Perspective
The chiaroscuro stem cell: a unified stem cell theory
Peter J. Quesenberry, Gerald A. Colvin, and Jean-Francois Lambert
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 popula-
tions. 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)
© 2002 by The American Society of Hematology
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laboratory and other laboratories and also from extensive unpublished, but abstracted, data from our laboratory.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 cytokinestimulated 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.
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 neutrophilmacrophage 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
From the Roger Williams Medical Center, Providence, RI.
Reprints: Peter J. Quesenberry, Center for Stem Cell Biology, Roger Williams
Medical Center, 825 Chalkstone Ave, Providence, RI 02908-4735; e-mail:
[email protected].
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.
4266
© 2002 by The American Society of Hematology
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
THE CHIAROSCURO STEM CELL
4267
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.
Figure 1. Classic hierarchical model of hematopoiesis. S represents stem cell;
P, progenitor; and D, differentiated cell.
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 T ⁄ 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 Lin⫺RhodullHodull–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 Lin⫺RhodullHodull
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 Lin⫺RhodullHodull 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 (T ⁄ ) 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 Lin⫺RhodullHodull 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
12
12
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 (Lin⫺Rhodull 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
Lin⫺RhodullHodull 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
Lin⫺RhodullHodull 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).
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.
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.
4268
QUESENBERRY et al
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
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 abovedescribed stem cell phenotypic shifts with cell cycle transit and the
concept of asymmetric divisions form the basis for the model
shown in Figure 3.
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
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.
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
Lin⫺RhodullHodull 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 lineagenegative 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
Lin⫺RhodullHodull and Lin⫺Sca-1⫹ hematopoietic stem cells with
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
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 Lin⫺Sca⫹ 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.
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.
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-proliferativepotential 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
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. , F, and 䉫 represent adhesion molecules.
THE CHIAROSCURO STEM CELL
4269
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.
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. KnaanShanzer79 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.
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
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.
4270
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
QUESENBERRY et al
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.
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.
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
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.
Acknowledgment
We wish to thank Dr Abby Maizel for his careful critique and
helpful suggestions.
References
1. Quesenberry PJ. Hematopoiesis and hematopoietic growth factors. In: Goldman L, Bennett JC,
eds. Cecil Textbook of Medicine. 21st ed. Philadelphia, PA: WB Saunders; 1999:834-840.
9. Suda T, Suda J, Ogawa M. Disparate differentiation in mouse hemopoietic colonies derived from
paired progenitors. Proc Natl Acad Sci U S A.
1984;81:2520-2524.
2. Quesenberry PJ, Colvin GA. Hematopoietic stem
cells, progenitor cells and cytokines. In: Beutler
E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn
U, eds. Williams Hematology. 6th ed. New York,
NY: McGraw-Hill; 2001:153-174.
10. Brady G, Iscove NN. Construction of cDNA libraries from single cells. Methods Enzymol. 1993;
225:611-623.
3. Guenechea G, Gan OI, Dorrell C, Dick JE. Distinct classes of human stem cells that differ in
proliferative and self-renewal potential. Nat Immunol. 2001;2:75-82.
4. Harrison DE, Astle CM. Short- and long-term multilineage repopulating hematopoietic stem cells in
late fetal and newborn mice: models for human
umbilical cord blood. Blood. 1997;90:174-181.
5. Akashi K, Traver D, Miyamoto T, Weissman IL. A
clonogenic common myeloid progenitor that gives
rise to all myeloid lineages. Nature. 2000;404:
193-197.
6. Borge OJ, Adolfsson J, Jacobsen AM. Lymphoidrestricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands. Blood.
1999;94:3781-3790.
7. McNiece IK, Almeida-Porada G, Shpall EJ, Zanjani E. Ex vivo expanded cord blood cells provide
rapid engraftment in fetal sheep but lack longterm engrafting potential. Exp Hematol. 2002;30:
612-616.
8. Wiesmann A, Kim M, Georgeias A, et al. Modulation of hematopoietic stem/progenitor cell engraftment by transforming growth factor beta. Exp Hematol. 2000;28:128-139.
11. Hodgson GS, Bradley TR. Properties of haemopoietic stem cells surviving 5-fluorouracil treatment: evidence for a pre-CFU-S cell? Nature.
1979:281:381-382.
12. Lerner C, Harrison DE. 5-Fluouracil spares hemopoietic stem cells responsible for long-term
repopulation. Exp Hematol. 1990;18:114-118.
13. Kay HEM. How many cell generations? Lancet.
1965:418.
14. Mintz B, Anthony K, Litwin S. Monoclonal derivation of mouse myeloid and lymphoid lineages
from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts. Proc Natl Acad
Sci U S A. 1984;81:7835-7839.
15. Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. 1986;45:917-927.
16. Capel B, Hawley RG, Mintz B. Long- and shortlived murine haematopoietic stem cell clones individually identified with retroviral integration markers. Blood. 1990;75:2267-2270.
17. Rosendaal M, Hodgson GS, Bradley TR. Organisation of haematopoietic stem cells: the generation-age hypothesis. Cell Tissue Kinet. 1979;12:
17-29.
18. Hodgson GS, Bradley TR, Radley JM. The organization of hemopoietic tissue as inferred from
the effects of 5-fluorouracil. Exp Hematol. 1982;
10:26-35.
19. Harrison DE, Lerner C, Hoppe PC, Carlson GA,
Alling D. Large numbers of primitive stem cells
are active simultaneously in aggregated embryo
chimeric mice. Blood. 1987;69:773-777.
20. Micklem HS, Lennon JE, Ansell JD, Gray RA.
Numbers and dispersion of repopulating hematopoietic cell clones in radiation chimeras as functions of injected cell dose. Exp Hematol. 1987;15:
251-257.
21. Harrison DE, Astle CM, Lerner C. Number and
continuous proliferative pattern of transplanted
primitive immunohematopoietic stem cells. Proc
Natl Acad Sci U S A. 1988;85:822-826.
22. Bertoncello I, Bradley TR, Hodgson GS. The concentration and resolution of primitive hemopoietic
cells from normal mouse bone marrow by negative selection using monoclonal antibodies and
dynabead monodisperse magnetic microspheres.
Exp Hematol. 1989;17:171-176.
23. Mulder AH, Viser JWM. Separation and functional
analysis of bone marrow cells separated by rhodamine 123 fluorescence. Exp Hematol. 1987;15:
99-104.
24. Wolf NS, Kone A, Priestley GV, Bartelmez SH. In
vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated
by sequential Hoechst-33342-rhodamine 123
FACS selection. Exp Hematol. 1993;21:614-622.
25. Neben S, Redfearn WJ, Parra M, Brecher G, Pallavicini MG. Short- and long-term repopulation of
lethally irradiated mice by bone marrow stem
cells enriched on the basis of light scatter and
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
Hoechst fluorescence. Exp Hematol. 1991;19:
958-967.
26. Nilsson SK, Dooner MS, Tiarks CY, Weier H,
Quesenberry PJ. Potential and distribution of
transplanted hematopoietic stem cells in a nonablated mouse model. Blood. 1997;89:4013-4020.
27. Ploemacher RE, Brons NHC. Cells with marrow
and spleen repopulating ability and forming
spleen colonies on days 16, 12 and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention. J Cell Physiol. 1988;136:
531-536.
28. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science. 1988;241:58-62.
29. Okada S, Nakauchi H, Nagayoshi K, Nishikawa
S, Miura Y, Suda T. In vivo and in vitro stem cell
function of c-kit and Sca-1-positive murine hematopoietic cells. Blood. 1992;80:3044-3050.
program of hematopoietic stem cells. Science.
2000;288:1635-1640.
46. Park IK, He Y, Lin F, et al. Differential gene expression profiling of adult murine hematopoietic
stem cells. Blood. 2002;99:488-498.
65.
48. Becker PS, Nilsson SK, Zhifang L, et al. Adhesion
receptor expression by hematopoietic cell lines
and murine progenitors: modulation by cytokines
and cell cycle status. Exp Hematol. 1999;27:533541.
66.
49. Berrios VM, Dooner GJ, Nowakowski G, et al.
The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic
stem cells. Exp Hematol. 2001;29:1326-1335.
50. Reddy GP, McAuliffe CI, Pang L, Quesenberry
PJ, Bertoncello I. Cytokine receptor repertoire
and cytokine responsiveness of Hodull/Rhdull stem
cells with differing potentials for G1/S phase progression. Exp Hematol. 2002;30:792-800.
31. Goodell MA, Brose K, Paradis G, Conner AS,
Mulligan RC. Isolation and functional properties
of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996;183:1797-1806.
51. Tada T, Tada M. Toti-/pluripotential stem cells and
epigenetic modifications. Cell Struct Funct. 2001;
3:149-160.
33. Vassort F, Winterholer M, Frindel E, Tubiana M.
Kinetic parameters of bone marrow stem cells
using in vivo suicide by tritiated thymidine or by
hydroxyurea. Blood. 1973;41:789-796.
34. Patt HM, Maloney MA, Lamela RA. Hematopoietic stem cell proliferative behavior as revealed
by bromodeoxyuridine labeling. Exp Hematol.
1980;8:1075-1079.
35. Hagan MP, MacVittie TJ. CFUs kinetics observed
in vivo by bromodeoxyuridine and near-UV light
treatment. Exp Hematol. 1981;9:123-128.
36. Hodgson GS, Bradley TR. In vivo kinetic status of
hematopoietic stem and progenitor cells as inferred from labeling with bromodeoxyuridine. Exp
Hematol. 1984;12:683-687.
37. Pietrzyk ME, Priestley GV, Wolf NS. Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo
BrdU infusion. Blood. 1985;66:1460-1462.
38. Bradford GB, Williams B, Rossi R, Bertoncello I.
Quiesence, cycling, and turnover in the primitive
hematopoietic stem cell compartment. Exp Hematol. 1997;25:445-453.
39. Pang L, Reddy PV, Quesenberry PJ. Are bone
marrow stem cells quiescent? [abstract]. Exp Hematol. 1999;27:106.
40. Nilsson SK, Dooner MS, Quesenberry PJ. Synchronized cell-cycle induction of engrafting longterm repopulating stem cells. Blood. 1997;90:
4646-4650.
41. Hendrikx PJ, Martens ACM, Hagenbeek A, Keij
JF, Visser JWM. Homing of fluorescently labeled
hemopoietic stem cells. Exp Hematol. 1996;24:
129-140.
42. Reddy GPV, Tiarks CY, Pang L, Quesenberry PJ.
Synchronization and cell cycle analysis of pluripotent hematopoietic progenitor stem cells. Blood.
1997;90:2293-2299.
43. Abkowitz JL, Galinelli D, Harrison DE, Gutturp P.
In vivo kinetics of murine hematopoietic stem
cells. Blood. 2000;96:3399-3405.
44. Frimberger A, McAuliffe CI, Werme KA, et al. The
fleet feet of haematopoietic stem cells: rapid motility, interaction and proteopodia. Br J Haematol.
2001;112:644-654.
45. Phillips RL, Ernst RE, Brunk B, et al. The genetic
64.
47. Prashar Y, Weissman SM. Analysis of differential
gene expression by display of 3⬘ end restriction
fragments of cDNAs. Proc Natl Acad Sci U S A.
1996;93:659-663.
30. Cheshier SH, Morrison SJ, Liao X, Weissman IL.
In vivo proliferation and cell cycle kinetics of longterm self-renewing hematopoietic stem cells.
Proc Natl Acad Sci U S A. 1999;96:3120-3125.
32. Becker AJ, McCulloch EA, Siminovitch L, Till JE.
The effect of differing demands for blood cell production on DNA synthesis by hemopoietic colonyforming cells of mice. Blood. 1965;26:296.
THE CHIAROSCURO STEM CELL
52. Forsberg EC, Downs KM, Christensen HM, Im H,
Nuzzi PA, Bresnick EH. Developmentally dynamic histone acetylation pattern of a tissuespecific chromatin domain. Proc Natl Acad Sci
U S A. 2000;97:14494-14499.
53. Miyata Y, Towatari M, Maeda T, Ozawa Y, Saito H.
Histone acetylation induced by granulocyte
colony-stimulating factor in a map kinase-dependent manner. Biochem Biophys Res Commun.
2001;283:655-660.
54. Bonifer C. Long-distance chromatin mechanisms
controlling tissue-specific gene locus activation.
Gene. 1999;2238:277-289.
55. Maes J, O’Neill LP, Cavelier P, Turner BM, Rougeon F, Goodhardt M. Chromatin remodeling at
the Ig loci prior to V(D)J recombination. J Immunol. 2001;167:866-874.
56. Jimenez G, Griffiths SD, Ford AM, Greaves MF,
Enver T. Activation of the beta-globin locus control region precedes commitment to the erythroid
lineage. Proc Natl Acad Sci U S A. 1992;89:
10618-10622.
57. Kontaraki J, Chen HH, Riggs A, Bonifer C. Chromatin fine structure profiles for a developmentally
regulated gene: reorganization of the lysozyme
locus before transactivator binding and gene expression. Genes Dev. 2000;14:2106-2122.
58. Hu M, Krause D, Greaves M, et al. Multilineage
gene expression precedes commitment in the
hemopoietic system. Genes Dev. 1997;11:774785.
59. Ford AM, Bennett CA, Healy LE, Navarro E,
Spooncer E, Greaves MF. Immunoglobulin
heavy-chain and CD3 delta-chain gene enhancers are DNAse I-hypersensitive in hemopoietic
progenitor cells. Proc Natl Acad Sci U S A. 1992;
89:3424-3428.
60. McConnell S, Kaznowski C. Cell cycle dependence of laminar determination in developing
neocortex. Science. 1991;254:282-285.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
61. Cosma M, Tanaka T, Nasmyth K. Ordered recruitment of transcription and chromatin remodeling
factors to a cell cycle and developmentally regulated promoter. Cell. 1999;97:299-311.
79.
62. Krebs JE, Kuo MH, Allis CD, Peterson CL. Cell
cycle related histone acetylation required for expression of the yeast H0 gene. Genes Dev. 1999;
13:1412-1421.
80.
63. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an
81.
4271
engraftment defect in normal hosts. Exp Hematol.
1995;23:461-469.
Peters SO, Kittler ELW, Ramshaw HS, Quesenberry PJ. Ex vivo expansion of murine marrow
cells with interleukin-3, interleukin-6, interleukin11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood. 1996;87:30-37.
Peters SO, Habibian HK, Vergilis K, Quesenberry
PJ. Effects of cytokines on stem cell engraftment
depends on time of evaluation post-marrow infusion. Int J Hematol. 1999;70:112-118.
Habibian HK, Peters SO, Hsieh CC, et al. The
fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med.
1998;188:393-398.
Fleming WH, Alpern EJ, Uchida N, Ikuta K, Spangrude GJ, Weissman IL. Functional heterogeneity
is associated with the cell cycle status of murine
marrow cells. J Cell Biol. 1993;122:897-902.
Orschell-Traycoff CM, Hiatl K, Dagher RN, Rice
S, Yoder MC, Srour EF. Homing and engraftment
potential of Sca-1⫹ lin- cells fractionated on the
basis of adhesion molecule expression and position in cell cycle. Blood. 2000;96:1380-1387.
Gothot A, Pyatt R, McMahel J, Rice S, Srour EF.
Functional heterogeneity of human CD34 (⫹)
cells isolated in subcompartments of the G0/G1
phase of cell cycle. Blood. 1997;90:4384-4393.
Gothot A, van der loo JC, Clapp DW, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34 (⫹)
cells in non-obese diabetic/severe combined immune-deficient mice. Blood. 1998;92:2641-2649.
Glimm H, Il-Hoan O, Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro
lose engraftment potential during their S/G2/M
transit and do not re-enter G0. Blood. 2000;96:
4185-4193.
Summers YJ, Heyworth CM, deWynter EA, Testa
NG. Cord blood G(0) CD34⫹ cells have a thousand-fold higher capacity for generating progenitors in vitro than G(1) CD34⫹ cells. Stem Cells.
2001;19:505-513.
Szilvassy SJ, Meyerrose TE, Grimes B. Effects of
cell cycle activation on the short-term engraftment properties of ex-vivo expanded murine hematopoietic cells. Blood. 2000;95:2829-2837.
Huttman A, Liu SL, Boyd AW, Li CL. Functional
heterogeneity within rhodamine 123 (lo) Hoechst
33342 (lo/sp) primitive stem cells revealed by pyronin Y. Exp Hematol. 2001;29:1109-1116.
Cerny J, Dooner M, McAuliffe C, et al. Homing of
purified murine lymphohematopoietic stem cells:
a cytokine-induced defect. J Hematother Stem
Cell Res. In press.
Yamaguchi M, Ikebuchi K, Hirayama F, et al. Different adhesive characteristics and VLA-4 expression of CD34 (⫹) progenitors in G0/G1 versus
S/G2/M phases of cell cycle. Blood. 1998;92:842848.
Lambert JF, Liu M, Baskaran NV, et al. Purified
hematopoietic stem cells show a major shift of
gene expression through cell cycle that coincides
with an engraftment nadir [abstract]. Exp Hematol. 2002;30:118.
Colvin GA, Lambert JF, McAuliffe CI, Quesenberry PJ. Stem/progenitor cell inversions: the
chiaroscuro stem cell model of hematopoiesis
[abstract]. Exp Hematol. 2002;30:128.
Knaan-Shanzer S. No hierarchy within the primitive hemopoietic stem cell compartment. Abstract
presented at: The New Stem Cells; September
18-19, 2000; Leiden, The Netherlands.
Kirkland M, Borokov K. A phase space model of
hemopoiesis [abstract]. Exp Hematol. 2002;30:
125.
Quesenberry PJ, Colvin GA, Lambert JF, et al.
The new stem cell biology. Trans Am Clin Climatol Assoc. 2002;113:182-207.