Hematopoiesis nature

The role of Smad signaling in hematopoiesis

Jonas Larsson1,2 and Stefan Karlsson*,1

1Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and The Lund Strategic Research Center for Stem CellBiology and Cell Therapy, Lund University, BMC A12, Lund 221 84, Sweden; 2Center for Regenerative Medicine and Technology,Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

The TGF-b family of ligands, including TGF-b, bonemorphogenetic protein (BMP) and activin, signal throughSmad pathways to regulate the fate of hematopoieticprogenitor and stem cells during development and post-natally. BMP regulates hematopoietic stem cell (HSC)specification during development, while TGF-b1, 2 and 3are not essential for the generation of HSCs. BMP4 canincrease proliferation of human hematopoietic progeni-tors, while TGF-b acts as a negative regulator ofhematopoietic progenitor and stem cells in vitro. Incontrast, TGF-b signaling deficiency in vivo does notaffect proliferation of HSCs and does not affect lineagechoice either. Therefore, the outcome of Smad signaling isvery context dependent in hematopoiesis and regulation ofhematopoietic stem and progenitor cells is more compli-cated in the bone marrow microenvironment in vivo than isseen in liquid cultures ex vivo. Smad signaling regulateshematopoiesis by crosstalk with other regulatory signalsand future research will define in more detail how thevarious pathways interact and how the knowledgeobtained can be used to develop advanced cell therapies.Oncogene (2005) 24, 5676–5692. doi:10.1038/sj.onc.1208920

Keywords: Smad signaling; hematopoiesis; stem cells

Introduction

TGF-b has been characterized as a well-known reg-ulator of hematopoiesis through detailed studies ofhematopoietic progenitors in vitro. A relatively recentdetailed review describes how TGF-b regulates prolif-eration and differentiation of hematopoietic precursorsin vitro, often as a negative regulator of proliferation(Fortunel et al., 2000). In this review, we will discusshow various Smad signaling pathways (TGF-b, bonemorphogenetic protein (BMP), activin) regulate hema-topoiesis in vitro and in vivo. Smad signaling participatesin the specification or generation of hematopoietic stemcells (HSCs) during development and continues toregulate the fate of HSCs and their progeny after theirgeneration during development and postnatally. We willdiscuss how genetic approaches using gain-of-function

or lack-of-function animal models can be used toinvestigate the role of Smad signaling in vivo and howthe findings obtained compare with earlier in vitrofindings using active ligands. The outcome of Smadsignaling is very context dependent and will often have adifferent effect in ex vivo cultures than in the bonemarrow (BM) microenvironment where many internaland external signals cooperate to regulate the fate ofHSCs and their progeny. The importance of signalingcrosstalk will be discussed as well as how the knowledgegenerated can be used in clinical medicine, for example,by expanding stem cells for advanced cell therapy in thefuture.

Ligands and receptors that activate Smad signalingpathways

TGF-b is the founding member and prototype of a largesuperfamily of structurally related peptide growthfactors. Other key members of this TGF-b superfamilyare the activins, BMPs and growth and differentiationfactors (Massague, 1998). There are three isoforms ofTGF-b in mammals, TGF-b1 (Derynck et al., 1985),TGF-b2 (de Martin et al., 1987; Madisen et al., 1988)and TGF-b3 (Derynck et al., 1988; ten Dijke et al.,1988), which are encoded by separate genes located atdifferent chromosomes. These isoforms share 70–80%amino-acid sequence identity, bind to the same receptorsand show very similar actions in in vitro culture systems.However, they have distinct expression patterns in vivoand mice lacking either TGF-b1, 2 or 3 overlapphenotypically very little, indicating that there are manyspecific functions for the different isoforms (see below).

Members of the TGF-b superfamily transduce theirsignals across the plasma membrane through transmem-brane serine/threonine kinase receptors. Ligand bindingto these signaling receptors is facilitated by certainaccessory receptors, for example, betaglycan and en-doglin, which have affinity for the ligand but nosignaling activity (Cheifetz et al., 1992; Lopez-Casillaset al., 1993). There are two classes of signaling receptors,type I and type II, both of which are required for signaltransduction (reviewed in Massague, 1998). Type Ireceptors, also known as activin receptor-like kinases(ALKs), have an inactive kinase domain, while type IIreceptor kinases are constitutively active. The ligand*Correspondence: S Karlsson; E-mail: [email protected]

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binds constitutively active type II receptors, whereby theinactive type I receptors are recruited to form aheterotetrameric complex in which the type II receptortransphosphorylates and activates the kinase domain ofthe type I receptor (Wrana et al., 1994). Activated type Ireceptor kinase subsequently phosphorylates intracellu-lar mediators known as the Smad proteins (Figure 1).

The activation of the receptors is tightly regulatedthrough ligand traps, soluble proteins that sequester theligand and block its access to membrane receptors.These include the TGF-b latency-associated polypep-tide, decorin and a2-macroglobulin, which bind to freeTGF-b, and follistatin, which binds to activins andBMPs (Shi and Massague, 2003). The regulation ofreceptor activation is also controlled by membrane-anchored proteins that act as coreceptors and promoteligand binding to the signaling receptors. Betaglycan,often referred to as the TGF-b type III receptor,promotes high-affinity presentation of TGF-b to thesignaling receptor and this is particularly critical forTGF-b2 (Derynck and Zhang, 2003; Shi and Massague,2003). Betaglycan also facilitates the binding of inhibinto activin receptors to block activin signaling. Endoglinis another membrane-anchored protein, structurallyrelated to betaglycan, which facilitates TGF-b signalingin endothelial cells (ECs). Although endoglin does notbind TGF-b by itself, mutations in endoglin and theALK1 receptor can give rise to hereditary hemorrhagictelangiectasia (HHT), indicating a common role forendoglin and ALK1 in maintaining normal vasculardevelopment. More recently, a role for endoglin inhematopoiesis has been suggested because endoglin is

expressed on HSCs (Chen et al., 2002). Antibodiesagainst endoglin can be used to highly enrich repopulat-ing HSCs by flow cytometry, sorting cells that areendoglin positive, Sca-1þ and Rhodamine-low (Chenet al., 2003). The role of endoglin, if any, in theregulation of HSCs is unknown.

Smad signaling pathways that regulate hematopoiesis

The Smad proteins can be divided into three differentgroups depending on their role in the signal transduc-tion: the receptor-activated Smads (R-Smads), thecommon-partner Smad (Smad4) and the inhibitorySmads (I-Smads) (Heldin et al., 1997). R-Smads becomephosphorylated by activated type I receptors, wherebythey oligomerize with Smad4 to form a heterodimericcomplex that is translocated into the nucleus (Figure 1).R-Smad/Smad4 complexes have been shown to interactdirectly with specific DNA sequences as well as withtranscription factors, coactivators and corepressors toregulate transcription of target genes in a cell-type-specific and ligand dose-dependent manner (Heldinet al., 1997; Derynck and Zhang, 2003; ten Dijke andHill, 2004). R-Smads and Smad4 bind to specific DNAsequences with a 100-fold lower affinity than theinteracting high-affinity, DNA-binding transcriptionfactors, yet their DNA binding (except Smad2) isrequired for transcriptional activation (Derynck andZhang, 2003). The I-Smads act as inhibitors of Smad-mediated signal transduction by interacting with thetype I receptor and inhibiting the phosphorylation ofR-Smads (Nakao et al., 1997), by recruiting E3-ubiquitin

Ligand

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Figure 1 Overview of the Smad signaling pathways. Ligands of the TGF-b superfamily transduce their signals throughheterotetrameric complexes of type I and type II receptors, which phosphorylate the intracellular R-Smad proteins. The inhibitorySmads are shown in red. The majority of the components shown are expressed on purified murine HSCs (Utsugisawa, Moody andKarlsson, unpublished studies). ActR-I is also referred to as ALK2, BMPRIA as ALK3, ActRIB as ALK4, TGF-bRI as ALK5 andBMPRIB as ALK6. ALK1 combines with TGF-bRII to mediate signals in ECs, but has not been shown to play a role in hematopoieticcells

Role of Smad signaling in hematopoiesisJ Larsson and S Karlsson

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ligases to degrade activated type I receptors, or by directdephosphorylation and subsequent inactivation of thetype I receptor (Shi and Massague, 2003; ten Dijkeand Hill, 2004). Alternatively, I-Smads may competewith Smad4 in binding R-Smads and thereby preventthe formation of the R-Smad/Smad4 complex (Hataet al., 1998).

Figure 1 describes the close relationship between theTGF-b, activin and BMP signaling pathways. TGF-bsignals through specific type I and type II receptorsknown as TbRI (or ALK5) and TbRII, respectively(Wang et al., 1991; Attisano et al., 1993; Ebner et al.,1993; Franzen et al., 1993). The activated TGF-breceptor complex phosphorylates the R-Smads Smad2and Smad3, which also act as mediators of the activinsignaling pathway (Heldin et al., 1997). In addition tosignaling via ALK5, it has been shown that TGF-bcan signal through another type I receptor (ALK1) inECs, which phosphorylates a different set of R-Smads:Smad1, Smad5 and Smad8 (Roelen et al., 1997; Luxet al., 1999; Oh et al., 2000). These Smads are theintracellular mediators of the BMP signaling pathway.As shown in Figure 1, the type I receptors ALK1, ALK2(ActRI), ALK3 (BMPRIA) and ALK6 (BMPRIB)phosphorylate Smad1, Smad5 and Smad8, whereasALK4 (ActRIB) and ALK5 (TGF-bRI) phosphorylateSmad2 and Smad3 (Derynck and Zhang, 2003; Shi andMassague, 2003; ten Dijke and Hill, 2004). All R-Smadsfrom both the activin and BMP groups use Smad4 as apartner to form a transcriptionally active complex. TheI-Smad, Smad7 inhibits the activity of all R-Smads,while Smad6 shows a more preferential inhibition of theBMP Smads (Itoh et al., 1998).

Specificity of signaling

This convergence in the mediation of signals from TGF-b superfamily members by using the Smad pathwayraises the question of how specificity in signaling fordifferent family members and different isoforms can beachieved. As mentioned above, expression of accessoryproteins like betaglycan and endoglin can affect signal-ing specificity. Different combinations of receptormolecules in the tetrameric receptor complexes allowdifferential ligand binding and a differential signalresponse. For example, activin receptor type II com-bines with ALK4 to mediate activin signals, but when itcombines with ALK3 or ALK6, it mediates signals fromBMP4 (Derynck and Zhang, 2003; Shi and Massague,2003). Heteromeric interactions of activated Smads mayalso determine signaling specificity. Most TGF-bresponses are mediated by Smad3 and Smad4, whereasactivin responses are mediated by Smad2 and Smad4(Derynck and Zhang, 2003). Some TGF-b responsesrequire a concomitant activation of Smad2 and Smad3together with Smad4 (Feng et al., 2000). The cellularcontext is also very important in determining theconsequences of Smad signaling responses since differ-ent cells express a variety of transcription factors andcofactors to determine whether a specific set of genescan be affected, activated or repressed. For example,

Smad3 cooperates with Runx proteins to activatetranscription in epithelial cells, whereas the samepromoters in mesenchymal cells are repressed (Allistonet al., 2001; Derynck and Zhang, 2003).

Duration and intensity of the signal

Upon signal stimulation, Smad complexes accumu-late in the nucleus where they remain for hours (tenDijke and Hill, 2004). The levels of the Smad complexesin the nucleus determine the nature and the durationof the signal. In the nucleus, the R-Smads aredephosphorylated and are disassociated from Smad4and exported from the nucleus. If the receptors areactive, Smad signaling continues, but if the receptorsare inactive, the dephosphorylated Smads accumulateover time in the cytoplasm and signaling stops(Inman et al., 2002; Xu et al., 2002). Apart fromnucleocytoplasmic shuttling of Smads, the durationand activity of Smad pathways can be regulated bydifferent receptor internalization routes. Clathrin-dependent internalization of receptors into earlyendosomes promotes Smad signaling, whereas inter-nalization via lipid raft-caveolar compartments contain-ing receptor bound to Smad7-ubiquitin ligasecomplexes leads to degradation of receptors (DiGuglielmo et al., 2003; Shi and Massague, 2003; tenDijke and Hill, 2004). Therefore, the internalizationpathway has implications for receptor availability andduration of the signal.

Smad-dependent and Smad-independent signalingcrosstalk with other pathways

Smads are the most well-characterized transducers ofTGF-b signaling so far, playing a critical role in mostTGF-b actions. Increasing evidence suggests, however,that TGF-b may also send signals though anotherintracellular signaling pathway, the mitogen-activatedprotein kinase (MAPK) pathway (reviewed in Massa-gue, 2000). The complexity of intracellular TGF-b signaltransduction is further demonstrated by the fact that theSmad pathway is believed to be part of a larger networkinvolving many other signaling pathways, whichare interacting in a so-called ‘signaling crosstalk’(Massague, 2000). Several kinase pathways inhibit orenhance TGF-b-induced nuclear translocation ofSmads. For example, the Erk MAPK pathway, stimu-lated by the activation of tyrosine kinase receptorsand/or Ras, inhibits ligand-induced nuclear transloca-tion of activated Smads (de Caestecker et al., 1998;Kretzschmar et al., 1999; Funaba et al., 2002; Derynckand Zhang, 2003). The Erk MAPK and calcium-calmodulin-dependent protein kinase II inhibit TGF-bsignaling through phosphorylation of Smads at phos-phorylation sites that are different from those thatare phosphorylated by ALKs (Derynck and Zhang,2003; Shi and Massague, 2003). TGF-b signaling canalso be independent of the Smad pathways (reviewed inDerynck and Zhang, 2003). Rapid activation (5–15 min)by TGF-b does not involve Smad-mediated trans-


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criptional responses and independence from Smadactivation has been supported by findings demon-strating MAPK activation in Smad4-deficient cells andcells that express dominant-negative Smads (Engel et al.,1999). Therefore, MAPK pathways can regulate Smadtranscriptional responses, but TGF-b can also activateErk, JNK (c-Jun N-terminal kinase) and p38 MAPKkinase pathways independent of Smad-mediated tran-scription. This complex crosstalk by TGF-b family ofligands with other signaling cascades has also beenextended to other signaling pathways (reviewed inDerynck and Zhang, 2003).

Hematopoiesis and HSCs

All blood cells can be generated from a common HSCthrough an extremely dynamic process called hemato-poiesis or blood cell formation. The generation ofdefinitive or adult-type HSC during development occursin the aorta-mesonephros-gonad region (AGM) of theembryo (Medvinsky et al., 1993; Cumano et al., 1996;Medvinsky and Dzierzak, 1996; Cumano et al., 2000),and more recently there have been reports that mayindicate that definitive HSC are also found in the yolksac and later in placenta (Yoder et al., 1997; Gekaset al., 2005). The fetal liver is seeded with HSC from theAGM and they are expanded there and generate a largeamount of progeny cells. Postnatally, hematopoiesistakes place in the BM where the HSCs as well as a

complex mix of dividing and maturing cells of differentlineages can be found. The process of hematopoiesis canbe described as hierarchical with the rare HSCs at thetop of the hierarchy giving rise first to progenitors andthen to precursors with single lineage commitment andending in terminally differentiated mature cells ofvarious lineages (Ogawa, 1993; Orkin, 2000). In betweenthe HSC and terminally differentiated cells, there is acontinuum of progenitors at different stages, which,depending on certain stimuli, can divide and progresstowards certain lineages (Figure 2). It is generallybelieved that hematopoietic development divides at anearly stage into a myeloid and a lymphoid branch. Thelymphoid branch gives rise to B, T and natural killercells, while the myeloid branch differentiates to all othercell types including erythrocytes. Indeed, two distinctprogenitor populations with either lymphoid or myeloidrestricted potential, the so-called common lymphoidprogenitors and common myeloid progenitors, havebeen isolated from mouse BM (Kondo et al., 1997;Akashi et al., 2000).

HSCs are pluripotent and should therefore be able togive rise to all hematopoietic lineages. They areoperationally defined as cells that can completelyreconstitute a recipient following BM ablation. Simi-larly, they must have the capacity to self-renew to giverise to other stem cells (Domen and Weissman, 1999).A distinction is often made between long-term repopu-lating HSCs (LT-HSCs) and short-term repopulatingHSCs (ST-HSCs) (Morrison and Weissman, 1994). The

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Figure 2 The hierarchy of hematopoietic cells. LT-HSC, long-term repopulating HSC; ST-HSC, short-term repopulating HSC; MPP,multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte/erythroidprogenitor; GMP, granulocyte–macrophage progenitor. The encircled pluripotent population, LT-HSC, ST-HSC and MPP are Lin�,Sca-1þ , c-kitþ as shown


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only way to test these criteria is through BM transplan-tation experiments into lethally irradiated recipients.

Although the HSC can only be absolutely definedthrough BM transplantation assays, advances in cellsorting have allowed near purification of murine HSC.Early committed progenitor populations, depicted inFigure 2, can also be purified (Spangrude et al., 1988;Morrison and Weissman, 1994; Kondo et al., 1997;Akashi et al., 2000). HSC are highly enriched in apopulation that is negative for lineage markers (Lin�)and positive for Sca-1 and c-kit (LSK cells) (Spangrudeet al., 1988). C-kit, which is the receptor for stem cellfactor (SCF), has a wider expression pattern than Sca-1,marking most multipotent progenitors (MPPs) (Lymanand Jacobsen, 1998). Sca-1 and c-kit are often usedtogether for positive selection of HSCs from Lin� cells.The LSK cells contain LT-HSC, ST-HSC and MPPs,which cannot repopulate recipients. Further enrichmentfor LT-HSC has been demonstrated by includingnegative selection for the CD34 marker. Indeed, LSKCD34� cells contain a very high proportion of HSCs andisolated single LSK CD34� cells have been transplantedand have given full long-term hematopoietic recon-stitution (Osawa et al., 1996). A further separationbetween LT-HSC, ST-HSC and MPP has recently beenmade by showing that repopulating HSC express thethrombopoietin (Tpo) receptor (c-mpl), whereas MPPdo not, and MPPs express Flt3, whereas pluripotentrepopulating HSC do not (Adolfsson et al., 2001; Yanget al., 2005a).

Regulation of hematopoiesis

Regulation of HSCs is governed by two intimatelyinvolved entities. One is the gene expression pattern inthe cell and the other is the composition of externalsignals from the BM microenvironment. Transcriptionfactors are internal signals that regulate gene expression,while external signals from the BM microenvironmentcan be mediated by cell–cell interactions, cell–extra-cellular matrix (ECM) interactions and by solublegrowth factors. Figure 3 demonstrates the various fateoptions upon alteration of internal or external signalsthat may promote or antagonize proliferation (self-renewal), quiescence, differentiation, migration, apop-tosis or malignant transformation.

A number of transcription factors have been shown tobe critical intrinsic factors for specification of HSCsduring ontogeny. Nascent HSC in the AGM regionexpress the hematopoietic transcription factors Runx-1,c-Myb, PU.1, SCL and LMO2 (Tavian et al., 1996;North et al., 1999; Orkin and Zon, 2002). Most of thesegenes have also been found to be essential forhematopoiesis using mouse knockout models. They arealso observed in chromosomal translocations in leuke-mias or cause leukemia when misexpressed or disruptedin the mouse (reviewed in Orkin, 2000). Studies inknockout mouse models have elegantly demonstratedcrucial roles of several transcription factors in earlyhematopoietic commitment steps and lineage outcome.For example, GATA-1 is required for erythroid and

megakaryocytic development and Pax-5 for B-celldevelopment (Pevny et al., 1991; Fujiwara et al., 1996;Shivdasani et al., 1997; Nutt et al., 1999; Rolink et al.,1999). The PU.1 and GATA-1 transcription factorshave both been shown to have concentration-dependenteffects on differentiation and lineage choice (Kulessaet al., 1995; DeKoter and Singh, 2000). Enforcedexpression of HoxB4 has been shown to increase theself-renewal potential of primitive murine and humanhematopoietic progenitors (Sauvageau et al., 1995;Antonchuk et al., 2002; Buske et al., 2002) anddeficiency of HoxB4 causes a mild proliferative defectin HSC (Bjornsson et al., 2003; Brun et al., 2004). Apartfrom transcription factors, there are also other intrinsicfactors within the HSCs that are important regulators ofstem cell fate. These include effector molecules in the cellcycle machinery. For example, high levels of cyclin D2have been detected in cycling HSCs, suggesting animportant role in HSC cycling, while studies of knock-out mice have shown that the cyclin-dependent kinaseinhibitor (CDKI) p21 acts to maintain HSCs in aquiescent state (Cheshier et al., 1999; Cheng et al., 2000).

Many hematopoietic growth factors have beenconsidered to be key external regulators of HSCs. SCF(also known as c-kit ligand) and its receptor c-kit as wellas Tpo and its receptor c-mpl are expressed on HSCsand efficiently promote survival of primitive murine andhuman primitive progenitors (Li and Johnson, 1994;Keller et al., 1995; Borge et al., 1996). They can alsosynergize with other growth factors, such as IL-3 andIL-6, to induce proliferation of these primitive cells andare therefore widely used in various in vitro culturesystems (Ramsfjell et al., 1996). Important roles in HSCregulation in vivo have been shown through analysis ofmice lacking either c-kit or c-mpl. These mice havesevere HSC deficiencies as demonstrated by reduced LT

apoptosis

self-renewal

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differentiation

development

malignanttransformation

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Figure 3 HSC fate options. Signals from internal and externalregulatory factors decide whether the HSC are maintained inquiescence, proliferate, undergo apoptosis, migrate out of the BMspace or develop a malignant clone that grows in an uncontrolledmanner


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repopulating ability following transplantation (Milleret al., 1996; Kimura et al., 1998). Flt3-ligand (FL) andits receptor Flt3 has been shown to promote in vitrogrowth of HSCs (Lyman and Jacobsen, 1998). However,a role of FL in regulating the most primitive HSCs isuncertain since Flt3 is not expressed on LTR-HSCs(Adolfsson et al., 2001). Stem cell homeostasis in theadult may also be under control of morphogens thatregulate early embryogenesis (Orkin and Zon, 2002).BMP4 maintains HSC in culture and sonic hedgehog(Shh) may amplify HSC through modulation of theBMP pathway (Bhatia et al., 1999; Bhardwaj et al.,2001). The Notch signaling pathway and the Wntpathway also regulate HSC homeostasis and bothactivation of Notch and stimulation by Wnt have beenshown to increase proliferation of HSC (Van Den Berget al., 1998; Varnum-Finney et al., 2000; Stier et al.,2002; Reya et al., 2003).

It is generally believed that maintenance of HSCs iscontrolled by a balance of positively and negativelyacting signals. Although a number of growth factorshave been identified that exert critical growth promotingeffects on HSCs, as described above, much less is knownabout negative regulation of HSCs. Apart from TGF-b(see below), both tumor necrosis factor-a (TNF-a) andinterferon-g (IFN-g) have been associated with negativeregulation of hematopoietic progenitors (Jacobsen et al.,1992; Maciejewski et al., 1995; Zhang et al., 1995; Yanget al., 2005b). Furthermore, it was recently shown thatTNF-a through activation of Fas inhibits self-renewal ofmurine LTR-HSCs, bringing out TNF-a and Fas asmajor negative regulators of HSCs (Bryder et al., 2001).

Smad signaling and specification of HSCs duringdevelopment

The first signs of hematopoiesis in the developing mouseembryos are the formation of blood islands fromextraembryonic mesoderm in the yolk sac at embryonicday 7.5 (E7.5) (Yoder, 2001). Blood islands arecomposed of primitive, nucleated erythrocytes sur-rounded by ECs. The close spatial and temporal

occurrence of erythrocytes and ECs in the blood islandshas led to the hypothesis that they have a commonmesodermal precursor, the ‘hemangioblast’ (Mikkolaand Orkin, 2002) (Figure 4). By E8–E8.5 precursorswith definitive hematopoietic potential arise in the yolksac as well as intraembryonically along the dorsal aortain a region called para-aortic splanchnopleura, whichlater forms the AGM (Cumano et al., 1996; Medvinskyand Dzierzak, 1996; Yoder et al., 1997). Shortlythereafter, hematopoiesis shifts to the fetal liver andaround the time of birth the BM becomes the majorsite of hematopoiesis. The mechanisms that control thesequential induction of mesoderm, formation of thehemangioblast and commitment of hematopoieticprecursors are still not well understood. An importanttool to investigate these early specification events ofHSCs, in addition to the study of embryos, has been theembryonic stem (ES) cell differentiation system. In theabsence of leukemia inhibiting factor, ES cells willaggregate and form the so-called embryoid bodies (EBs)that are composed of all three germ layers. These can bedifferentiated further in a manner that mimics severalaspects of normal embryonic development in utero,including formation of hemangioblasts from mesoder-mal precursors and the generation of primitive anddefinitive hematopoietic cells (Lacaud et al., 2001)(Figure 4).

A large number of studies in several different specieshave implicated a key role of BMP4 in mesoderminduction and hematopoietic commitment (reviewed inSnyder et al., 2004). For example, studies using Xenopusembryos and explant assays have demonstrated thatBMP4 together with either basic fibroblast growthfactor or activin A can induce the formation of redblood cells from non-mesodermal structures (Huberet al., 1998). Furthermore, hematopoietic commitmentinduced by the GATA binding transcription factorsGATA-1 and GATA-2 required intact BMP signaling(Maeno et al., 1996; Huber et al., 1998). Mice lackingBMP4 die between E7.5 and E9.5 exhibit severe defectsin mesoderm formation and the embryos that survive upto E9.5 show defective blood islands (Winnier et al.,1995). Studies using differentiation of mouse ES cellssupport these findings as BMP4 induces the formation

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Figure 4 Specification of lineages during development. HSCs are formed from the mesodermal germ layer through an intermediateprecursor with both endothelial and hematopoietic potential, the hemangioblast. The Smad signaling pathways critically regulateseveral aspects of the specification and subsequent expansion of HSCs


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of mesoderm and hematopoietic precursors and inhibi-tion of BMP signaling impairs hematopoietic develop-ment in this setting (Johansson and Wiles, 1995; Parket al., 2004). In addition, it was recently demonstratedthat BMP4, in combination with cytokines, pro-motes hematopoietic differentiation of human ES cells(Chadwick et al., 2003). While BMP4 appears to have acrucial instructive role for the induction and formationof blood cell precursors, other factors may regulate theirsubsequent expansion. Vascular endothelial growthfactor (VEGF) has been shown to mediate efficientexpansion and differentiation of BMP4 induced hema-topoietic progenitors (Park et al., 2004). Interestingly,the VEGF effect is inhibited by TGF-b and augmentedby activin A implicating these family members in theregulation of committed blood precursors during devel-opment (Park et al., 2004) (Figure 4).

The original studies of mouse embryos lacking TGF-b1 demonstrated severe defects in the yolk sacs,including malformed vascular structures and an almostcomplete absence of red blood cells leading to deatharound E10 (Dickson et al., 1995). The apparent lack ofhematopoiesis in these embryos suggested a critical rolefor TGF-b1 in directing hematopoietic developmentduring embryogenesis, a notion that was furthersupported from studies of TbRII-deficient embryos(Dickson et al., 1995; Oshima et al., 1996). However,the mechanism behind the lack of hematopoietic cellswas investigated in more detail using TGF-b signaling-deficient embryos lacking TbRI. In striking contrast tothe severe yolk sac anemia, which was present also inthese embryos, a large increase in numbers of erythroidcolony-forming cells (CFCs) could be detected whenyolk sac cells were assayed in vitro (Larsson et al., 2001).These findings demonstrated that induction and com-mitment of hematopoietic precursors during develop-ment are intact in the absence of TGF-b signaling. Thus,in contrast to BMP4, TGF-b1 does not seem to bean important regulator of the earliest specificationevents of hematopoietic commitment. The increasednumbers of erythroid progenitors suggested, however,that TGF-b has an inhibitory function in the earlyexpansion of committed hematopoietic precursorswhich is consistent with the studies mentioned aboveusing ES cell differentiation (Park et al., 2004).

The role of Smad5 in embryonic hematopoiesis hasbeen studied using embryos and ES cells with targeteddisruptions of the Smad5 gene. Disruption of the Smad5gene in mice leads to a phenotype very similar to thatobserved in knockouts for the TGF-b receptors withsevere defects in yolk sac circulation and subsequentdeath around midgestation (Chang et al., 1999; Yanget al., 1999). Yolk sacs from these embryos hadincreased numbers of high-proliferative potential col-ony-forming cells (HPP-CFCs) with enhanced replatingpotential (Liu et al., 2003). Importantly, similar findingswere also demonstrated in EBs derived from in vitrodifferentiated Smad5�/� ES cells (Liu et al., 2003).These EBs had increased numbers of blast colony-forming cells (BL-CFCs), which are believed to be the invitro equivalent of hemangioblasts in addition to a

marked increased frequency of HPP-CFCs. These datasuggest that Smad5 transduces signals that inhibit theearly specification events of hematopoietic precursors aswell as their subsequent expansion (Liu et al., 2003).These findings are somewhat confusing since Smad5 isprimarily thought to be involved in the BMP signalingcascade and BMP4 has quite an opposite role in thisregard. However, Smad5-deficient HPP-CFCs displayeddecreased sensitivity to TGF-b1 inhibition, suggesting arole of Smad5 in mediating TGF-b signals in this setting(Liu et al., 2003). Furthermore, in another study,inhibition of Smad5 was shown to neutralize theinhibitory effects of TGF-b on primitive hematopoieticprogenitors (Bruno et al., 1998). Thus, Smad5 may be anadditional mediator of TGF-b signaling in hematopoie-tic cells (apart form Smad2 and Smad3), but thepotential mechanisms for this are not understood. Ithas been shown that Smad5 differs from the otherBMP-regulated Smads (Smad1 and Smad8) in its DNA-binding abilities and more resembles the TGF-b-specificSmad3 in those aspects (Li et al., 2001). The alternativeTGF-b type I receptor, ALK1, can transduce TGF-bsignals through Smad1, Smad5 and Smad8 in ECs(Roelen et al., 1997; Lux et al., 1999; Oh et al., 2000). Anintriguing possibility would be that the accessoryreceptor endoglin could play a role in this regard.Endoglin and ALK1 seem to mediate highly similarresponses as mutations in the genes encoding eitherendoglin or ALK1 have been directly linked to a humandisease called HHT, which is an autosomal dominantdisorder, characterized by recurrent hemorrhages andvascular dysplasia in multiple organs (McAllister et al.,1994; Johnson et al., 1996). While ALK1 is notexpressed in HSCs (our unpublished observations),endoglin was recently shown to be selectively expressedin the most primitive fraction of hematopoietic cells(Chen et al., 2002).

Smad signaling in adult HSCs

TGF-b signaling regulates multiple aspects of hemato-poiesis and exerts a wide range of responses in cells at alldifferentiation stages of the hematopoietic hierarchy.Here, we have focused our discussion mainly on the roleof TGF-b and Smad signaling in growth regulation ofHSCs. For a detailed review on TGF-b signaling inhematopoiesis in general see Fortunel et al. (2000).

TGF-b1 ligand stimulation of HSCs and progenitors

A critical role for TGF-b in the regulation of HSCs andprogenitor cells was demonstrated more than 15 yearsago. The original findings showed potent inhibition byTGF-b1 on the growth of early MPPs, while moremature progenitors were unaffected (Ohta et al., 1987;Keller et al., 1988). A large number of studies on bothhuman and murine cells have supported these originalfindings of potent growth inhibitory actions on earlyhematopoietic progenitors (Ottmann and Pelus, 1988;Sing et al., 1988; Keller et al., 1990; Jacobsen et al.,


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1991a). However, the effects on more mature progenitorcells are complex and depend on the presence of othergrowth factors. For example TGF-b inhibits IL-3-induced granulocyte–macrophage (GM) colony forma-tion, while GM-CSF-induced GM colony formation isstimulated (Ruscetti and Bartelmez, 2001). Thus, theeffects of TGF-b in these in vitro systems are dependenton the differentiation stage of the target cells and theactions of other cytokines. Studies on purified murineHSCs showed that TGF-b inhibits the initial celldivisions of these cells demonstrating direct regulatoryeffects on HSCs (Sitnicka et al., 1996). Similar directinhibitory actions of TGF-b have also been demon-strated in purified human primitive progenitor cells(Garbe et al., 1997; Batard et al., 2000). The effects ofTGF-b on hematopoiesis have also been studied in vivo.TGF-b1 was injected into the femoral artery andpotently inhibited multipotent hematopoietic progeni-tors in the BM (Goey et al., 1989).

The mechanism of TGF-b action on hematopoieticprogenitors is not fully understood, but the effects are inpart due to modulation of cytokine receptors. TGF-bdownmodulates expression of the receptors for IL-1,GM-CSF, IL-3, G-CSF and SCF (Dubois et al., 1990;Jacobsen et al., 1991b; Dubois et al., 1994). The growthinhibitory effects by TGF-b are mainly thought to beexerted by cell cycle arrest. This is supported by findingsdemonstrating reversibility in the growth inhibitoryactions of TGF-b, suggesting that TGF-b delaysproliferation rather than exerting irreversible negativeeffects such as induction of apoptosis (Sitnicka et al.,1996; Batard et al., 2000). However, a number of reportshave shown involvement of TGF-b also in apoptosis ofBM progenitors. In fact, both apoptotic and antiapop-totic effects of TGF-b have been described (Jacobsenet al., 1995; Veiby et al., 1996; Dybedal et al., 1997).Thus, TGF-b may regulate growth of hematopoieticprogenitors through effects on both cell cycling andapoptosis.

A relative drawback when interpreting effects ofexogenous ligand stimulation involves the difficulty ofruling out toxic effects from very biopotent moleculeslike active TGF-b. The actions of TGF-b are known tobe strongly contextual and it is possible that factors likethe presence or absence of serum or the degree ofcytokine stimulation used for in vitro culture maystrongly affect the biological responses observed.Although soluble growth factors can be intravenouslyinjected into mice to test also their in vivo functions, it isvery difficult to control the concentrations and distribu-tion of the growth factor or the efficiency of antibodyneutralization. Interpretations of experiments involvinginjection of active TGF-b in vivo are also complicated bythe fact that active TGF-b is a very potent growth factorwith a short half-life and is generated from the morestable latent TGF-b form (Taipale et al., 1994) (Mungeret al., 1997; Crawford et al., 1998; Fortunel et al., 2000).Further, the levels and spatial distribution achievedfrom exogenous sources of active TGF-b could very wellexert effects that are not relevant in the cells physiolo-gical context. Since practically all cell types, including

those of hematopoietic origin, express TGF-b receptors,they are also likely to respond to exogenous TGF-bstimulation.

TGF-b ligand neutralization

The role of endogenous TGF-b has been investigated inseveral studies using TGF-b neutralizing antibodies orantisense oligonucleotides to block TGF-b signaling invitro. Such treatment of early human hematopoieticprogenitors releases these cells from quiescence andenhances the frequency of colony formation (Fortunelet al., 1998; Hatzfeld et al., 1991). Similar effects havebeen demonstrated in the murine system. Culture ofmouse BM cells in the presence of an anti-TGF-bantibody resulted in increased long-term in vivo repo-pulation ability of these cells, suggesting that interferingwith TGF-b signaling may cause expansion of HSCs invitro (Soma et al., 1996). These studies have implied animportant role of endogenous TGF-b signaling inmaintaining quiescence of HSCs. The effects of TGF-bneutralization have further been evaluated in severalstudies using retroviral gene transfer to hematopoieticcells. Release from quiescence is a prerequisite forsuccessful gene transfer with oncoretroviral vectors anda number of studies have demonstrated increasedretroviral transduction efficiency of both murine andhuman primitive hematopoietic progenitors followingneutralization of TGF-b (Hatzfeld et al., 1996; Imbertet al., 1998; Yu et al., 1998). However, when evaluatingresults from such studies, a particular concern is thecommon use of high concentrations of serum in theculture medium. Indeed, when levels of latent and activeTGF-b1 were quantified in serum used for hematopoie-tic cultures, significant levels of both forms could bedetected (Dybedal and Jacobsen, 1995). Thus, observedeffects could at least partly be due to neutralization ofexogenous TGF-b in the medium rather than blockedendogenous signaling. Therefore, experimental ap-proaches involving loss of function may be preferredto determine the physiological role of a particularsignaling pathway.

Genetic approaches for inactivation of TGF-b signaling inHSCs

While numerous in vitro studies have suggested a criticalrole of TGF-b in negatively regulating proliferation ofhematopoietic stem- and early progenitor cells, the roleof endogenous TGF-b signaling in hematopoiesis in vivohas until recently remained largely unknown. Althoughgenetic manipulation of hematopoietic cells can be usedeffectively to study fate decisions in hematopoietic cellsin vitro, genetic perturbation of Smad signaling offers aparticularly promising approach to study the role ofSmad pathways in the function and fate options ofhematopoietic cells in vivo. Gain-of-function approachescan be used through retroviral transgenesis of BM cellsfollowed by transplantation to restrict the phenotype tothe hematopoietic system (Sauvageau et al., 1995) orthrough the use of transgenic mice overexpressing


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ligands, mutant receptors or Smads. Overexpression ofthe TGF-b ligands has been used to study liver fibrosisand kidney pathology (Sanderson et al., 1995), butstudies using overexpression of ligands can generatecomplex phenotypes because many effects are not cellautonomous. The regulation of T-cell development andfunction was effectively studied by targeting expressionof a dominant-negative TGF-b receptor II to T cells inmice (Gorelik and Flavell, 2000; Lucas et al., 2000).However, for studies of HSCs, overexpression intransgenic mice has been used to a quite limited extentmostly because good tissue-specific promoters for theHSC compartment are not available, and even if theywere, they could cause a lethal phenotype duringdevelopment and their use would then be limited todevelopmental hematopoiesis. Inducible systems havebeen developed to study genetic regulation of hemato-poiesis (Bjornsson et al., 2001), but this approach, whilepromising, has not been applied so far to study theSmad signaling pathway in hematopoietic development.

Lack-of-function studies using gene knockout micemay be the most accurate way to study the physiologicalrole of a certain gene or regulatory pathway in vivo.However, conventional knockout models have somelimitations. For example, knocking out genes that areimportant during development often results in embryo-nic or early lethality. This has been a commonphenotype for many genes associated with stem cellregulation, like TGF-b and its receptors (Shull et al.,1992; Kulkarni et al., 1993; Kaartinen et al., 1995;Oshima et al., 1996; Sanford et al., 1997; Larsson et al.,2001). Important insights into the role of TGF-b inregulating the immune system came from the TGF-b1knockout mice (Shull et al., 1992; Kulkarni et al., 1993).These animals exhibited two phenotypes: around 50%of the mice died during embryogenesis from failed yolksac circulation as discussed above (Dickson et al., 1995),while the others survived beyond birth, due to maternaltransfer of TGF-b1 via the placenta (Shull et al., 1992;Kulkarni et al., 1993; Letterio et al., 1994). Survivingmice developed a rapid wasting syndrome shortly afterbirth and died at an age of 3–5 weeks exhibiting massiveinflammatory infiltrates (mainly lymphocytes andmacrophages) and tissue destruction in several organs,particularly the heart and lungs. The early lethality andmassive inflammatory response made it very difficult tocarry out detailed studies of hematopoiesis and HSCfunction in the TGF-b1 knockout mice. The Cre/loxPconditional knockout system has become an importanttool to bypass embryonic lethality and to direct a geneknock out to a certain tissue, cell type or time pointduring development and in the adult mouse (Sauer,1998). The success of this system is highly dependent onspecific and efficient Cre delivery from transgenic mice.The IFN-inducible MxCre transgenic mouse has be-come a very useful tool for Cre delivery in thehematopoietic system because it expresses Cre in HSCupon induction with IFN (Kuhn et al., 1995; Horcheret al., 2001; Brakebusch et al., 2002; Gerber et al., 2002).Thus, this system allows for accurate studies of steady-state hematopoiesis in adult mice or the behavior of

HSCs under stress, for example, during recovery afterBM transplantation or serial transplantation.

HSCs in conditional knockout mice for TGF-b receptors

The physiological role of TGF-b signaling in adulthematopoiesis has been assessed using conditional Cre/loxP knockout mice for the TGF-b receptors I and II.By mating the animals to the inducible MxCretransgenic mice, the consequences of disrupted TGF-bsignaling could be studied in adult hematopoiesis. Asexpected, since both TbRI and TbRII are believed to benecessary for TGF-b signal transduction, knockoutinduction of either TbRI or TbRII caused a completeblock in TGF-b signaling and the phenotypes in thesemice were indistinguishable (Leveen et al., 2002; Larssonet al., 2003). The induction of receptor-knockout inadult mice lead to a lethal disorder by 8–10 weeks afterinduction, in many ways similar to the phenotype inTGF-b1-deficient mice (Leveen et al., 2002). Histo-pathological examination showed multiple inflamma-tory lesions in several organs. Surprisingly, however,and unlike the TGF-b1 mice, BM from these receptor-deficient animals, taken immediately after induction,caused inflammation and death by 6–9 weeks post-transplantation when transferred in limited numbers tonormal C57BL/6 recipients. The findings demonstratedthat TGF-b receptor-deficient cells of hematopoieticorigin, most likely T cells, could induce multifocalinflammatory disease in a dominant manner (Leveenet al., 2002). However, before the onset of disease allhematopoietic parameters were normal in the inducedknockout mice (Larsson et al., 2003). Transplantation ofBM cells to the immune-deficient nude mice bypassedthe inflammatory disease and provided a model to studythe LT repopulation kinetics of knockout HSCs.Surprisingly, when the proliferation kinetics of TGF-bsignaling-deficient HSCs were measured by competitiverepopulation in this model, no difference compared tocontrols was observed, neither in primary nor insecondary recipients (Larsson et al., 2003). The sameapplied when HSCs were further challenged underconditions of severe hematopoietic stress triggered bymyeloablative treatment or serial transplantations(Larsson et al., 2005). Thus, these experiments clearlysuggested that endogenous TGF-b signaling does notplay a critical role in regulating growth and maintenanceof HSCs in vivo. This is in sharp contrast to thenumerous reports demonstrating very potent effects ofblocking TGF-b in these cells in vitro and could possiblybe explained by differences in the experimental ap-proach to interfere with TGF-b signaling: transientblock using antibodies or antisense oligonucleotidesversus permanent genetic deletion of the receptors.However, when TGF-b signaling-deficient HSCs frominduced receptor-knockout mice were assayed in vitrowith low cytokine stimulation (SCF alone), these cellshad a significantly increased recruitment to prolifera-tion, consistent with other in vitro studies. This suggeststhat differences in the in vitro and in vivo environmentare the main reason for the different outcomes between


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these experimental settings. Cells in the hematopoieticmicroenvironment, including BM stromal fibroblasts,osteoblasts and ECs, present growth regulatory signalsto HSC and play an important role for HSC regulationin the BM environment (Calvi et al., 2003; Zhang et al.,2003) (Figure 5). In addition, interactions between ECMcomponents and membrane bound molecules on HSCssuch as the integrins allow for adhesion and homing ofcirculating HSCs to the BM microenvironment (Potoc-nik et al., 2000). These interactions are also thought totransduce signals to the intracellular space, and act tomaintain the HSCs in a quiescent state (Krause, 2002)(Figure 5). Thus, there are numerous signaling entities inthe in vivo microenvironment that are not present instandard in vitro culture systems. The extreme expansionpotential of HSCs requires a tight regulatory networkwith backup mechanisms in order to minimize the riskof uncontrolled growth and differentiation. Crosstalkand compensatory mechanisms from other signalingpathways are therefore likely to be more pronounced invivo and may very well explain the differences betweenour findings in vivo and those from in vitro studies. Animportant observation when the growth kinetics ofknockout HSCs were studied in vitro under serum-freeconditions was that increased proliferation recruitmentof HSCs only was evident under low stimulatoryconditions (SCF alone) and could not be observed whenadditional cytokines were added to the medium (Lars-son et al., 2003). This suggests that endogenous TGF-bsignaling, in this experimental setting, only executessignificant antiproliferative effects when positive actingsignals are kept at low levels. The same phenomenonwas seen when primitive human progenitors overexpres-sing a dominant-negative mutant of the TGF-b type IIreceptor were grown under similar conditions withvarying cytokine stimulation (Fan et al., 2002). To-gether, these observations demonstrate the contextdependency of TGF-b signaling in HSCs and how

activation of several signaling pathways can result ininterference. It is not clear, however, whether this occursat the level of intracellular signaling through specificcrosstalk between signaling pathways or whether thecellular responses from positively acting signals simplydominate over the effects from inhibitory signals, in thiscase endogenous TGF-b signaling. Similar mechanismscould apply in vivo. The degree of cytokine stimulationwithin the BM compartment is highly dependent on thelevel of hematopoietic stress. Thus, under conditionsrequiring high demands on the hematopoietic system,such as the recovery from irradiation or cytotoxictreatment, the signals within the BM microenvironmentare quite different than under normal steady-statehematopoiesis. Thus, the behavior of HSCs followingtransplantation as studied in the receptor-knockoutmice may not be representative for normal steady-stateconditions. It will therefore be important to createmodels where development and maintenance of TGF-bsignaling-deficient HSCs can be followed over a longperiod of time under normal steady-state conditionswith less stimulation.

The case of TGF-b2

The vast majority of studies of TGF-b signaling inhematopoiesis have specifically investigated the role ofthe TGF-b1 isoform. Although some in vitro studieshave suggested inhibitory effects of TGF-b2 and TGF-b3 on the growth of primitive progenitors, the roles ofthese TGF-b isoforms in the regulation of hematopoiesishave remained mostly unclear (Ohta et al., 1987;Ottmann and Pelus, 1988; Jacobsen et al., 1991a).However, very recent work has demonstrated anunexpected positive regulatory role of TGF-b2 in HSCsand progenitors. Langer et al. (2004) studied prolifera-tion of primitive hematopoietic progenitor cells andfound a biphasic dose response to TGF-b2 where low

HSC

Osteoblasts

Fibroblasts

Endothelial cells

cytoskeletonintegrins

membrane boundgrowth factors

Bone

soluble growth factors

ECM

Figure 5 The principal regulatory entities within the HSC niche in the BM microenvironment. Stromal fibroblasts, osteoblasts andECs as well as ECM components present growth regulatory signals to HSCs


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concentrations exerted stimulatory effects, while higherconcentrations were inhibitory. Interestingly, the re-sponse to TGF-b2 was also determined by geneticbackground with extensive variation between mousestrains. A quantitative trait locus (QTL) was identifiedfor the TGF-b2 effects on HSC proliferation thatoverlapped with a previously reported QTL-regulatingHSC frequency (Langer et al., 2004). Furthermore,frequency, proliferation and repopulation ability of HSCsin heterozygous knockout mice for TGF-b2 were lowercompared to wild type, demonstrating the significance ofthe overlapping QTLs (Langer et al., 2004). Thus, thefindings suggest that TGF-b2, as opposed to TGF-b1,has an important role as a positive regulator of HSCs invitro and in vivo. The distinct properties of TGF-b1 andTGF-b2 in the regulation of HSCs are puzzling and raisethe question how their divergent responses are executedat the molecular level given the fact that the isoformsshare significant sequence homology and signal throughthe same receptors. It has been shown that TbRII as wellas the accessory receptors endoglin and betaglycan bindTGF-b1 and TGF-b2 with different affinities, whichcould help explain the intrinsically different effects of thetwo isoforms in HSCs (Cheifetz et al., 1992; Lopez-Casillas et al., 1993; Rodriguez et al., 1995). However, themolecular mechanism behind the fundamentally differentresponses for the two isoforms and whether differentSmads are involved in transducing their signals remainsunknown.

The BMPs in adult hematopoiesis

Apart from the important role in specification of HSCsduring development, described above, the BMPs and inparticular BMP4 have been shown to regulate prolifera-tion of adult HSCs. Treatment of highly enrichedhuman HSCs (CD34þCD38�Lin�) with BMP2 andBMP7 inhibited proliferation similar to TGF-b1 (Bhatiaet al., 1999). However, using relatively high concentra-tions, BMP4 enhanced survival and increased theengraftment in nonobese diabetic-severe immunodefi-ciency (NOD/SCID) mice following ex vivo culture(Bhatia et al., 1999). Thus, BMP4 may act as animportant positive regulator of both proliferation andsurvival of HSCs. Interestingly, it was recently demon-strated that BMP4 signaling is important in mediatingShh-induced proliferation of primitive human progeni-tors (Bhardwaj et al., 2001). Noggin, a specific inhibitorof BMP4, inhibited Shh-induced proliferation, butinhibition of Shh had no effect on BMP4-inducedproliferation (Bhardwaj et al., 2001). Similar associa-tions between Shh signaling and BMP4 have beendemonstrated in Drosophila. Both BMP and Shhrepresent pathways that are well conserved betweenembryonic settings and adult tissue stem cells as well asbetween different species, and it will certainly be animportant task to further elucidate the molecularmechanisms behind the complex signaling crosstalk thatoccurs between these and other pathways.

Little is known about the in vivo role of BMPs inhematopoiesis mainly due to early lethality of knockout

mice for the ligands and receptors. Conditional knock-out mice for the BMP type I receptor, BMPRIA,revealed however, an important, yet indirect role ofBMP signaling in regulation of HSCs by influencing theosteoblasts in the stem cell niche (Zhang et al., 2003).The absence of BMPRIA caused increased numbers ofHSC-supporting osteoblastic cells lining the bone sur-face, which in turn led to an increase in the numbers ofHSCs in these mice (Zhang et al., 2003). A role for BMPsignaling in stress erythropoiesis in the spleen hasrecently been described (Lenox et al., 2005)

Interfering with the entire Smad pathway

Since Smad7 has been shown to block all Smadsignaling pathways, enforced expression of Smad7 canbe used to greatly reduce or eliminate Smad signaling inHSC. Recently, overexpression of Smad7 in humanCD34þ hematopoietic cells was found to augmentmyeloid differentiation at the expense of lymphoidcommitment (Chadwick et al., 2005). The Smad7protein is produced in Lin� hematopoietic cells andSmad7 mRNA is expressed in purified LSKCD34�

murine HSC (unpublished observations). When BMcells overexpressing Smad7 are transplanted into irra-diated recipients, the repopulation activity is increased,and upon serial transplantation, there is a significantincrease in the regeneration of all hematopoietic lineages(Blank et al., 2004). This demonstrates that enforcedexpression of Smad7 can increase self-renewal of HSC invivo. In contrast, BM cells overexpressing Smad7 in astroma-free ex vivo liquid culture do not exhibitincreased proliferative capacity. Rather, the prolifera-tion is reduced (Blank et al., 2004). The findings indicatethat a total block in Smad signaling increases self-renewal of HSC in the BM microenvironment and thatsignaling crosstalk, perhaps mediated by the cells in theBM HSC niches, will increase self-renewal in Smadsignaling-deficient HSC. These findings demonstratethat the outcome of HSC fate decisions mediated byall Smad signaling pathways is context dependent,as is seen with specific TGF-b signaling (Larssonet al., 2003).

Involvement of TGF-b in hematopoietic malignancies

While alterations in TGF-b pathway genes havecommonly been found in many epithelial neoplasms,for example, pancreatic cancer and colon cancer(Villanueva et al., 1998; Grady et al., 1999), mutationalinactivation involving the TGF-b signaling pathway isuncommon in leukemias and other hematologicalmalignancies (reviewed in Kim and Letterio, 2003). Apotential role of TGF-b as a tumor suppressor has alsobeen demonstrated in knockout mice. Heterozygousknockout mice for TGF-b1 get increased numbers oflung and liver tumors upon exposure to carcinogenicstimuli (Tang et al., 1998) and homozygous knockoutmice for Smad3 live to adulthood, but spontaneouslydevelop metastatic colorectal cancer, clearly supporting


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a role of the TGF-b signaling pathway in tumorsuppression (Zhu et al., 1998). In contrast, we havenot been able to detect any signs of hematologicalmalignancies in conditional knockout mice for TGF-breceptor I or transplanted BM recipients, where theknockout is induced in HSC just prior to transplanta-tion (Larsson et al., 2003). These findings suggest thatdeficient TGF-b signaling alone is not sufficient toinduce neoplastic transformation in hematopoietic cells.

In an interesting recent study, the Smad3 proteincould not be detected in fresh samples from patientswith T-cell acute lymphocytic leukemia (ALL). Themechanism for the Smad3 deficiency is not known sincethe Smad3 mRNA was present and no mutations couldbe detected in the MADH3 gene (Wolfraim et al., 2004).However, T-cell leukemogenesis was promoted in micewith haploinsufficiency of Smad3 that were completelydeficient in p27Kip1 (Wolfraim et al., 2004). Thesefindings are interesting because the p27Kip1 gene isfrequently mutated in pediatric ALL, due to transloca-tions and deletions or germline mutation (Komuroet al., 1999; Takeuchi et al., 2002). Impaired TGF-bsignaling in hematologic malignancies can also becaused by suppression of Smad-dependent transcriptionresponses by Tax, Evi-1 and AML1/ETO oncoproteins(Pietenpol et al., 1990; Kurokawa et al., 1998; Jaku-bowiak et al., 2000). These findings agree with thenotion that loss of TGF-b responsiveness in hematologicneoplasms is commonly caused by altered expression ofSmad signaling coactivators or corepressors or due tomutations in TGF-b target genes (Kim and Letterio,2003). In contrast, primary mutations in TGF-b path-way genes, for example, in TGF-b receptors or Smad4,are rare in hematological malignancies, although singlecases have been reported (Knaus et al., 1996; Schiemannet al., 1999; Imai et al., 2001).

Stem cell expansion and development of cell therapy

Blood and marrow transplantation (BMT) is a well-established therapeutic modality to treat malignancieslike leukemias and lymphomas and genetic disordersthat affect the hematopoietic system. As it can be achallenge to find donors who have compatible histo-compatibility antigens for the patients who need BMT,umbilical cord blood (CB) is used increasingly as asource of HSC due to the common availability of CBcells and the diversity of histocompatibility genehaplotypes that are available in the banked CB samples(Gluckman et al., 1989). The number of HSC in each CBsample is limited and it would, therefore, greatlyincrease the applicability of this stem cell source if theCB stem cells could be expanded ex vivo prior totransplantation. Stem cell expansion can also theoreti-cally be accomplished in vivo following engraftment ofthe transplanted cells, but this approach may involvemore risks than ex vivo expansion unless the growthstimuli used in vivo can be carefully controlled (reviewedin Attar and Scadden, 2004; Sorrentino, 2004). How-ever, recent interesting findings suggest the possibility of

increasing the number of stem cell niches in the BMmicroenvironment through alterations in BMP signal-ing. Mice with a conditional knockout of the BMPRIAreceptor exhibit an increase in trabecular bone and anincrease in the number of HSCs (Zhang et al., 2003).The increased number and/or size of HSC niches wasmediated through osteoblast–HSC contacts. Similarly,transgenic mice expressing parathyroid hormone inosteoblasts showed increased number of osteoblasts intrabecular bone and an increase in the size of the HSCpool through activation of the Notch pathway in HSCgenerated by the osteoblast–HSC association (Calviet al., 2003).

Stem cell expansion ex vivo will be easier to developand involves fewer risks than in vivo expansion becausethe exact culture conditions can be controlled bydefining the components in the media and transientmodulation of regulatory pathways can be employed.However, even this approach is a challenge sincesuccessful stem cell expansion involves symmetricdivisions of HSCs where the progeny cells are bothHSC (Attar and Scadden, 2004). More commonly,HSCs grown in vitro undergo asymmetric divisionswhere one HSC and a more differentiated progenitor isproduced, or even worse, a symmetric division whereboth progeny cells have lost their HSC potential.

Since the transition from quiescence to proliferationof HSC is regulated by a balance between positive andnegative regulators, the strategies for stem cell expan-sion should involve activation of regulators thatencourage HSC self-renewal and inhibit pathways thatmediate quiescence, differentiation or apoptosis of theHSC. The classical hematopoietic growth factorsincrease HSC survival and differentiation, but self-renewal is only induced to a limited effect. Owing to therecent advances in understanding the mechanisms thatcontrol HSC self-renewal, novel approaches to expandstem cells can now be developed (Attar and Scadden,2004; Sorrentino, 2004). In order to accomplish this,several different signaling pathways, transcription fac-tors and cell cycle regulators have to be simultaneouslymodulated to achieve the desirable effect (Figure 6).

There is a potential role for modulation of Smadsignaling to increase self-renewal of HSC and expandstem cells. The findings that BMP4 enhances prolifera-tion of human HSC as described above indicate thatBMP4 may be used in stem cell expansion protocols.Since TGF-b1 inhibits proliferation of primitive pro-genitors and perhaps HSC, antibodies against TGF-b1have been used as a strategy to enhance HSC prolifera-tion and engraftment of transplanted mouse recipients.Preliminary findings have suggested that antisensetowards TGF-b can enhance the rate and absolute levelof engraftment in mice (Bartelmez et al., 2000), althoughthe mechanism of the proliferative effect is not clearsince TGF-b signaling-deficient HSC (that are deficientin signaling from all TGF-b isoforms including TGF-b2)do not exhibit increased proliferation in vivo (Larssonet al., 2003). Interestingly, inactivation of TGF-b inserum-free medium did not enhance HSC proliferationwithout simultaneous downregulation of p27 (Dao


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et al., 1998). However, there may be potential use forinactivation of this negative regulator in future clinicalstem cell expansion protocols. It is not clear at presentwhether inactivation of other negative HSC regulatorycytokines like IFN-g or TNF-a may be useful for clinicalstem cell expansion, but clear inhibitory effects of thesefactors have been demonstrated in murine HSC(Jacobsen et al., 1992; Maciejewski et al., 1995; Zhanget al., 1995; Bryder et al., 2001; Yang et al., 2005b).

Modulation of Smad signaling pathways alone maynot be sufficient to achieve effective stem cell expansion,but may be effective if used in concert with theactivation of key positive regulatory pathways thatpromote HSC self-renewal. These include the transcrip-tion factor HoxB4 and the Wnt and Notch pathways.When HoxB4 is expressed in HSC ex vivo, over 40-foldnet expansion of HSC was generated in 10–14 days(Antonchuk et al., 2002). Further, modified HoxB4proteins have been used successfully to stimulateproliferation of human and murine HSC ex vivo(Amsellem et al., 2003; Krosl et al., 2003). The solubleform of the HoxB4 protein is actively taken up by HSCand can be used without concerns about insertionalmutagenesis caused by HoxB4 retroviral vectors or thepotential adverse effects that continuous production ofHoxB4 in HSC might generate in vivo. Developmentalcues that activate Notch and Wnt signaling in HSCmay also be useful to increase HSC self-renewal ex vivo.Wnt3A can expand murine repopulating HSC andWnt5A injection into NOD/SCID mice repopu-lated with human hematopoietic cells increased thereconstitution and number of primitive hematopoietic

cells. Similarly, a soluble form of the Notch ligand,Jagged 1, can stimulate growth of human HSCs andmay therefore be used to expand stem cells ex vivo(Karanu et al., 2000).

The approaches described above involve the use ofsoluble proteins (or potentially matrix-fixed ligands,when required) to stimulate self-renewal of HSC andminimize differentiation since implementation of clinicalstem cell expansion using these approaches is relativelystraightforward provided that the proteins can beefficiently produced. Direct genetic modification canalso be used using viral gene expression vectors todownregulate inhibitory pathways or for upregulationof pathways that stimulate self-renewal. Downregula-tion of CDKIs may be a feasible approach to increaseproliferation and self-renewal of HSC. Downregulationof p21 in CD34þ ,CD38� human hematopoietic pro-genitors led to increased proliferation of primitivehematopoietic cells in vitro and in vivo (Stier et al.,2003) and HSC deficient in p18 exhibit increased self-renewal in vivo (Yuan et al., 2004). Viral vectorscontaining small interfering RNAs against p21 andp18 can be used to transduce HSC and may be able torelease them from quiescence and increase proliferationand perhaps self-renewal divisions. Similarly, Smad7vectors may be able to increase self-renewal of HSCin vitro if crosstalk pathways that help Smad7 to mediatethe self-renewal effect in vivo can be identified and usedin concert with enforced expression of Smad7. Theseapproaches require viral vector-mediated gene transferto HSC for efficient gene delivery. Ideally, the vectorsshould be nonintegrating and mediate transient geneexpression for safety reasons (Baum et al., 2003; Hacein-Bey-Abina et al., 2003; Woods et al., 2003). However,integrating vectors may be considered if the expressionof the gene can be regulated upon demand (Bjornssonet al., 2001) and the vector design includes chromatininsulators and other safety-design features meant tominimize the risk of insertional oncogenesis (Baumet al., 2003).

Effective stem cell expansion can only be achieved ifmultiple gene and signaling pathways are modulated inconcert as described above for TGF-b and p27 (Daoet al., 1998) and the recent observation that p21deficiency enhances the ability of HoxB4 to increaseself-renewal (Miyake et al., 2004). Figure 6 shows aschematic picture of how stem cells may be expanded inthe future using modulation of many genetic pathwaysincluding signaling pathways, transcription factors andinternal cell cycle regulators. It may even be possible tomodify these genetic pathways with small moleculedrugs that modulate gene expression as has been shownin the modulation of Wnt signaling and the expressionof p21 and HoxB4 (Sato et al., 2004; Bug et al., 2005;De Felice et al., 2005).

Conclusion

Smad signaling plays an important role in the regulationof hematopoiesis, both in the specification of HSCs and

Sonic Hedgehog Wnt

BMP4Notch

HoxB4Smad7

TGF-β1

p21

p18

Stimulators

Negative regulators

Figure 6 Modulation of stem cell signals to achieve stem cellexpansion. Several different signaling pathways, transcriptionfactors and cell cycle regulators will have to be simultaneouslymodulated to achieve efficient expansion


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in the regulation of their behavior postnatally. Usinggenetic models, new knowledge has been emergingabout the role of Smad signaling in vivo. While TGF-bis an important negative regulator of HSCs in vitro, it isclear that disruption of TGF-b signaling in vivo has alimited impact on stem cell fate decisions. Similarly,overexpression of Smad7 in vivo increases the self-renewal of HSC and their regenerative capacity follow-ing BM transplantation, whereas the same cells exhibitreduced proliferative capacity in vitro. These findingsemphasize the complex regulation of stem cells in theBM microenvironment and the many internal andexternal signals that cooperate to regulate homeostasisof stem and progenitor cell populations. The finaloutcome of stem cell fate decisions is regulated by acomplex crosstalk of many signaling pathways, tran-scription factors and cell cycle regulators, and dysregu-lation of hematopoiesis is frequently seen when morethan one pathway is altered in vivo. However, it is easierto regulate stem cell fate in vitro when liquid culturemedia can be well defined and ligands, antibodies andgenetic manipulation can be used to alter fate options ofstem cells and their progeny. The emerging knowledgeabout how Smad and other signaling pathways regulate

stem cell behavior may be used in the future to developadvanced cell therapies, for example, through stem cellexpansion in vitro. The benefits for patients are obvioussince many patients that lack a suitable BM donor canpotentially be treated with stem cell sources that areinadequate for treatment of adult patients at present.

Acknowledgements

We would like to thank Ann CM Brun and Ulrika Blankfor designing several figures and David Scadden for enthu-siastic support. We are indebted to our colleagues, JenniferMoody, Ulrika Blank, Goran Karlsson and Sofie Singbrantfor critical reading of the manuscript and for helpfulsuggestions. SK is supported by grants from The SwedishCancer Society, The European Commission (CONSERT), TheSwedish Gene Therapy Program, The Swedish MedicalResearch Council, The Swedish Children Cancer Foundation,A Clinical Research Award from Lund University Hospital,The Joint Program on Stem Cell Research supported by TheJuvenile Diabetes Research Foundation and The SwedishMedical Research Council. JL is supported by a fellowshipfrom The European Molecular Biology Organization. TheLund Stem Cell Center is supported by a Center of Excellencegrant in life sciences from the Swedish Foundation forStrategic Research.

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