Blood, 15 December 2002, Vol. 100, No. 13, pp. 4266-4271
PERSPECTIVE
The chiaroscuro stem cell: a unified stem cell theory
Peter J. Quesenberry,
Gerald A. Colvin, and
Jean-Francois Lambert
From the Roger Williams Medical Center, Providence, RI.
 |
Abstract |
Hematopoiesis has been considered hierarchical in nature,
but recent data suggest that the system is not hierarchical and is, in
fact, quite functionally plastic. Existing data indicate that
engraftment and progenitor phenotypes vary inversely with cell cycle
transit and that gene expression also varies widely. These observations
suggest that there is no progenitor/stem cell hierarchy, but rather a
reversible continuum. This may, in turn, be dependent on shifting
chromatin and gene expression with cell cycle transit. If the phenotype
of these primitive marrow cells changes from engraftable stem cell to
progenitor and back to engraftable stem cell with cycle transit, then
this suggests that the identity of the engraftable stem cell may be
partially masked in nonsynchronized marrow cell populations. A general
model indicates a marrow cell that can continually change its surface
receptor expression and thus responds to external stimuli differently
at different points in the cell cycle.
(Blood. 2002;100:4266-4271)
| |
Introduction |
We present here a different view of early
hematopoiesis. This is not the standard dogma of many investigators in
the field, but is built upon solid work by many laboratories over many
years. This is a perspective, and thus naturally reflects the biases of
the authors. These, in turn, have a basis in many published studies
from our laboratory and other laboratories and also from extensive
unpublished, but abstracted, data from our laboratory. It is
appropriate to consider this contribution as speculative in nature.
The term chiaroscuro refers to the treatment of light and
shade in painting. Our current view of the changing phenotype of the
marrow stem cell suggests a chiaroscuro nature to this picture.
In general, models of stem cell regulation have been
hierarchical.1,2 A primitive stem cell, with great
potential, gives rise to a proliferating progenitor pool, which in turn
gives rise to recognizable differentiated cells. During this process,
proliferative potential is lost, while specific differentiated features
are acquired. Presumptively, but without definite proof, there is also
self-renewal at the most primitive stem cell level, and this is also
lost with differentiation. Many data exist to support such a
hierarchical model. Marrow cells have been separated with short- and
long-term repopulation potential3,4 and progenitors have
been characterized as exclusively committed to the production of
restricted progeny.5,6 In addition, the clear expansion of
different progenitor types in cytokine-stimulated in vitro culture with
a loss of long-term engraftment capacity speaks to the existence of a
progenitor hierarchy, at least at the more differentiated progenitor
levels.7,8 A model encompassing these features is shown in
Figure 1.
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| Figure 1.
Classic hierarchical model of hematopoiesis.
S represents stem cell; P, progenitor; and D, differentiated
cell.
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This model does not fit with all published data. For instance, the
"daughter cell" or paired-progenitor experiments of Suda and
colleagues9 indicate that a primitive progenitor cell can make totally different lineage choices during one cell cycle transit, such that, for example, one daughter cell forms an erythroid colony while the other daughter (or sister) cell forms a neutrophil-macrophage colony. Out of a total of 387 pairs evaluated, 68 pairs of colonies consisted of dissimilar combinations of cell lineages. Admittedly, these studies were carried out using spleen as a cell source with erythropoietin and an uncharacterized conditioned media as a source of
stimulation. In addition, others have reported high degrees of
congruence in the phenotype of individual colonies observed after
replating single cells from colony starts.10 Despite these caveats, the elegant experiments of Suda and colleagues9
would seem to weigh against an ordered hematopoietic stem cell hierarchy.
An intrinsic component of this hierarchical model is that the
most primitive hematopoietic stem cell is a quiescent cell in G0 and a fount of potential without differentiated
characteristics. It has generally been believed that primitive
hematopoietic stem cells were dormant or quiescent and were thus
protected from depletion or exhaustion.11,12 On
the basis of a number of transplant studies, a clonal succession model
has been proposed.13 This model proposes that the
production of blood cells is maintained sequentially by one or just a
few lymphohematopoietic stem cells, the residual stem cells remaining
dormant. This model is consistent with reports evaluating the
contributions of individually marked hematopoietic stem cells in
transplant studies.14-16 This scenario formed the basis
for the concept that hematopoietic stem cells are hierarchically
ordered based on their relative quiescence.17,18 Other
investigators have presented data indicating that hematopoietic stem
cells are functioning concurrently, continuously cycling and
contributing to blood cell production.19-21 This latter
view suggests that hematopoietic stem cell quiescence is relative, the
cells being relatively quiescent compared with more differentiated progenitor cells, but not dormant, often proceeding through cell cycle
and undergoing cell division. Certainly, stem cells, as evaluated at
any one point in time, are relatively quiescent. Our laboratory's
standard stem cell separation is based on this relative quiescence,
separating lineage-negative, rhodamine dull and Hoechst dull
(Lin
RhodullHodull)
cells,22-27 although other separations based on Sca-1 and
c-kit epitopes include a small percentage of proliferating
cells.28-30 In studies on Hoechst-stained side population
marrow stem cells, Goodell et al31 determined that between
1% and 3% of these cells were in S/G2/M. Previous studies
with cell cycle-specific killing drugs did not distinguish between
cells that are truly dormant and cells that are either in prolonged
cell cycle or intermittently entering and exiting cell cycle at a slow
rate. In previous studies of hematopoietic cell cycling the cells
analyzed were populations with functional characteristics of late
progenitors.32-37 Bone marrow repopulating ability had
been assessed with 7 days of bromodeoxyuridine (BrdU) labeling
and a turnover time of more than 11.5 days was demonstrated.36 Labeling after 32 days of exposure to BrdU
was assessed by one group.37 They showed a
T1/2 of 21 days for colony-forming unit
spleen (CFU-S) on day 14, but in this study long-term
repopulating cells were not assessed.
Bradford and colleagues, studying long-term repopulating cells as
represented by
LinRhodullHodull-separated
murine marrow cells and using in vivo exposure to BrdU, evaluated the
proliferative history of these stem cells over longer periods of time
in vivo.38 They studied mice continuously exposed to BrdU
in their drinking water and then assessed the percentage of
LinRhodullHodull marrow stem
cells labeling over different time intervals of BrdU administration.
BrdU is incorporated into DNA during DNA synthesis, and thus labeling
with this agent is an accurate measure of whether a cell has transited
S phase while progressing through the cell cycle. These researchers
isolated LinRhodullHodull cells
from these BrdU-exposed mice. They found that 60% (± 14%) of these
primitive stem cells were labeled after 4 weeks and showed a time to
50% labeling (T1/2) of 19 days. Cheshier and
colleagues, using a different mouse strain and a different stem cell
separation, confirmed these data; they showed more rapid labeling of
stem cells.30 We evaluated whether these data could be
explained by DNA damage and repair, rather than proliferation,
and after extensive studies came to the conclusion that the BrdU
incorporation did, in fact, indicate proliferation of these primitive
stem cells over time.39 We studied BALB/c mice and
LinRhodullHodull cells in a
fashion identical to that of Bradford et al38 and obtained
virtually identical results. Thus, when the cycling status of stem
cells is approached over a longer time frame, it appears that these
relatively quiescent cells are either in a prolonged active cell cycle
or, perhaps more likely, repeatedly entering and leaving the cell
cycle. The latter possibility is supported by studies showing that
transplanted stem cells rapidly enter cell cycle40,41 and
that primitive marrow stem cells are easily induced into cell cycle on
in vitro cytokine exposure.42 Abkowitz and
colleagues,43 using stochastic modeling and computer
simulation to study the replication, apoptosis, and differentiation of
murine hematopoietic cells, estimated that murine hematopoietic stem cells replicated (on average) every 2.5 weeks. When this approach was
used in cats, heterozygous for glucose-6-phosphate dehydrogenase, different estimates of hematopoietic stem cell replication rate (1 per
8.3 to 10 weeks) were derived. Thus this present work, along with the
work of others, indicates that virtually all primitive marrow stem
cells, while relatively quiescent, pass through the cell cycle over a
varying period of time, depending on mouse strain and possibly the
specifics of the stem cell separation.
| |
Stem cell genes and functions |
In a similar vein, while these cells do not express terminal
differentiation functions, they robustly express certain stem cell
functions. They are highly motile, showing very rapid directed movement
and rapid membrane deformation on cytokine exposure.44 These cells also express a large number of stem cell-specific genes.
Phillips et al45 have reported on 2119 nonredundant gene products from fetal liver hematopoietic stem cells, using a subtracted cDNA library to generate a micro array chip. They have identified several genes specific to fetal liver hematopoietic cells. In addition,
when comparing fetal hematopoietic cells with adult hematopoietic cells
(LinRhodull Hodull
c-kit+Sca-1+), they found several genes that
were coexpressed on fetal and adult stem cells as well as genes
specific for either fetal or adult stem cells. Park et
al46 have also reported on murine hematopoietic stem cells
gene profiling. They used a 5000-cDNA array obtained by subtraction of
cDNA from lineage-positive cell populations and studied both
hematopoietic adult stem cells and multipotent progenitors (with
minimal self-renewal capacity). Genes primarily expressed in stem cells
were transcription factors, RNA binding proteins, chromatin modifiers,
and protein kinases. In differential gene display studies using a
gel-based method that uses the display of 3' end fragments of cDNA
generated by cutting with specific enzymes,47 we have
identified a total of 637 differentially expressed genes in murine
LinRhodullHodull cells compared
with differentiated (Lin+) cells (S. Weissman et al,
unpublished data, 2001). These genes include 411 unknowns and
19 different gene categories. These categories included transcription
factors (22), protein synthesis regulators (11), surface proteins (11),
mitochondrial sequences (10), RNA metabolism proteins (10), signaling
pathway factors (9), and cytokines (8). In separate studies, we have
also shown that
LinRhodullHodull marrow
cells express adhesion proteins48,49 and cytokine
receptors50 on the cell surface. These data indicate a
different picture of the primitive marrow stem cell than is usually
envisioned
a functional cell in which a large number of stem
cell-specific, as opposed to differentiated, functions are manifest
(Figure 2).
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| Figure 2.
Stem cell-specific functions.
This figure illustrates proteopodia membrane extensions and adherence
to a mesenchymal stromal cell (M). Surface-based symbols illustrate
cell surface-based receptors.
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In addition, work by others has indicated that marrow-derived stem
cells may express lineage-specific genes prior to
commitment.51-53 These studies indicate that primitive
hematopoietic stem cells express a wide variety of genes, including a
number of transcription factors. Presumptively, transcription factor
access to DNA is a major determinant of such gene expression.
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Cell cycle transit and chromatin modulation |
Chromatin remodels during cell cycle progression. In previous
studies chromatin modulation at the B-globin and lysozyme gene loci
were evaluated54-57 and myeloid specific
cis-regulatory elements showed a specific chromatin pattern
at the lysozyme locus in myelomonocytic cells at different
differentiation stages. This chromatin pattern was also found in
multipotent hematopoietic progenitors, but it disappeared when cells
differentiated into the erythroid lineage. Evaluation of the chromatin
structure of multipotent hematopoietic cells indicated that the
lineage-associated genes, globin, myeloperoxidase, immunoglobulin H
(IgH), and CD3
have accessible control regions before
unilineage commitment.55-59 Other work indicates that
chromatin remodeling factors may be recruited in one phase of the cell
cycle for ultimate action in a later phase, which in turn results in changes in transcriptional programs. Consistent with the model of a
fluctuating cell cycle-based stem cell phenotype and asymmetric division is the proposal of McConnell and Kaznowski60 that
cell cycle passage could determine the fate of cells derived from stem cell division and renew stem cell multipotency. Studying laminar determination in the developing neocortex, these investigators transplanted embryonic neural progenitor cells into older host brains.
The fate of the transplanted neurons correlated with the position of
the progenitors in the cell cycle at the time of transplantation. Daughters of cells transplanted in S phase migrated to layer 2/3, while
progenitors transplanted later in the cell cycle produced daughters
that were committed to their normal deep-layer fate. These studies
indicate that the cell fate of the immediate stem cell progeny is
restricted by environmental signals shortly after S phase; passage
through the next S phase then restores multipotency. These observations
might be explained by alterations in chromatin structure during DNA
replication. Such alterations could have marked effects on gene
expression and therefore on cell fate.
This hypothesis is supported by studies in yeast61,62
showing that chromatin-remodeling factors are recruited during
M/G1 and chromatin is modeled in the next G1.
Thus a reasonable sequence of events would be chromatin remodeling with
cytokine-induced cell cycle transit leading to varying levels of DNA
access to transcription factors, followed by alterations in patterns of gene expression.
In models of stem cell-cycle progression and division, there are
several possibilities. The stem cell could have a symmetric division
with the production of 2 identical stem cells, a symmetric division
with the production of 2 differentiating cells, or an asymmetric
division with the production of 1 stem cell and 1 differentiated cell.
Most models assume that, on a population basis, stem cells would show
asymmetric division; the other alternatives would lead to either
hyperproliferation or stem cell exhaustion. These events might also be
modulated by cellular apoptosis. The above-described stem cell
phenotypic shifts with cell cycle transit and the concept of asymmetric
divisions form the basis for the model shown in Figure
3.
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| Figure 3.
Fluctuating stem cell phenotype and genotype with cell
cycle transit.
Chromatin modulation and resulting changes in access of transcription
factors to different control regions. Shown is an asymmetric division
in which a stem cell (S) produces a phenotypically similar stem cell
and a cell (D) destined for terminal differentiation. Cylinders
represent DNA chromatin coverage; boxes, active transcription
factors.
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Changing engraftable multipotent stem cell phenotype with cell
cycle transit |
We have been investigating the phenotype of marrow stem
cells as they progress through the cell cycle under cytokine
stimulation. Initial studies showed that cytokine stimulation in vitro
with interleukin-3 (IL-3), interleukin-11 (IL-11), interleukin-6
(IL-6), and steel factor for 48 hours resulted in a loss of engraftment capacity at 3 to 24 weeks and that longer-term engraftment was most
strongly affected.63-65 Early engraftment, at 1 to 3 weeks, was either modestly augmented or unaffected. Subsequent studies evaluated the engraftment capacity of BALB/c or C57BL/6J marrow cells
cultured in IL-3, IL-6, IL-11, and steel factor in nonadherent Teflon
bottles every 2 to 4 hours from 24 to 80 hours of
culture.66 Separate studies had determined the cell cycle
status of LinRhodullHodull marrow
cells under the same cytokine and culture conditions.42 These hematopoietic stem cells isolated on the basis of quiescence progressed in a highly synchronized fashion through the cell cycle, entering S phase at about 18 hours and completing the first population doubling at about 36 to 38 hours. These studies showed dramatic and
reversible fluctuations in 8-week and 6-month engraftment capacity at
2- to 4-hour intervals, with a nadir and recovery appearing in the
first cell cycle transit. Cell cycle-related shifts in the functional
and surface phenotype of both murine and human primitive hematopoietic
stem cells have been observed by a number of investigators. Fleming and
colleagues showed functional heterogeneity associated with the cell
cycle status of murine stem cells.67 They found that
purified lineage-negative Sca-1+ and thy-1low
cells showed decreased engraftment in S/G2/M as compared
with G1. Orschell-Traycoff et al68 showed that
when engrafting phenotypes of Sca-1+Lin stem
cells were Hoechst fractionated into G0 /G1 or
S/G2/M, cells with long-term engraftment capacity were
found in G0/G1. Gothot et al,69
studying primitive human progenitor in bone marrow and
peripheral blood, found multipotent progenitors in G0 or
early G1, while lineage-restricted granulomonocytic
progenitors were more abundant in late G1. These
investigators used Hoechst 33342 and pyronin to isolate cells. The same
group found that the repopulating capacity of human mobilized
peripheral blood CD34+ cells in nonobese diabetic/severe
combined immunodeficient mice (NOD/SCID ) was increased in
G0 as opposed to G1.70 In a
similar vein, Glimm and colleagues, studying human cord blood with
lymphomyeloid repopulating activity in NOD/SCID mice, showed that
transplantable stem cell activity was restricted to the G1
fraction, even though colony forming cells (CFCs) and long-term culture
initiating cells (LTC-ICs) were equally distributed between
G0/G1 and S/G2/M.71 G0 cells had (and were defined by) reduced K167 and cyclin
D expression and low Hoechst expression. CD34+ cord blood
cells stimulated with steel factor, thrombopoietin, and Flt-3 ligand
showed functional differences between G0 and G1
cell cycle phases, as reported by Summers et al.72 They
showed a 1000-fold increase in granulocyte-macrophage colony forming cells (GM-CFCs) in G0 as compared with
G1, a 250-fold increase in burst-forming unit-erythroid
(BFU-e), and a 600-fold increase in CD34+ cells.
Finally, in studies of murine hematopoietic stem cells, using 2 different stem cell separative approaches, long-term in vivo
engraftment was shown to be better in G0 than in
G1 and in G0/G1 than in
S/G2/M.73,74
Studies of adhesion protein cell-surface expression on
LinRhodullHodull and
LinSca-1+ hematopoietic stem cells with cell
cycle transit showed fluctuations of 
-4, -5, -6, 
-1,
L-selectin, and platelet-endothelial cell adhesion molecule-1
(PECAM-1) at different points in the cell cycle.48,49 A probable causal role for these fluctuations
in the engraftment fluctuations was shown by homing studies in which it
was found that CFDA-SE (5- (and -6)-carboxyfluorescein
diacetate succinimidyl ester)-labeled
LinSca+ stem cells cultured in IL-3, IL-6,
IL-11, and steel factor showed a marked depression in homing at a time
when engraftment and -4 were also markedly depressed (48 hours of
culture).75 A schematic summarizing the results with
marrow cells cultured in IL-3, IL-6, IL-11, and steel factor is shown
in Figure 4. These results indicated that
engraftment was reversibly lost at late S/early G2 and
recovered in the next G1.
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| Figure 4.
Fluctuating engraftment phenotype with cytokine-induced
cell cycle transit.
Based on studies of murine marrow stem cells stimulated with IL-3,
IL-6, IL-11, and steel factor.31,32 Engraftment is lost in
late S early G2 and recovers in the next
G1.  ,  , and  represent adhesion
molecules.
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In addition, Yamaguchi et al,76 studying
CD34+ human progenitors, showed different adhesive
characteristics and very-late antigen 4 (VLA-4) expression in
the G0/G1 and S/G2/M phases of the
cell cycle. A wide variety of other genes, including cytokine receptors, were also modulated with cycle progression.50
Other gene families that were either down-regulated or up-regulated in
purified murine hematopoietic stem cells after 48 hours' culture in
IL-3, IL-6, IL-11, and steel factor included genes involved in energy
metabolism, cell cycle regulation, signaling pathways, transcription
fate, cytoskeleton, apoptosis regulation, membrane trafficking, RNA
metabolism, and chromatin.77 In addition, a total of 246 unknown genes were modulated. These studies indicate that a large
variety of genes are modulated with cell cycle transit, presumably
underlying the shifting stem cell phenotype seen with cell cycle
transit (Figure 5). All together, these
studies showed a reversible fluctuating engraftment phenotype
associated with cell cycle phase position, which is associated with a
loss of homing and a modulation of the stem cell genotype.
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| Figure 5.
Cell cycle-related fluctuations in gene expression.
The primitive marrow stem cells express a large variety of genes, and
that expression changes with cell cycle transit. S represents
engraftable stem cell; D, differentiated cell.
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Stem/progenitor cell inversions |
Differentiation was also assessed with cell cycle transit, this
time with different cytokines and with 2 different culture systems.
Unseparated BALB/c or C57BL/6J murine marrow cells were cultured in
Teflon bottles or in microgravity rotating wall vessels in the presence
of thrombopoietin, FLT3, and steel factor, and progenitor and
engraftable stem cell levels were determined at various times in
culture corresponding to different points in stem cell-cycle
transit.78 These studies showed that there were marked and
reversible increases in progenitor cell (high-proliferative-potential colony-forming cell [HPP-CFC] or colony-forming unit culture
[CFU-C]) numbers during the first cell cycle transit. Remarkably,
these increases in progenitor cells were tightly linked to decreases in
engraftment capacity (8-week competitive engraftment into lethally irradiated mice), and generally, when the progenitor numbers returned to baseline there was a reciprocal change in engraftment capacity. A
total of 20 marked increases in HPP-CFC or CFU-C numbers were observed,
and in all but one case this increase was linked to a significant
decrease in engraftable stem cells. These progenitor/stem cell shifts
were significant at P < .0001. With longer times in cytokine culture, the progenitors expanded, indicating that the immediate kinetics were not those that might be seen in the setting of
progenitor exhaustion. In a smaller number of observations, when the
number of progenitors was decreased, the number of engraftable stem
cells was elevated or at baseline. In general, in these experiments, when engraftable stem cell levels decreased and then returned to
baseline, they did not show increases. This is consistent with an
asymmetric-division stem cell model. We have termed these phenomena "stem cell inversions." Others have reported similar findings. Knaan-Shanzer79 has proposed that there is "no hierarchy
within the primitive hemopoietic stem cell compartment." This
investigator, studying umbilical cord blood cells, showed that
primitive
CD34+CD33,38,71
NOD/SCID repopulating cells were interconvertible with the less primitive
CD34+CD33+,38+,71+
cells in culture. These latter showed practically "no NOD/SCID repopulating activity." The less primitive cells could give rise to
the more primitive cells. Finally, Kirkland and Borokov80 have developed a phase space model of hemopoiesis and stem cell proliferation and differentiation, which is used to describe the differentiative state of cells in 2 or more dimensions. In this model
the stem cell population is viewed as a continuum, rather than being
composed of discrete states.
Such a model includes the possibility that some daughter cells will
have greater "stemness" than the parent cells in a renewing model.
This is also consistent with our observations of stem/progenitor cell
inversions. A schematic encompassing these observations is presented in
Figure 6.
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| Figure 6.
Stem cell/progenitor cell inversion.
As marrow stem cells progress through the cell cycle, there is an
inverse correlation between progenitor and stem cells. When progenitors
increase, engraftable stem cells decrease, and this is
reversible.
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The stem cell continuum |
These studies indicate that previous views of the stem cell
hierarchy, at least at the more primitive stem/progenitor cell levels,
may be untenable. Rather than a hierarchical transition from stem to
progenitor cell, it appears that a fluctuating continuum exists, in
which the phenotype of these primitive marrow cells shifts from one
state to another and back. Presumptively, these phenotypic shifts are
based on chromatin remodeling associated with cell cycle transit, which
reversibly alters the surface phenotype of the stem cell, which then
determines its response to environmental stimuli. This model would also
be consistent with an asymmetric model of stem cell regulation in
steady state hematopoiesis in which one daughter cell of a division
returns to its base primitive (stem/progenitor) cell phenotype, while
the other daughter cell commits to a differentiation pathway or an
apoptotic death.
| |
The masked stem cell hypothesis |
If the phenotype of the stem cell reversibly modulates with cell
cycle transit, with a predominantly progenitor phenotype at one point
in the cycle and a predominantly engraftable multipotent stem cell
phenotype at another point in the cycle, then estimates of long-term
engraftable stem cell incidence in a cell population are probably
underestimates. In essence, the identity of the stem cell would be
masked at certain points in the cycle, and in a nonsynchronized
population of cells, at any one point in time, a number of true stem
cells would not be detectable. This could be termed the "masked" or
"stealth" stem cell concept.
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A unified stem cell theory |
The present model builds upon a large base of previously
accomplished work.81 It suggests that cell cycle transit,
a fundamental characteristic of primitive hematopoietic stem cells, is
associated with a continually changing stem cell phenotype and that, at
least at the more primitive stem/progenitor cell levels, a fluctuating continuum, rather than a stem cell/progenitor hierarchy, may exist. Thus, outcomes would be determined by the changing cell cycle surface
phenotype of the stem cell, that is, its receptor expression, and the
delivered environmental stimuli, that is, fluid-phase or membrane-based
cytokines, adhesion protein, or other ligands.
This provides, ultimately, a very flexible system for hematopoietic
regulation, in which multiple different outcomes could occur
sequentially, dependent on cell cycle phase and specific microenvironment. One can speculate that such flexibility might also
hold for the recently described transdifferentiation of marrow stem
cells to nonhematopoietic cells in different tissues, although at
present there are no data addressing this point. This theory is
presented in model form in Figure 7.
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| Figure 7.
The chiaroscuro stem cell model.
As early marrow stem cells move through cell cycle transit, chromatin
coverage changes, resulting in activation of different transcription
regions and thus different gene expression. This change underlies the
reversibly shifting phenotype of stem cells. Here stem cells are shown
altering their phenotype from hematopoietic engraftable stem cell (S)
to hematopoietic progenitor 1 (P1) to hematopoietic
progenitor 2 (P2) with different phenotypes (eg,
proteopodia) and back to hematopoietic engraftable stem cell. Cylinders
represent DNA chromatin coverage; boxes, active transcription
factors.
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Acknowledgments |
We wish to thank Dr Abby Maizel for his careful critique and
helpful suggestions.
| |
Footnotes |
Submitted April 26, 2002; accepted July 19, 2002.
Prepublished
online as Blood First Edition Paper, August 22, 2002;
DOI 10.1182/blood-2002-04-1246.
Supported by grants PO1 DK50222-02, PO1
HL56920-04, and RO1 DK27424-19.
Reprints: Peter J. Quesenberry, Center for Stem Cell
Biology, Roger Williams Medical Center, 825 Chalkstone Ave, Providence,
RI 02908-4735; e-mail: pquesenberry{at}rwmc.org.
| |
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Abstract
Introduction
