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Blood First Edition Paper, prepublished online March 11, 2015; DOI 10.1182/blood-2014-12-570200
Hematopoietic Stem Cells: Concepts, Definitions and the New Reality
Connie J Eaves
Terry Fox Laboratory, British Columbia Cancer Agency and Department of Medical Genetics,
University of British Columbia, Vancouver, BC, Canada
Running title: Mouse hematopoietic stem cells
Correspondence: Dr. C. J. Eaves
Terry Fox Laboratory
675 West 10th Avenue
Vancouver, BC
Canada V5Z 1L3
Phone: 604-675-8122
Fax: 604-877-0712
Email: [email protected]
1
Copyright © 2015 American Society of Hematology
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Abstract
Hematopoietic stem cell (HSC) research took hold in the 1950’s with the demonstration that
intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production. Attempts to quantify the cells responsible led to the discovery
of serially transplantable, donor-derived, macroscopic, multi-lineage colonies detectable on the
spleen surface 1-2 weeks post-transplant. The concept of self-renewing multi-potent HSCs was
born, but accompanied by perplexing evidence of great variability in the outcomes of HSC selfrenewal divisions. The next 60 years saw an explosion in the development and use of more
refined tools for assessing the behavior of prospectively purified subsets of hematopoietic cells
with blood cell-producing capacity. These developments have led to the formulation of
increasingly complex hierarchical models of hematopoiesis and a growing list of intrinsic and
extrinsic elements that regulate HSC cycling status, viability, self-renewal and lineage outputs.
More recent examination of these properties in individual, highly purified HSCs and analyses of
their perpetuation in clonally-generated progeny HSCs have now provided definitive evidence of
linearly transmitted heterogeneity in HSC states. These results anticipate the need and use of
emerging new technologies to establish models that will accommodate such pluralistic features
of HSCs and their control mechanisms.
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Historical Beginnings
We all like stories that have a beginning to capture our interest, a middle to sustain it, and an end
that brings closure, but also harks to a future. The story of hematopoietic stem cells (HSCs) fits
well into such a framework. The origin of blood cells, first in the developing embryo, and then
later throughout life, has intrigued scientists, caregivers and patients for centuries. As for many
advances, a combination of serendipity and the opportunistic exploitation of new tools have been
important determinants of progress. For the HSC field, the development of atomic weaponry in
the first half of the 20th century proved to be a game-changing event. It galvanized interest in
understanding how ionizing radiation damages normal tissue and whether the effects of a lethal
dose could be abrogated by a medically applicable intervention. The microscope helped to reveal
the bone marrow to be one of the most radiosensitive of all tissues, but this tool proved
inadequate to address the question of rescue.
In fact, HSC research as a quantitative science emerged as a by-product of other investigative
strategies seeking to determine how the consequences of myeloablation might be overcome. The
seminal discovery was the finding of transplantable multi-potent adult bone marrow cells with
clonally demonstrable hematopoietic activity - a finding that evolved from experiments showing
that an intravenous transplant of normal adult mouse bone marrow cells could protect recipients
from a lethal dose of radiation1 by replacing the destroyed blood-forming system with a new and
sustained source of lymphoid and myeloid cells.2 These observations established the presence in
the bone marrow of adult mice of cells with long-term hematopoietic repopulating activity. This
finding, in turn, sparked the idea that the original cells with this repopulating activity might then
be characterized and even quantified based on the mature cells they could produce in
myeloablated recipients (Figure 1A).
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Lessons from the First Clonal Assays for Transplantable Hematopoietic Cells
An early experimental design to pursue this idea involved transplanting decreasing numbers of
adult mouse bone marrow cells into lethally irradiated recipients. The goal was to determine the
minimal number of cells that would then protect the hosts.3 Interpretation of the results of this
early type of limiting dilution assay (LDA) experiment (Figure 1A) was based on the assumption
that a finite number of repopulating cells would have to be injected to enable the recipients to
survive. As is now widely recognized, many subsequent studies have shown that the validity of
this assumption is confounded by the presence in many hematopoietic organs of multiple,
distinct types of transplantable hematopoietic cells with different, apparently predetermined,
regenerative properties.4 Nevertheless, this first experimental attempt to measure the frequency
of hematopoietic cells with radio-protective activity is noteworthy in its introduction of an
objective biological endpoint to quantify a population of cells that could not be uniquely
identified by any directly evident feature.
These early LDA experiments also served as the launch pad for a key derivative observation.
This was the detection of distinct “nodules” that become macroscopically visible on the surface
of the spleen in irradiated mice injected with low doses of syngeneic bone marrow cells 9-14
days previously5 (Figure 1A). Importantly, the finding that the number of these nodules was
linearly related to the number of cells injected over a 10-fold range prompted the idea that their
formation might offer an alternative - more direct (but still functional) - method to quantify
hematopoietic cells with extensive and rapid mature blood cell-producing potential. These initial
observations were then followed by a remarkable series of experiments that established the
following core principles.
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•
The large spleen nodules generated in such experiments are true clones, each derived from a
single parental cell (hence its designation as a colony-forming unit, or CFU5), as shown using
injections of bone marrow cells carrying different genetic (chromosomal) markers.6
•
Many of the spleen colonies thus obtained consist of mixtures of mature cells of the myeloid
(erythroid, megakaryopoietic and granulocyte/macrophage (GM)) lineages.7 Later genetic
experiments showed that the cells that produce spleen colonies are derived from cells that can
also produce lymphoid progeny, thus proving the maintenance in adult mice of individual
cells with all of these differentiation potentialities.8
•
Some of these spleen colonies also contain daughter cells that generate similar
macroscopically visible, multi-lineage colonies in the spleens of secondary irradiated
recipients.9 The revelation of this behaviour at the single cell level was the birth of the
concept of “self-renewal” – now widely considered to be a defining “stem cell” property in
multiple tissue contexts.
•
The numbers and types of mature and primitive cells present in individual spleen colonies
varies widely and independently.9,10 This diverse behavior led to the concept of stochastic
variables underlying the type(s) of progeny generated by individual primitive hematopoietic
cells. This idea was captured by the descriptor “hemopoiesis engendered randomly”
(“HER”), in contrast to “hemopoietic inductive microenvironment” (“HIM”) suggesting an
alternative explanation for the observed variable behavior seen.11,12
•
Most of the cells in the bone marrow of adult mice that produce these spleen colonies are
quiescent (i.e., in a dormant or G0 state). Thus, in the absence of any unusual perturbation,
they are resistant to drugs or other treatments that specifically target dividing cells.13,14
5
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Together these findings demonstrated the maintenance in the bone marrow of adult mice of rare,
mainly quiescent, hematopoietic cells that originate from cells that also have lymphoid potential
and can be activated to proliferate and regenerate all known myeloid lineages. The concept that
all of the different major types of blood cells are derived throughout normal adult life from a
common pool of self-renewing HSCs through a hierarchical differentiation process was thus
introduced (Figure 2A). The existence of mechanisms that produce extensive heterogeneity in
their self-renewal and differentiation behavior was also recognized. These findings also served to
establish the importance of clonogenic assays to detect and quantify cells that are not directly
identifiable and the need for new functional names for these cells5 – to confer on them the
specificity that no morphologically-based, or indeed any direct descriptor, could provide. At the
same time, this nomenclature side-stepped the unresolved issue of their heterogeneous behavior.
Early In Vitro Colony Assays Identify Downstream Progenitors
Despite the important insights afforded by these early experiments, their restricted application to
mouse cells precluded testing whether similar principles would apply to processes governing
human hematopoiesis. Over the next 2 decades, however, an expanding capability for growing
mammalian cells in vitro and the recognized power of clonal assays enabled the development of
protocols that allowed in vitro hematopoietic colony assays to be developed, first for mouse cells
and, soon thereafter, for their human counterparts.15-17 The first such colonies obtained were
found to consist of GM cells. This finding had 2 possible explanations. One was that the in vitro
GM colonies were incomplete reflections of progenitors with a broader differentiation potential;
for example, as displayed by the cells that form spleen colonies in vivo (CFU-S). The other
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possibility was that the in vitro GM colonies originated from a previously unidentified progenitor
cell with restricted GM differentiation potential (a “CFU-GM”). Efforts to distinguish between
these alternatives revealed that the in vitro GM colonies are generated from a distinct type of
progenitor whose numbers under regenerative conditions (e.g., within a spleen colony) make it
appear closely related to CFU-S,18 but whose physical and biological properties are largely
different.19,20 These findings provided the first evidence for a stage of differentiation
intermediate between HSCs (measured as CFU-S at that time) and the terminal stages of blood
cell differentiation described by sequential, morphologically defined changes (Figure 2B).
The discovery of in vitro conditions that enable the detection of erythroid, megakaryocyte and
mixed/multi-lineage colonies soon followed. In each case, measurements of the frequencies,
parent-progeny relationships, growth requirements, and biological properties of the initial
clonogenic cells reinforced the concept that they represent separate intermediate stages of
hematopoietic differentiation (Figure 2B),21,22 analogous to the transit-amplifying populations
recognized histologically in other tissues by their more obvious spatial arrangement.
A strong correlation between multiple changing properties in cells that produce progressively
smaller single-lineage colonies in vitro provided further support for a model in which the
execution of erythroid, GM and megakaryocytic differentiation pathways constitute well coordinated, multi-step processes that span many cell divisions (>10 cell cycles to account for the
largest sizes of single lineage colonies typically obtained). At the same time, this co-ordination
had to be sufficiently flexible to generate a progressive loss of correlation within individual
spleen colonies in the numbers of progenitors that generate increasingly smaller colonies in
vitro.23,24
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HSC Detection in Clonal Assays for Long-term Repopulating Cells (LTRCs)
Subsequent studies revealed that spleen colony formation is obtained from a biologically more
heterogeneous population than was originally appreciated, and one that does not overlap
extensively with cells with long-term repopulating ability (hereafter referred to as long-term
repopulating cells, or LTRCs). Detailed time course analyses showed that both the appearance
and disappearance of individual spleen colonies is very rapid, with a precise timing determined
by biologically distinct subsets of CFU-S.25,26 Subsequent cell separation experiments
demonstrated that immediate radioprotection and long-term repopulating ability are not
necessarily vested in cells with the same physical properties.27 Thus a need to devise an assay
specific for LTRCs became apparent.
Two complementary strategies were then pursued. Both adopted endpoints indicative of a more
prolonged production of mature cells than is obtained in either the CFU-S or short-term in vitro
clonogenic cell assays.28 The in vivo “competitive repopulating-unit” (CRU) assay relies on the
ability of the environment of a myelosuppressed or genetically compromised host to support the
full spectrum of potential growth and differentiation possibilities of transplanted input cell(s)
(Figure 1A). The second approach incorporates features of the in vivo marrow environment, but
in an in vitro context, thus offering the possibility that a similar approach might be applicable to
human cells.
The mouse CRU assay was designed to enable the sensitive and specific detection within
heterogeneous populations of a single cell that is able to regenerate and maintain long-term blood
cell production in vivo (Figure 1A). This required identifying conditions that would support the
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initial as well as the lifelong survival of all recipients while still maintaining a useful sensitivity
of detecting the cells of interest. Early experiments showed that this could be achieved by
providing another source of cells with transient repopulating activity as well as a minimal source
of genetically distinct LTRCs – i.e., in numbers just sufficient to keep the recipient alive longterm. The latter manoeuvre, which determines the sensitivity of the assay, can be met using
various ways to deplete or genetically compromise the other LTRCs present in the host. CRUs
frequencies in the test cell suspension are then quantified using a LDA transplant strategy. To
achieve specificity for detecting LTRCs exclusively, a test-cell derived contribution to the WBCs
present 4-6 months post-transplant of at least 1% is required.
The term CRU and the assay used to detect CRUs should not be confused with the “competitive
repopulation assay” introduced into the field much earlier.29 This older approach quantifies
LTRC activity (not numbers) by comparing a similarly long-term contribution of a genetically
distinct test population to the total blood cell content of lethally irradiated recipients that have
been co-transplanted with a large (and radio-protective) number of HSCs and transient
reconstituting cells. This latter experimental design is much more efficient than the CRU assay
as the number of recipient mice required is much smaller. This assay is also particularly useful
for determining whether a given manipulation has had a gross effect on LTRCs by comparing
them to a “control” cell population. However, it does not discriminate between effects on LTRC
numbers and their clonal outputs, which can vary (e.g., as with aging30) and are independently
measured parameters in the CRU assay.28
The second in vitro approach also incorporates an endpoint of prolonged hematopoiesis. In this
case, the cell of origin is variably referred to by the operational terms: long-term cultureinitiating cell (LTC-IC) or cobblestone area-forming cell (CAFC), according to the indicator of
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continuing hematopoiesis used. The development of this approach was made possible by the
initial discovery that prolonged growth of primitive mouse hematopoietic cells can be achieved
when adult bone marrow cells are cultured at 33oC in the presence of steroids on a competent
(self-formed or provided) stromal feeder layer.31-33 The separate provision of a stromal feeder
layer is critical to enable input LTC-IC/CAFC frequencies to be quantified using LDA
principles. To confer specificity for very primitive cells, evidence of continuing hematopoietic
cell production for at least 4-6 (mouse) or 6-12 (human) weeks is required. Culture conditions
can also be varied to affect the types and lineages of cell(s) produced, as well as the duration of
their generation. Such modifications can provide specificity for detecting cells with lymphomyeloid differentiation potential or with in vivo assayed LTRCs.28
HSC Purification
Methods to prospectively separate viable cells based on their different physical (size and
density), biochemical (enzymatic) and surface antigen profiles have enabled distinct subsets of
mouse (and human) hematopoietic cells with different repopulating kinetics and activity to be
identified, quantified and further characterized.4 These findings have now led to a widely
accepted hierarchical model of hematopoiesis in which the orderly and co-ordinated loss of
multi-lineage differentiation and durable proliferative potential by an initial compartment of
LTRCs can be traced by accompanying changes in their surface and metabolic phenotypes
(Figure 2C).34,35
Advances in the development of robust methods for isolating mouse LTRCs at high purities
typically combine the use of Rhodamine-123 and/or Hoechst 33342 efflux measurements (to
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isolate Rho- SP+ cells), or antibodies against 2 SLAM markers (CD48 and CD150) in
combination with those that isolate Lin-Sca1+Kit+CD34-Flt3- or EPCR+ phenotypes (to give
LSK34-FL-48-150+ or ESLAM cells, respectively).36-40 The purities of “durable” LTRCs in these
populations (~20-30% using a stringent definition of serial reconstituting activity, not just
persisting chimerism at 4-6 months) are sufficient to analyze their clonal outputs directly from
single-cell transplants (Figure 1C). The results thus obtained indicate that all of these purification
strategies isolate the same, or very similar, cell populations.30,38,39,41-43 Interestingly, very few of
the markers generally used to purify LTRCs contribute to their functional properties, suggesting
that new, more relevant markers may still be awaiting discovery.
In the meantime, the powerful LTRC purification strategies currently available are already being
used in combination with analytical methods that can be applied to small numbers of cells to
identify unique molecular features (or unique combinations of molecular features) of LTRCs.
These analyses have uncovered numerous types of chromatin regulators, including members of
many transcription factor complexes and their downstream targets, as well as regulators of the
metabolome, protein synthesis and mediators of signaling responses to external cues.44-49 Indeed,
the multiplicity of entities that appear to have non-redundant roles has been disconcerting in
suggesting the lack of a singular pathway or state that regulates the maintenance of LTRC
potential, or that distinguishes these cells from derivatives that appear irreversibly destined to
differentiate in available assay systems. Rather, current findings suggest the involvement of
highly complex and possibly alternative molecular networks controlling LTRC self-renewal that
may require systems approaches to elucidate.50,51 Indeed, one might advance the somewhat
heretical idea that intracellular states responsible for different HSC self-renewal potentialities
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may not be well matched to cellular phenotypes that display different transplant-derived
endpoints of repopulating activity.
HSC/LTRC Heterogeneity and the “New Reality”
As noted, broad ranging heterogeneity in the self-renewal and differentiation behavior of
individual multi-potent hematopoietic cells became evident from early analyses of the cellular
composition of individual spleen colonies.7,9,10,24 Later examination of clones generated in vitro
from multi-potent cells in the presence of soluble factors (and hence more homogeneous
environments) confirmed the display of an equivalent degree of heterogeneity in the clonal
outputs of multi-potent cells, including progeny detectable as CFU-S.52-54 The results of these
later studies argue strongly against a deterministic role of external cues as the major regulators of
the self-renewal and lineage potentialities of primitive hematopoietic cells. These findings do
not, however, exclude the possibility that exposure to external factors can have deterministic
influences on LTRC outputs, as has been suggested.55,56 Undoubtedly, a complete understanding
of the mechanisms involved in regulating the defining properties of HSCs will likely require a
separate analysis of intrinsic and extrinsic parameters that can alter their viability and cycling
status, as well as their lineage options and self-renewal activity (Figure 3A).
LTRC self-renewal control. The development of a protocol to expand LTRCs ex vivo without
predisposing the cells to leukemic transformation has long been a driving goal in the field of
hematology. A simple view of the needs for such a protocol would be to achieve conditions that
promote LTRC survival and proliferation as well as their self-renewal. The ability of external
factors to promote the survival and alter the cycling status of primitive hematopoietic cells has
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also been appreciated for a long time, as exemplified by the pioneering studies of CFU-S cycling
control both in vivo and in vitro,13,57 later extended to functionally identified LTRCs.58 An ability
of external factors to modulate LTRC self-renewal has been inferred from their ability to
enhance LTRC outputs in transplanted mice59 (assuming that such expansions are not due to a
decreased rate of LTRC death or to an increased proportion of cycling LTRCs). Assessments of
highly purified LTRCs to defined factors in vitro allow requirements for their viability and
cycling activity to be directly visualized.60,61 The exploitation of this latter approach has now
provided conclusive robust evidence that LTRC self-renewal, mitogenesis and survival can be
independently regulated (Figure 3B).62
Nevertheless, the large (>500-fold) variations in self-renewal of individual LTRCs seen in vivo
when these are tracked clonally and measured through at least 2 serial transplants support the
operation of an underlying stochastic process.41,63 On the other hand, analysis of clones produced
from the first division progeny of stringently defined LTRCs have shown both variable (in
vitro)62,64 and more restricted (in vivo)63 patterns of self-renewal behavior in paired daughter
cells. The latter findings suggest LTRC self-renewal responses to external stimuli may reflect
variable cell-intrinsic elements that can be sustained through multiple cell generations. Such an
inference is noteworthy in that it departs from the traditional concept of a singular shared
“ground state” of LTRC self-renewal ability.
Superimposed on these sources of heterogeneity in HSC self-renewal probabilities are those that
are incurred during development. Documented differences in key properties of primitive fetal
and adult hematopoietic cells date back several decades. These include differences in both the
cycling state of these cells in situ13,65 and the self-renewal activity they display post-transplant.6668
Recent studies have confirmed that these developmental changes also apply to stringently
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defined mouse LTRCs, and appear to be co-ordinated by a process that causes an abrupt switch
in these same 2 properties (cycling status and self-renewal ability) between 3 and 4 weeks after
birth.58,69 These changes are associated with changes in the responsiveness of LTRCs to Steel
factor (SF)70 and a differential expression of (or dependence on) a panoply of chromatin
regulators. The latter include a demonstrated selective dependence of fetal LTRCs on members
of the Polycomb2 complex (Eed and Ezh2),71,72 Sox17,73 and Lin28-regulated Hmga2.74,75
Conversely, a selective dependence of adult LTRC self-renewal and/or survival on Bmi176 and
Tel/Etv6,77 has been reported, and a selective dependence of adult LTRC quiescence/dormancy
on E47-regulated p21,78-80 Pbx1,81 Gfi-1,82 and C/ebpa.83,84
LTRC differentiation control. Very little is known about the molecular mechanisms that
establish the hematopoietic lineage potential of LTRCs. Past models have generally assumed that
LTRCs are created in the embryo with an unbiased ability to activate all hematopoietic lineage
programs. Indeed, clonal tracking of the progeny of fetal LTRCs indicate this to be generally the
case.41 However, it is also well established that primitive multi-potent fetal and adult
hematopoietic cells produce lymphoid progeny with different molecular features and biological
properties.85-87 Subsequent studies have shown that at least some of these changes are determined
at the level of fetal and adult LTRCs,88 and by the same developmentally regulated decline in
Lin28b expression and consequent post-natal “switch” effect that alters LTRC self-renewal and
proliferation control, albeit via different downstream effectors.74,75
The first definitive indication of specific patterns of lineage outputs sustained through multiple
self-renewal divisions of adult LTRCs came from tracking the clonal progeny regenerated from a
single LTRC through successive serial transplants.89 These observations have since been
confirmed in numerous experiments.38,41-43,90 The stability of these clone-specific lineage output
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preferences through many LTRC self-renewal divisions argues strongly in favor of some type of
preserved epigenetic signature as the responsible mechanism. Clone-specific differentiation
patterns associated with LTRCs with durable self-renewal ability are either “balanced” in their
output of lymphoid and myeloid progeny, or “lymphoid-deficient”. The latter type of LTRC is
also commonly referred to in the literature as “myeloid-biased” or “-skewed”. However, these
latter terms are misleading, since the myeloid outputs characteristic of such clones are not
different from those produced by LTRCs that produce a balanced contribution of myeloid and
lymphoid cells to the circulating pools of these mature blood cells. Rather, it is the reduced
production of lymphoid cells that gives the clone a myeloid-biased appearance.38,41 The other
types of LTRCs produce reduced contributions of mature myeloid cells without corresponding
increases in lymphoid cell production and thus are best referred to as myeloid-deficient rather
than lymphoid-biased, for the same reasons. Interestingly, the same spectrum of lineage output
patterns has been documented even under conditions where many clones are generated within a
single recipient and tracked individually using powerful vector-mediated DNA barcoding
strategies.91-94 Statistical analysis of the various lineage output patterns seen have suggested that
these may reflect the existence of a defined repertoire of LTRC subsets with discrete,
predetermined lineage options (rather than a continuum),38,95 hence the suggested use of
objectively assigned terms for them (i.e., α, β, γ, δ, Figure 2E). The finding that myeloiddeficient clones also do not contain sufficient progeny LTRCs to reconstitute secondary
recipients38 is notable as it suggests a possible mechanistic link between LTRC retention of
myeloid and self-renewal potential.
It has been reported that LTRCs with different outputs can be selectively enriched according to
their phenotype (i.e., increased or decreased expression of various proteins, like CD150,39,42
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CD41,96 CD22997 and vWf98). These findings support the operation of cell-intrinsic mechanisms
determining the specific differentiation programs that characterize individual LTRCs. However,
these markers have proven to be more discriminatory for LTRCs with durable activity than for
LTRCs with a particular lineage output profile. It is also important to note that some secondary
clones generated from serial transplants of single-cells,41 or from paired daughter cells obtained
from purified LTRCs stimulated to divide in vitro,62,64,90 have not shown convincing evidence of
a preserved differentiation profile. Likewise, the distribution of LTRCs categorized according to
the different lineage options they display has been found to change during development and
aging30,41,99 and be subject to extrinsic modulation.43 Thus, although the molecular mechanisms
involved in predetermining the specific lineage output properties of individual LTRCs remain
elusive, they do not appear to overlap entirely with those that control LTRC cycling
activity/dormancy or self-renewal potential.
Initial models of HSC differentiation in vivo invoked the principle of a relatively fixed order and
timing of sequential lineage restriction events beginning with a separation of lymphoid and
myeloid programs (Figure 2A-C). This concept is also now being eroded by more sensitive
analyses of the cell types produced from individual LTRCs and other multi-potent cells. For
example, different phenotypes with persisting GM differentiation potentialities in combination
with other different lineage options have now been reported (Figure 2C and 2D).100-104
Additional deviations from a uni-linear branching model of hematopoietic cell differentiation
from a single original compartment include examples of a very rapid loss of LTRC properties
with full retention of viability and proliferative responsiveness in vitro62 or a rapid differentiation
of their progeny into mature cells in vivo.90,105 Also of note is the suggestion that the
unexpectedly accelerated differentiation profiles exhibited by some cells with an LTRC
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phenotype may be related to their particular “poised” or “primed” gene expression state – as
reflected in their expression of certain lineage-specific genes at a low level.98,106,107
Physiological relevance of LTRCs. The number of serially transplantable LTRCs in normal
adult mice is maintained at a relatively high level (~3,000 per mouse assuming a total bone
marrow content of 2x108 cells). These numbers gradually increase with age,99 with a low but
measureable and continuous rate of entry into a cycling state.108 The contribution of an
individual LTRC to the total output of any mature cell lineage would thus be anticipated to be
highly variable given the many cell generations and time likely separating it from most of its
ultimately generated mature progeny. A lack of correlation in the clonal representation of cells at
these 2 ends of the hematopoietic hierarchy would thus be predicted, as has recently been
documented.109 This finding does not, however, imply that LTRCs are superfluous to the longterm integrity of mature blood cell production after adulthood is reached. To address this latter
issue requires an inducible LTRC-specific lineage tracing approach and new markers (such as
those recently suggested110,111) that might be used to design the required reporters. In addition,
there is a compelling need to gain a better understanding of the molecular mechanisms that
confer on cells the unique properties of LTRCs detected by transplantation assays so that these
might be enhanced for clinical applications.
Of related interest is the growing list of mature myeloid cells produced before birth from Mybindependent embryonic hematopoietic sources that persist throughout life and are not normally
reconstituted from adult LTRCs whose generation is Myb-dependent.112 These include the
microglia of the brain,113 adult Langerhans cells,114 and the Kupffer cells of the liver.115
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Looking Forward
The era of predicting biological behavior from knowledge of the molecular mechanisms that
define cell types and how they respond to external cues is upon us. An impending challenge in
the HSC field is the extent of biological heterogeneity that requires molecular elucidation at the
single-cell level. Compounding this challenge are issues related to the essential averaging
process inherent in historically used procedures for performing molecular analyses. Fortunately,
many new and powerful tools for interrogating individual cells in diverse populations are now
becoming available. These include advances in cellular reprogramming and molecular profiling
together with new bioinformatic programs to infer cell states and relationships.116-119 We can thus
anticipate that current 2-dimensional concepts of how specific primed blood “lineage programs”
are activated may soon be replaced by more pluralistic models. These, in turn, will likely require
significant revision of present ideas about how molecular mechanisms permit, ignore or direct
changes with biological sequelae, and how these may relate to cell types currently defined by
surface phenotypes and growth endpoints. However, the reward may be the generation of models
of HSC states and their differentiation that predict how to modify biological outcomes, the
molecular trajectories that may be involved, and the speed of their attainment. These issues are
not just of academic interest. They may create paradigms relevant to other tissues, in addition to
being critical drivers of future methods to detect and treat disease with greater effectiveness.
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Acknowledgements
The preparation of this review was aided by a grant from the Terry Fox Run. The author also
acknowledges helpful discussions with A. Eaves and a group of talented past and present trainees
whose ideas and experimental results have contributed to many of the ideas presented.
C. Eaves reviewed the literature and wrote the paper.
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Figure Captions
Figure 1 Historical sequence of methods used to detect and quantify mouse HSCs in vivo.
A. Development of LDA approaches to identify the transplantable cells that can rescue mice
permanently from radiation-induced lethality by regenerating the inactivated blood-forming
system of the host. B. Genetic approaches to track HSCs by detection of their long-lived clonal
outputs in transplanted recipients. Random sites of vector integration into the DNA of the
regenerated progeny of transduced transplanted cells were the first unique DNA identifiers
used.120,121 More recently, uptake of a single vector encoding a short unique “barcode” sequence
from a diverse vector library has been used as a clonal tracking strategy.91-94 C. Advances in
LTRC purification enabling single-cell transplants to reveal the diversity of long-term clonal
WBCs outputs of individual HSCs as previously suggested by limiting dilution transplants and
vector-marking experiments. Data shown are for purified Rho-SP+ LTRCs (HSCs) adapted from
Uchida et al122 (with permission from Experimental Hematology.) BM = bone marrow, L+M =
lymphoid + myeloid, LDA = limiting dilution assay, CFU-S = colony-forming unit-spleen, HSC
= hematopoietic stem cells, CRU = competitive repopulating unit, mo = months, LAM-PCR =
linear amplification mediated polymerase chain reaction, Rho = Rhodamine-123, SP = side
population.
Figure 2 Hierarchical models of HSC self-renewal and differentiation.
A-E. Increasingly complex models of HSC differentiation hierarchies reflecting historically
changing information about the numbers and types of lineages obtained from single mouse
“HSCs” in various in vitro and in vivo systems. A. Initial view showing all lymphoid and all
27
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myeloid potentialities as the first lineage groupings to be segregated. B. A more detailed view of
the compartmentalization of intermediate, lineage-restricted progenitor subsets based on their
behavior in short-term in vitro colony assays and properties allowing their separate isolation. C.
Map of early lineage restriction events based on the predominant functional activities of
particular cell phenotypes (redrawn from Figure 1 of Seita & Weissman in Wiley Interdiscip Rev
Syst Biol Med.35 D. Concept of multiple origins of GM cells derived from examination of the
lymphoid and myeloid activities elicited from different subsets of mouse hematopoietic cells in
vitro. The hierarchy shown in this case has been adapted from Figure 1E of Kawamoto et al in
Immunol Rev.104 E. Differences in lineage potentialities exhibited by LTRCs with durable
(serially transplantable) self-renewal activity (HSCs) as reported by Dykstra et al38 and Benz et
al41. HSC = hematopoietic stem cell, CFU = colony-forming unit, S = spleen; BFU-E = burstforming unit-erythroid, E = erythroid, GM = granulocyte/macrophage, Mk = megakaryocyte, NK
= natural killer; DC = dendritic cell; T = T-lymphoid; B = B-lymphoid; LTRC = long-term
repopulating cell, STRC = short-term repopulating cell.
Figure 3 HSC viability, mitogenesis and self-maintenance can be separately regulated by
different external cues.
A. Schema showing the multiple responses that can affect HSC numbers. B. Demonstration that
different extrinsic conditions can separately regulate the survival, proliferation and self-renewal
responses of highly purified, durable LTRCs assessed in single-cell cultures. Shown are
examples of different in vitro conditions in which full LTRC activity of the surviving input cells
was maintained for 7 days either in the absence of cell death or division, or under different
28
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conditions that variably kept the input cells alive but maximally stimulated mitogenesis of the
survivors. Also shown are conditions that supported full survival and mitogenesis of the same
input cells, but with substantial loss of their original LTRC activity (redrawn from data published
in Wohrer et al62 with permission from Cell Reports). HSC = hematopoietic stem cell, ESLAM =
EPCR+CD48-CD150+, FBS = fetal bovine serum, EP = erythropoietin, SF = Steel factor, CM =
UG26 stromal cell conditioned medium, LDA = limiting dilution assay, LTRC = long-term
repopulating cell.
29
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Figure 1
Cytogenetically marked
BM transplants produce
L+M chimerism (1950’s)
Quantitation of
radioprotective cells by
LDA experiments (1960)
% dead mice
A
Spleen colony
assay
(1961)
1
0
0
14 days
3
7
CFU-S
Injected cell no.
Quantitation of HSC by LDA:
the CRU assay (1989)
Radioprotect hosts
but still maximally stimulate
injected HSC
Detection of HSC
using DNA inserts as
clonal markers (1985-present)
C
HSC purification makes
single-cell transplants
feasible (2000’s)
UV blue
B
Longer chimerism endpoint :
(positive mice- contain ≥1% test
cell-derived WBCs at ≥ 4 mo)
UV red
Southerns (1980’s)
LAM-PCR (1990’s)
% donor-derived
WBCs
100
Purified
Rho-SP+
cells
10
Next generation
sequencing (2010’s)
≥4 mo
1
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Figure 2
A
B
Initial hierarchical concept
Steps highlighted by clonal assays
CRU
HSC
myeloid
lineages
lymphoid
lineages
thymus
C
BFU-Mk
BFU-E
BM
lymph nodes
red blood
cells
CFU-E
Steps identified by phenotype changes
D
CFU-G
CFU-Mk
CFU-M
Myeloid-based lineage restriction
METB (HSC)
GEMMkTB (iTRC)
MTB/CMLP
ME/CMEP
ProNK
ProT
ProB
EP
E
GP
MP
ProDC
HSC heterogeneity in lineage options
GEMMkTB (β-LTRC)
GEMMk
MT
CLP
GMP
MkP
GEMMkTB (α-LTRC)
TB (γ/δ-LTRC)
GEMMk (STRC)
lymph nodes
granulocytes/ platelets
B-cells
monocytes
T-cells
GEMMkTB (LTRC)
MkEP
thymus BM
CFU-GM
red blood
cells
granulocytes/ platelets
B-cells
monocytes
T-cells
CMP
lymphoid
lineages
CFU-S / CFU-GEMM
E
M
M
T
M
MB
M
M
B
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Figure 3
A
HSC expansion
self-renewal
quiescence
death
proliferation
or
differentiation
HSC decline
Colony
Colony In vivo LDA of
formation durable LTRC
information vitro: in 7-day cultures:
B
UG26 CM &
SF+IL11
100
80
% of
TOTAL input
ESLAM cells
that divided
FBS+SF
CM+SF+IL‐11
+IL3+IL6+Ep
in vitro:
>95%
>95%
>95%
>95%
8x increase
10x decrease
36%
36%
10x decrease
94%
94%
Not tested
60
SF+IL11
SF+IL‐11
40
20
UG26 CM
CM alone
Hours in culture
1 ESLAM cell
per well
FBS+SF+IL3
+IL6+Ep
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Prepublished online March 11, 2015;
doi:10.1182/blood-2014-12-570200
Hematopoietic stem cells: concepts, definitions and the new reality
Connie J. Eaves
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