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The Brain-Bone-Blood Triad: Traffic Lights for Stem-Cell

Homing andMobilization

TsveeLapidot1 and Orit Kollet1

1Immunology Department, Weizmann Institute, Rehovot, Israel

Navigation of transplanted stem cells to their target organs is essential for clinical bone marrow reconstitution. Recent

studies have established that hematopoietic stem cells (HSCs) dynamically change their features and location, shifting

from quiescent and stationary cells anchored in the bone marrow to cycling and motile cells entering the circulation.

These changes are driven by stress signals. Bidirectional migrations to and from the bone marrow are active processes

that form the basis for HSC transplantation protocols. However, how and why HSCs enter and exit the bone marrow as

part of host defense and repair is not fully understood. The development of functional, preclinical, immune-deficient

NOD/SCID (non-obese diabetic-severe combined immunodeficiency) mice transplantation models has enabled the

characterization of normal and leukemic human HSCs and investigation of their biology. Intensive research has

revealed multiple tasks for the chemokine SDF-1 (stromal cell-derived factor-1, also known as CXCL12) in HSC

interactions with the microenvironment, as well as the existence of overlapping mechanisms controlling stress-

induced mobilization and enhanced HSC homing, sequential events of major physiological relevance. These processesentail dynamically interacting, multi-system aspects that link the bone marrow vasculature and stromal cells with the

nervous and immune systems. Neural cues act as an external pacemaker to synchronize HSC migration and

development to balance bone remodeling via circadian rhythms in order to address blood and immune cell production

for the physiological needs of the body. Stress situations and clinical HSC mobilization accelerate leukocyte

proliferation and bone turnover. This review presents the concept that HSC regulation by the brain-bone-blood triad via

stress signals controls the bone marrow reservoir of immature and maturing leukocytes.

IntroductionIt is not the strongest of the species that survives, nor the most

intelligent that survives. It is the one that is the most adaptable to

change (L.C. Megginson, 1963, commenting on the studies of

Charles Darwin). The light is what guides you home, the warmth

is what keeps you there (attributed to Ellie Rodriguez).

The potential of hematopoietic stem cells (HSCs) to repopulate ablated

bone marrow is the basis for their definition as true stem cells. They are

dynamic and versatile cells with changing phenotypes. These primitive

progenitor cells can modulate their motility, location, and cell cycle

status. They respond to signals from their microenvironment as well as

from remote organs. Such modulations include intensive changes in the

repertoire of cytokines and their cell surface receptors, adhesion

molecules, proteolytic enzyme activation, and cytoskeletal rearrange-

ment. The changes are aimed at facilitating hematopoietic stem and

progenitor cell (HSPC) migration and development, which enables

their transformation from quiescent, tissue-anchored cells to motile,

proliferating cells. These processes are also associated with the cell

differentiation needed for the replenishment of the blood on demand

with new cells with a finite life span, which is required for host defense

and repair mechanisms.

The terms homing and mobilization are used to describe the in

vivo migration of circulating HSPCs into their physiologic site of

hematopoiesis, the bone marrow, and their enhanced exit from this

organ back to the blood, respectively. HSPCs egress from the bone

marrow during steady-state homeostasis is at a very low rate.

However, stress conditions such as bleeding, inflammation, and

injury greatly amplify and intensify this process. These processes

are mimicked by clinical mobilization, in which HSPCs are

recruited from the bone marrow to the circulation by means of

chemotherapy induction and repeated cytokine stimulation to ex-

pand and harvest HSPCs for clinical transplantations.

HSPC mobilization and enhanced homing share various regulators

and overlapping mechanisms showing that these two processes are

sequential events with physiological relevance. Both directional

migration processes to and from the bone marrow involve adhesion

to the vascular wall and crossing over the endothelial blood-bone

marrow barrier. Recently, multiple studies have established that

murine HSPCs are preserved in their primitive phenotype within the

bone marrow. These cells are in close contact with various stromal

cells, including endosteal bone-lining osteoblasts, endothelial and

peri-arterial reticular cells, as well as nestin-positive cells, all

robustly expressing the chemokine SDF-1, which is essential for

maintaining murine HSC quiescence.1 These nursing microenviron-

ments, defined as stem cell niches, protect HSCs and provide

them with signals that maintain their quiescent, slow-dividing and

stationary state.14 Detachment of HSCs from these niches is

believed to be associated with their entry into the cell cycle,

proliferation, and differentiation, which is also accompanied by

increased migration and recruitment to the circulation (as discussed

in 57).

The nervous system, which is the bodys master regulator, has been

implicated in controlling the immune system (discussed in 8). Neural

and immune cells also regulate HSPC motility and development,

both directly via secretion of neurotransmitters and myeloid cyto-

kines and indirectly by way of bone remodeling processes. These

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include bone formation by osteoblasts, bone resorption by oste-

oclasts, circadian rhythms, and the dynamic nature of the stem-cell

niches. The current understanding of leukocyte production on

demand, the regulation of HSPC homing and mobilization, and the

central role of the brain-blood-bone triad in these processes will be

discussed herein.

Essential Tools for Human HSPC Research:

Functional, Preclinical, Immune-Deficient Mouse

ModelsUnderstanding the mechanisms that regulate human HSPC homing

and mobilization in vivo required the development of animal

models for human cell engraftment. The development of functional,

preclinical models of transplanted, immune-deficient NOD/SCID

(non-obese diabetic-severe combined immunodeficiency) mice pro-

moted significant progress in normal and leukemic human HSC

research. Particularly, these models enabled the generation of

chimeric mice engrafted with human HSPCs, which provided

mechanistic insights into their phenotypic characterization, as well

as the identification of molecules participating in the regulation of

HSPC homing, retention, proliferation, differentiation, and mobili-

zation. The SCID, NOD/SCID, and the later-derived immune-

deficient murine models constitute an experimentally relevant

microenvironment for human HSPCs due to their immunodefi-

ciency, which makes them permissive for transplanted human cells.

In addition, high homology and partial cross-reactivity exist be-

tween human and murine regulating factors, such as cytokines,

chemokines, adhesion molecules, and enzymes. This enhances the

success of transplanted human cells in these mice. NOD/SCID-

repopulating human HSPCs, mostly defined as CD34/CD38-/low/

CXCR4 cells, can home in the bone marrow of sub-lethally

irradiated NOD/SCID hosts and imbue the mouse with a high

degree of multi-lineage hematopoiesis. This process can be repeated

in secondary transplanted recipient mice.5 However, these models

still suffer xenogeneic limitations, because HSPCs in mice do not

always produce the same results as in humans. For example, human

cord blood CD34 cells engraft in these mice better than bone

marrow or mobilized CD34 cells. Chimeric, immune-deficient

mice have also been used for establishing both homing and

mobilization assays that identify and characterize molecular and

cellular components participating in human HSPC motility pro-

cesses. The NOD/SCID mouse model was also established as a

functional, preclinical model for leukemic HSPC cells obtained

from some acute myeloid leukemia (AML) and precursor B-acute

lymphocytic leukemia (ALL) patients. The severity of leukemia in

transplanted mice correlates highly with the aggressiveness of the

disease from the derived patient, and thus can be used to predict

clinical outcomes. Finally, NOD/SCID mice are also used to

monitor homing of human stromal progenitors. After certain

manipulations, human CD44 cells could navigate to the murine

bone marrow via interactions with selectins.9

Non-motile, Tissue-Anchored Quiescent HSPCs:Bone Marrow ResidentsHSPCs reside primarily in the bone marrow in contact with supportive

stromal cells, which maintain their undifferentiated phenotype and

developmental potential. Proliferation, differentiation, and release to

the circulation during the steady state occur at low levels, providing the

body with short-lived hematopoietic cells as well as immature and

maturing leukocytes as part of host immunity. Stress signals accelerate

and intensify these processes dramatically, producing leukocytes on

demand as part of host defense and repair10 and to address the bodys

immediate needs during emergency situations.10

Various molecular anchors keep HSPCs in contact with stromal

cells in the bone marrow.4 The chemokine SDF-1 plays a central

role in HSPC regulation, with a dose, tissue, and context depen-

dency. SDF-1 is expressed by murine bone marrow osteoblasts,

endothelial, reticular, nestin-positive, and other stromal cell types.

At the homeostatic basal expression level, SDF-1 acts via its major

receptor, CXCR4, as a survival factor for bone marrow HSPCs,

inducing their quiescence and retention. Mouse embryos that do not

express SDF-1 or CXCR4 have multiple lethal defects, including

the lack of bone marrow seeding by HSPCs. Human and murineSDF-1 are cross-reactive, which explains why human HSPC can

home in and be retained within the bone marrow of transplanted

NOD/SCID mice (as discussed in 1,5) HSPCs residing in the bone

marrow express a wide array of adhesion molecules for attachment

to the stromal supportive network. For example, immature human

CD34 cells express CD44 at high levels while retained in the bone

marrow.11,12 This adhesion molecule anchors HSPCs to endothelial

cells of human bone marrow blood sinusoids or endosteal osteo-

blasts via CD44s major ligand, hyaluronan. Proteolytic enzymes,

such as the cell-surface MT1-MMP (membrane type 1 matrix

metalloproteinase), cleave CD44, thus negating its function. These

proteolytic enzymes are also expressed by immature human CD34

HSPCs and by various myeloid cells.12 During steady state, these

interactions are tightly controlled by RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), which inhibits MT1-MMP

and MMP-2/9 activity. Human CD34 progenitors residing in the

bone marrow do not secrete MMP-2/9, while circulating progenitors

do produce these enzymes (discussed in5). These processes are also

controlled by RECK, which is also expressed by human CD34

progenitors.12 Several other molecules, such as 1-integrin and

Rac1/Rac2,13 have also been implicated in regulating HSPC reten-

tion. Mechanisms mediating the retention of human CD34HSPCs

in the bone marrow are illustrated in Figure 1A.

Stress Signals: The Impetus for HSPC Migration andDevelopmentStress conditions due to bleeding, inflammation, or DNA damage

introduce dramatic systemic and local changes, which have a large

impact on HSPC regulation. These changes include detachment of the

progenitors from their bone marrow docking sites, cell cycle entry,

increased motility, and recruitment. These cells follow the stress signals

via the circulation toward the injured tissues. Interestingly, long-term

repopulating HSCs extensively proliferate in response to in vivo

bacterial infection. Both infection and interferon (IFN)- administra-

tion also facilitate HSPC mobilization, demonstrating an important role

of IFN- in HSPC quiescence and mobilization.14

Inflammation is also associated with increased production of

granulocyte colony-stimulating factor (G-CSF), which triggers

HSPC mobilization. This physiologic effect is clinically utilized to

expand and mobilize HSPCs from the bone marrow to the circulat-

ing blood in order to harvest progenitors for stem-cell transplanta-

tions. Experiments in parabiotic mice with a shared circulation

demonstrate that HSC mobilization and homing are sequential

events that are both triggered and enhanced by G-CSF, since HSPC

mobilization by repeated G-CSF stimulation also leads to increased

homing and engraftment of the partner mouses bone marrow.57

The bone marrow is a highly vascularized organ. Sinusoidal vessels

are believed to be the site of HSPC exchange between the bone

marrow and the circulating blood. Thus, endothelium integrity and

the regulation of its permeability are pivotal in the endotheliums

role as the gatekeeper of the blood-bone marrow barrier. Bone

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marrow endothelium permeability is increased by total body

irradiation, chemotherapy, and inflammation (as discussed in 3,5) all

of which enhance HSPC bidirectional transmigration.

HSPC Mobilization: Extensive RecruitmentMobilization by repeated G-CSF stimulation requires awakening

the quiescent HSPCs, generating guiding signals, locally repressing

the inhibitory attachment machinery, and gaining motility. Stress

signals induce the detachment of HSCs from their docking sites and

inhibit bone marrow retention-inducing factors. These signaling

molecules can be delivered via the circulating blood from a remote

site in an endocrine-regulated fashion. However, stress signals can

also be transmitted in a paracrine-regulated fashion to HSPC via

stromal cells within the neighboring bone marrow vicinity. The

chemokine SDF-1 also provides a pivotal guiding signal. The basal

expression of SDF-1, which retains HSPC quiescence during the

steady state, is reduced during mobilization. Studies by our group

and others have shown that G-CSF-induced mobilization is associ-

ated with a transient increase in SDF-1 levels, which is then

followed by reduced production and secretion and cleavage of this

chemokine in the bone marrow by a host of proteolytic en-

zymes.6,11,15,16 In a complex series of events, bone marrow HSPCs

increase their CXCR4 expression and motility. Inflammatory sig-

nals also expand and activate mature bone-resorbing osteoclasts. In

addition to their traditional role in bone turnover, these cells are also

implicated in HSPC release and mobilization. Particularly, cathep-

sin K, the enzymatic hallmark of bone degradation by osteoclasts,

can also degrade SDF-1, osteopontin, and the membrane-bound kit

ligand, all of which are molecules that regulate HSPCs. 17

Thrombolytic agents are also involved in G-CSF mobilization.18

Interestingly, G-CSF stimulation leads to a reduction in SDF-1

production in the bone marrow due to morphological and functional

changes of the endosteal osteoblasts, major producers of this

chemokine that are also involved in signaling from the nervous

system.16,19 These events are followed by the proteolytic deactiva-

tion of SDF-1 and, concomitantly, HSPC proliferation and differen-

tiation. While SDF-1 is reduced in the bone marrow and CXCR4 is

increased, the level of CXCR4 in the circulation does not change

significantly. Bone marrow HSPCs become more motile, gain

CXCR4 expression, and the proteolytic machinery is activated (as

discussed in 11). In parallel, human CD34 cell adhesion via CD44

is now down-regulated due to CD44s cleavage by MT1-MMP. This

activation of human MT1-MMP in chimeric NOD/SCID mice is

made possible by the down-regulation of its inhibitor, RECK. 12 All

of these adaptations act in synergy to facilitate HSPC egress from

the bone marrow. The levels of MT1-MMP expression by myeloid

cells in the patients blood circulation are correlated with clinical

mobilization, and mobilized human CD34 cells have reduced

CD44 expression.12 In recent studies, the CXCR4 antagonist

AMD3100 (plerixafor) was shown to rapidly mobilize human

CD34 HSPC. AMD3100 synergizes with G-CSF to increase

HSPC motility.20,21 This process also involves innate immunity

components such as the complement cascade.22 It is interesting that

the mobilization of HSPCs and endothelial progenitors share

common mechanisms.23,24

One of the pathways that G-CSF activates in HSPC is the reactive

oxygen species (ROS) signaling pathway. By activating the c-Met/

HGF (hepatocyte growth factor) axis, G-CSF increases production of

ROS, which induces HSPC mobilization. Accordingly, ROS inhibition

reduces both G-CSF-induced mobilization and the enhanced motility

of HSPC,25 demonstrating the importance of ROS as a regulator of

enhanced HSPC migration, proliferation, and differentiation, leading to

reduced long-term stem cell repopulation. However, this transient

increase in ROS is reversible, and the long-term repopulation potential

can be restored by in vitro ROS inhibition (as discussed in 25).

Guided by stress-induced signals, HSPCs are recruited to injured

sites and tissues. We have shown that local damage induced in the

Figure 1. (A) Retention of human CD34 HSPCs via SDF-1/CXCR4 interactions, CD44/HA interactions, and inhibition of MT1-MMP and MMP2/9 by

RECK, all leading to tissue-anchored, quiescent CD34HSPCs. (B) G-CSF-induced mobilization of human CD34HSPCs via activation of

osteoclasts, proteloytic enzymes (including up-regulation of surface MT1-MMP), cleavage of CD44 and SDF-1, and CXCR4 up-regulation, leading toCD34HSPC proliferation, differentiation, and increased recruitment to the circulation. (C) Homing of human CD34 HSPCs in transplanted,

immune-deficient NOD/SCID mice preconditioned with total body irradiation. Increased SDF-1 levels in the murine bonemarrow endotheliumand

endosteum region attract human CD34/CXCR4HSPC, while CXCR4-/lowHSPCs are mostly trapped in the circulation

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liver of NOD/SCID chimeric mice increased the expression of

SDF-1 in the injured liver. This increase caused the recruitment of

human CD34 progenitors from the engrafted murine bone mar-

row. The human cells were observed localizing in close proximity to

SDF-1-expressing bile duct epithelial cells. Interestingly, SDF-1

expression in the human liver is dramatically up-regulated by

hepatitis C virus infection and in the murine liver by irradiation,

which suggests that orchestrated changes induced by stress signals

can potentially participate in host defense and repair by as-yet-

unknown mechanisms (as discussed in 26).

The bone marrow microstructure in general, and the trabecular

endosteum region in particular, are critical regulators of HSPC fate

and retention. Upon osteoclast malfunction, both steady-state re-

lease and G-CSF-induced progenitor mobilization are preferentially

reduced.17 Osteoclast perturbations can be genetically introduced in

young PTP KO (protein tyrosine phosphatase-epsilon knockout)

females or can be induced by anti-osteoclast drug therapy, which

imposes changes in bone turnover.17

G-CSF administration activates osteoclasts, which were recently shown

to be involved in mediating the increased proliferation of bone marrow

stromal progenitors.27 These observations suggest that osteoclasts

participate notonly in bone resorption and HSPC mobilization, but alsoin the regulation of the bone marrow-supporting niches. Similarly, mice

lacking CD45 have a distorted morphology and a reduction in

osteoclast activity, resulting in mild osteopetrosis. These mice display

extramedullar hematopoiesis and abnormal bone microstructure, mainly

reflected in modified distribution and density of the bone trabecules,

implicating reduced levels of murine HSPCs in the bone marrow.

CD45 knockout mice poorly mobilize HSPCs in response to G-CSF,

RANKL (receptor activatorfornuclear factorB ligand), andAMD3100

stimulation.28 Mechanisms of G-CSF-induced mobilization of human

CD34HSPCs in NOD/SCID mice are illustrated in Figure 1B. Taken

together, these studies demonstrate the essential function of balanced

osteoblast/osteoclast regulation and the bone marrow microenviron-

ment to support and allow the mobilization process. These results

suggest that via their progeny, osteoclasts, neutrophils, and other

myeloid cells, HSCs indirectly participate in regulating the dynamic

bone marrow microenvironment, the immature stromal and hematopoi-

etic pool size, and the mobilization process.

Several other mobilizing agents, such as antibodies for VLA-4

(very-late antigen-4) and IL-8 (interleukin-8), have been identified;

however, in this review, we chose to focus on those acting via the

SDF-1/CXCR4 axis, which is a pivotal regulator of HSPC function.

Homing: The Active Crossing of the Blood-Bone

Marrow Barrier and Retainment in the Bone MarrowMicroenvironmentClinical protocols of bone marrow transplantations are dependent

on the homing skills of the transplanted cells. Intravenously infused

into the peripheral blood, HSPCs find their way to the bone marrow

and lodge there to initiate hematopoiesis and bone marrow reconsti-

tution, which is a multi-step, coordinated process.

The homing of human CD34 HSPCs to immune-deficient murine

bone marrow requires preconditioning of the host mice with total body

irradiation or other means of ablation, such as chemotherapeutic-

induced DNA damage. This induces a dramatic elevation of SDF-1

production by bone marrow stromal cells within 1 to 2 d leading to

activation of proteolytic enzyme machinery. Navigating human HSPCs

can successfully migrate to the murine bone marrow if CXCR4 is

functionally expressed on the CD34 cell surface (as discussed in 5).

Expression of this receptor is dynamic and can be up-regulated by short

term, 1- to 2-d pre-stimulation of CD34 cells ex vivo with SCF (Stem

Cell Factor), IL-6, HGF, and other cytokines. Homing of human

CD34 HSPCs in transplanted NOD/SCID mice is regulated by the

availability of both SDF-1 as the guiding signal and of CXCR4 as the

detector of this signal. Importantly, the migration potential of a

patients CD34-enriched cells toward a gradient of SDF-1 in vitro is

also correlated with their repopulation potential in clinical autologous

transplantations. These results suggest that the repopulation potential ofhuman CD34 cells in patients and in chimeric NOD/SCID mice is

dependent on CXCR4 signaling (as discussed in 5). CXCR4 is also

functionally expressed by many malignant human cells and is a poor

prognostic factor for AML patients.

Bone marrow endothelial cells produce and present SDF-1, as well as

its major receptor, CXCR4. Endothelial CXCR4 binds this chemokine

and functionally transfers it from the circulation via the vessel lumen

into the bone marrow. Bone marrow endothelial CXCR4 transfer of

SDF-1can also facilitate the homing of transplanted human CD34

HSPCs to the murine bone marrow.29 Interestingly, preliminary results

reveal that SDF-1 injected directly into the bone was transferred by

endothelial cells and released into the circulation in a CXCR4-

dependent manner, facilitating rapid murine HSPC mobilization.11 This

feature of the endothelial blood-bone marrow barrier is believed to

constitute a major vectorial regulator directing HSPCs in or out of the

bone marrow. This vectorial regulation follows SDF-1s dynamic

fluctuations due to stress-induced enhanced production in the bone

marrow or in other organs. In agreement with these observations, the

inhibition of CD26, a peptidase with SDF-1 cleavage activity, im-

proves homing and engraftment of human CD34 progenitors in

transplanted, immune-deficient mice.30,31

Similarly to HSPC mobilization, bone marrow homing also requires an

active crossing of the blood-bone marrow barrier, namely the vascular

wall. Many molecules expressed by endothelial cells take an active part

in this process. HSC attachment to the vascular lumen requires binding

of selectins and integrins to their receptors, which facilitates cell

adhesion to the vessel wall under blood flow shear stress prior to their

crossing. Presentation of SDF-1 by endothelial cells mediates increased

expression and activation of the HSPC adhesion machinery, which

involves cytoskeleton rearrangement to enable cell spreading (as

discussed in 5). Anchors required for docking in the bone marrow

include CD44, which is also expressed by human CD34 HSPCs.

CD44 is required for cell spreading and adhesion to hyaluronan and

osteopontin, both of which are expressed by blood vessel walls and

along the endosteum, and are implicated together with SDF-1 and

membrane-bound SCF in murine HSC homing and repopulation.3234

Indeed, blocking CD44 function prevents homing of immature human

CD34 cell to the murine bone marrow and spleen (as discussed in 5).

Mechanisms regulating the homing of human CD34 HSPCs to the

NOD/SCID mouse bone marrow are illustrated in Figure 1C.

Activation of the adhesion machinery is dynamic and requires a

tight regulation to allow HSPC attachment and detachment, both of

which are essential for cell migration. For example, genetic

modulation of CD45 leads to a constant and highly adhesive HSPC

phenotype due to Src hyperactivation, which prevents their normal

homing to the bone marrow of wild-type hosts.28 Lodging of

homing HSPCs to the bone marrow endosteum also requires sensing

of the calcium levels,35 which links bone turnover with the

regulation of murine stem cell homing. Intracellular rho-GTPases

are also important regulators of HSC motility and adhesion. 13



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The Brain-Blood-Bone Triad: A Circular Hierarchy

Regulator via Dynamic InteractionsThe nervous system via its various pathways has been implicated as

a major regulator of the immune system. However, activated

leukocytes, including lymphocytes and lipopolysaccharide-stimu-

lated macrophages, also secrete neurotransmitters that exert bifunc-

tional effects on the immune cells and provide feedback to the

nervous system. The bone marrow is highly innervated, with nerve

fibers running along the brachiated blood vessels and into the bone

marrow parenchyma. Thus, catecholamines produced by the sympa-

thetic system can be delivered to the bone marrow by the blood

circulation, but some are also secreted from nerve endings directly

in the bone marrow to act on bone marrow resident cells in a

paracrine fashion.8,36 Accumulated data depict a major role for

signals coming from the sympathetic system in the regulation of

HSPC retention, homing, and mobilization, which include direct

and indirect effects exerted via the bone-remodeling processes.

Human CD34 HSPCs dynamically express catecholaminergic

receptors, with higher expression observed by the more primitive

CD34CD38-/low subset. Repeated stimulation with the mobiliz-

ing myeloid cytokines G-CSF and GM-CSF (granulocyte-macro-

phage colony stimulating factor) also increases this expression.

Therefore, mobilized human CD34 cells isolated from the periph-eral blood have higher catecholaminergic receptor expression

compared with tissue-anchored bone marrow CD34 progenitor

cells. These receptors play a functional role in the homing process,

because stimulation with neurotransmitters triggers in vitro CD34

cell proliferation and increased motility and engraftment capabili-

ties, as measured in chimeric NOD/SCID mice. Moreover, cat-

echolaminergic stimuli also enhance the expression of the membrane-

bound enzyme MT1-MMP and SDF-1-directed migration.36,37

The Wnt canonical signaling pathway and beta-catenin stabilization are

also activated by this catecholaminergic stimulation to take part in the

homing process. Interestingly, we found that these neural signals can

also induce murine HSPC mobilization.37 Indeed, antagonizing the

Wnt signaling pathway with Dickkopf-1 during steady-state homeosta-sis mobilized both hematopoietic and endothelial murine progenitor

cells.23,38 In contrast, during alarm situations and repeated G-CSF-

induced mobilization, inhibition of Wnt signaling inhibits mobiliza-

tion.37 Driving forces of HSPC mobilization also include the suppres-

sive effects that G-CSF stimulation exerts on bone-lining osteoblasts

and their capacity to produce SDF-1. This requires peripheral beta-

adrenergic signals.19

Sympathetic signals also control bone-remodeling processes, which

constitute part of the HSPC paracrine regulatory milieu, including bone

formation by osteoblasts and degradation by osteoclasts. Nerve endings

with norepinephrine secretion capacity are observed in close proximity

to osteoblasts and osteoclasts near the bone-growing plates, at regions

enriched with HSPCs. Bone mass and remodeling processes aimed atmaintaining bone integrity are known to be regulated by signals from

the nervous system. Particularly, beta-adrenergic signals that are

involved in HSPC migration and mobilization tilt the remodeling

balance toward osteoclast activation and osteoblast suppression. For

example, parathyroid hormone regulates bone turnover and in vivo

HSPC expansion and mobilization (as discussed in 8).

The emerging relationship among the nervous, immune, and

bone-maintaining systems may allow us to conceive a well-

orchestrated hierarchal network by which the body functions in the

context of HSPC regulation. The evidence that exogenous signals

transmitted by light/dark cycles are detected by the nervous system,

which then translates these circadian cues to neurotransmitter and

hormone secretion, both major regulators of HSPCs and their

microenvironment, places the nervous system at the top of this

regulatory hierarchy. SDF-1, the major HSPC chemokine, is

produced and secreted in the bone marrow in association with

circadian rhythms in a mechanism that includes beta3-adrenergic

(AdR) activity, which is not yet fully understood.8,39 HSPC release

from the bone marrow to the circulation in steady-state conditions is

also synchronized with and follows bone marrow SDF-1 produc-

tion, with anti-phase fluctuations.39 Accordingly, expression ofCXCR4 by human bone marrow CD34 HSPC cells also demon-

strates a circadian oscillation pattern.40 The effects of immune cells

and bone disorders on the nervous system demonstrate the circular,

bidirectional manner by which this network exerts its regulation.

Concluding Remarks and Future DirectionsThe nervous system is the fastest detection and message-delivery

framework, which makes it capable of immediate response to environ-

mental changes throughout the body. This regulatory network demon-

strates multidirectional streaming. Thus, exogenous stimuli and other

stimuli originating from the immune system, bone, bone marrow, and

the stromal microenvironment all regulate HSPCs. However, the

nervous system dominates the brain-bone-blood triad.

Prospectively, the major question is what does the body gain by

responding to circadian signals in controlling bone turnover and HSPC

trafficking? This question may be answered by seeing these body

defense tools as constantly working at low gear to maintain homeo-

static balance. Bone maintenance requires continuous formation and

degradation. HSC regulation requires their quiescence and retention in

the bone marrow, but also their migration, self-renewal, differentiation,

and recruitment to the circulation. These opposing processes may

require an on/off switch that reliably inverts the regulatory machinery

in opposite directions. To continuously keep this network active, there

could be a need for repetitive, low rates of mild, stress-like signals that

are generated in constant intervals. These intervals are provided by

circadian peaks, serving as an external pacemaker to synchronize blood

cell production, bone turnover, and immunity. These circadian rhythmsalso keep the body ready and well trained for dynamic and acute

changes, including intensified stem-cell migration and development,

which are triggered by stress signals during acute situations. This

concept is relevant to clinical mobilization and homing during stem-

cell transplantation. Future protocols may harness these insights to

increase the success of HSPC transplantation by taking into consider-

ation normal circadian rhythms, the timing of G-CSF and AMD3100

treatments, and unique features of the patients bone, immune, and

neural status.

AcknowledgementsWe regret that due to the citation limit of 40 references published

during the last 5 years, we could not cite and discuss many

important studies on stem-cell homing and mobilization.

DisclosureConflict-of-interest disclosure: The authors declare no competing

financial interests.

Off-label drug use: None disclosed.

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