haematopoietic stem cells

Review Article

Haematopoietic stem cells{

Dominique Bonnet*Cancer Research UK, London Research Institute, London, UK

*Correspondence to:D. Bonnet, PhD, HaematopoieticStem Cell Laboratory, CancerResearch UK, London ResearchInstitute, 44 Lincoln’s Inn Fields,London, WC2A 3PX, UK.E-mail: [email protected]

Abstract

Considerable efforts have been made in recent years in determining the composition of the cell

types that constitute the human haematopoietic stem cell (HSC) compartment. These studies have

emphasized the heterogeneity of the human HSC in terms of proliferative and self-renewal

capacities. Recent studies have indicated that CD34 is not the universal marker of all human

HSCs. New markers for purifying HSCs have been described. A number of genes that regulate

the formation, self-renewal, or differentiation of HSCs has been identified. The elucidation of the

molecular phenotype of the HSC has just begun. Finally, an unexpected degree of developmental

or differentiation plasticity of HSC has emerged. This review summarizes all the recent advances

made in the human HSC field and examines the impacts that these discoveries may have both

clinically and in understanding the organization of the human haematopoietic system. Copyright

# 2002 John Wiley & Sons, Ltd.

Keywords: haematopoietic stem cell (HSC); xenotransplantation model; NOD/SCID; fetal

sheep; regulation; plasticity; transdifferentiation

Introduction

Stem cell populations from a variety of tissues offergreat promise for tissue regeneration, cell-based trans-plantation therapies, and the eventual development ofclinically effective gene therapy protocols [1–3]. Thebest characterized stem cells are those responsible forhaematopoiesis. All the experimental strategies andconceptual paradigms that are applicable to stem cellsin general were defined first in this system [4]. The hall-mark properties of haematopoietic stem cells (HSCs)are as follows: HSCs have the ability to balance self-renewal against differentiation cell fate decisions; theyare multipotent, a single stem cell producing at leasteight to ten distinct lineages of mature cells; they havean extensive proliferative capacity that yields a largenumber of mature progeny; HSCs are rare, with afrequency of 1 in 10 000 to 100 000 total blood cells;they are slowly cycling in a steady-state adult haema-topoietic system. Thus, determining the compositionand relationship of the cell types that constitute thehuman stem cell compartment may help both toidentify the cellular and molecular factors that governnormal and leukaemic stem cell development and toadvance clinical applications of transplantation, genetherapy, stem cell expansion, and tumour cell purging.Furthermore, recent studies have provided compellingevidence that the adult stem cells may have a pre-viously unsuspected degree of developmental or differ-entiation plasticity (see the review in this issue byPoulsom et al. and reviews in refs [5–11]). These recent

findings have shattered the existing dogma that onlyembryonic stem cells are capable of giving more thanone tissue.

In this review, we present a brief summary of themost recent findings in the field of human HSCs,discussing the assays used so far, the cellular and mole-cular phenotypes of HSCs, and the regulatory mechan-isms involved. We will finish with a short overview onrecent results on HSC plasticity and the potential cli-nical implications of this new property.

Characterization of HSCs

Surrogate in vitro assays

A number of in vitro assays have been described thatassess primitive human progenitors. These include long-term culture-initiating cell (LTC-IC) assays, whichdetect primitive cells capable of giving rise to colony-forming cells (CFCs) after 5 weeks of culture on com-petent feeder layers [12,13]; cobblestone area-formingcell (CAFC) assays [14,15]; and extended-LTC-IC (E-LTC-IC) assays [16]. The E-LTC-IC defines a smallsubpopulation of LTC-IC, which has more extensiveproliferative capacity and can be maintained for up to10 weeks in culture. These assays enumerate primitivemyeloid progenitors, but not cells with multi-lineagedifferentiation or self-renewal potential. More recently,several groups have developed cultures that allow thedifferentiation of single human LinxCD34+ cells intocells with myeloid, natural killer (NK), B-lymphoid,dendritic, and/or T-lymphoid phenotypes, showing thata single cell can differentiate in vitro into multiplelineages [17–19]. However, none of these assays is ableto generate secondary primitive progenitors that again

{Note: Cancer Research UK, London Research Institute comprisesthe Lincoln’s Inn Fields and Clare Hall Laboratories of the formerImperial Cancer Research Fund following the merger of the ICRFwith the Cancer Research Campaign in February 2002.

Journal of PathologyJ Pathol 2002; 197: 430–440.Published online 30 May 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 /path.1153

Copyright # 2002 John Wiley & Sons, Ltd.

have multi-lineage differentiation potential (i.e. self-renewal potential). Using a stroma-based culture sys-tem supplemented with early-acting cytokines, Verfaillie’sgroup defined a very primitive human progenitor, themyeloid–lymphoid initiating cells (ML-ICs), capable ofgenerating multiple secondary progenitors that havethe ability to reinitiate long-term multi-lineage haema-topoiesis [20].

In vivo assays

A conclusive way to assay stem cells is based on theircapacity to repopulate the entire hematopoietic systemin conditioned recipients after transplantation [21]. Inmice, the phenotype and function of haematopoieticstem cells (HSCs) have been characterized using com-petitive in vivo repopulation assays [22–25]. As suchrepopulation assays cannot be performed in humans,surrogate in vivo and in vitro assays are used to evalu-ate human HSCs. In an attempt to develop in vivoanimal models for human haematopoiesis, severalgroups have transplanted human cells in xenogeneictransplant recipients, such as fetal sheep [26,27] orimmune-deficient mice [28,29].

Non-obeses diabetic–severe combined immunodeficient

(NOD/SCID) mouse model

The engraftment of normal human haematopoieticcells in immunodeficient mice provides an assay thatmeasures the repopulating capacity of human stemcells. Dick’s group has shown that intravenous injec-tion of human bone marrow (BM) [30] or cord blood[31] into severe combined immunodeficient (SCID)mice resulted in the engraftment of primitive cells thatproliferated and differentiated in the murine BM, pro-ducing large numbers of LTC-ICs, CFCs, immatureCD34+Thy.1+, CD34+CD38x cells, and mature mye-loid, erythroid and lymphoid cells. The primitive cellsthat initiated the graft were operationally defined asSCID repopulating cells (SRCs). Kinetic experimentsshowed that only 0.1% of the injected CFCs and LTC-ICs were detectable in the murine BM 2 days post-transplant and that there was a large expansion ofthese cells, as well as primitive CD34+ cells, over thenext 4 weeks, implying their production from a moreprimitive cell [32]. Since this pioneering work, a num-ber of research groups have confirmed these results[33–35].

Most of the earlier studies used SCID mice, whichwere not ideal, as they still possessed significant antigen-non-specific immunity. As a result, high cell doses wererequired to overcome any residual host resistance,ruling out the development of quantitative assays andany purification strategies. A new mouse strain, createdby crossing the SCID gene onto the non-obese diabetic(NOD) background, proved to be a better recipient.Compared with SCID mice, this new strain of mice –NOD/Lt-Sz-Scid/Scid (NOD/SCID) – appears to havelower NK and complement activities, and a defect inmacrophages [36]. A lower number of cells (ten to

20-fold less) were necessary to engraft NOD/SCIDmice, when compared with SCID mice. Overall, thesemice showed high levels of engraftment for normal andleukaemic human transplants and, more importantly,enabled engraftment with lower cell doses, renderingpurification strategies possible [29,37,38]. The onlylimitation of these NOD/SCID mice is their inabilityto support human T-cell development. On the con-trary, beige-nude-SCID (Bnx) mice allow T-cell devel-opment, but not B-cell differentiation [39,40]. Othermouse strains have also become available recently: b2-microglobulin knockout/NOD/SCID, Rag1 knockout/NLD, and the nude/NOD/SCID mice [41–43]. Thecapabilities of each of these new strains still need toexplored.

The fetal sheep HSC assay

This assay, based on the permissive environment of theearly gestational age fetus, aims at the development ofa large animal model of human haematopoiesis insheep. The preimmune sheep fetus assay allows thelong-term engraftment and multi-lineage expression ofhuman HSCs in the absence of irradiation or other mye-loablative therapies [44–46], possibly due to reducedNK cells and the preimmune status in early sheepgestation [47]. An essential feature of this model isthat human HSCs primarily engraft host marrow andpersist for long periods into post-natal life [44–46],showing multi-lineage expression and biological respon-siveness to human cytokines [45]. The multi-lineageexpression included T- and B-lymphoid cells [48].Furthermore, this model is relatively specific of thehuman HSC pool. Indeed, while both CD34+CD38+

and CD38x subpopulations engraft the sheep, onlyprimary recipients engrafted with CD34+CD38x cellsexhibited long-term persistence of human cells, whereasCD34+CD38+ cells persist for a short period of timeonly and were unable to engraft into a secondaryrecipient. Although not ideal, the human/sheep xeno-graft model is comparable to the NOD/SCID assay; inparticular, the sheep model does not require myelo-ablation, while it allows prolonged follow-up studiesafter birth. However, widespread utilization of thismodel is hindered by its high costs.

Isolation and purification of HSCs

Haematopoietic stem cells have been enriched using avariety of techniques, including density centrifugation,activation and/or cell-cycle status, and surface antigenexpression, but no unique characteristics have beenfound to identify these elusive cells specifically. Animportant point in the isolation of HSCs is the one-to-one correspondence between physically purified cellsand their potential ability to function as stem cells.

Cell surface markers

Systematic functional analysis of haematopoietic cellsexpressing a particular cell surface antigen or other

Haematopoietic stem cells 431

Copyright # 2002 John Wiley & Sons, Ltd. J Pathol 2002; 197: 430–440.

markers has led to the identification of rare popula-tions highly enriched for stem cell (SRC and/or LTC-IC) activity. HSCs do not express many of the surfaceantigens (‘lineage markers’) that are characteristic ofterminally differentiating haematopoietic cells. Thus,the removal of such lineage-positive cells leaves asuspension of predominantly immature cells.

CD34 as the universal marker of HSCs

CD34 was discovered originally as the result of astrategy to develop antibodies that recognize smallsubsets of human marrow cells, but not mature bloodand lymphoid cells [49]. The discovery of the sialomu-cin CD34 as a haematopoietic cell surface antigen hastransformed and accelerated studies on human haema-topoietic development. Cell surface expression of theCD34 antigen has rapidly become the distinguishingfeature used as the basis for the enumeration, isolation,and manipulation of human stem cells, because CD34is down-regulated as cells differentiate into moreabundant mature cells [50,51]. Despite this extensiveuse, the normal function of the CD34 molecule inhaematopoiesis has remained enigmatic. Studies havedescribed its potential role in cell adhesion and in thehoming process [52]. Human and murine CD34 homo-logues are highly conserved in their protein codingregions [53,54]. The cytoplasmic domains of the humanand mouse proteins share 90% amino acid identity; thetransmembrane and C-terminal regions of the extra-cellular domains are also well conserved, with 73–82%amino acid identity. The N-terminal portions of theextracellular domains are the least conserved regions ofthe molecule (45% amino acid identity). The expressionpattern of CD34 is also conserved between human andmouse. Thus, in addition to being expressed selectivelyon stem cells and early progenitors during human[49,55,56] and murine [53,54] haematopoiesis, bothmouse and human CD34 are expressed outside thehaematopoietic system on vascular endothelial cells[57,58] and some fibroblasts [53,59]. This distributionsuggests a function outside haematopoiesis. Transplantstudies in several species, including baboons and mice,have shown that long-term marrow repopulation canbe provided by CD34+ selected cells. Thus, all relevantclinical and experimental protocols are designed forCD34+ cells enriched by a variety of selection methods.However, several recent studies have suggested thatthere may be human and murine stem cells that do notexpress CD34.

Other stem cell markers

CD133 represents the human homologue of prominin5 transmembrane glycoproteins (PROML 1) [60–63].Several studies have shown the presence of CD133 cellsthat co-express CD34, c-kit, and other cell surfacemarkers [64,65]. Taken together, these studies clearlyindicate that CD133 represents a significant cell surfacemarker for the identification of human HSCs, but itremains unclear whether the use of this marker

provides any distinct advantage over CD34 expression.Further details on the expression of CD133 expressionin human stem cells can be found in the recent reviewby Bhatia [66].

Another recent marker allowing the isolation ofhuman HSCs is the vascular growth factor receptor 2(KDR) [67]. The KDR+ cell fraction, essentially Linx,is largely present in populations enriched for HSCs,namely CD34+CD38x, CD90+, and CD117low cells.It has been reported that CD34+KDR+ is highlyenriched in putative HSCs (SRC and E-LTC-IC orCAFC). Conversely, haematopoietic progenitors withno self-renewal activity are restricted to and highlyenriched in the CD34+KDRx cell fraction. Severalother markers have proven useful in further dividingthe population into more functionally homogeneouspopulations, e.g. CD90, CD117, and CD38 [68,69].Further details on the procedures of isolation of theseHSCs can be found in the review article by Thomaset al. [70].

Side population

In 1996, Goodell et al. reported a new method ofobtaining enriched populations of HSCs from adultmouse bone marrow [71]. This procedure exploits theability of HSCs to efflux the fluorescent dye, which,like the activity of P-glycoprotein (encoded by theMDR gene), is verapamil-sensitive [71]. The Hoechst33342 low cells thus isolated were called side popu-lation (SP) cells and were found to have the sameLin xScal+CD34x phenotype independently identifiedin adult murine HSCs [72]. SP cells have since beenidentified in adult bone marrow from several speciesincluding humans [73]. To date, a description of thefunctional activities of human SP cells in normal indi-viduals has been limited to an in vitro study of cordblood [74] and more recently to an in vivo study ofhuman fetal liver [75]. In this latter study, it was demon-strated that SP cell are present in the secondxtrimesterhuman fetal liver. These cells include all transplantableHSC activity detectable in NOD/SCID mice and alsoother more differentiated haematopoietic cell types [75].More recently, Sorrentino’s group established the linkbetween Bcrp1/ABCG2 expression and the SP pheno-type [76].

Heterogeneity of the human HSCcompartment

Initially, it was assumed that in humans, only cellsexpressing CD34 would display HSC activity, as thefrequency of CD34+ cells is now commonly used toanticipate the adequacy of clinical haematopoietic celltransplants. Recently, however, several groups includ-ing ours have provided evidence of various types ofhuman HSCs that do not express detectable levels ofCD34. Xenograft repopulation assays using fetal sheepand immunodeficient mice have been crucial for theidentification of human CD34x stem cells, as little or

432 D. Bonnet


no clonogenic cell (CFC) or LTC-IC activity wasobserved within the human LinxCD34x cell popula-tion. Using the sheep xenograft model, Zanjani et al.[77] showed that LinxCD34x cells contained stem cellscapable of long-term repopulation and multi-lineage

differentiation in vivo. Moreover, these cells were alsoable to repopulate secondary recipients, attesting to theextensive self-renewal potential of the engrafting cells.The fact that large numbers of CD34+ cells werefound in repopulated sheep suggests that the stem cellswithin the LinxCD34+ cell fraction are more primi-tive than CD34+ cells. Even though the possibility ofslight contamination by CD34+ cells could not be

ruled out, this seems unlikely based on limiting dilu-tion analysis.

Using the NOD/SCID model, the presence of anovel human haematopoietic repopulating cell that isdevoid of lineage-specific markers and of the CD34antigen has also been reported [78]. This population isnot only distinct from LinxCD34+ cells in the absenceof CD34, but also in the lack of HLA-DR and Thy. 1(CD90) markers. In addition to phenotypic differences,

several additional lines of evidence functionallydistinguish this novel stem cell population. WhileLinxCD34x cells have limited haematopoietic activityin the CFC and LTC-IC assays, LinxCD34+CD38x

cells are highly clonogenic in these assays. Further-more, the in vitro response to growth factor stimulationof LinxCD34x cells is clearly distinct from that ofLinxCD34+CD38x cells [6]. LinxCD34xCD38x cells

were unable to proliferate or increase their clonogeniccapacity under culture conditions known to induceproliferate or increase their clonogenic capacity underculture conditions known to induce proliferative anddifferentiative responses in LinxCD34+CD38x cells.Similar to the sheep model, the development ofCD34+ cells, as well as the more differentiated pro-

geny in vivo, suggests that CD34x cells might be moreprimitive than the CD34+ stem cells. Thus, it can beconcluded that the CD34x SRC found within theLinxCD34xCD38x cell fraction represents a novelrepopulating cell within the human haematopoietichierarchy. The fact that the LinxCD34x cell fractionfrom mice and humans contains repopulating cells

indicates an evolutionary conservation of this novelstem cell population [74,77–79]. The identification ofCD34x SRCs within the LinxCD34x subfractionestablishes that the human HSC compartment is morecomplex than previously recognized. However, it is notknown whether CD34x stem cells are important clini-cally. Our current knowledge on the nature of theprecise relationship between CD34x and CD34+ stemcells is illustrated in Figure 1 and has been reviewedrecently [80].

Regulation of the HSC

Over the years, several models have been advancedproposing that haematopoietic lineage determination isdriven extrinsically (through growth factors, stroma orother external influences) [3,81–84], intrinsically (asdescribed in stochastic models) [85,86], or both [87,88].Within the haematopoietic microenvironment, earlyprogenitors are maintained in specific compartmenta-lized niches, where they interact with other cell typesand components of the extracellular matrix [89,90].The microenvironment has been reported to influencesurvival, proliferation, and differentiation [91,92]. Morerecently, it has been suggested that the primary func-tion of these extrinsic signals, including growth factors,is to support the survival and development of committed

Figure 1. Human haematopoietic stem cell hierarchy

Figure 2. Evolving view of adult haematopoietic stem cellcapacity



cells, whereas lineage commitment can be attributedto cell intrinsic mechanisms [85,86,88,93]. Overall, itappears that the regulation of haematopoiesis is theresult of multiple processes involving cell–cell andcell–extracellular matrix interactions, the action ofspecific growth factors and other cytokines, as well asintrinsic modulators of haematopoietic development.

Role of transcription factors in the regulation ofHSC fate

Ultimately, all signal transduction pathways convergeat the level of gene expression where positive and nega-tive modulators of transcription interact and delineatethe pattern of genes expressed by the cell and itsoverall haematopoietic response. As such, transcriptionfactors represent the nodal point of the control ofhaematopoiesis [88,94,95]. It is the alternative expres-sion of specific combinations of transcription factorsthat determines the survival, proliferation, commit-ment, and differentiation responses of haematopoieticprogenitors to such signals, whether they arise fromextrinsic or intrinsic regulatory factors. Increasingevidence suggests that different families of transcrip-tion factors regulate the developmental programme ofstem cells [95] and when their expression is disrupted,leukaemic proliferation is initiated [96]. The mostimportant specific transcription factors identified todate that act at the earliest stage of blood formationare members of the GATA and SCL/tal-1 gene families[95]. GATA-1/2 are the earliest haematopoietic specificfactors to be expressed during mesodermal inductionand in amphibians, expression can be used to defineregions of the ventral mesoderm that give rise tohaematopoietic progenitors. Mice in which the SCL/tal-1 gene was disrupted do not generate definitivehaematopoietic cells, suggesting that SCL/tal-1 func-tions very early in haematopoietic development at thestage of specification of ventral mesoderm to a bloodcell fate [95,97]. Disruption of the GATA-1 and LMO-2/rbtn-2 genes also severely impairs early erythroidlineage development. The fact that the most frequenttargets of chromosomal translocations in acute leukae-mias are genes that encode transcription factorsemphasizes the critical role of these master regulatorymolecules in the control of blood development [96,98].Translocations that inappropriately activate transcrip-tion factor genes in ALL and AML show remarkablespecificity for haematopoietic cells blocked in definedstages of differentiation [98]. This property suggeststhat the different oncoproteins produced by chromo-somal translocations interfere specifically with trans-criptional networks that normally function in concertwith growth factors and their receptors to regulatehaematopoiesis.

Finally, a number of myeloid transcription factorshave been identified through their involvement inleukaemias, either as a result of abnormal expression,such as PU.1 and WT-1 [99,100], or through theirinvolvement at the site of a consistent chromosomal

translocation (examples include AML1, CBFb, andSCL/Tal.1) [96,98]. The technique of gene disruptionhas been used to confirm the role of these specificfactors in myeloid development [95]. Several transcrip-tion factors that are expressed in haematopoietic cellsand play a role in leukaemogenesis (chromosomal trans-locations) contain a homeo-box DNA binding region.HOX gene family members encode DNA-bindingtranscription factors characterized by a conserved60-amino acid homeo-domain which is homologousto the Drosophila homeo-box proteins and also plays acrucial role in mammalian embryonic axis formation[101]. Human HOX genes are organized on differentchromosomes in four major clusters, A, B, C, and D,each of which consists of nine to 12 tandem genes[102]. While their role in embryonic axis formationhas been well studied, the role that they play in regula-ting haematopoiesis and leukaemogenesis is less clear,although several recent studies point to a major role[103,104]. Furthermore, further detailed analysis of Hoxgene expression in functionally distinct subpopulationsof CD34+ cells has shown that genes, primarilylocated at the 3k end of the clusters (e.g. HOX B3 andB4), are preferentially expressed in the subpopulationcontaining the most primitive haematopoietic cells[104]. Overexpression of Hox B4 has been shown tofavour self-renewal over differentiation [105,106].

Some initial clues to the regulatory relationshipsbetween transcription factors are coming. For example,three recent studies over the past year have reportedthe direct physical interactions and cross-antagonismsof PU.1 and GATA-1 [107–109]. Additional examplesof cross-antagonisms between lineage-specific trans-cription factors, or their co-factors, are beginning toemerge and reveal novel mechanisms (see the review byCantor and Orkin [110]). Thus, there appears to be adynamic balance of forces that ultimately determinethe phenotype of a cell.

Role of mesodermal inducing factors in theregulation of HSC fate

As indicated above, amongst the approximately 50different chromosomal translocations that have beencloned, the majority involve transcription factors.However, there are numerous other molecules forwhich there is reasonable information to warrant con-sideration for roles in stem cell regulation. One of theseis the collection of mammalian Notch molecules, theirligands Delta-like, Jagged family, the fringe family ofsignalling modifiers, as well as the variety of down-stream regulators [111]. Notch can affect cell fate byregulating transcription directly via association withnuclear factors and can thus affect the growth anddifferentiation of HSCs. Incubation of human HSCswith soluble Notch ligands, Jagged 1, has recentlybeen shown to drive in vitro HSC self-renewal. Wheninjected into NOD/SCID mice, Jagged 1-treated HSCswere capable of reconstituting lymphoid and myeloiddifferentiation [112]. Furthermore, the constitutive

434 D. Bonnet


expression of Notch 1C using a retroviral vector is capa-

ble of generating a clonal population of immortalized

HSCs [113].Members of the TGF-b superfamily and those that

act through the TGF-b pathway also appear to be very

prominent [114,115]. TGF-b itself is a potent inhibitor

of murine stem cells and human LTC-ICs and appears

to be a major regulator that keeps stem cells in a

quiescent state [116]. Interestingly, treatment of human

LTC-ICs with anti-TGF-b can induce them to enter

the cell cycle [117]. Recently, it has been demonstrated

that bone morphogenetic protein (BMP-2 and BMP-4)

and activin A are potent ventralizing factors and

inducers of haematopoietic tissue, and that BMP-4

and GATA-2 can function in two adjacent germ layers,

mesoderm and ectoderm, to participate in blood cell

formation during embryogenesis [118]. We reported

that BMPs are capable of regulating the proliferation

and differentiation of highly purified primitive human

haematopoietic cells. Populations of LinxCD34+-

CD38x cells isolated from human haematopoietic

tissue were shown to express the BMP type I receptors

ALK-3 and ALK-6, and their downstream transducers

SMAD 1, 4 and 5. Soluble BMP-2, BMP-4, and

BMP-7 induced dose-dependent responses in human

LinxCD34+CD38x cord blood cells, as determined by

changes in proliferation, clonogenicity, cell surface

phenotype, and multi-lineage repopulation capacity in

NOD/SCID mice (SCID-repopulating cells; SRCs).

Similar to TGF-b, treatment of purified cells with

BMP-2 or BMP-7 at high concentrations inhibited

proliferation, yet maintained primitive CD34+CD38x

phenotype and SRC activity. In contrast, low concen-

trations of BMP-4 induced the proliferation and

differentiation of LinxCD34+CD38x cells, whereas

at higher concentrations BMP-4 extended the period in

which ex vivo cultured cells maintained repopulating

function. This study illustrates a novel role for the

BMP pathway and suggests that this family of mor-

phogens continues to play an important role in human

blood stem cells beyond haematopoietic tissue specifi-

cation [119].Other factors that play a role in mesodermal tissue

include basic fibroblast growth factor (bFGF), which

has been shown by Allouche to be involved in the

proliferation and differentiation of numerous cell types

including those in the haematopoietic lineage [120].

bFGF is expressed mostly in tissues of mesoderm and

neuroectoderm origin, and plays an important role in

mesoderm induction, together with TGF-b. bFGF is

expressed and produced by bone marrow stromal cells,

as well as by cells from several mature peripheral blood

lineages. FGF-receptors (FGF-Rs) are expressed on

nearly every cell of haematopoietic origin tested so far

and bFGF can regulate haematopoiesis, by acting on

stromal cells, early and committed haematopoietic

progenitors, and mature blood cells. It synergizes with

haematopoietic cytokines, or antagonizes the negative

regulatory effects of TGF-b.

The genetic programme of the HSC

As mentioned above, several molecules have beenshown to play a role in several aspects of haemato-poetic development, but the elucidation of the mole-cular phenotype of the HSC has just begun [121,122].Indeed, key aspects of the stem cell regulation arelikely to be emergent properties of interacting path-ways and networks, the elucidation of which requiresan extensive description of the genetic programmeavailable to the stem cell. Recently, using a highly sen-sitive single-cell RT-PCR approach, or a quantitativereal-time PCR method, it has been demonstrated thatHSCs co-expressed several lineage-restricted gene sets[123,124]. These data indicate the complexity of geneexpression from which distinct patterns must emergeduring the lineage commitment process by selective up-regulation of the relevant gene cohorts, accompaniedby the inactivation of others. To take into account thisnew concept of regulation, a model for the molecularnature of the uncommitted stem cell ‘state’ has recentlybeen postulated [125]. This model proposes that a HSCpossesses a molecular ‘ground state’ composed of lowlevels of transcripts normally associated with thefunction of various mature cell lineages. According tothis model, a commitment process would involve theselection and amplification of an appropriate subset ofthe available transcriptional programme and possiblerepression of the remainder. Interestingly, the featuresof the ‘ground state’ model are consistent with theclinical description of gene expression promiscuity incertain mixed leukaemias. The features of the groundstate model fit well with the stochastic model of stemcell commitment.

Engraftment of HSC

In a transplantation context, a stem cell is definedretroactively as a biological activity that can give riseto substantial measurable numbers of mature cells.Similarly, the presence of a single clonotypic marker indonor-derived cells of different lineages defines multi-potentiality. Thus, following transplantation into therecipient, HSCs, in order to engraft and reconstitutethe bone marrow functions, must home to and lodge inthe specialized niches of the BM microenvironment. Atpresent, only partial understanding of the cellular andmolecular mechanisms governing homing exists. It isbelieved that an intricate process involving interac-tions between adhesion molecules and their counter-receptors expressed on HSCs and endothelial cellsdirects the cells. In many aspects, the homing of HSCsduring transplantation mimics the natural movementof these cells during ontogeny. The direct involvementof particular adhesion molecules in homing has beenelucidated [126,127]. Recently, Zanjani et al. demon-strated that VLA-4 played a central role in the homingand engraftment of transplanted human cells to theBM of sheep fetuses [128]. Another ligand receptor



pairing, SDF-1–CXCR4, has also been implicated inselectively directing the homing of HSCs to the BM.The engraftment of human cells in NOD/SCID micewas prevented by treatment with antibodies againstCXCR4. Furthermore, the expression of CXCR4 onCD34+ human HSCs has suggested a role for thesechemoattractants in the homing process [129]. How-ever, Rosu-Myles et al. recently demonstrated thatCXCR4 expression on human HSCs was not requiredfor effective stem cell repopulation function [130].Redundancy between different chemoattractant mole-cules may be responsible for this discrepancy (for moredetails see the reviews in refs 131 and 132), Whilemore is known about homing, less is understood aboutHSC niches [133].

Recently, the spatial organization of subpopulationsof haematopoietic cells following syngeneic transplan-tation in mice has been investigated. The studydemonstrated that the spatial distribution of trans-planted cells is not a random process; candidate stemcells exhibited selective lodgement in the endostealregion of the bone [134].

HSC plasticity

The notion of adult stem cell plasticity has alreadybeen discussed in this issue by Poulsom et al. and canbe found in different recent review articles [5–11].Thus, we will restrict our comments here on the recentstudies involving HSCs, including some of our recentwork on HSC plasticity using the NOD/SCID model.

Several striking observations have been reportedwhich are beginning to raise questions about the tradi-tional view of HSC biology (illustrated in Figure 2).For example, some observations have challenged thedogma that HSCs are committed solely to the haema-topoietic lineage [135–142]. In particular, reports haveindicated that the bone marrow of adult rodents con-tains cells with the capacity to give rise to hepatocytes,muscle tissue, or even neurons. Lagasse et al. estab-lished that some haematopoietic stem cells present inadult bone marrow co-purified with stem cells thatgave rise to hepatocytes, supporting the new conceptthat somatic adult stem cells can change cell fate [143].However, these reports were based on adult rodentstem cell populations, which may differ from humanstem cells in their capacity to give rise to multipletissues. Nevertheless, the studies by Theise et al. andAlison et al. based on patients who received bonemarrow transplants, indicate that some human adultstem cells present in bone marrow also have the abilityto give rise to hepatocytes [144,145]. These two studiesdid not distinguish whether HSCs, mesenchymal stemcells or hepatocyte stem cells circulating in the bonemarrow were responsible for the observed liver engraft-ment.

We reported recently the potential of a highlypurified population present in adult bone marrow andumbilical cord blood (human C1qRp

+ stem cells) to

differentiate in vivo into hepatocytes (Danet et al.,in preparation). These data provide the first directdemonstration that a highly purified and phenotypi-cally defined human adult stem cell population canrepopulate the bone marrow and differentiate in vivointo functional hepatocytes, using the NOD/SCIDmouse model. Not only HSCs, but also mesenchymalstem cells (MSCs) present within the adult humanmarrow possess remarkable plasticity. Kopen et al.showed that cells that gave rise to neurons and gliawere derived from cultures of adherent bone marrowstroma, suggesting that they included MSCs [146].More recently, Verfaillie’s group identified mesodermalprogenitor cells (MPCs), which have the capacity todifferentiate into osteoclasts, chondrocytes, adipocytes,skeletal myoblasts, and endothelial cells [147,148]. Inthe light of the recent identification of a single mousebone marrow-derived stem cell with multi-organ andmulti-lineage engraftment [149], additional experimentswill be required to determine the full developmentalcapacity of marrow-derived stem cells.

Conclusion

Despite the lack of definitive proof of plasticity inmany of the present studies, the use of HSCs for thepotential treatment of human diseases such as liverdiseases and muscular dystrophy represents an excitingnew therapeutic strategy. HSCs represent a safe andaccessible source of stem cells that can be geneticallymanipulated and may thus prove to be an ideal vehiclefor delivering therapeutic genes to other organs. TheNOD/SCID xenotransplant model will play an impor-tant role in evaluating this potential.

A more detailed and systematic analysis of differentsomatic stem cell types based on their gene expressionprofiles and functional properties is needed for a betterunderstanding of the nature of stem cells and theirdevelopmental plasticity.

The study of HSCs has become extremely exciting,with new insights into HSC biology being reported ona weekly basis. HSCs may be more plastic than pre-viously appreciated and assumptions about theirphenotype may be under revision. We are entering avery exciting area of investigation where dogma willevolve rapidly and new techniques will open up newexperimental approaches.

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