSPECTIVE
A ligand-receptor signaling threshold model of stem cell
differentiation control: a biologically conserved mechanism
applicable to hematopoiesis
Peter W. Zandstra,
Douglas
A. Lauffenburger, and
Connie J. Eaves
From the Institute of Biomaterials and Biomedical
Engineering and Department of Chemical Engineering and Applied
Chemistry, University of Toronto, Toronto, Ontario,
Canada; Biotechnology Process Engineering Center,
Massachusetts Institute of Technology, Cambridge, MA; Terry Fox
Laboratory, British Columbia Cancer Agency and Department of Medical
Genetics, University of British Columbia, Vancouver, British Columbia,
Canada.
 |
Abstract |
A major limitation to the widespread use of hematopoietic stem
cells (HSC) is the relatively crude level of our knowledge of how to
maintain these cells in vitro without loss of the long-term multilineage growth and differentiation properties required for their
clinical utility. An experimental and theoretical framework for
predicting and controlling the outcome of HSC stimulation by exogenous
cytokines would thus be useful. An emerging theme from recent HSC
expansion studies is that a net gain in HSC numbers requires the
maintenance of critical signaling ligand(s) above a threshold level.
These ligand-receptor complex thresholds can be maintained, for
example, by high concentrations of soluble cytokines or by
extracellular matrix- or cell-bound cytokine presentation. According to
such a model, when the relevant ligand-receptor interaction falls below
a critical level, the probability of a differentiation response is
increased; otherwise, self-renewal is favored. Thus, in addition to the
identity of a particular receptor-ligand interaction being important to
the regulation of stem cell responses, the quantitative nature of this
interaction, as well as the dynamics of receptor expression,
internalization, and signaling, may have a significant influence on
stem cell fate decisions. This review uses examples from hematopoiesis
and other tissue systems to examine existing evidence for a role of
receptor activation thresholds in regulating hematopoietic stem cell
self-renewal versus differentiation events.
(Blood. 2000;96:1215-1222)
© 2000 by The American Society of Hematology.
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Introduction |
Transplantation of hematopoietic stem cells (HSC)
has an established and unique position in the treatment of human
disease. This promising approach is limited, however, by a lack of
knowledge about how to maintain these cells in vitro without loss of
the very long-term multilineage growth and differentiation properties required for their clinical utility. A theoretical framework for predicting and controlling the outcome of HSC stimulation by exogenous cytokines would be highly useful. Although the extent to which individual HSC are biologically amenable to cytokine-determined alteration of their fates remains to be clarified,1,2 data from some in vivo studies has been interpreted as indicating that sustained HSC self-renewal, resulting in a significant net expansion of
HSC numbers, may be constrained by the tissue
environment.3-6 Moreover, the observation that cytokines
such as interleukin (IL)-3 and tumor necrosis factor (TNF) have both
been shown to exert HSC differentiation-inducing activities under
certain conditions in vitro7,8 or that (murine) HSC
self-renewal probabilities may be increased by infection with Friend
murine leukemia virus9 or by overexpression of
HOXB4,10 have renewed interest in the possibility of
defining in vitro conditions that might allow the controlled
manipulation of HSC self-renewal. However, neither the conditions under
which these manipulations may be exploited nor their mechanisms of
action at the cellular level have been defined. Even modest advances in
this area could have an important medical impact, for example, in cord
blood transplantation and gene therapy.
The introduction of quantitative and specific assays for hematopoietic
cells capable of long-term multilineage repopulation in
vivo11-16 has been key to assessing the magnitude of
changes in HSC populations in vivo and in vitro. The use of these
assays has established that a significant net expansion of HSC occurs during ontogeny17 and that this process can be reactivated
in the adult during marrow regeneration.3,4,18
Identification of in vitro conditions that will support a similar
degree of HSC self-renewal activity has proven to be a major challenge,
although recently, some success toward achieving this goal has
occurred.6,12,15,19-24 Clues to why these groups have begun to succeed may lie in certain common features of their
methodologies. These include the initiation of cultures with relatively
low concentrations of cells that are enriched in their HSC content,
frequent replacement of the medium, interactions of the HSC with
particular stromal cell types, or the use of very high concentrations
of selected cytokines. There are obviously certain intrinsic properties
that can influence the magnitude of HSC amplification detected,
including CXCR425 and VLA-426 expression and
other parameters that may fluctuate during the cell cycle to
specifically affect HSC engraftment in vivo,27,28 as well
as factors that may limit the ultimate proliferative potential
displayed by a given HSC without affecting its undifferentiated state
(eg, telomere shortening29). However, an emerging theme
from HSC expansion studies is that a net gain in HSC numbers requires
the maintenance of some critical signaling ligand(s) above a threshold
level. According to such a signaling-threshold model of stem cell
differentiation control, when a relevant ligand-receptor interaction is
kept above this threshold level, differentiation continues to be
suppressed. When this threshold level is not maintained, the
probability of a differentiation response being activated is increased.
Thus, not only may the identity of a particular receptor-ligand
interaction be important to the regulation of stem cell responses, the
quantitative nature of this interaction may also have a significant
influence on stem cell fate. In this review, we will examine some of
the evidence that receptor activation thresholds, achieved through
interaction with either soluble or surface-immobilized ligands, can
regulate stem cell self-renewal versus differentiation responses.
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Signaling complex threshold control of stem cell fate |
Evidence from hematopoiesis
Several lines of evidence support the idea that
quantitative changes in receptor/ligand signaling complex numbers may
modulate HSC fate. Our recent comparison of the cytokine dose-response relationship for the amplification of hematopoietic progenitors (detected as colony-forming cells [CFC]) and HSC (detected as long-term culture-initiating cells [LTC-IC]) provided an important clue that these responses might involve pathways distinct from (or
additional to) those responsible for blocking apoptosis and stimulating
S-phase entry. The relevant key finding in these studies was the
observation that to maximize LTC-IC expansion, it is necessary not only
to stimulate cells with particular cytokines,30 but also
to present these cytokines at very high concentrations
in fact more
than 10-fold higher than the concentrations of the same cytokines that
are sufficient to maximize the expansion of CFC numbers in the same
cultures.8 Experiments were then performed with single
CD34+CD38
cells to determine whether the
reduced LTC-IC production at low cytokine concentrations was simply due
to a failure of these conditions to stimulate a subset of
CD34+CD38 cells with high LTC-IC generating
potential. The results did not support such a model. We found no
difference in the number of cells initially stimulated to divide (clone
frequency) nor in the total number of progeny they generated (clone
size distribution) under the 2 cytokine conditions, despite the
expected difference in the number of progeny with LTC-IC activity
(Figure 1). Similarly Bennaceur-Griscelli
et al31 observed an enhanced preservation of LTC-IC in the
presence of a stromal cell line, which was also independent of cellular
survival and proliferation.
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| Figure 1.
Clone formation and progenitor expansion from
single cell cultures of CD34+CD38 adult human
bone marrow cells.
CD34+CD38 cells were isolated and cultured as
single cells in serum-free medium in the presence of 300 ng/mL stem
cell factor, Flt-3 ligand, and 60 ng/mL IL-3 (High) or 30 ng/mL stem
cell factor, Flt-3 ligand, and 6 ng/mL IL-3 (Low) for 10 days before
analysis. Analysis consisted of determining the number of clones
produced under each condition and then performing LTC-IC and CFC assays
on a pool of each set of clones generated under the same condition.
These studies showed that although the same number of clones and CFC
were generated in the High versus the Low cytokine concentrations, the
net generation of LTC-IC was dramatically affected by changes in
cytokine concentration. Taken together these results suggested that
self-renewal versus differentiation, not self-renewal versus survival,
was being modulated. Results are from reference 8.
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More recently, Ramsfjell et al32 have confirmed and
extended our studies by showing that the cytokine concentrations
required to amplify hematopoietic cells with extended LTC-IC (ELTC-IC) are significantly higher than the cytokine concentrations required to
amplify LTC-IC that produce CFC after shorter periods of time. Using
cell division tracking studies, they further showed that the observed
cytokine concentration effect was independent of the rate or extent of
cell proliferation.
These results are, of course, not the only ones to show that the
differentiation of primitive hematopoietic cells can be affected by the
types or levels of cytokines used to stimulate them. Metcalf et al
first showed almost 20 years ago that the proliferative activity and
lineage commitment of bipotent granulocyte-macrophage progenitors could
be shifted according to the concentration and order of
granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage
colony-stimulating factor (M-CSF) to which the cells were initially
exposed.33,34 More recently, evidence of a
differentiation-inducing effect of IL-5 or thrombopoietin, in
combination with steel factor, on murine progenitor cells was
presented.35 Extended "self-renewal" of erythroid
progenitors stimulated by insulin-like growth factor rather than
insulin36 and their accelerated differentiation by
exposure to transforming growth factor (TGF)-
37 have
also been reported. Finally, IL-3 concentration-dependent control of proliferation and differentiation of murine FDCP-mix
cells38,39 and a concentration-dependent ability of IL-3
and IL-1 to suppress the self-renewal of murine stem
cells40 have been observed.
A further message from a large number of studies is the ability of
particular feeder cell types to improve stem cell maintenance in
vitro.23,31,41,42 Although these observations may indicate the production by these feeders of novel cytokines or nontoxic inhibitors of differentiation, a capacity to stimulate more prolonged signaling by sustained receptor activation may also be
important.43 In studies with (predominantly)
membrane-bound steel factor (SF), prevention of internalization
prolonged tyrosine kinase activation and the half-life of the SF
receptor on the surface of the responsive cells.43,44
Transmembrane SF is a more potent stimulant of primordial germ cell
survival in vitro than soluble SF and, in vivo, the SLd
mutation (which causes only a soluble form of SF to be
produced45) results in significant hematopoietic and
developmental abnormalities including severe anemia, sterility, and
progenitor cell defects.46 In fact, the positive effects
of cell surface-bound ligand presentation may derive from both the
high effective local concentration achieved47 and the
inhibition of receptor internalization that occurs after soluble
ligand-receptor interactions.48
It is interesting to note that thresholds in receptor
expression/activation have also been shown to be important in T- and B-cell development/lineage commitment.49-51 In fact,
similar to our studies with HSC, it is not the presence or absence of a
particular receptor that acts as a developmental switch, but the
relative levels of surface expression that appear to govern
developmental potential.51 Particularly interesting in
this regard are data that show a clear difference in the sensitivity of
responses of mature and immature B-cells.50 In these
studies, low activation signals to immature B cells (induced by low
antigen concentrations) resulted in clonal elimination
(differentiation), whereas higher signal thresholds were required for
clonal expansion. In the T-cell system, Smith52 has shown
that the cell cycle progression of T cells can be predicted based on
changes in IL-2 concentration, IL-2 receptor density, and the duration
of receptor activation. Particularly noteworthy is the finding that
gaussian distributions in cycle progression times closely correlate
with parallel differences in IL-2 receptor expression, even within
otherwise identical clonal T-cell populations. This suggests that the
rate-limiting step in the IL-2-stimulated expansion of T-cell
populations is the interaction of IL-2 with its receptor. From studies
of the responses of separated T-cell subpopulations isolated on the
basis of their individual IL-2 receptor densities, evidence has been
obtained to indicate that some finite number of ligand/receptor
interactions must occur before the cell replicates its
DNA.53 Under the same conditions, this threshold may be
reached earlier in cells expressing a high number of receptors than in
cells expressing a low number of receptors. Moreover, a recent study
has shown that the potency of IL-2 for stimulation of T-cell
proliferation is enhanced by a ligand mutation that reduces its
endocytic degradation54 (thereby resulting in prolonged
receptor stimulation).
Evidence from nonhematopoietic systems
The role of inductive membrane-associated or soluble
concentration gradients in activating distinct genetic programs during embryonic development and tissue specification is also well documented (for recent reviews see references 55 and 56). Cell secreted
factorsmorphogensform concentration gradients over distances of
more than 300 µm and thereby elicit positional information that
dictates tissue patterning. Current evidence indicates that these
gradients are relatively stable and may involve the diffusion of
soluble factors across many cells, as well as juxtacrine57
and transcytotic58 cell relay. Examples of molecules that
form gradients resulting in spatially distinct tissue specification
during vertebrate development include members of the TGF- family
(ie, activin, TGF-, bone morphogenic proteins [BMPs]). Mesoderm
development in Xenopus is induced by treating presumptive
ectoderm with activin. Low concentrations result in the induction of
hematopoietic tissue, whereas very high concentrations induce the
development of notochord.59 The ability of cells to
respond to many factors, or factor complexes with overlapping
activities, as well as the existence of complex intersecting
concentration gradients of these factors suggests that relative
concentrations of factors, not just their absolute magnitudes, are
important. The observation that dorsal-ventral patterning in
Drosophila is positionally mediated by a morphogen called
short gastrulation (Sog) and its interaction with decapentaplegic (Dpp), a secreted protein that belongs to the family of BMPs, exemplifies the types of elaborate mechanisms for the control of
signaling thresholds that exist.60 Similarly, lineage
determination during neural crest stem cell differentiation is
instructively influenced by the timing and relative dosage of growth
factor encountered (reviewed in Morrison et al61).
Recently, much attention has been directed toward the possible
roles of fibroblast growth factor (FGF) signaling in early mammalian
development. For example, signaling by the FGF receptor is required for
the normal development of multiple organs during embryogenesis62 and may be spatially and directionally
modulated by secretion and presentation of FGF by the extraembryonic
trophectoderm.63 Further complexity is generated by the
presence of membrane-bound and secreted receptor isoforms and by the
interaction of FGF ligands with heparin sulfate proteoglycans on the
cell surface and extracellular matrix.64 The availability
of multiple, highly developed ways of controlling the local
concentration of FGF in fetal tissues points to the importance of a
threshold-based mechanism of FGF control of developmental processes.
Perhaps the most experimentally accessible model for the
threshold-dependent regulation of stem cell self-renewal and
differentiation is provided from in vitro studies of embryonic stem
(ES) cell responses. A well-established feature of ES cells is their
ability to be maintained in culture in an undifferentiated state in the presence of high concentrations of cytokines from the IL-6
family,65 the biologic action of which is mediated by
multiple subunit cell surface receptors that share the gp130 protein.
In fact, the concentration of the IL-6 type of cytokines required to
prevent ES cell differentiation is dependent on the identity of the
ligand.66 Like the HSC regulation by different
concentrations of soluble cytokines mentioned above, analysis of ES
cell responses to leukemia inhibitory factor (LIF) indicates that
changes in extracellular ligand concentration directly influence the
probability of differentiation independent of effects on the rate of ES
cell proliferation.67 Our more recent studies using the J1
and D3 ES cell lines have extended these results by showing that the
potency of the mitogenic stimulus required to maintain ES cell
pluripotency may be related to the numbers of LIF receptors expressed
by each ES cell line.81 In fact, in ES cells, as in PC12
neuronal cells, the level of receptor occupancy appears to determine
the self-renewal versus differentiation decision.67
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A ligand-receptor signaling threshold (LIST) model of stem
cell regulation |
What might be the mechanisms by which high levels of cytokines
selectively promote HSC self-renewal? We know that the continual stimulation of responsive cells by cytokines is a dynamic process with
significant changes occurring over many different time scales. Formation of ligand-receptor complexes results in the recruitment and
activation of specific intracellular molecules that then initiate different signaling pathways. At any specific point in time and spatial
position, the number of ligand-receptor complexes per cell depends on 2 variablesthe number of unoccupied receptors available and the ligand
concentrationand one parameterthe ligand-receptor binding affinity.
However, the 2 variables can change as a function of time or spatial
position (or both), so that the number of ligand-receptor complexes can
consequently change. It is also conceivable that accessory molecular
factors, either inside (eg, signaling intermediates) or outside (eg,
receptor agonists/antagonists) the cell can influence the parametric
value of the ligand/receptor binding affinity. Thus, mechanisms that
govern cell receptor number and ligand concentration can be predicted
to correspondingly govern whether or not the resulting
complex-activated signal(s) remain(s) above a threshold level.
If binding affinity is sufficiently great that the ligand/receptor
complex remains stable during the usually relatively brief time
required to internalize the receptor (about 10 minutes),68 the ligand may be rapidly depleted from the extracellular milieu by
cellular endocytic degradation of receptor-ligand
complexes.48,54,69 This internalization and degradation of
receptors can also result in their down-regulation.48,70
Simultaneously, on the time scale of a cell division cycle, newly
synthesized or recycled receptors (or both) can typically be
re-expressed on the cell surface.71,72 Sustained signal
propagation, itself, can result in protein-mediated desensitization of
the complex to further signaling73 or perhaps the
depletion of intracellular signaling intermediates. Thus, some metric
characterizing the number of ligand-receptor complexes per cell is
likely to determine the types and magnitude of the signals activated.
(Indeed, it might be important to further characterize the homogeneity
of ligand-receptor complexes on the cell surface and between the cell
surface and intracellular compartments, because signaling pathway
fluxes may also be altered by differences in these
distributions.72) Hence, by careful quantitation of
changes in receptor levels to improve understanding of the parameters
that influence these changes, it may be possible to develop rational
strategies to enhance HSC self-renewal divisions.
It is important to note that the kinetics of ligand-stimulated
receptor-mediated action depend exquisitely on the molecular properties
of the receptor-ligand pair. Our investigations into the kinetics of
epidermal growth factor (EGF) binding to its receptor are consistent
with the concept that information essential for regulating cellular
changes can be found in both the magnitude and the persistence of the
cytokine signal (not simply the presence of the signal per
se).74 When the response of wild-type cells was compared
with cells expressing a carboxy-terminal truncated EGF receptor mutant
that is deficient in ligand-induced internalization,75 proliferation rather than death of the 2 cell types was similar only
when EGF depletion was minimized by medium
replenishment,76 or when a mutant EGF with a significantly
reduced binding affinity was used.71,77 These results
suggest that a dynamic integration of the kinetics of receptor binding,
internalization, and degradation may be important to relate cellular
proliferation/death decisions to the number of ligand-receptor
complexes activated. Similar findings have emerged more recently for
control of T-lymphocyte proliferation by IL-2.54,69
Moreover, results showing than a similar model is applicable to the
regulation of hepatocyte differentiation by IL-6 and soluble IL-6
receptor (sIL-6R) (and "hyper-IL-6"a fusion protein of Il-6 and
sIL-6R78) have been reported.70 Taken
together, these findings support the application of these concepts to
other systems where proliferation may be associated with a change in
cellular phenotype.
The model we are proposing (summarized diagrammatically in Figure
2) envisages that the fraction of cells
undergoing a self-renewal division depends on the numbers of signaling
ligand-receptor complexes as compared to a given threshold level. If a
cell is expressing sufficiently high numbers of the relevant receptors
and is exposed to sufficiently high concentrations of the cognate
ligands, so that the number of ligand-receptor signaling complexes is
above a certain threshold, the probability that the cell will then
undergo a self-renewal division will be high. Conversely, if a cell
expresses too few receptors, or if its receptors have been sufficiently down-regulated, or if the cognate ligand concentrations are
sufficiently depleted, the number of ligand/receptor signaling
complexes is likely to fall below this threshold, with the consequence
that the proportion of cells in the population that then undergo a self-renewal division will be significantly reduced. In either situation, the threshold comparison must be to some time-integration of
the ligand-receptor complex numbers and resulting signal transduction. One especially interesting prediction from this model, with great potential for technologic applications, is that presentation of the
ligand in a mode that minimizes ligand depletion or receptor down-regulation can potentially enhance the likelihood that
suprathreshold complex levels will be maintained, hence the probability
of self-renewal cell divisions occurring will be maximized. This
prediction has, as implied previously, been borne out in the cases of
EGF and IL-2 in the regulation of proliferation responses of
fibroblasts and T lymphocytes, respectively.54,69,71
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| Figure 2.
A ligand-receptor signaling threshold (LIST) model of
stem cell differentiation control.
Examples of different mechanisms by which proliferating cells can move
from a net loss in the numbers of undifferentiated cells (scenario A),
to a net gain of undifferentiated cells (scenario B), as well as the
reverse of this process, are listed. See text for further
discussion.
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Measurements of low (but present) numbers of cytokine receptors
on hematopoietic progenitors79 further substantiates this model by allowing spatially or temporally controlled changes in ligand
densities to result in greater relative changes in the fraction of
occupied receptors.80 (The temporal variance in the number
of receptors bound at a constant ligand concentration is inversely
proportional to receptor number, ie, if the cell has only one receptor,
it fluctuates between the bound and unbound state at a binding
constant-dependent rate.68) This model may also go a long
way to explaining the heterogeneity of responses of seemingly identical
cell populations to the same culture conditions. For example, given the
order of magnitude differences in gp130 receptor numbers on ES cells,
it is likely that, even at the highest LIF concentration, a proportion
of cells may not be able to form sufficient numbers of ligand-receptor
complexes to elicit a self-renewal division. This hypothesis is
consistent with observations by our group,81 as well as
others67 in various ES cell lines.
The existence of overlapping domains in many cytokine receptors may
also be consistent with this model. Here we view self-renewal versus
differentiation as a simple yes/no response, despite the fact that the
events leading up to this decision must be at least as complex as the
numbers of cytokines to which the cell may be sensitive. Because the
signaling threshold is determined by receptor expression or
availability, the effective ligand concentration, and the particular
binding properties of the ligand/receptor pair, multiple scenarios may
result in an intensity of signaling required to promote a
"self-renewal" cell division. An experimental example of this can
be found once again in the ES cell model, where the concentration of
LIF, oncostatin M, and ciliary neurotrophic factor required to prevent
differentiation differ significantly, even though these cytokines use
the same gp130 transmembrane molecule for signaling.66
Because each cytokine in this family has its own binding properties,
but uses a limited number of common receptor subunits, the balance
between competition for these different receptor subunits and ligand
concentration may provide a mechanism for the differential control of
cell responses to microenvironmental changes. Results showing that this
family of cytokines may be organized as exchangeable
modules,82 and the hypothesis that even for a particular
cytokine, concentration-dependent changes in receptor complex
stochiometry83 (each with their own signaling capacity)
may exist, implicates additional levels of control that may influence
how thresholds are (or are not) reached.
Of course, this model cannot be the whole story. First of all, the fact
that very high concentrations of cytokines are required to
differentially stimulate cells with low numbers of receptors suggests
that the formation of active ligand-receptor complexes may be limited
by the diffusion of low affinity partners in the plasma
membrane.80 Other factors, such as receptor clustering or
asymmetrical localization of other critical components are also likely
to be important in allowing cells to respond to changes in the
concentrations of protein signals over several orders of magnitude.84
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In vivo mechanisms for controlling stimulation levels |
Although many of the previously discussed studies show that
multipotential cells have the capacity to differentially respond to a
wide range of concentrations of soluble ligands, multiple mechanisms,
including autocrine,85-87 juxtacrine47,88 and
exocrine89 stimulation, may be responsible for achieving
the same level of control in vivo (Figure
3). In fact, recent studies of autocrine cell signaling support the role of this mechanism in the robust control
of ligand concentrations in the cellular
microenvironment.90 As suggested earlier for SF, high
levels of receptor activation can be obtained when responsive cells
react with ligands that are expressed as transmembrane elements on the
surface of other cells, or attached to extracellular matrix
components.47 The prevalence transmembrane growth factors
on primitive hematopoietic cells that can be converted to soluble
factors by proteolytic processing or membrane shedding, without losing
their biologic activity, provides yet another mechanism for the local
control of cellular stimulation.91 Taken together, this
suggests a model whereby transmembrane, soluble and extracellular
matrix-bound cytokines provide a dynamically variable array of stimuli
that can be stored in the cellular microenvironment.92
Understanding the response patterns of cells to dynamic changes in such
arrays could be key to developing an ability to predict the kinetics of
cell behavior in different culture systems.
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| Figure 3.
Examples of mechanisms that cells can use for the in
vivo for the control of effective ligand concentrations and receptor
expression (and thus the level of receptor-ligand complex activation).
(1) Preventing or diminishing ligand/receptor complex internalization
through interactions between extracellular matrix (shown) or
cell-surface bound ligands; (2) autocrine ligand secretion; (3)
interactions with proteins secreted by other cells (either locally or
systemically); (4) ligand interactions with agonistic or antagonistic
soluble receptors (shown) or nonreceptor cytokine binding proteins (eg,
uromodulin); (5) receptor internalization/synthesis; and (6)
proteolytic cleavage of surface-bound receptors (shown) and/or ligands.
Each of these mechanisms may determine whether a particular (threshold)
level of receptor ligand activation is achieved. See text for further
discussion.
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The fact that serum levels of Flt3L are disregulated during
leukemogenesis and transplantation,93,94 along with the
documented ability of high concentrations of this cytokine to promote
stem cell self-renewal,8 provides further evidence that
changes in exogenous levels of particular cytokines may be important in regulating HSC differentiation in vivo. Similarly, the physiologic relevance of changes in the relative concentrations of other
cytokines in regulating hematopoietic progenitors in vivo can be
inferred from the altered serum cytokine levels that correlate with
conditions of HSC recovery. For example, 2- to 3-fold changes in serum
LIF and IL-3 concentrations in the autologous transplantation setting have been associated with an induction of HSC cycling and
differentiation caused by myeloablation.95 Conversely,
increased serum SF and Flt3L concentrations correlate with increased
numbers of hematopoietic progenitors in some patients with aplastic
anemia,89,93 and administration of these cytokines has
been reported to have a therapeutic effect in this
disease.96 It is important to note that the changes in
progenitor numbers (both relative and absolute) in these and other
studies are due to changes in the serum concentrations (not their
presence or absence) of cytokines relative to untreated controls.
The differential expression of soluble and surface bound isoforms of
many cytokines and their receptors is another way by which stimulatory
levels of critical factors may be controlled. Indeed, the modulatory
effects of cytokines that have been shown to be important in the
regulation of embryonic and HSC responses, including
FGF,97 LIF,98 SF,43 and
Flt3L,99 can be regulated in this manner. In some cases at
least, such changes are found to influence the ability of cytokines to
bind to membrane receptors and subsequently generate a
response.100 Although many examples of this regulation
exist, the fact that soluble receptors generally retain their ligand
binding ability and can act as either competitive inhibitors (eg, IL-1,
IL-2, G-CSF101) or as positive effectors (sIL-6R102,103), gives an insight into the complex
processes that have evolved to regulate ligand-stimulated thresholds.
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Molecular mechanisms for differentially transducing
activation thresholds |
Although evidence strongly suggests that threshold levels of
receptor activation induced by soluble and bound factors can modulate
the in vitro and in vivo responses of unspecialized cells, what has not
been clarified are the downstream mechanisms by which such cells
perceive and differentially respond to different levels of receptor
activation. The idea that certain intracellular signaling thresholds
stimulated by exogenous cytokines are important to cell fate decisions
is not new.74 Receptors with intrinsic or associated
tyrosine kinase activity are known to be capable of alternatively
eliciting proliferative or differentiation responses in
factor-dependent cell lines104-106 and it is likely that
levels of signaling intermediates represent key determinants in these decisions. For example, both the duration and magnitude of
extracellular signal-regulated kinase (ERK) activation by nerve growth
factor (NGF) and EGF in the PC12 neuronal cell line influence whether these cells will proliferate or differentiate.107 The fact
that a given cytokine can elicit different outcomes in the context of
different expression levels of the corresponding receptor supports the
view that a quantitative metric for signaling (eg, ERK activation) helps govern the biologic outcome in this system.108
Recent studies of the regulation of ES cell self-renewal and
differentiation suggest this system holds much promise for further analysis of this process. A critical step in connecting receptor occupancy to the genetic programs involved in ES differentiation is
signaling though the JAK/STAT (janus kinase/signal transducer and
activator of transcription) pathway. Evidence that intracellular STAT3
activation is involved in LIF-mediated changes in ES cell self-renewal
is provided by the observation that a threshold level of STAT3
activation is essential for this response.67 Dimerization of gp130 by LIF induces both the Ras-mitogen activated protein kinase
(MAPK) and JAK/STAT pathways in ES cells.109 The results of Raz et al67 suggest that it is the level of activated
STAT3 that is critical to maintaining a block of ES cell
differentiation, MAPK activity being predominantly associated with mitogenesis.
Importantly, this type of signaling regulation also has an in
vivo parallel. In the preimplantation embryo, asymmetrical localization of STAT3 correlates with LIF concentration gradients during the morula
stage of development.110 These concentration gradients may
be achieved by incorporation of locally produced LIF into the
surrounding extracellular matrix100 and may allow temporal and spatial control of differentiation decisions during this early stage of development. A prevalence of gp130-mediated responses in other
stem cell systems, including HSC47,48,102,111-113 along with the documented importance of the JAK/STAT signaling pathway in
hematopoiesis114 suggest that regulation of stem cell
differentiation decisions by this family of cytokines may be widely
conserved. Genetic evidence of common mechanisms regulating mammalian
embryonic development, tissue patterning, and adult HSC
differentiation115 provides further support for the
generality of a model in which the numbers of effective ligand/receptor
interactions control differentiation decisions.
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Conclusion |
Several fundamental questions must be answered before it will be
feasible to usefully predict and control HSC responses to exogenous
cytokines on other than empirical grounds. In particular, a better
understanding of how specific cytokines may alter the fate of
mitogenically activated HSC is needed. This is likely to require
knowledge of both the dynamics of changes in stem cell populations occurring over prolonged periods, as well as the
cellular fate outcomes of individual stem cell divisions.
These issues will be key to the design of bioreactors in which
cytokine-mediated expansion of HSC populations would be achieved. It
has been proposed that the successful use of cytokines in bioprocessing applications will require the control of 2 attenuation mechanisms, cytokine depletion and receptor down-regulation,71,116,117
Measurements of cytokine depletion rates by hematopoietic
cells48,17,119 underscore the importance of monitoring and
appropriately regulating the concentrations of cytokines considered
necessary for optimizing the growth of HSC in culture. Strict
monitoring of cytokine concentrations is likely to be most important
where a decrease in the cytokine concentration is known to affect the
desired cellular response, or if the rate of cytokine depletion is
high. This may be particularly important in mixed cell systems typical
of hematopoietic expansion cultures, where cell type-specific rates of
cytokine depletion (and secretion) have been
demonstrated.118
Additional studies are required to determine if modulation of the
strength of receptor-induced signaling, possibly caused by differences
in the timing, amount, or duration of receptor stimulation, can be
linked to the activation of distinct programs of gene activity. A
critical aspect of this analysis will be to measure temporal changes in
the nuclear and cytoplasmic concentrations of intracellular
intermediates such as the phosphorylated STAT proteins. Such
information would not only facilitate the development of more
controlled HSC expansion processes, it will undoubtedly also offer new
insights into the fascinating biology of HCS self-renewal mechanisms.
| |
Footnotes |
Submitted October 29, 1999; accepted February 14, 2000.
Supported by a National Science Foundation Engineering Research Center
(ERC) grant to the Biotechnology Process Engineering Center at the
Massachusetts Institute of Technology and the National Cancer Institute
of Canada (NCIC) with funds from the Terry Fox Run and P01HL 55435 from
the National Institutes of Health (USA). P.W.Z. held a Natural Sciences
and Engineering Research Council of Canada postdoctoral fellowship;
C.J.E. is a Terry Fox Cancer Research Scientist of the NCIC.
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: Peter W. Zandstra, Institute of Biomaterials and
Biomedical Engineering, Rm 407, Roseburgh Bldg, 4 Taddle Creek Rd,
Toronto, Ontario M5S 3G9, Canada; e-mail.
zandstra{at}ibme.utoronto.ca.
| |
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Abstract
Introduction
