the haemopoietic stem cell

REVIEW

The Haemopoietic Stem Cell:Between Apoptosis and Self Renewal

Faris Q. Alenzia*, Badi Q. Alenazib, Shamweel Y. Ahmada, MohamedL. Salemc, Ali A. Al-Jabrid, and Richard K.H. Wysee

aDepartment of Clinical Laboratory Sciences, College of Applied Medical Sciences, KingSaud University, Saudi Arabia; bDepartment of Pediatrics, College of Medicine, King SaudUniversity, Riyadh, Saudi Arabia; cDepartment of Surgery and Hollings Cancer Center,Medical University of South Carolina, USA, and Department of Zoology, Faculty ofScience, Tanta University, Egypt; dDepartment of Microbiology and Immunology, Collegeof Medicine, Sultan Qaboos University, Oman; eDepartment of Surgery, HammersmithCampus, Imperial College of Science, Technology and Medicine, London, UK

Self renewal and apoptosis of haemopoietic stem cells (HSC†) represent major factors thatdetermine the size of the haemopoietic cell mass. Changes in self renewal above or belowthe steady state value of 0.5 will result in either bone marrow expansion or aplasia, respec-tively. Despite the growing body of research that describes the potential role of HSC, thereis still very little information on the mechanisms that govern HSC self renewal and apoptosis.Considerable insight into the role of HSC in many diseases has been gained in recent years.In light of their crucial importance, this article reviews recent developments in the under-standing of the molecular, biological, and physiological characteristics of haemopoietic stemcells.

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YALE JOURNAL OF BIOLOGYAND MEDICINE 82 (2009), pp.7-18.Copyright © 2009.

STEM CELL: BACKGROUND ANDPROPERTIES

The most primitive haemopoietic cellsare the haemopoietic stem cells (HSC).They are defined as cells with a high poten-tial for self renewal and possess the capacityfor dividing into identical copies of them-selves without forming any newly differen-

tiated features. Because most mature bloodcells have a very short life span, the impor-tance of HSC in sustaining the life of themammal — for example, through its abilityto self renew — is very critical. Stem cellsin both embryonic and adult tissues are de-fined by their ability to undergo self renewaland differentiation in a balanced state with-

*To whom all correspondence should be addressed: Faris Q. Alenzi, PhD, Associate Profes-sor of Immunology and Consultant Immunologist, Department of Clinical Laboratory Sci-ences, College of Applied Medical Sciences, King Saud University, PO Box 422 AlKharaj11942, Saudi Arabia; E-mail: [email protected].

†Abbreviations: HSC, haemopoietic stem cells; HEV, high endothelial venules; H-CAM, hom-ing-associated cell adhesion molecule; SP, side population activity; CFU-GEMM, colony-forming units granulocyte, erythroid, monocyte, and megakaryocyte; CFU-G, colony-formingunit granulocyte; CFU-M, colony-forming unit megakaryocyte; pSR, probability of self re-newal; pDiff, probability of differentiation; BM, bone marrow; ROS, reactive oxygen species;ESC, embryonic stem cells; Epo, erythropoietin; FasL, Fas ligand; NBM, normal bone mar-row; RMGT, retroviral-mediated gene transfer; CML, chronic myelogenous leukemia.

out depleting the stem cell pool. If a progen-itor can divide (and in certain circumstancescan generate a secondary colony), it does notmean that it is capable of self renewal. Allmyeloid progenitor cells, except HSC, willterminally differentiate within two months orsooner. In contrast, HSC are capable of main-taining haemopoiesis during the life of theanimal or longer if transplanted. So self re-newal implies immortality at least within areasonably long period of time, even as faras lifespan. All other progenitors that eventu-ally extinguish do not self renew; all theirdaughters are of a progressively decreasedquality (in terms of proliferative potential)and, therefore, cannot be considered ascopies of the original cell. This topic is cur-rently under huge debate.

HSC are capable of differentiating intoat least eight cell lines. The balance betweenself renewal and differentiation is consideredto be critical to the maintenance of stem cellnumbers [1-3]. More importantly, stem cellsin general are proposed to have a major rolein curing many degenerative diseases andcancers [4-7]. Significant efforts have beenmade in recent years in understanding themechanism governing HSC generation, selfrenewal, proliferation, and commitment. Yetunderstanding the overall process is still farfrom complete and largely hypothetical. Agrowing body of research suggests that path-ways regulating the self renewal of normalstem cells are dysregulated in cancer stemcells, resulting in continuous expansion ofself renewal and tumor development and,therefore, offering hope that new cancer ther-apies may emerge via this approach [4-10].

Most stem cells are in G0 phase of thecell cycle, and, therefore, only a small num-ber of stem cells are responsible for stem cellmaintenance and for producing mature cellsat any specific time [3,11]. Experimentalwork has indicated that a single primitiveprogenitor may survive in a quiescent statefor more than two weeks in culture before itdivides [1-2,12-14].

Stem cells are characterized by the ex-pression of CD34 and Thy1 and absence ofCD38, CD33, and HLA-DR [15-17]. Thesecells also lack expression of a great number

of markers that are expressed on matureblood cells (lineage negative), and that line-age negativity is as important as other criteriafor identifying and isolating these cells.Human HSC are CD34+, while murine HSCare CD34-. CD34 is a transmembrane glyco-protein, expressed in immature haemopoieticcells, fibroblasts, vascular endothelium, andhigh endothelial venules (HEV) [18-19].CD34 is a stem and progenitor cell marker inhumans. It contains two sites for serine/thre-onine phosphorylation by protein kinase C(PKC) and a tyrosine phosphorylation site,implying a possible role in signaling [20]. Inaddition, endothelial CD34 binds to thelectin-like adhesion molecule L-selectin [21].Surprisingly, however, experimental work onCD34 deficient mice has revealed no majorabnormalities either in haemopoiesis or in in-teractions of haemopoietic progenitor cellswith stromal cells [22]. It is well establishedthat even more primitive progenitor cells arepresent within the CD34 negative fraction[23]. Additionally, haemopoietic progenitorcells express adhesion molecules such as L-selectin [24], integrins [25], and homing-as-sociated cell adhesion molecule (H-CAM)[26]. Alternative methods to isolate HSC in-clude side population activity (SP) or rho-damine efflux, both of which were recentlysuperseded by new markers such as Slamf1(CD150), or endoglin.

COMMITTED PROGENITOR CELLS:BACKGROUND AND PROPERTIES

This compartment includes stem cellprogeny that have been identified by theirability to form colonies of morphologicallyrecognizable cells in semi-solid cultures. Inaddition, progenitor cells are characterizedphenotypically by expression of CD38,CD33, and HLA-DR, and a greater propor-tion of them are in an active cell cycle com-pared with stem cells. It is generally assumedthat progenitor cells can not self renew. How-ever, considerable self renewal recently hasbeen demonstrated experimentally in vitro byreplating CFU-GM into secondary cultures[27]. This now has provided an informativeclonogenic assay for demonstrating self re-

8 Alenzi: Haemopoietic stem cell

newal capacity [3]. The size of the progeni-tor’s cell compartment is governed by thebalance between the cell gain (self renewal)and the cell loss (apoptosis). Therefore, animbalance in either of these parameters willresult in an elevation or a fall in the progen-itor cell mass. The colony-forming unitsgranulocyte, erythroid, monocyte, andmegakaryocyte (CFU-GEMM) are interme-diate between stem cells and single lineageprecursors. They are capable of producinggranulocytes, erythrocytes, monocytes, andmegakaryocytes. The more committed pro-genitor cells have only single lineage poten-tial. These are colony-forming unitgranulocyte (CFU-G), colony-forming unitmacrophage (CFU-M), burst-forming uniterythroid (BFU-E), and colony-forming unitmegakaryocyte (CFU-Meg) that producegranulocytes, macrophages, erythrocytes, ormegakaryocytes, respectively [28-30].

MODELS OF HSC SELF RENEWAL

Stochastic theoryStochastic is a term applied to processes

that appear random but have associated un-derlying probability distribution. In the con-text of haemopoiesis, the fate of individualcells cannot be predicted. An important fea-ture of stochastic processes is that they areassociated with an overall probability of anevent. Therefore, in haemopoiesis, there is aprobability of self renewal (pSR) and a prob-ability of differentiation (pDiff). When cellsdivide, the values of pSR and pDiff will be0.5 for steady state haemopoiesis [31]. Thereis often a misconception that stochasticprocesses are random and, therefore, thatstem cells can not respond to environmentalfactors. It is sometimes assumed that sto-chastic processes are regulated intrinsically.

In 1961, Till and McCulloch developedan assay that involved inbred strains of mice,in which the donor and recipient are geneti-cally identical [32]. The recipient mice areirradiated to ablate all haemopoietic cells.Bone marrow cells from donor mice are theninjected into the recipient mice. After 10days, the mice are sacrificed, and the number

of spleen colonies are counted. The numberof colonies forming on the spleen of lethallyirradiated mice correlated with the numberof bone marrow (BM) cells transplanted, anddifferent lineages were represented in the de-veloping colonies. It is known from otherworks that not all CFU-S are capable of giv-ing rise to secondary colonies. Other workersfound that replating of erythroid colonies andblast cell colonies showed a similar distribu-tion to that seen in CFU-S [33-34]. Abkowitzand colleagues have shown that stochastic ef-fects may occur in haemopoiesis in vivo [35].

Deterministic model of haemopoiesis

This model suggests that decisions toself renew or differentiate are entirely con-trolled and that responses to a particular setof circumstances will be predetermined[12,36]. Morrison and Weissmann suggesteda model based on the purification of threemultipotent progenitor cells that had differ-ent self renewal capacities. The progenitorsfrom the most primitive population showeda greater self renewal capacity than thosefrom less primitive progenitors. They con-cluded that self renewal is a deterministicevent based on the fact that cell separationcould predict self renewal potential [37]. Inthe deterministic model, the haemopoiesisprocess is controlled by extrinsic factorssuch as cell-cell interactions within thehaempoietic micro-environment or sig-nalling by cytokines, and, therefore, the cellfate is determined. These obstacles havebeen reviewed by Metcalf and Enver andHeyworth and Dexter [38-39].

Gordon and Blackett have proposed thestochastic model with observations that cellswith different self renewal capacities couldbe separated into subpopulations. They pro-posed that self renewal was a stochasticevent, but the probability of this processcould be modified by extrinsic factors [11].

KINETIC MODELS OF STEM CELLREGULATION

Two theories have been proposed to ex-plain the ability of stem cells to maintain life-long haemopoiesis. The first suggests that the

9Alenzi: Haemopoietic stem cell

embryo has sufficient stem cells to maintainhaemopoiesis throughout life. However, thisconcept fails to explain the ability of a bonemarrow transplant (consisting of a smallfraction of the total marrow) to restorehaemopoiesis to normal levels after the re-cipient has been exposed to myeloablativetherapy [40]. The second theory is that asmall number of stem cells are able to sustainlifelong haemopoiesis because they are ca-pable of self renewal when they divide. Forsteady state haemopoiesis, the probability ofself renewal must be 0.5 and the probabilityof differentiation and/or loss by apoptosismust also be 0.5 [13-14]. Asymmetric divi-sion indicates a stem cell when it divides intoone new stem cell and one differentiated cell,while symmetrical division is when one stemcell divides into two stem cells and anotherdivides into two differentiated cells. Bothmodels can account for steady statehaemopoiesis. However, only the lattermodel can account for expansion or recoveryof the stem cell pool following damage.

Several investigators have shown thatthe probability of stem/progenitor cell selfrenewal is not fixed. Metcalf demonstratedthat G-CSF decreases the replating ability ofthe WEHI-3B colony-forming cells [41]. Ad-ditionally, Lewis and colleagues have re-ported that cytokines modify the self renewalkinetics of primary granulocytic and ery-throid progenitor cells [42-44]. The controlof stem cell numbers can be regulated by theloss of apoptosis, as well as by differentia-tion, from the stem cell population. However,there is a paucity of information on thispoint. Despite the wide acceptance and beingpublicized repeatedly at scientific meetings,still there is a need to re-examine some of thebasic concepts in haemopoiesis. Quesenberryand Lowry suggested previously the impor-tant need to bring current models of stem cellself renewal and differentiation into line withexperimental observations [45-46]. This ne-cessity is further emphasized by the discov-ery that haemopoietic stem cells maydifferentiate into non-haemopoietic lineagessuch as muscle and neural cells [47-48].

The concept of stem cell compartmen-talization is a cornerstone of HSC self re-

newal [49]. There are various known factorsthat contribute to stem cell functionality, in-cluding second messenger systems, tran-scription factors, and the type and numberof growth factor receptors expressed. The in-teraction of these factors will determine thepotential responses of those cells to both in-ternal and external signals. Probability ofself renewal is decreased as HSC progressestoward maturity. Ogawa and colleagues [50]pointed out that the heterogeneity of HSCand the inability to measure the self renewalprobability of an individual HSC remainamong the most significant limitations. It isimpossible to measure precisely the self re-newal capacity of an individual HSC at thesame time as measuring its differentiationcapacity. Therefore, the focus should not beon individual cells, but perhaps rather onpopulations of stem cells by using tech-niques that have been developed byJankovic et al. [51]

While progenitor cells do not have thesame self renewal capacity as the parentstem cells, they retain some capacity for fur-ther divisions. Therefore, the CFU-GM andBFU-E progenitor assays are very usefultools for studying haemopoiesis, and consid-erable evidence has been accumulated thatthis might be the case [52]. Till and col-leagues developed a technique to detectHSC, and it is widely believed that it detectsnot only early but also more mature progen-itor cells [32]. It has been demonstrated thatreplating colonies from murine blast cellcolonies and committed progenitors such asCFU-GM and BFU-E results in secondarycolony formation [27,33-34]. Additionally,murine B cell progenitors can undergo selfrenewal in the presence of stromal cells andinterlukin-7 (IL-7) [53].

RELATIONSHIP BETWEENAPOPTOSIS AND SELF RENEWAL(I.E., PROLIFERATION)

HSCs are defined by their ability both toself renew and to give rise to any of thehaemopoietic cell lineages throughout thelifetime of an animal [54]. To maintainhaemopoietic homeostasis in a host, the HSC


numbers need to be precisely regulated. Thefate decisions, including life and death andself renewal and differentiation, of HSCs areimportant processes that regulate the num-bers and lifespan of the HSC pool in a host.Defects in these processes can contribute tohaemopoietic insufficiencies and the devel-opment of haemopoietic malignancies [55].Failure of HSCs to self renew during aging isbelieved to depend on several intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) factors. Products of numerousgenes that are involved in either DNA-dam-age responses or longevity-related signalingcontribute to the maintenance of the HSCself renewal capacity. Several intrinsic fac-tors have been identified as potential mech-anisms underlying the self renewal of HSC.

Cell cycle checkpoints induced by telom-ere dysfunction represent one of the major invivo tumor suppressor mechanisms provokingage dependent decline in self renewal and re-generation of tissues and organs. Since stemcells are continuously proliferating throughouttheir lifetime, most stem cell compartmentsexpress telomerase. In stem cells, however,the level of telomerase activity is not sufficientto maintain telomere length during aging. Ex-haustion of replicative potential of telomeraseappears to be at least partially dependent onthe cell cycle regulatory component of theDNA damage response. To overcome telom-ere activity, stem cells appear to have tighterDNA damage checkpoint control in compari-son to somatic cells. These enhanced check-point responses may have a detrimentalimpact on stem cell function by causing in-creased sensitivity toward senescence orapoptosis induced by telomere shortening[56]. Stresses on stem and progenitor cellpools, in the form of telomere shortening orother genome maintenance failures, have beenshown to lower tissue renewal capacity andaccelerate the appearance of senescence.Therefore, long-term stem and progenitor cellpotential depends on both the genome mainte-nance mechanisms that counter DNA damageand the cell cycle checkpoint responses todamage [57-58].

In addition to checkpoint factors, pro-teins that control the normal cell cycle have

been shown to regulate stem cell fate. Cdk2is a major regulator of S phase entry, is acti-vated by mitogenic cytokines, and has beensuggested to be involved in antigen-inducedapoptosis of T lymphocytes. Analysis of theHSC compartment in mice deficient in Cdk2revealed normal proportions of stem cellsand progenitors. Furthermore, a competitivegraft experiment on HSC deficient in Cdk2showed normal renewal and multilineagedifferentiation [59], indicating that Cdk2 isnot required for proliferation and differenti-ation of HSC in vivo. In vitro analyses, how-ever, consider Cdk2 to be a major player inproliferation and apoptosis in HSCs. There-fore, further studies are required to analyzethe contribution of this factor and a potentialtarget for therapy.

Reactive oxygen species (ROS) also in-fluence stem cell fate. It has been demon-strated that long-term self-renewing HSCsnormally possess low levels of intracellularROS. If intracellular ROS levels become ex-cessive under pathological conditions, theycause senescence or apoptosis of stem cellsand a failure of their self renewal. Correctionof the intracellular levels of ROS in HSC bytreatment with an antioxidant that scavengesROS can rescue HSC self renewal [60].Defining the molecular mechanisms thatgovern the ROS regulation and strategiesthat can control the excess levels of ROScould lead to the significant improvement indesigning novel therapeutic approaches forhaemopoietic diseases, regenerative medi-cine, and the prevention of leukemia.

There are several transcription factors,such as Foxo3a and Zfx, that play a role instem cell self renewal. For example, Foxo3ais a forkhead transcription factor that actsdownstream of the PTEN/PI3K/Akt path-way. It has been found in mice deficient inFoxo3a that the frequency of HSC is lessthan normal; the number of colony-formingcells is lowered and the ability of HSC tosupport long-term reconstitution ofhaemopoiesis is impaired; HSC showed in-creased phosphorylation of p38MAPK, anupregulation of ROS; there is defectivemaintenance of quiescence; and there is in-creased sensitivity to cell-cycle-specific


myelotoxic injury [61]. Taken together,these results demonstrate that Foxo3a is animportant intrinsic factor role for maintain-ing the HSC pool.

Recent studies have shown that deletionof Zfx, another transcription factor, inmurine HSC and embryonic stem cells(ESC) impairs their self renewal, resultingin increased apoptosis and upregulation ofstress-inducible genes. By contrast, Zfx di-rectly activated common target genes, in-cluding ESC self-renewal regulators Tbx3and Tcl1, in ESC and HSC [62]. These stud-ies identify Zfx as another factor that is crit-ical for self renewal in embryonic and adultstem cells.

Evidence has accumulated concerningthe participation of certain proliferation pro-moting factors (such as oncogenes) that arealso able to act as potent triggers of apopto-sis. Such factors include GM-CSF, Epo,Flt3, and Notch.

Recent experimental evidence also hassuggested there may be a positive relation-ship between apoptosis and proliferation innormal haemopoiesis. For example, Tray-coff et al. [63] showed that cord blood andNBM CD34+ cells with a high number ofcell divisions in short-term cultures in vitroare associated with an increase in the per-centage of apoptotic CD34+ cells. It hasbeen demonstrated that both GM-CSF de-pendent and G-CSF dependent cell lines un-dergo rapid cell death after removal of therelevant CSF [64]. On the other hand, mod-els proposed by Koury [65] imply that a re-duction in apoptosis might result in anincrease in proliferation, potentially result-ing in an inverse relationship between celldeath and cell division. Evidence for a linkbetween apoptosis and proliferation (i.e.,self renewal) in haemopoiesis remains un-clear; we hypothesize that cell proliferation(i.e., self renewal) and cell apoptosis arelinked in normal haemopoietic tissues andthat this relationship may be abnormal inchronic myeloid leukemia (CML).

A possible role for apoptosis is illus-trated by the relationship between erythro-poietin (Epo) levels and erythropoieticactivity in vivo in mice. When the Epo con-

centration is elevated due to an increase indemand (such as anaemia), many Epo-de-pendent progenitor cells that would other-wise rapidly undergo apoptosis will survive.If Epo levels are decreased by hyper-transfu-sion, Epo-dependent progenitor cells thatwould normally survive will die rapidly anderythrocyte production is reduced [66]. Thishypothesis is supported by results usinghyper-transfused mice, which show contin-ual production of erythroid progenitor cellsbut with no increase in their number follow-ing the cessation of erythropoiesis, thus in-dicating the direct involvement of cell death[66].

Flt3 is a member of the receptor tyro-sine kinase family, which plays a critical rolein maintenance of haemopoietic homeosta-sis. Recently, it has been found that thehuman HSC in both the bone marrow andthe cord blood that are capable of long-termreconstitution in xenogeneic hosts uniformlyexpress Flt3. Detailed analysis of Flt3 ex-pression shows that it is expressed not onlyin early lymphoid progenitors, but also inprogenitors continuously along the granulo-cyte/macrophage pathway, including thecommon myeloid progenitor and the granu-locyte/macrophage progenitor [67]. Thesestudies showed further that an intact Flt3signaling pathway is required to preventHSC stem and progenitors from sponta-neous apoptotic cell death. This role of Flt3has been suggested, at least in part, to theup-regulating of Mcl-1, which is an indis-pensable survival factor for haemopoiesis.

Notch is another receptor that regulatesdiverse cell fate decisions during develop-ment and is reported to promote murineHSC self renewal. Recent in vitro studieshave shown that constitutive expression ofactive human Notch 1 intracellular domainin human blood CD34+ cells induced a re-duction in the proliferation of the number ofCD34+ cell populations, coinciding with in-hibited cell cycle kinetics and up-regulationof p21 mRNA expression and induced apop-tosis [68]. The results of this study show thatactivation of the Notch signaling pathwayplays an important role in regulation of theproliferation and survival of adult stem cells.


In addition to oncogenes, tumor suppres-sors like p21 influence stem cell division. Weinvestigated the role of p21 in normal HSCfrom p21 knockout mice. We found that p21deletion increases the growth rate of coloniesand the multiplication of haemopoietic pro-genitor cells in vitro. We also found an in-crease in apoptotic percentage on primaryhaemopoietic cells from mice (unpublishedobservations). Therefore, in order for a trans-formed haemopoietic cell to launch a cloneand result in leukemia, it is of importance forsome defect to provide the cell with an in-creased probability of self renewal. This isbecause a self renewal probability greaterthan 0.5 will not result in clonal expansion;rather, it will attain maintenance of cell num-ber (SR = 0.5) or result in clonal loss (SR <0.5). These preliminary unpublished resultssuggest that p21 has the potential to act as atumor suppressor gene in the myeloid line-age. Additionally, it is now confirmed thatp21 is attributed with a role in regulating selfrenewal (i.e., proliferation) and apoptosis.

Apoptotic factors, such as BCL-2, Fas,and Fas ligand (FasL), also control HSCproliferation. Domen et al. produced Bcl-2transgenic mice that over-expressed Bcl-2.They found that the HSC from WT micedied after growth factor withdrawal,whereas HSC from Bcl-2 transgenic miceremained viable. More importantly, HSCfrom Bcl-2 transgenic mice proliferatedmore rapidly and extensively (in the pres-ence of a cocktail of factors including IL-1,IL-3, IL-6, SCF, Flt3L) than those of WT.Additionally, there was a delay in cell cycleentry. The most dramatic difference betweenWT and Bcl-2 transgenic mice was revealedwhen HSCs were cultured in the presence ofSCF. Only 20 percent of WT HSC remainedviable after one week; whereas, HSC fromBcl-2 transgenic mice showed enhanced sur-vival and more vigorous proliferation. Bcl-2 over-expression and SCF/c-kit signalingwere found to be sufficient for HSC prolif-eration; however, it should be noted that pro-liferation also resulted in differentiation ofmyeloid progenitor cells [69-70].

Fas and FasL are also apoptotic factorsinvolved in erythriod differentiation. Peschle’s

group has demonstrated the possible involve-ment of Fas and FasL in the regulation of ery-thropoiesis and immunohistochemistry ofnormal bone marrow (NBM) samples and de-termined that several immature erythroblastsundergo apoptosis in vivo. These resultsshowed that erythroid blast express Fas;whereas, more mature cells express FasL.These findings suggest the existence of a neg-ative regulatory feedback between mature andimmature erythroid cells. Accordingly, the in-teraction of Fas and FasL might represent anapoptotic control mechanism for erythro-poiesis, contributing to the regulation of redblood cell homeostasis [71]. Furthermore, wehave shown that Fas, FasL, and caspase acti-vation are likely to play an important role inthe regulation of myelopoiesis [72].

Alenzi and colleagues [73] showedgreater frequencies of myeloid progenitorcells (CFU-GM) in lpr and gld mice BM(Fas and FasL knockout mice, respectively)compared to wild-type (WT) mice marrow(p = 0.0008). The self renewal (i.e., prolifer-ation capacity) was also significantly greaterfor lpr and gld CFU-GM compared to WTCFU-GM. Retroviral-mediated gene trans-fer (RMGT) of Fas (apoptotic gene) into lprmarrow reduced CFU-GM self renewal(proliferative capacity) to WT levels. There-fore, Fas is likely to play an important role inregulating myeloid progenitor cell self re-newal. This raises the possibility thatFas/FasL are linked to self renewal (i.e., pro-liferation capacity) in mice. Therefore, ourresults suggest that the Fas/FasL pathwayand caspase activation inhibit progenitor cellproliferation and promote differentiation.They support proposals that caspase activa-tion may have non-apoptotic functions in theregulation of haemopoiesis [74-75]. Thus, itcan be inferred that the presence of fullyfunctional genes that regulate both cell pro-liferation (i.e., self renewal) and apoptosiswill maintain the balance between the rateof cell division and apoptosis of any (cell)population in vivo. Therefore, malfunctionin, or loss of, any of these genes (or inappro-priate DNA or RA splicing) may lead to anincrease in their self-replication (i.e., self re-newal).


We investigated whether changes in thelevel of apoptosis due to Fas/FasL deficien-cies cause changes in the number and kinet-ics of HSC. For this, mice with mutations inFas or FasL were used to investigate howthis change may affect the self renewal ofclonogenic haemopoietic cells. It has beenshown previously that haemopoietic activityis increased in lpr mice that have Fas muta-tions [76], but this was done only at the levelof the number of CFU-GM and not in termsof their proliferative activity. The results ob-tained differ from those of Schneider et al.[77], who did not show a significant increasein the CFU-GM frequency in the bone mar-row of lpr mice, although they found no dif-ference in BM cellularity. Instead, theyfound increased levels of extramedullaryhaemopoiesis, especially in the spleen,which is a site of haemopoiesis in normalmice. Our results are in line with those ofTraver et al. [78], who showed that lpr mar-row contains a significantly greater numberof myeloid progenitor cells compared to WTmice.

Our unpublished results concerning theself-renewal (i.e., proliferation) by NBMprogenitors are in line with Marley and col-leagues [79], who showed that the self re-newal values of CML progenitors aresignificantly greater than those of normalmarrow (p = 0.0001). There was a relativelylow frequency of apoptotic CD34+ cells inprogenitors grown from NBM with a signif-icant difference between the NBM and CMLsamples. We have accumulated a largernumber of CML samples and found a signif-icantly increased level of self renewal (i.e.,proliferation capacity) in CML compared toNBM (n = 100, p = 0.01). There was evi-dence of a difference in the self renewal (i.e.,proliferation capacity) between progenitorsgrown from NBM or CML samples. The fre-quency of apoptotic cells in CML progeni-tors was dramatically increased compared tonormal samples that agree that apoptosis isreduced in CML. These data are consistentwith those of Thiele et al. [80-86] and otherswho found a considerable level of apoptosisin CD34+ cells harvested from CML pa-tients. The correlation between apoptosis

and proliferation was fairly strongly positive(unpublished observations).

NON-APOPTOTIC ROLE OF FASAlthough several in vivo and in vitro

studies have demonstrated variable sensitiv-ities of haemopoietic precursors to Fas-me-diated apoptosis, HSC, at least in murinemodel, induced to express Fas after treat-ment with TNF-α showed reduction in theirengraftment potential [87]. The induced Fasexpression also decreased the self renewalof highly purified progenitors [87]. Further-more, murine HSC with best haemopoieticreconstituting potential express Fas;whereas, ~50 percent of the colony-formingcells in spleen-derived lineage-negative(lin–) progenitors were resistant to Fas liga-tion [87]. These data suggest that Fas ex-pression in HSC is not a negative factor inHSC apoptosis. Recent experiments sug-gest, however, that Fas can improve the en-graftment of HSC. With this regard, it wasfound that haemopoietic progenitors thathomed successfully to the BM showed amarked up-regulation in the expression ofthe Fas and FasL and were more resistant toinduction of apoptosis [88-89].

It also was found in a recent study thatFas is capable of transducing growth signalsin haemopoietic progenitors, after trimeriza-tion of this receptor [90]. Therefore, identifi-cation of factors that can up-regulate Fasexpression in HSC can be of particular im-portance to HSC transplantation. Inflamma-tory cytokines, in particular TNF-α and IFN-γinduced in response to environmental stressfactors, are a potent inducer of Fas expressionon the HSC and haemopoietic progenitors[91-94]. The acquisition of HSC to apoptoticsignals upon Fas up-regulation can help sus-tain viability of progenitors under stress con-ditions in case of allogeneic haemopoieticgrafts into myeloablative recipients. Ectopicexpression of FasL in HSC also showed bet-ter survival. For example, it has been reportedthat FasL-modified lin(-) BM can kill Fas-ex-pressing T cells in vitro and that transplantingof allogeneic FasL(+) lin(-) BM into recipientmice treated with nonmyeloablative condi-


tioning regimen resulted in an enhancedshort-term engraftment [95].

CLINICAL RELEVANCEIn CML, HSC was found to have defec-

tive self renewal and apoptosis. Much atten-tion is now focused on the identification ofthe features and biological properties of thecells responsible for generating and main-taining this neoplastic clone in patients withCML on their relationship to specific func-tions of the BCR-ABL gene product and onthe development of better experimental mod-els of the human diseases. From such an ap-proach, we can hope that this information,likely to have a greater impact on the treat-ment of CML, will finally be forthcoming.Improved understanding of apoptosis geneshas introduced an entirely new modality fortreating leukemias and lymphomas. Al-though these anti-apoptotic mechanisms arecurrently obscure, many of these new devel-opments will be implemented rapidly andsoon will play an important role inchemotherapeutic strategies in the treatmentof cancer. Such strategies are likely to radi-cally change the management of patientswith hematological malignancies.

CONCLUSIONCell population size is determined by a

balance between cell loss (apoptosis and dif-ferentiation) and cell gain (proliferation andmitosis). In normal haemopoiesis, these fac-tors are controlled so that steady-state kinet-ics are preserved. In contrast, in myeloidleukemias, myeloid expansion can be ex-plained by an increase in proliferation (selfrenewal), and reduced apoptosis. There havebeen huge recent advances in research intostem cells. We are currently in a phase wherethese developments may be used preciselytoward the successful attainment of the useof “intelligent” therapeutics for many dis-eases, such as CML.

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