Catecholaminergic neurotransmitters regulatemigration and repopulation of immature human CD34+

cells through Wnt signaling

Asaf Spiegel1, Shoham Shivtiel1, Alexander Kalinkovich1, Aya Ludin1, Neta Netzer1, Polina Goichberg1,Yaara Azaria1, Igor Resnick2, Izhar Hardan3, Herzel Ben-Hur4, Arnon Nagler3, Menachem Rubinstein5 &Tsvee Lapidot1

Catecholamines are important regulators of homeostasis, yet their functions in hematopoiesis are poorly understood. Here we

report that immature human CD34+ cells dynamically expressed dopamine and b2-adrenergic receptors, with higher expression

in the primitive CD34+CD38lo population. The myeloid cytokines G-CSF and GM-CSF upregulated neuronal receptor expression

on immature CD34+ cells. Treatment with neurotransmitters increased the motility, proliferation and colony formation of

human progenitor cells, correlating with increased polarity, expression of the metalloproteinase MT1-MMP and activity of the

metalloproteinase MMP-2. Treatment with catecholamines enhanced human CD34+ cell engraftment of NOD-SCID mice through

Wnt signaling activation and increased cell mobilization and bone marrow Sca-1+c-Kit+Lin– cell numbers. Our results identify

new functions for neurotransmitters and myeloid cytokines in the direct regulation of human and mouse progenitor cell migration

and development.

Hematopoietic stem cells are a rare population of cells that continu-ously produce all mature blood cell lineages ‘on demand’ whilemaintaining a small pool of undifferentiated stem and progenitorcells1–3. Stem cells are identified and characterized functionally by theirin vivo ability to repopulate the bone marrow of ablated recipients.Functional in vivo assays for normal and leukemic human stem cellsby intravenous transplantation into sublethally irradiated immune-deficient mice have been developed and include nonobese diabetic,severe combined immunodeficient (NOD-SCID) mice4–6.

The release of progenitor cells from the bone marrow to theperipheral blood occurs constitutively in steady-state conditions, inwhich very low numbers are circulating in the peripheral blood7.These low numbers are greatly amplified during ‘alarm’ situations aspart of host defense and repair mechanisms8,9. Clinical protocols inwhich stem cell mobilization is induced by chemotherapy or cyto-kine treatment mimic this situation; granulocyte colony-stimulatingfactor (G-CSF) is the most commonly used agent for this10–12.Mobilization involves the proliferation of stem and progenitor cellsand the activation of matrix metalloproteinases (MMPs) that facili-tate cell motility and consequent egress from the bone marrowreservoir to the circulation across the physical extracellular matrixand endothelial barriers13–15.

Dopamine is the main catecholamine neurotransmitter in themammalian brain. It also serves as a precursor of norepinephrineand epinephrine, the chief neurotransmitter of the sympatheticnerve system and the principal adrenomedullary hormone, respec-tively. The bone marrow is highly innervated with both nonmyeli-nated and myelinated nerve fibers16, including noradrenergicsympathetic nerve fibers that store a large amount of dopamine17.The effect of the catecholamines is mediated by the activation ofsecond messengers through their interaction with specific G protein–coupled receptors18. The adrenergic receptors constitute a family ofclosely related proteins, including the b2-adrenergic receptor, whichis found on many organs, including smooth muscles, skeletalmuscles and liver. Five dopamine receptors have been identified,subcategorized into the DR-1 family (DR1 and DR5) and the DR-2family (DR2–DR4)19. The mobilization of mouse progenitor cellsfrom the bone marrow has been shown to be indirectly regulated bynorepinephrine through the suppression of bone-lining osteoblastsand downregulation of the chemokine SDF-1 (also called CXCL12)expressed by these cells20.

Wnt proteins constitute a large family of secreted signaling mole-cules that are expressed in diverse tissues and influence manyprocesses in development21. The activation of canonical Wnt signaling

Received 22 June; accepted 13 August; published online 9 September 2007; doi:10.1038/ni1509

1Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. 2Department of Bone Marrow Transplantation & Cancer Immunotherapy,Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel. 3Department of Hematology & Bone Marrow Transplantation, Chaim Sheba Medical Center,Tel-Hashomer 52621, Israel. 4Assaf Haroffe Medical Center, Tsrifin 70300, Israel. 5Department of Molecular Genetics, The Weizmann Institute of Science,Rehovot 76100, Israel. Correspondence should be addressed to T.L. (tsvee.lapidot@weizmann.ac.il).

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results in the accumulation of cytoplasmic b-catenin, leadingto upregulation of gene expression22. The canonical Wnt signalingpathway is an important regulator of mouse hematopoietic stemcell function. Exposure of mouse and human hematopoieticprogenitor cells to conditioned media containing Wnt proteinsresults in an increase in the formation of immature coloniesin vitro23,24. In addition, purified Wnt proteins and viruses withgenes encoding activated b-catenin enhance the self-renewal of mousestem cells in vitro and their in vivo repopulation25,26. TheWnt pathway is also required for the maintenance of mouse progeni-tor cells, as expression of the Wnt inhibitor axin leads to inhibitionof stem cell proliferation and viability in vitro and reducedreconstitution in vivo27. The effects of Wnt signaling on mousestem cells have been recapitulated in the NOD-SCID xenotrans-plant model, in which in vivo delivery of conditioned mediumenriched with Wnt5a results in increased engraftment by humanrepopulating cells28.

Here we examine direct involvement of the nervous system in theregulation of hematopoietic progenitor cells. We find that immaturehuman CD34+ cells and primitive CD34+CD38lo cells (includingCD38– cells, called ‘CD38lo’ cells here) expressed receptors for neuro-transmitters that were involved in the regulation of their proliferationand motility and repopulation of NOD-SCID mice through activationof the Wnt signaling pathway. Using a physiological mouse model, wedemonstrate that in vivo treatment with epinephrine resulted inexpansion of the Sca-1+c-Kit+Lin– population in the bone marrowand consequent mobilization of immature progenitor cells and maturecells to the peripheral blood.

RESULTS

Immature human CD34+ cells express dopamine receptors

To determine a possible function for the nervous system in the directregulation of human progenitor cells, we evaluated the cell surface

expression of dopamine receptors. We focused on DR3 and DR5 asrepresentative receptors of each subfamily. Flow cytometry showedthat enriched human CD34+ cells in cord blood and mobilizedperipheral blood CD34+ cells expressed both DR3 and DR5 on theirsurfaces. Notably, the more primitive CD34+CD38lo cell populations(which were enriched for hematopoietic stem cells) had higherexpression of both receptors than did the more differentiatedCD34+CD38hi cells (Fig. 1a). Furthermore, human CD34+ cells hadvariable expression of cell surface dopamine receptors depending onthe cell source and cytokine exposure. We found only low expressionon unstimulated CD34+ cells derived from steady-state adult bonemarrow or cord blood, but we detected higher expression on bonemarrow and mobilized peripheral blood CD34+ cells obtained fromG-CSF-treated healthy donors (Fig. 1b,c). This finding indicates apotential direct function for neuronal regulation in stress-inducedsituations leading to the accelerated proliferation and recruitment ofprogenitor cells.

We next applied a functional in vivo model of G-CSF-inducedmobilization in NOD-SCID chimeric mice previously engraftedwith human cord blood cells. Similar to our results obtained withenriched CD34+ cells obtained from humans, we also foundhigher expression of cell surface dopamine receptors by primitivehuman CD34+CD38lo bone marrow cells (relative to that of themore differentiated CD34+CD38hi cells) in the chimeric mice.Furthermore, in vivo G-CSF treatment led to an increase in DR3and DR5 expression by the primitive human CD34+CD38lo cellpopulation (Fig. 1d).

To further evaluate the direct effect of G-CSF on the hemato-poietic progenitor cells, we tested dopamine receptor expression inresponse to in vitro stimulation of enriched CD34+ cells with G-CSF.Incubation of cord blood CD34+ cells with G-CSF upregulated theirsurface expression of both DR3 and DR5 (Fig. 1e). Similarly, wedetected upregulation of dopamine receptor expression after treating

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Figure 1 Increased dopamine receptor expression in G-CSF-treated human CD34+ cells. (a) Cell surface expression of DR3 and DR5 by mobilized human

CD34+ cells. Right, expression of DR3 and DR5 on the more mature CD34+CD38hi (R1) and more primitive CD34+CD38lo (R2) cell populations. Dashed

lines indicate control cells stained with secondary antibody only (goat anti-rabbit). (b) Expression of DR3 and DR5 on human cord blood (CB) and mobilized

(Mob) CD34+ cells. 2nd Ab, secondary antibody (goat anti-rabbit). (c) Flow cytometry of the cell surface expression of DR3 and DR5 on steady-state or

in vivo G-CSF-treated human bone marrow (BM) CD34+ cells. MFI, mean fluorescence intensity. (d) Expression of DR3 and DR5 on human CD34+ cells

recovered from the bone marrow of untreated or G-CSF-treated chimeric NOD-SCID mice, presented as percent mean fluorescence intensity relative

to that of CD34+CD38hi cells from untreated mice. (e) Expression of DR3 and DR5 on cord blood CD34+ cells after 3 d of stimulation with G-CSF relative

to that of untreated control cells (set as 100%). *, P o 0.05. Data are representative of at least three experiments (a), are one representative of six

independent experiments (b) or are the mean ± s.e.m. of at least five (c), three (d) or four (e) experiments.

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steady-state bone marrow CD34+ cells with G-CSF in vitro (datanot shown). These results collectively indicate a direct function forneuronal regulation of human progenitor cells through myeloidcytokine stimulation during stress situations, such as G-CSF-inducedmobilization, which are known to enhance the proliferation andmotility of hematopoietic stem and progenitor cells. To furtherexamine this hypothesis, we tested the effect of the neurotransmitterdopamine on each of these processes.

Dopamine induces migration and MT1-MMP, MMP-2 expression

First we tested the effect of dopamine on the in vitro migration ofimmature human CD34+ cells. The placement of dopamine in thelower chamber of transwells significantly increased the migration ofcord blood CD34+ cells pretreated with GM-CSF (Fig. 2a). We did notdetect this dopamine-induced migration in cells not pretreated withGM-CSF (data not shown). As dopamine is highly oxidative, we useddopamine agonists, particularly in treatments requiring longer incu-bation periods. Cytoskeleton rearrangement is a vital part of cellmotility; thus, we did immunocytochemical analysis of CD34+ cellmorphology after cells had adhered to extracellular matrix compo-nents. The percentage of human cord blood CD34+ cells withpolarized morphology and cellular elongation doubled in the presenceof the dopamine receptor agonists SKF and 7-OH-DPAT relative tothat of control untreated cells (Fig. 2b). This increase was alsoaccompanied by clustering of DR5 in the membranes of polarizedcells (Fig. 2c).

To elucidate the mechanisms underlying dopamine-regulated cellmotility, we assessed the expression of membrane-associated type 1matrix metalloproteinase (MT1-MMP), originally identified as aninvasion-promoting enzyme. We chose MT1-MMP because reportsindicate that it is important in human progenitor cell homing andG-CSF-induced mobilization29,30. Flow cytometry showed increasedMT1-MMP cell surface expression on mobilized human CD34+ cellstreated with SKF or 7-OH-DPAT (Fig. 2d). Moreover, we noted a 60%increase in MMP-2 activity after treatment with these dopaminereceptor agonists (Fig. 2e). These results collectively indicate involve-ment of dopamine and its receptors in the regulation of MMPexpression and activity in human CD34+ progenitor cells, thusaffecting their motility and trafficking.

Dopamine agonists increase clonogenic capacity and repopulation

Next we assessed the neuronal regulation of human CD34+

cell proliferation. The dopamine receptor agonists SKF and 7-OH-DPAT both augmented cord blood CD34+ colony formationonly in the presence of the myeloid cytokines GM-CSF and G-CSF(Fig. 3a). Moreover, the proliferative effect of 7-OH-DPAT on cordblood CD34+ cells treated with GM-CSF was dose dependent(Fig. 3b). As stem cells are characterized functionally by theirability to repopulate and engraft the bone marrow of recipients, wesought to determine whether in vitro treatment with these agonistsinfluenced the in vivo repopulation potential of CD34+ cells. Westimulated G-CSF-mobilized CD34+ cells (with high dopaminereceptor expression) ex vivo with SKF or 7-OH-DPAT (withoutmyeloid cytokines) and transplanted the cells into NOD-SCIDmice. This treatment resulted in a twofold increase in engraftmentin the mouse bone marrow relative to that of untreated controlcells. Furthermore, we noted a 50% decrease in engraftment whenthe cells were pretreated with the dopamine receptor antagonistclozapine (Fig. 3c), suggesting an important direct function forthe neurotransmitters in augmenting in vivo repopulation. Similarly,we noted an increase in engraftment when cord blood CD34+

cells were treated in vitro with the dopamine receptor agonist7-OH-DPAT (compared with cells treated only with GM-CSF).Notably, we found the agonistic effect only in cells also pretreatedwith GM-CSF before transplantation (Fig. 3d), further strengtheningthe idea that myeloid cytokines are involved in the regulation ofdopamine receptor expression on immature CD34+ cells and theirconsequent repopulation potential.

Adrenergic neurotransmitters regulate motility and expansion

Adrenergic stimulation is a chief mechanism by which the body reactsto stress situations. Having established the idea of crosstalk betweenG-CSF-induced stress and dopamine in the regulation of the prolif-eration and motility of hematopoietic progenitor cells, we nextassessed whether the regulation of CD34+ cells by neurotransmittersis a broader phenomenon. We investigated the effects of the catechol-amines epinephrine and norepinephrine as well as expression of theircognate b2-adrenergic receptor on human cord blood and mobilizedCD34+ cells. Flow cytometry showed that human CD34+ cells

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Figure 2 Dopamine receptor agonists increase the polarization and motility of CD34+ cells. (a) Transwell

migration of human cord blood CD34+ cells (pretreated overnight with 5 ng/ml of GM-CSF) toward 10 nM

dopamine in the lower chamber. (b) Quantification of cord blood CD34+ cells with an elongated and highly

polarized morphology in untreated control cells and after treatment with SKF or 7-OH-DPAT. (c) Microscopy of cells immunolabeled for DR5

(green) and stained for polymerized actin (red). Arrows indicate clustering of DR5 in polarized cells. Scale bar, 10 mm. (d) Cell surface expression of

MT1-MMP after treatment with 1 mM SKF or 7-OH-DPAT. (e) MMP-2 activity in conditioned media of CD34+ cells after treatment with 1 mM SKF

or 7-OH-DPAT. Numbers below lanes indicate band intensity, analyzed by densitometry (AU, arbitrary units). *, P o 0.05, compared with control

(no dopamine in lower chamber (a) or untreated cells (b,d,e)). Data are the mean ± s.e.m. of four (a), three (b) or five (d) experiments or are representative

of three (c) or seven (e) independent experiments.

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expressed the b2-adrenergic receptor (Fig. 4a). As with the dopaminereceptor, G-CSF-mobilized CD34+ cells had higher expression of theb2-adrenergic receptor than did cord blood CD34+ cells. To confirmthat this resulted from a direct effect of G-CSF on the hematopoieticprogenitor cells, we treated cord blood CD34+ cells with G-CSFin vitro. Indeed, b2-adrenergic receptor expression was upregulatedafter treatment with G-CSF (Fig. 4b).

We next evaluated the effect of norepinephrine on the in vitromigration of human CD34+ cells. Placement of norepinephrine in thelower well increased the migration of both cord blood (pretreated withGM-CSF to increase b2-adrenergic receptor expression) and mobilizedCD34+ cells in a dose-dependent way (Fig. 4c). To further elucidatethe involvement of neurotransmitters in motility, we tested forpossible crosstalk with MMPs. We detected a 30% increase in MT1-MMP expression and a sixfold increase in MMP-2 activity afterstimulation with epinephrine or norepinephrine, suggesting involve-ment of adrenergic neurotransmitters in expression and activity ofMMPs (Fig. 4d,e). We further assessed the effect of adrenergicneurotransmitters on human CD34+ cell colony formation andrepopulation. Norepinephrine augmented the colony formation ofboth human GM-CSF-treated cord blood CD34+ cells and G-CSF-mobilized CD34+ cells in a dose-dependent way (Fig. 4f).

We next determined whether in vitro treatment with adrenergicneurotransmitters increased the in vivo repopulation potential ofCD34+ cells. When we treated cord blood CD34+ cells with a highconcentration (1 mM) of epinephrine together with GM-CSF, wenoted a 30-fold increase in engraftment relative to that of controlcells treated with GM-CSF alone (Fig. 4g). The results were highlyvariable and donor specific, most probably because of sensitivities tothe combination of myeloid cytokines and neurotransmitters. We thenreduced the concentration of epinephrine to 10 nM and noted areduced effect on engraftment but a more homogeneous distribution.Transplantation of ex vivo GM-CSF-treated cord blood CD34+ cellsstimulated with epinephrine (10 nM) or norepinephrine (1 mM)resulted in increased engraftment efficiency (Fig. 4h). Similarly, weobtained an increase in engraftment when we treated mobilizedCD34+ cells (which already had high expression of the b2-adrenergicreceptor and therefore were not treated with GM-CSF in vitro) withepinephrine or norepinephrine (Fig. 4i). Furthermore, when wetransplanted mice with a limiting number of cord blood CD34+-

enriched cells (1 � 105 cells per mouse), short ex vivo treatment withGM-CSF plus epinephrine (10 nM) or norepinephrine (1 mM)resulted in successful engraftment of 64% (9 of 14) or 88% (7 of 8)of the mice, respectively. In contrast, control cells treated withGM-CSF alone (1 � 105 cells per mouse) engrafted only 44% (4 of9) of mice. Moreover, among highly engrafted mice (over 10%engraftment) transplanted with 1 � 105 cord blood CD34+ cells permouse, pretreatment in vitro with GM-CSF plus epinephrine resultedin engraftment similar to or even higher than that of mice trans-planted with double the amount of control cord blood CD34+ cellspretreated with GM-CSF alone (2 � 105 cells per mouse; Fig. 4j). Ofnote, adrenergic stimulation did not impair differentiation,because engrafted mice showed normal hematopoietic lineagedevelopment, as determined by CD19 and CD45 expression, as wellas normal progenitor development, as determined by CD34 andCD38 expression (data not shown). These results collectivelysuggest involvement of adrenergic neurotransmitters in the regulationof hematopoietic stem and progenitor cell motility, proliferationand repopulation.

Epinephrine induces proliferation and mobilization in vivo

Having established the idea that catecholamine neurotransmitters areinvolved in regulating the motility and development of immaturehuman CD34+ cells, we next tested whether a similar effect was alsopresent in vivo in a physiological mouse model. For this, we testedwhether in vivo neurotransmitter stimulation induced cell mobiliza-tion. We first noted that treatment with epinephrine (50 mg per mouseonce daily for 6 d) resulted in more leukocytes in the peripheral bloodon day 6 (Fig. 5a). Moreover, we detected a 2.5-fold increase in thespontaneous in vitro migration of bone marrow mononuclear cells inthe epinephrine-treated mice (Fig. 5b); this may facilitate cell egressfrom the bone marrow to the periphery. Of note, stimulation ofadrenergic receptors with epinephrine led to enhanced mobilization ofimmature colony-forming cells to the peripheral blood (Fig. 5c),increasing both colony frequency and number. Progenitor cell pro-liferation in the bone marrow reservoir is a prerequisite for cellegress and stress-induced mobilization31. We therefore compared theability of primitive bone marrow cells to proliferate in vivo inresponse to adrenergic stimulation. After 6 d of epinephrine treatment(50 mg/mouse/day), we noted a 2.3-fold increase in the stem

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Figure 3 Dopamine receptor agonists increase the clonogenic progenitor content and engraftment potential of CD34+ cells. (a) Number of colonies per

1 � 103 seeded cord blood CD34+ cells treated with 1 mM SKF or 7-OH-DPAT with (+) or without (–) the addition of GM-CSF (5 ng/ml) or G-CSF

(100 ng/ml), relative to that of untreated control cells (set as 100%). Average colony numbers for control cells: 47 ± 5 (without G-CSF or GM-CSF),

69 ± 11 (with GM-CSF) and 58 ± 5 (with G-CSF). (b) Number of colonies per 1 � 103 seeded cord blood CD34+ cells treated with 7-OH-DPAT

(concentration, horizontal axis) relative to that of control cells treated with GM-CSF (set as 100%; average colony numbers, 86 ± 13). (c) Bone marrowengraftment of NOD-SCID mice injected with mobilized human CD34+ cells (2 � 105 to 4 � 105 cells/mouse) that were left untreated (Control) or were

incubated with 1 mM SKF, 7-OH-DPAT or clozapine before injection. (d) Bone marrow engraftment of NOD-SCID mice injected with human cord blood CD34+

cells (2 � 105 to 3 � 105 cells/mouse) that were left untreated (Control) or were incubated with 1 mM 7-OH-DPAT (with (+) or without (–) GM-CSF) before

injection. *, P o 0.05, compared with control. Data are the mean ± s.e.m. of at least three (a–c) or five (d) experiments.

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cell–enriched Sca-1+c-Kit+Lin– population in mouse bone marrow. Incontrast, treatment with the b2-adrenergic antagonist propranololreduced the Sca-1+c-Kit+Lin– population in the bone marrow by65% (Fig. 5d). These results collectively suggest involvement of theadrenergic neurotransmitters in the regulation of hematopoieticprogenitor cell motility and proliferation in physiological conditions.

Neurotransmitter stimulation activates canonical Wnt signaling

Given our results showing that stimulation with neurotransmittersincreased the colony formation and repopulation capacities of imma-ture human CD34+ cells, we next sought to delineate the molecularmechanism underlying this phenomenon. The canonical Wnt signalingpathway is an important regulator of hematopoietic stem cell prolif-eration. We therefore determined whether Wnt signaling was involvedin the neuronal receptor–induced proliferation of hematopoieticprogenitor cells. Indeed, we found that stimulation of cord blood

CD34+ cells with GM-CSF and epinephrine led to accumulationof b-catenin in the cells (Fig. 6a). Notably, we did not detectstabilization of b-catenin and subsequent accumulation whenwe neutralized Wnt activity with conditioned medium enrichedwith the cysteine-rich domain of Frzb (CRD-Frzb), whichcontains a Wnt-binding domain32. Stimulation of mobilized humanCD34+ cells with SKF or 7-OH-DPAT resulted in a four- or sevenfoldincrease in b-catenin expression in the cells, respectively (Fig. 6b).We did not detect stabilization of b-catenin and increasedcellular protein abundance when we neutralized Wnt activity withCRD-Frzb (Fig. 6b).

MT1-MMP has been shown to be a direct target in the Wntsignaling pathway in both colorectal cancer cells and normal humanmesenchymal stem cells33,34. Therefore, we tested if the increasedexpression of MT1-MMP after stimulation with dopamine receptoragonists was mediated by the Wnt signaling pathway. The increased

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Figure 4 Adrenergic neurotransmitters regulate CD34+ cell motility and

proliferation. (a) Expression of b2-adrenergic receptor on human cord blood (CB)

or mobilized CD34+ cells. (b) Expression of b2-adrenergic receptor on cord blood

CD34+ cells after 3 d of stimulation with G-CSF, relative to that of untreated

control cells (set as 100%). (c) Transwell migration of human cord blood CD34+

cells (pretreated with GM-CSF) or G-CSF-mobilized CD34+ cells toward

norepinephrine (NE). Average migration of control cells (no treatment in lower

well): 1.6% ± 0.4% (mobilized) and 1.1% ± 0.3% (cord blood). (d) Cell surface

expression of MT1-MMP after treatment with epinephrine (Epi) or norepinephrine.

(e) MMP-2 activity in the conditioned medium of mobilized CD34+ cells after

treatment with epinephrine (10 nM) or norepinephrine (1 mM). Numbers below

lanes indicate band intensity, analyzed by densitometry. (f) Number of colonies per

1 � 103 seeded cord blood or mobilized CD34+ cells treated with 1 nM or 10 nM

norepinephrine. Average colony numbers for untreated control cells: 52 ± 9 (cord blood) and 32 ± 6 (mobilized). (g) Engraftment of NOD-SCID mice

injected with human cord blood CD34+ cells treated with GM-CSF (2 � 105 cells/mouse) and incubated with 1 mM epinephrine (Epi) or not (Control) beforeinjection. Average engraftment with control cells: 1.7% ± 1.4%. (h) Engraftment of NOD-SCID mice injected with human cord blood CD34+ cells pretreated

with GM-CSF (2 � 105 cells/mouse) and incubated with 10 nM epinephrine, 1 mM norepinephrine or neither (Control) before injection. (i) Engraftment

of NOD-SCID mice injected with human mobilized peripheral blood CD34+ cells (2 � 105 to 4 � 105 cells/mouse) that were incubated with 10 nM

epinephrine, 1 mM norepinephrine or neither (Control) before injection. In h,i, each dot represents a single mouse and percents in graphs indicate average

engraftment frequency. (j) Engraftment of NOD-SCID mice injected with 1 � 105 or 2 � 105 human cord blood CD34+ cells treated with GM-CSF alone

(Control) or with GM-CSF plus epinephrine. Numbers above outlined areas indicate percent human CD45+ cells. SSC, side scatter. *, P o 0.05, compared

with controls. Data are from one representative of seven (a) or five (e) independent experiments, are the mean ± s.e.m. of five (b) or at least four (c,f), six (d)

or seven (g) independent experiments, or are from at least five (h), at least six (i), or two (j) independent experiments.

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MT1-MMP expression noted after treatmentof mobilized peripheral blood CD34+ cellswith 7-OH-DPAT was prevented in cells trea-ted with both 7-OH-DPAT and the Wnt-neutralizing CRD-Frzb-enriched conditionedmedium (Fig. 6c). Furthermore, the increasedMT1-MMP expression noted after treatmentwith epinephrine was ablated in cells alsotreated with the soluble Wnt antagonistDKK-1 (Fig. 6d). We further tested the invol-vement of Wnt signaling functionally by asses-sing progenitor clonogenic capacity. Althoughtreatment with norepinephrine or 7-OH-DPAT enhanced clonogenic capacity ofhuman cord blood CD34+ cells, we detectedno increase when we added DKK-1 (Fig. 6e).We also tested the ability of human CD34+

cells to repopulate the bone marrow of NOD-SCID mice. Notably, the increased engraft-ment noted after stimulation of enriched bone marrow CD34+ cellswith GM-CSF and norepinephrine was ablated when Wnt signalingwas inhibited by the addition of conditioned medium enriched withCRD-Frzb (Fig. 6f). These results collectively suggest that stimulationby dopamine or adrenergic neurotransmitters leads to activation ofthe Wnt signaling pathway and accumulation of b-catenin, directlyregulating the proliferation, MT1-MMP upregulation and increasedrepopulation potential of immature human CD34+ cells.

DISCUSSION

Our study has indicated an important function for neurotransmittersin regulating three key processes of human CD34+ and mouse Sca-1+c-Kit+Lin– progenitor cells: motility, proliferation and repopulation.Here we have demonstrated a direct influence of catecholamines ofthe sympathetic system (dopamine, epinephrine and norepinephrine)on hematopoietic progenitor cells, which dynamically expressedreceptors for these neurotransmitters in stress-induced situations.

When stimulated by neurotransmitters ofthe adrenergic system, human CD34+ pro-genitor cells had active motility phenotypes,including cell polarity and MMP secretion,as well as increased proliferation. Receptorexpression and subsequent responses to neuro-transmitters was further enhanced by costi-mulation with the myeloid cytokines G-CSFor GM-CSF as part of the host defense andrepair pathways.

Cell motility is a hallmark of hematopoieticstem and progenitor cell function and is anessential component of homeostasis, acceler-ated stress-induced ‘alarm’ situations and clin-ical mobilization protocols. Our results haveshown that the neurotransmitters norepi-nephrine and dopamine serve as chemoattrac-tants and increase the migration potential ofimmature human CD34+ cells. By modulatingthe chemotactic activity of hematopoietic pro-genitor cells, neurotransmitters may beinvolved in the recirculation of these cellsamong the bone marrow, blood and otherorgans. Our findings demonstrated increasedMT1-MMP expression and MMP-2 activityafter neurotransmitter stimulation, suggestingthat neuronal regulation may serve as anadditional way in which the egress, recruit-ment and mobilization capacities of progeni-tor cells are directly regulated.

The cytokines G-CSF and GM-CSF arecommonly used to accelerate myelopoiesisin clinical protocols to minimize the sideeffects of cancer chemotherapy, such as

a

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Figure 5 Epinephrine induces progenitor cell proliferation, motility and in vivo mobilization. (a) Number

of leukocytes in the peripheral blood of mice treated for 6 d with epinephrine or PBS (Control).

(b) Transwell migration of bone marrow mononuclear cells from mice treated for 6 d with epinephrine

(50 mg/mouse/day) or PBS (Control). Average spontaneous migration of control cells: 1.6% ± 0.5%.

(c) Colony formation by peripheral blood mononuclear cells from mice treated for 6 d with epinephrine

(50 mg/mouse/day) or PBS (Control). CFC, colony-forming cells. (d) Flow cytometry of primitive

undifferentiated Sca-1+c-Kit+Lin– (SKL) cells in the bone marrow of mice treated with epinephrine

(50 mg/mouse/day), propranolol (Prop) or PBS (Control). MNCs, mononuclear cells. *, P o 0.05,

compared with control. Data are the mean ± s.e.m. of four (a,c) or at least two (d) independent

experiments with four or more mice in each or are the mean ± s.e.m. of six or more experiments (b).

Human CD45

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Figure 6 Neurotransmitters activate the canonical Wnt signaling pathway. (a) Flow cytometry of

b-catenin expression by cord blood CD34+ cells treated with GM-CSF alone (5 ng/ml; Control) or with

GM-CSF (5 ng/ml) and epinephrine (10 nM), with or without conditioned medium enriched with Wnt-

binding CRD-Frzb. (b) Flow cytometry of b-catenin expression in mobilized CD34+ cells left untreated

(Control) or treated with SKF or 7-OH-DPAT, with or without conditioned medium enriched with CRD-

Frzb. (c) Flow cytometry of MT1-MMP expression on mobilized CD34+ cells left untreated (Control)

or treated with 7-OH-DPAT, with or without conditioned medium enriched with CRD-Frzb. (d) Flow

cytometry of MT1-MMP expression in mobilized CD34+ cells left untreated (Control) or treated withepinephrine, with or without DKK-1 (200 ng/ml). (e) Number of colonies per 1 � 103 seeded cord

blood CD34+ cells treated with norepinephrine (1 mM), 7-OH-DPAT (1 mM) and/or DKK-1 (200 ng/ml),

relative to that of untreated control cells (set as 100%; average colony numbers (control): 113 ± 36).

(f) Engraftment of NOD-SCID mice injected with human cord blood CD34+ cells treated with GM-CSF

alone (Control) or also incubated with norepinephrine, with or without conditioned medium enriched

with CRD-Frzb, before injection. Numbers above bracketed lines indicate percent human CD45+ cells.

*, P o 0.05, compared with control. Data are from one representative of three (a–d) or two (f)

independent experiments or are the mean ± s.e.m. of at least three independent experiments (e).

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myelosuppression and peripheral blood cytopenia35. Despite frequentclinical use of myeloid cytokines, the precise mechanisms underlyingtheir mode of action are not fully understood. On the basis of ourresults, we propose that myeloid cytokines such as G-CSF and GM-CSF have additional direct functions in the regulation of humanCD34+ progenitor cells. These cytokines upregulate the expression ofneuronal receptors on hematopoietic progenitor cells, thus augment-ing their response to neurotransmitters, leading to enhanced prolifera-tion and motility of human CD34+ progenitor cells, repopulation ofmouse bone marrow and egress of progenitor cells to the circulation.

It has been shown that G-CSF-induced mobilization of mouseprogenitor cells requires peripheral adrenergic signals. According tothe proposed model, released norepinephrine controls G-CSF-inducedosteoblast suppression and consequent downregulation of SDF-1 in thebone marrow, leading to progenitor mobilization20. The cell mediatorsof the G-CSF effect remain unknown36,37. Our study has broadenedthe understanding of the function of the sympathetic nervous systemin hematopoiesis, demonstrating that bone marrow innervation reg-ulates the retention as well as the proliferation and recruitment ofhematopoietic progenitor cells not only indirectly through its effects onbone-lining osteoblasts and bone-degrading osteoclasts38 but alsothrough a direct effect on hematopoietic progenitor cells.

The notch, hedgehog and Wnt pathways have been associated withboth human and mouse hematopoietic stem cell function25,27,28,39,40.Our results suggest that the Wnt signaling pathway mediates theeffects of neurotransmitters on the population expansion and motilityof immature human CD34+ cells by increasing the abundance ofb-catenin. This is in line with reports showing that in vitro treatmentof human CD34+ cells with sonic hedgehog39 or in vivo treatment ofhuman SCID-repopulating cells with Wnt5a-conditioned mediumincreases their engraftment in transplanted NOD-SCID mice28.Furthermore, inhibitors of the Wnt signaling pathway lead to inhibi-tion of mouse stem cell growth in vitro and reduced reconstitutionin vivo25. Reports have shown that permanent excess activation ofcanonical Wnt signaling impairs hematopoietic stem cell function byblocking mouse stem cell differentiation, resulting in widespreadabnormalities and death of mice due to anemia41,42. These studiescollectively demonstrate that the Wnt–b-catenin pathway is animportant regulator of stem cells and hematopoiesis. However, theprecise conditions, such as the strength (dose) and duration (short-term stimulation versus long-term, constitutive overexpression) of theb-catenin signal, as well as synergy with additional microenviron-mental factors, remain to be determined43.

Clinical transplantation of CD34+ cells is an effective method fortreating hematopoietic disorders, including malignancy and immu-nodeficiency. However, several clinical limitations remain, includingthe unavailability of matched allogeneic donors and the suboptimalcollection of CD34+ cells for autologous transplantation from elderlypatients and cancer patients who have been extensively treated withchemotherapy and therefore have poor mobilization of bone marrowcells. Umbilical cord blood cells have great potential as an alternatesource of transplantable progenitor cells; however, because low num-bers of cells are collected from each graft, these progenitor cells are ofonly limited use. The expansion of such cell populations would beadvantageous for increasing stem and progenitor cell numbers forautologous or allogeneic transplantation. Our results suggest treat-ment with neurotransmitters and myeloid cytokines might serve as aprotocol for enhancing progenitor cell mobilization, proliferation andrepopulation in clinical transplantation protocols.

In conclusion, our study has provided new insights into themechanisms regulating the retention, proliferation and migration of

human CD34+ and mouse Sca-1+c-Kit+Lin– progenitor cells, suggest-ing that interactions between these cells and their bone marrowmicroenvironment include regulatory signals exerted by neurotrans-mitters. Our data have demonstrated an important and direct func-tion for the nervous system in regulating the proliferation and motilityof hematopoietic progenitor cells, together with myeloid cytokines, aspart of host defense and repair, producing leukocytes ‘on demand’.These interactions bear clinical relevance and identify a new approachfor the ex vivo and in vivo manipulation of immature human CD34+

cells and the more primitive CD34+CD38lo cells.

METHODSHuman CD34+ cells. Human cord blood was obtained from full-term

deliveries. Adult G-CSF-mobilized peripheral blood and bone marrow samples

(normal or G-CSF treated) were obtained from healthy donors for clinical

transplantation. All human cell samples were obtained after informed consent

was received and were used in accordance with procedures approved by the

Human Experimentation and Ethics Committees of the Weizmann Institute.

Mononuclear cells were isolated from samples by standard separation by

density-gradient centrifugation on Ficol-Hypaque (Pharmacia Biotech) and

were washed in PBS. Enrichment of human CD34+ cells was achieved by

magnetic bead separation with the MACS cell isolation kit and the autoMACS

magnetic cell sorter (Miltenyi Biotec) according to manufacturer’s instructions,

producing a purity of over 95%.

Chemotaxis assays. Chemotaxis was assessed with transwells (6.5-mm dia-

meter and 5-mm pore; Corning) as described44. Cord blood CD34+ cells (1 �105 cells) with or without pretreatment overnight with GM-CSF (5 ng/ml) were

placed in the upper chamber, and medium supplemented with ascorbic acid

(Sigma) and with or without dopamine (10 nM; Sigma) or norepinephrine

(10 nM or 1 mM; without ascorbic acid) was placed in the lower chamber.

Migration was stopped after 2 h and migrating cells were counted with a

FACSCalibur (Becton Dickinson).

Colony-forming unit assay. Semisolid cultures were used for counting of

human progenitor cells as described45. CD34+ cells (1 � 103 cells/ml) were

plated in 0.9% (wt/vol) methylcellulose (Sigma), 30% (vol/vol) FCS, stem cell

factor (50 ng/ml), interleukin 3 (5 ng/ml), GM-CSF (5 ng/ml; R&D Systems)

and erythropoietin (2 U/ml; Ortho BioTech). In some experiments, GM-CSF

was not added to the semisolid culture or was replaced with G-CSF (100 ng/ml;

Roche). Where indicated, DKK-1 (200 ng/ml; R&D Systems) or SKF (1 mM,

unless indicated otherwise; SKF-38393 hydrochloride; Sigma), 7-OH-DPAT

(7-hydroxy-2-(di-n-propylamino)tetralin hydrobromide; Sigma) or norepi-

nephrine (10 nM; Sigma) was added to the culture. Cultures were incubated

at 37 1C in a humidified atmosphere containing 5% CO2 and were assigned

scores 14 d later for morphological criteria.

Immunocytochemical staining. Enriched cord blood CD34+ cells (2 � 105 to

3 � 105 cells per well) treated with SKF or 7-OH-DPAT (Sigma) were plated

for 2 h at 37 1C on hyaluronic acid–coated coverslips. Samples were processed

for microscopic observation as described46. Adherent cells were fixed with

3% (wt/vol) paraformaldehyde (Merck) and were made permeable in 0.5%

(vol/vol) Triton X-100 (Sigma-Aldrich). Samples were indirectly immuno-

labeled at 24 1C in a humidified chamber with rabbit polyclonal antibody to

human DR5 (324408; Calbiochem) and Alexa 488–conjugated goat antibody to

rabbit immunoglobulin G (secondary antibody; A11034; Molecular Probes).

Polymerized actin was detected with tetramethylrhodamine isothiocyanate–

phalloidin (Sigma). After being labeled, cells were mounted in Elvanol (Mowiol

4-88; Hoechst). Immunofluorescence images were acquired with scientific-

grade charge-coupled device camera and were processed by the DeltaVision

system with Resolve3D software (Applied Precision).

Engraftment. Engraftment experiments were done as described6. NOD-SCID

mice were bred and maintained at the Weizmann Institute. All experiments

with animals were approved by the Weizmann Institutional Animal Care and

Use Committee. Recipient mice were irradiated with a sublethal dose (350 cGy)

from a cesium source 24 h before injection of cells. Mobilized human CD34+

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cells (2 � 105 to 4 � 105 cells/mouse) were treated for 1–3 d with SKF (1 mM),

7-OH-DPAT (1 mM), epinephrine (10 nM) or norepinephrine (1 mM) before

being injected intravenously into NOD-SCID mice. Cord blood CD34+ cells

were treated for 1–3 d with SKF (1 mM; 2 � 105 to 3 � 105 cells/mouse), 7-OH-

DPAT (1 mM; 2 � 105 to 3 � 105 cells/mouse), epinephrine (10 nM or 1 mM;

1 � 105 or 2 � 105 cells/mouse) or norepinephrine (1 mM; 2 � 105 cells/

mouse) and then were injected intravenously into NOD-SCID mice. Unless

otherwise indicated, cord blood CD34+ cells were pretreated with GM-CSF

before transplantation. In some experiments, cells were also pretreated with

conditioned medium enriched with CRD-Frzb32. Recipient mice were killed

5–6 weeks after transplantation for assessment of human cell engraftment. Bone

marrow cells (flushed with a syringe from femur and tibia bones) were collected

and were resuspended into single-cell suspensions. Engraftment frequency was

determined as the percent of CD45+ human cells among total mouse bone

marrow cells. Engraftment of less than 0.2% was excluded from analysis.

In vivo adrenergic stimulation. These experiments used C57BL/6 mice

(Harlan) and were approved by the animal care committee of the Weizmann

Institute. Mice (8 weeks old) received a daily intraperitoneal injection of PBS or

of epinephrine, norepinephrine or propranolol (2.5 mg per kg body weight per

day for each; Sigma) for 6 consecutive days and were killed 1–2 h after the last

injection. Bone marrow was obtained from femurs and tibias and the bones

were flushed with PBS. Peripheral blood from mice asphyxiated by CO2

inhalation was collected by cardiac aspiration into heparinized tubes. Peripheral

blood and bone marrow mononuclear cells were isolated from samples

by standard density-gradient centrifugation separation on Ficol-Hypaque

(Pharmacia Biotech) and were washed in PBS. The percent Sca-1+c-Kit+Lin–

cells was assessed by staining of bone marrow mononuclear cells as described9.

Peripheral blood mononuclear cells (2 � 105) were seeded in semisolid cultures

as described45. Colonies were assigned scores 7 d later under an inverted

microscope according to morphological criteria. Migration of bone marrow

mononuclear cells (2 � 105) was assayed with transwell chambers.

Human cell mobilization. Mobilization in NOD-SCID mice previously

engrafted with human cord blood mononuclear cells (20 � 106 cells/mouse)

was achieved as described45. At 5 weeks after transplantation, mice received a

daily subcutaneous injection of G-CSF (300 mg filgrastim per kg body weight

in 250 ml 0.9% (wt/vol) NaCl and 5% (vol/vol) FCS, pH 4.55) for 5 consecutive

days and were killed 4 h after the final injection. Bone marrow mononuclear

cells were isolated from samples by standard separation on Ficol-Hypaque

(Pharmacia Biotech) and cells were stained for dopamine receptor expression

on CD34+ cells.

Flow cytometry analysis. The phenotypes of human cells were examined by

immunostaining with one- or two-step staining procedures, followed by flow

cytometry on a FACSCalibur (Becton Dickinson) with CellQuest software.

Single-cell suspensions were prepared in PBS containing 0.01% (wt/vol)

sodium azide and 1% (vol/vol) FCS. Human plasma and mouse immuno-

globulin G were used for blockade of human and mouse Fc receptors,

respectively. Neuronal receptor expression was determined by staining with

rabbit antibody to human b2-adrenergic receptor (ab13300; Abcam) or rabbit

antibody to human DR3 324402; or DR5 (324408; both from Calbiochem),

followed by secondary Alexa Fluor 488–conjugated goat antibody to rabbit

(anti-rabbit; Molecular Probes). In some experiments, neurotransmitter recep-

tor expression was determined after 3 d of treatment of cord blood CD34+ cells

with G-CSF (100 ng/ml; filgrastim). Engraftment of human cells was assessed

by staining with fluorescein isothiocyanate–conjugated mouse antibody to

human CD45 (1QP-12YF; Becton Dickinson). In mobilization experiments,

dopamine receptor expression on bone marrow human CD34+ cells was tested;

cells were triple stained with rabbit antibody to human DR3 or DR5

(Calbiochem) followed by Alexa Fluor 488–goat anti-rabbit (secondary anti-

body; Molecular Probes) and phycoerythrin-conjugated mouse antibody to

human CD38 (HB-7; Becton Dickinson) and allophycocyanin-conjugated anti-

CD34 (581; Becton Dickinson). In experiments testing b-catenin expression,

cells were fixed with 3% (wt/vol) paraformaldehyde, were made permeable with

0.1% (vol/vol) Tween, were washed in PBS and were stained with anti-

b-catenin (C2206; Sigma) followed by Alexa Fluor 488–conjugated goat

anti-rabbit (secondary antibody; Molecular Probes). Cells treated with second-

ary antibody only served as the control. MT1-MMP expression was assessed by

staining with rabbit antibody to human MT1-MMP (Chemicon International)

followed by Alexa Fluor 488–conjugated goat anti-rabbit (secondary antibody;

Molecular Probes). In some experiments, CD34+ cells were treated with

conditioned medium enriched with CRD-Frzb or DKK-1 (200 ng/ml; R&D

Systems) before MT1-MMP staining.

Gelatin zymography. CD34+ cells were incubated for 48 h at 37 1C in serum-

free RPMI medium (at a density of 2 � 106 cells/ml) with or without SKF

(1 mM), 7-OH-DPAT (1 mM), epinephrine (10 nM) or norepinephrine (1 mM).

Equal volumes of conditioned media were then collected for zymographic

analysis of MMP-2 activity as described44. Conditioned medium from HT1080

cells (human fibrosarcoma cells) served as a positive control for MM-2 activity.

Statistical analysis. Significance was determined by analysis with the paired,

two-tailed Student t-test.

ACKNOWLEDGMENTSWe thank L. Abel for assistance; E. Tzahor (Weizmann Institute of Science) forCRD-Frzb-enriched conditioned medium; and A. Globerson and S. Berrih-Aknin for discussions and critical review of the manuscript. Supported byAres-Serono, the Gabriella Rich Center for Transplantation Biology, the IsraelScience Foundation (796/04) and the Helen and Martin Kimmel Institute forStem Cell Research at the Weizmann Institute of Science.

AUTHOR CONTRIBUTIONSA.S. designed and did experiments, analyzed data and wrote the manuscript;S.S., A.K., A.L., N.N., Y.A. and P.G. did experiments and analyzed data; I.R.,I.H., H.B.-H. and A.N. provided human blood and bone marrow cells; M.R.provided advice on experimental design and manuscript preparation; andT.L. designed the research and wrote the manuscript.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-textHTML version of the paper at http://www.nature.com/natureimmunology

Published online at http://www.nature.com/natureimmunology

Reprints and permissions information is available online at http://npg.nature.com/

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