Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 345-347
Lineage Commitment and Maturation in Hematopoietic Cells: The
Case for Extrinsic Regulation
By
Donald Metcalf
From The Walter and Eliza Hall Institute of Medical Research, Royal
Melbourne Hospital, Victoria, Australia.
 |
INTRODUCTION |
THE TWO SPECIFIC QUESTIONS at issue in
the present discussion are (1) whether extrinsic signaling by
hematopoietic regulators can influence the occurrence or nature of the
differentiation commitment decisions a cell makes and (2) whether these
regulators can initiate or influence maturation events in the progeny
of committed cells. These are questions of practical importance if hematopoiesis is to be selectively manipulated in clinical emergencies by the use of hematopoietic regulators.
| |
LINEAGE COMMITMENT |
Commitment can be defined as the decision a cell makes to enter, or
generate progeny that enter, a particular maturation lineage at some
future time. This decision need not necessarily be accompanied by any
immediate change in morphology or expression of novel membrane proteins
or regulator receptors.
The existence of a lineage-committed state in many hematopoietic
progenitor cells is substantiated by the ability of such cells, when in
semisolid cultures, to generate colonies of a single lineage even when
stimulated by a mixture of growth factors that should have permitted
cells of other lineages to survive and proliferate had they been
generated in the clones.1
In embryonic development, the principle is firmly established that
commitment is extrinsically regulated by position effects or inductive
gradients and does not occur by random chance. There is clear evidence
that commitment events in the initiation and continued production of
hematopoietic populations follow the same principle.2
Hematopoietic commitment depends on, and is presumably initiated
by, activation of a succession of nuclear transcription factors, beginning with SCL/TAL-1 and LMO2 and then involving more
lineage-restricted transcription factors such as GATA-1 or
PU-1.3 The extrinsic agents responsible for activation of
these nuclear transcription factors are at present unknown.
The earliest of these events occur in cells not expressing
hematopoietic receptors and are thus not subject to control by such
regulators. However, there are hematopoietic precursors such as stem
cells and multipotential and bipotential progenitor cells that are
still able to make commitment decisions and that do express receptors
for many, but not all, regulators.4 Can these commitment decisions be modulated by the action of particular regulators? The
window of opportunity for such regulators is quite narrow. The
commitment of single-lineage progenitor cells appears to be irreversible and not subject to change by the action of hematopoietic growth factors. Thus, insertion of the erythropoietin receptor into
macrophage precursors from the marrow or fetal liver allows erythropoietin to stimulate macrophage colony formation, without the
development of erythroid cells in such colonies.5
Conversely, insertion of the macrophage colony-stimulating factor
(M-CSF) receptor into erythroid precursors allows M-CSF
then to stimulate the formation of pure erythroid
colonies.6
For cells still able to make commitment choices, it is unfortunate that
documentation of commitment usually requires a readout involving the
production of maturing cells, because these two processes may be
unrelated in their molecular nature. An essential requirement in such
studies is that all subsequent progeny of the cell under study be able
to be accounted for, because apparent commitment may be spurious if
selective survival has occurred.
Recloning studies on dividing blast or multipotential colony-forming
cells indicated that commitment is an asymmetrical event, that
virtually random combinations of committed progeny can be generated,7,8 and that lineage commitment does not follow a
structured sequential pattern. However, the stochastic (random) nature
of these events does not permit the conclusion that the pattern of
commitment is incapable of being skewed by extrinsic signaling. Indeed,
the growth factors used to stimulate the initial cell divisions might
themselves have imposed a particular pattern of commitment.
In this context, the behavior of biopotential granulocyte-macrophage
progenitor cells suggested that hematopoietic regulators could
influence commitment decisions. When one daughter was cultured with one
growth factor and the other with a differing growth factor, such as
granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF,
the lineage of the progeny generated appeared to be significantly
influenced by the growth factor initially used.9 Because
both GM-CSF and M-CSF can support the proliferation of granulocytes and
macrophages,10 there seems little risk that selective loss
of progeny cells biased the results observed.
A somewhat similar outcome was observed when blast colonies were
stimulated to develop by the combination of stem cell factor (SCF) and
interleukin-3 (IL-3). Compared with the content of
eosinophil progenitors in SCF-stimulated blast colonies, the SCF/IL-3
combination resulted in a significant increase in the proportion of
eosinophil-committed progenitors.11 However, in this
example, the colonies were large and it is difficult to exclude
selective loss of eosinophil progenitors in SCF-stimulated colonies
because SCF has little action on eosinophil precursors.
A comparable study failed to obtain convincing evidence that the
pattern of progenitor cell formation in developing human multipotential
colonies could be altered when using various combinations of growth
factors.12
In the case of the multipotential cell line FDCP-Mix A4, selective
lineage commitment appeared to be achieved by particular growth
factors,13 but in this study the key experiments were performed using suspension cultures and the progeny could not be traced
back to individual cells.
There are two experimental systems in which the induced change is
undoubtedly due to the action of extracellular regulatory factors. The
difficulty lies in establishing whether either system involves the
occurrence of genuine differentiation commitment.
Cells of murine leukemic lines WEHI-3B or M1 grow autonomously as
undifferentiated blast cells. The cells are highly clonogenic (>80%
to 90%) and self-renewal at cell division is essentially 100%. When
cultured in the presence of granulocyte colony-stimulating factor
(G-CSF) for WEHI-3B cells or of IL-6, leukemia inhibitory factor
(LIF), or oncostatin M for M1 cells, self-renewal is
markedly and irreversibly suppressed and the few progeny produced in
clonal cultures exhibit maturation in the macrophage, or less often the granulocytic, lineage.14,15 This response is described as
differentiation induction. However, it is not clear that the leukemic
and normal stem cell systems are necessarily biologically equivalent or
that the molecular basis for the changes is necessarily the same.
Suppression of leukemic self-renewal often is accompanied by cell
death, unlike commitment in normal stem cells. Furthermore, the
leukemic cell lines appear not to be multipotential; thus,
suppression of self-renewal is not associated with much evidence of
lineage commitment choices, although with agents such as IL-6 acting on
M1 or WEHI-3B cells, exclusive macrophage maturation can be
striking.15
In the second system, blood monocytes or culture-generated macrophages
can be converted to phenotypically and functionally distinct dendritic
cells by culture with GM-CSF plus tumor necrosis factor 
(TNF) or
IL-4.16 The system is highly reproducible and does not
overtly involve selective cell death. The question is whether this
phenotypic switch warrants the label of differentiation commitment?
Studies using truncated or mutated receptors have identified a
C-terminal domain in the G-CSF,17,18 thrombopoietin
(TPO),19 and GM-CSF15 receptors
whose loss or reduced function results in failure to suppress
self-renewal in leukemic cells or failure to induce maturation in
leukemic or immortalized cells. However, analysis using different leukemic cell lines has suggested that the domain signaling suppression of self-renewal need not be identical to the domain inducing
maturation,15 suggesting that these processes may differ in
their signaling requirements in different cell types. If so, the
compelling data on regulator-inducible suppression of self-renewal,
particularly in leukemic cell lines, may not be transferrable to the
commitment process occurring in normal stem cells.
With appropriate immortalized cell lines, regulators such as G-CSF
clearly can suppress self-generation and induce granulocytic generation.13,17 However, is this genuine commitment, as
defined by a decision to enter a particular lineage, or is it merely
that suppression of self-renewal allows the activation by default of a
pre-existing maturation program that was previously suppressed by
self-generation? The same question can be raised for the various leukemic cell line models.
Possible differences between lineage commitment, differentiation
induction, and phenotypic switching will only be resolved when the
molecular events and genetic basis of the various changes are
established.
| |
MATURATION |
When a hematopoietic regulator is used to stimulate colony formation,
maturation accompanies the cell proliferation and is commonly assumed
to be due to the action of the regulator. However, there is no fixed
link between the number of cell divisions and the progress of
maturation. Indeed, high growth factor concentrations increase colony
size with an accompanying delay in maturation to postmitotic
cells.1
Despite this, there are multiple lines of evidence indicating that
hematopoietic regulators play an active role in maturation. A
C-terminal region of the G-CSF receptor has been shown to be required
for granulocyte maturation in cell lines17,18 and the
maturation arrest of congenital neutropenia is often associated with
mutations in the C-terminus of the G-CSF receptor.20 TPO provides a clear example of the mandatory action of a regulator in
achieving maturation. Although IL-3 is a better proliferative stimulus
for megakaryocytes than is TPO, only TPO can achieve full cytoplasmic
maturation and platelet formation.21 Another obvious
example of hematopoietic regulator action is the distinct phenotypic
difference imposed on the morphology and behavior of macrophage colony
cells when part of the colony is subsequently stimulated by M-CSF and
part stimulated by GM-CSF.22
However, it has been questioned whether hematopoietic regulators are
necessary to initiate maturation. The receptor insertion studies
referred to earlier indicate that the actual maturation programs
executed are not dependent on the particular hematopoietic regulator
used as the proliferative stimulus. Furthermore, when growth factor was
withdrawn from cultures of an immortalized cell line, but cell survival
was ensured by overexpression of Bcl-2, many cells were able to undergo
substantial maturation.23 A similar phenomenon can be
observed in clonal cultures of normal marrow cells when developing
clones are transferred to factor-free cultures. Cell division ceases,
but some maturation to granulocytic or macrophage cells can be
observed.24 However, in these experiments it is unclear
what earlier role may have been played by the growth factor before it
was withdrawn. Can growth factors initiate maturation programs that
remain arrested while active cell division is being stimulated but are
able to become activated after factor withdrawal and proliferation
ceases?
| |
THE IN VIVO PROBLEMS |
In vivo, there are clear examples of what appears to be a lack of any
rational control of differentiation commitment, resulting in an
apparently bizarre inefficiency in the process of hematopoiesis. Inactivation of the genes for G-CSF or TPO leads to selective deficiencies in mature granulocytes or platelets25,26 that can be corrected by the injection of G-CSF or TPO. However, in both
cases, depletion of the lineage-specific growth factor results in a
severe depletion of progenitor cells in all lineages25,27 and, conversely, the injection of either factor results in an equally
inappropriate expansion of progenitor cells in all
lineages.28,29 The pattern is strongly reminiscent of the
cloning results from blast colonies that suggested that commitment is
random and may not be subject to extrinsic control. In vitro studies
have indeed shown that an agent such as G-CSF, when acting with SCF,
amplifies the production of many inappropriate cells whose further
proliferation cannot be stimulated by this combination.30
Because other growth factors are present in the body, these may have
synergized with the agent being studied to produce the results
observed. These observations do not therefore actually exclude the
possibility that an agent can induce a more selective extrinsic
modulation of differentiation commitment.
| |
CONCLUSIONS |
Hematopoietic commitment is likely to be extrinsically regulated, but
there is only limited evidence, and probably only a limited
opportunity, for hematopoietic regulators being involved in these
events. The major extrinsic agents initiating commitment may in fact be
novel molecules yet to be detected and possibly with no proliferative
actions on hematopoietic cells. A variety of evidence implicates
hematopoietic growth factors as playing an important role in some
aspects of maturation. However, once established, maturation programs
seem not to be qualitatively altered by the particular growth factor
used to activate mature cell production.
| |
FOOTNOTES |
Supported by the Carden Fellowship Fund of the Anti-Cancer Council of
Victoria, the National Health and Medical Research Council, Canberra,
and the National Institutes of Health Grant No. CA-22556.
Address reprint requests to Donald Metcalf, MD, The Walter
and Eliza Hall Institute of Medical Research, PO Royal
Melbourne Hospital, 3050 Victoria, Australia.
| |
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Introduction
References
Introduction
