A RT I C L E S

738 VOLUME 5 NUMBER 7 JULY 2004 NATURE IMMUNOLOGY

Although cancer is often regarded as overproliferation of neoplastic cellsthat do not differentiate properly, it has been known for more than acentury that solid tumors and leukemias show heterogeneity of cellularmorphology. However, morphology and function are difficult to link,and consequently little is known of the cells that maintain the neoplasm.The idea that only a minor subpopulation of so-called ‘cancer stem cells’is responsible for maintenance of the neoplasm emerged about 50 yearsago1, with the best evidence coming from the discovery that the mostacute myeloid leukemia (AML) blasts do not proliferate and only aminor proportion (∼ 1%) of human leukemic cells are clonogenic prog-enitors (AML colony-forming units (AML-CFU))2,3. However, parallelstudies of normal clonogenic progenitors showed that most are notrepopulating hematopoietic stem cells (HSCs), calling into questionwhether AML-CFU represent a true leukemic stem cell (LSC)4.Conclusive evidence for the existence of LSCs came from the identifica-tion of a very rare population of human SCID leukemia-initiating cells(SL-ICs; 1 per 1 × 106 leukemic blasts) that were capable of propagatingacute myeloid leukemia in a xenograft transplant system developed forleukemic and normal stem cells5–8. In this system, SL-ICs generatedleukemic grafts that were highly representative of the original patient’sdisease, having both identical blast morphology and dissemination pro-files. Cell purification, based on cell surface markers that allow enrich-ment for normal stem and progenitor cells, demonstrated that theCD34+CD38– fraction from a large number of AML samples was highlyenriched for SL-ICs, but none were found in any other fraction, includ-ing the CD34+CD38+ fraction8–10. As AML-CFU are contained in the

CD34+CD38+ fraction, these studies provided functional proof that theAML clone is organized as a hierarchy that originates from SL-ICs,which produce AML-CFU and leukemic blasts. Normal HSCs, as mea-sured by repopulation of nonobese diabetic–severe combined immuno-deficient (NOD-SCID) mice, are also found exclusively in theLin–CD34+CD38– fraction4,11,12. Our hypothesis was that the similarcell surface phenotype between cell fractions highly enriched for normalHSCs and SL-ICs from AML samples derived from a wide diversity ofleukemic subtypes in terms of their differentiation properties indicatedthat LSCs originated from the HSC pool rather than the committedprogenitor pool13. However, one limitation of this comparison is thatthe leukemogenic process disrupts cell differentiation, making a directlink based on cell surface markers between LSCs and a representativestage of normal hematopoietic development tenuous. Indeed, some dif-ferences between normal HSC and LSC surface marker expression havebeen identified14–17. Thus, the only comparison that is reliable must bebased on functional stem cell properties.

The essential feature common to all stem cells is the capacity for self-renewal after cell division, with the production of at least one daughtercell that has stem cell properties identical to those of the parent18. Theprogressive loss of self-renewal capacity is probably the mechanismunderlying the existence of multipotential murine HSCs with short-term and long-term repopulation capacity. Earlier clonal tracking stud-ies showed that the human HSC pool is similarly composed ofshort-term and long-term SCID-repopulating cells (SRCc)19. If LSCsand HSCs are related as we predict, the LSC pool should be similarly

Division of Cell and Molecular Biology, University Health Network, and Department of Molecular Genetics and Microbiology, University of Toronto, 620 UniversityAvenue, Toronto, Ontario, M5G 2C1, Canada. 1These authors contributed equally to this work. Correspondence should be addressed to J.E.D.(jdick@uhnres.utoronto.ca).

Published online 30 May 2004; doi:10.1038/ni1080

Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacityKristin J Hope1, Liqing Jin1 & John E Dick

Emerging evidence suggests cancer stem cells sustain neoplasms; however, little is understood of the normal cell initially targeted and the resultant cancer stem cells. We show here, by tracking individual human leukemia stem cells (LSCs) in nonobesediabetic–severe combined immunodeficiency mice serially transplanted with acute myeloid leukemia cells, that LSCs are notfunctionally homogeneous but, like the normal hematopoietic stem cell (HSC) compartment, comprise distinct hierarchicallyarranged LSC classes. Distinct LSC fates derived from heterogeneous self-renewal potential. Some LSCs emerged only in recipientsof serial transplantation, indicating they divided rarely and underwent self-renewal rather than commitment after cell divisionwithin primary recipients. Heterogeneity in LSC self-renewal potential supports the hypothesis that they derive from normal HSCs.Furthermore, normal developmental processes are not completely abolished during leukemogenesis. The existence of multiplestem cell classes shows the need for LSC-targeted therapies.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urei

mm

unol

ogy

A RT I C L E S

NATURE IMMUNOLOGY VOLUME 5 NUMBER 7 JULY 2004 739

complex. Although LSCs are typically regarded as homogenous, nostudies to our knowledge have directly addressed whether cancer stemcells or LSCs are homogeneous or functionally heterogeneous.Theoretically, all cancer stem cells must have self-renewal potential tocontinuously regenerate the neoplastic clone; however, the self-renewalof individual human cancer stem cells from any neoplasm has neverbeen established, to our knowledge. Here we demonstrate that the LSCpool in AML is composed of distinct LSCs that are hierarchically orga-nized because of heterogeneity in longevity of the produced clone andself-renewal capacity.

RESULTSLentivirus vector transduction of SL-ICsUse of retrovirus vector–mediated clonal tracking and the NOD-SCIDxenotransplant system to assay SRC has provided insight into the compo-sition of the stem cell compartment and has shown that the normalhuman HSC pool is composed of individual HSCs that differ in theirrepopulation and self-renewal capacity19. To directly compare LSCs andHSCs using a similar clonal tracking approach, we first attempted to‘mark’ AML cells by transduction with retrovirus vectors followed bytransplantation into NOD-SCID mice. However these marking studieswere unsuccessful (data not shown) because of poor SL-IC survival in theculture conditions required to initiate cell cycling of quiescent SL-ICs20–22.In contrast, lentivirus vectors effectively transduce normal SRCs afterovernight exposure of these cells to viral particles in cytokine- and serum-free media23–25. Transduction of AML cells with a lentivirus vector con-taining a gene encoding enhanced green fluorescent protein (EGFP)resulted in expression of EGFP in 40.5% ± 6.4% of the total cells and38.0% ± 11.1% of clonogenic AML-CFU. This proportion of transducedcells was maintained for 28 d in suspension culture, suggesting that prim-itive leukemic progenitors were stably transduced (data not shown).

To determine whether the rare LSC population, as measured with theSL-IC assay, could be efficiently transduced by lentivirus vectors andpreserved in minimal culture conditions, we transduced cells from fivedifferent AML donors and then injected them into sublethally irradi-ated NOD-SCID mice at a dose of 5 × 106 to 10 × 106 cells per mouse.We assessed the kinetics of growth of marked and unmarked AML cellswithin NOD-SCID mice by serial bone marrow aspiration at 4, 8 and12 weeks after transplantation (Fig. 1). The median proportion ofhuman engraftment within the mouse bone marrow was maintainedbetween 4 weeks (40%) and 12 weeks (49%), and the percentage ofmarked human cells over time remained relatively constant (49–46% at4 and 12 weeks, respectively; Fig. 1a,d). The human cells isolated fromthe bone marrow of engrafted mice showed aberrant expression ofmyeloid markers and had an abnormal blast-like morphology typical ofthe leukemic blasts of the original donor (Fig. 2). Moreover, wedetected only AML-CFU and not normal hematopoietic progenitors byclonogenic assay of the bone marrow of engrafted mice (data notshown). In keeping with past findings, no normal HSCs engraftedNOD-SCID mice when peripheral blood from AML patients was transplanted8,9,13,26. Normal SRCs, if present, always generate a highproportion (>75%) of CD19+ B cells as well as multiple lineages ofmyeloerythroid clonogenic progenitors. These data indicate thatlentivirus vectors efficiently transduce SL-ICs in conditions that per-mit their survival and allow repopulation of NOD-SCID mice.

Functional heterogeneity of SL-ICsThe engraftment kinetics of each specific patient sample was highlyreproducible within cohorts of mice and over multiple experiments,although each donor sample generated a distinct engraftment pattern(data not shown). These patient-specific engraftment kinetics suggestthat the SL-ICs from each sample had intrinsically unique repopula-tion capacities. In addition, these different patterns of engraftment‘predict’ heterogeneity in the repopulation potential of SL-ICs.

To directly assess the activity of individual transduced SL-ICs, weused clonal analysis by Southern hybridization of DNA extractedfrom serial femoral aspirates, as well as from the whole bone mar-row, spleen and other organs of recipient mice. Of four mice withrepresentative repopulation dynamics (Fig. 3a), in mouse 1 wedetected a unique clone at the 4-week time point; however, at latertimes we did not find this clone. In this same mouse, many newclones arose at 12 weeks that were not detected at 4 or 8 weeks. Inmouse 2, one clone was detected at 4 weeks only; a unique clone, at8 weeks only; and many later clones, at the final time point. In mice3 and 4, clones making a dominant contribution at early points per-sisted until 12 weeks, despite the emergence of several new clones.In all AML samples tested, we noted clones that contributed tran-siently as well as stable clones. We called these ‘short-term SL-ICs’and ‘long-term SL-ICs’, respectively. We also used integration siteanalysis to investigate the dissemination of leukemic clones to sitesoutside the bone marrow and found that many clones in the bonemarrow were also present in the spleen and other tissues (Fig. 3b).These data show substantial heterogeneity in the contribution thatindividually marked SL-ICs made to the graft.

Individual SL-ICs have different self-renewal capacitiesTo determine the mechanism that might underlie the functionalheterogeneity of SL-ICs, we assessed the self-renewal capacity ofindividually marked SL-ICs. Self-renewal is a defining property ofstem cells18, and preliminary work has suggested that as a popula-tion, SL-ICs have increased self-renewal capacity compared withthat of normal SRCs13. ‘Serial transplantation’ is the best measure of

a

b

c

d

e

f

Figure 1 Efficient transduction of SL-ICs. (a–c) Proportion of human leukemiccells in the bone marrow of transplanted primary (a), secondary (b) andtertiary (c) NOD-SCID mice. Each circle represents data from an individualmouse and horizontal bars indicate the median value. (d–f) Kinetics of thecontribution of EGFP+ cells in the human leukemia graft in primary (d),secondary (e) and tertiary (f) mice. Values represent mean ± standard error.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urei

mm

unol

ogy

A RT I C L E S

740 VOLUME 5 NUMBER 7 JULY 2004 NATURE IMMUNOLOGY

self-renewal. To accomplish this, we injected the bone marrow froman engrafted NOD-SCID primary mouse into two secondary miceand from a secondary recipient into two tertiary recipients. We didtransplantations only when the donor graft was greater than 10%human cells. Flow cytometric analysis of engraftment at 12 weeks in arepresentative serial transplantation experiment demonstrated a highproportion of transduced human cells in the bone marrow of pri-mary, secondary and tertiary mice (Fig. 4a). Of 47 secondary recipi-ents, 40 were repopulated with both marked and unmarked cells overthe 12-week engraftment period (Fig. 1b,e). Transplantation into ter-tiary recipients also yielded high percentages of both marked andunmarked leukemic cells in the bone marrow of 23 of 27 tertiary mice(Fig. 1c,f). The presence of many EGFP+ cells in both secondary andtertiary mice attests to the efficient transduction of SL-ICs withextensive self-renewal capacity.

To characterize the self-renewal potential of individual SL-ICs, we didintegration site analysis of the serial aspirates and whole bone marrowfrom secondary and tertiary recipients (Fig. 4b). Some clones from pri-mary mice were present in the secondary recipients, providing conclu-sive proof that the long-term SL-ICs giving rise to these clones musthave undergone self-renewal in the primary mouse. In many cases, bothof the two secondary recipients transplanted with leukemic cells from

the same primary mouse contained common clones that were alsodetected in the primary donor (Fig. 4b, primary mice 1, 2 and 6). SL-ICscapable of repopulating both secondary recipients may have moreextensive self-renewal capacity than those SL-ICs in only one secondaryrecipient. We created Venn diagrams to demonstrate which proportionof the 169 tracked clones was unique to primary mice, could be found inserial secondary and tertiary mice having undergone serial transplanta-tion or first appeared only after serial transplantation (Fig. 4c). Manycases showed many clones present in the primary mouse and not ineither of the secondary recipients, suggesting that short-term SL-ICclones and some of those tentatively identified as long-term SL-ICsbecause they were still present at 12 weeks after transplant in primarymice had reduced self-renewal capacity, preventing their detection insecondary mice (Fig. 4c). We also noted individual clones that con-tributed persistently in primary mice but produced only a transient graftin secondary mice (Fig. 4d). This result is evidence of the generation of short-term SL-ICs from long-term SL-ICs, which was probablybecause of the intrinsic stochastic nature of self-renewal, in which thetwo daughter cells at a specific cell division both commit rather thanself-renewing, resulting in eventual clonal extinction18. We found fewer clones after tertiary transplants, indicating that the extensive self-renewal capacity necessary for repopulation of tertiary recipients is

a b

c d

e f

Figure 2 Differential characteristics of human grafts in NOD-SCID micetransplanted with normal cord blood or AML cells. Phenotype andmorphology of human cells isolated from the bone marrow of NOD-SCIDmice engrafted with either unsorted lineage-depleted cord blood cells (CB;a,c,e) or AML cells (b,d,f). (a–d) Flow cytometric analysis of the expressionof CD19 and CD15 (a,b) and CD33 and CD14 (c,d). (e,f) May-GrunwaldGiemsa–stained cytospin preparations.

a bFigure 3 In vivo kinetic activity of individualSL-IC clones in NOD-SCID mice as determinedby integration site analysis. (a) Kinetics ofindividual SL-IC clones in the bone marrow of primary NOD-SCID mice. Gray arrowheads,clones present at multiple time points; blackarrowheads, clones present at only late timepoints; open arrowheads, representative clonespresent at only early time points. Mice 1 and 4were transplanted with cells from patient 5;and mice 2 and 3, with cells from patient 4.Femur, femoral aspirates; BM, bone marrow.4w, 8w and 12w, 4, 8 and 12 weeks,respectively. (b) Dissemination of transducedSL-IC clones into tissues at 12 weeks aftertransplantation in a representative mousetransplanted with cells from patient 3.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urei

mm

unol

ogy

A RT I C L E S

NATURE IMMUNOLOGY VOLUME 5 NUMBER 7 JULY 2004 741

restricted to a small proportion of the SL-IC compartment (Fig. 4b, pri-mary mice 6 and 7). These studies indicated that heterogeneity in theself-renewal capacity of SL-ICs underlies the differences in clonallongevity and that only a relatively small proportion of SL-ICs haveextensive self-renewal potential and aggressively drive AML growth.

We identified a small proportion of clones that were unique to sec-ondary mice and were not detected in primary mice (Fig. 4b, primarymice 2, 5, 6 and 7), and in one case we detected a new clone in a tertiaryrecipient (Fig. 4b, primary mouse 7). Within each of the cohorts of sec-ondary mice transplanted with one of four different patient samples,we noted at least one secondary mouse that showed this pattern ofclonal emergence, indicating that although they are infrequent, suchSL-ICs were a common component of AML. Because we collected only10–20% of the entire bone marrow compartment for secondary trans-plant, it is likely that these marked SL-ICs proliferated to some extent inprimary mice but remained below the limit of detection. SL-ICs aregenerally quiescent21,22,27, and we speculate that this rare SL-IC classmust cycle very slowly, and/or after division both daughter cells remainslowly proliferating. We called these ‘quiescent long-term SL-ICs’.Alternately, it is possible this SL-IC class has the same rate of cell cyclingas other classes, but both daughter cells self-renew and remain as SL-ICs rather than differentiating to become rapidly expanding progenitor

populations that yield a large clone size. After secondary or tertiarytransplantation, the quiescent long-term SL-ICs, because of stochasticprocesses or the stimulation caused by transplantation, divide to gener-ate at least one daughter cell that is committed to the proliferatingprogenitor pool, resulting in a clone detectable by Southern blot analy-sis. We determined the cumulative numbers of clones with differentself-renewal capacities from multiple mice transplanted with cells fromeach patient (Fig. 4c). In all cases, SL-ICs detectable only in secondaryor tertiary mice were the most infrequent of all the SL-IC classes.

DISCUSSIONOur study has provided direct evidence of self-renewal of individual can-cer stem cells and for the existence, at least for AML, of distinct LSCclasses that are organized as a hierarchy. The mechanism that creates thehierarchical structure of the LSC pool derives from heterogeneity in theself-renewal potential of individual LSCs. A model consistent with ourexperimental results includes three distinct classes of SL-ICs, which wehave called short-term, long-term and quiescent long-term SL-ICs.Clonal tracking conclusively demonstrated that long-term SL-ICs(defined by persistence for 12 weeks in primary mice) gave rise to short-term SL-ICs (defined by transient repopulation at only 4 or 8 weeks) aftertransplantation into secondary mice, and quiescent long-term SL-ICs

b d

a c

Figure 4 Heterogeneous self-renewal capacity of transduced SL-ICs. (a) Flow cytometric analysis of the proportion of human cells in the bone marrow of mice afterserial transplantation in one representative experiment. Numbers in upper right corners indicate percentage of EGFP+ cells in the human (h) CD45+ population. (b) Heterogeneity in the self-renewal of transduced SL-ICs assessed by integration site analysis. Because of the large number of bands, only representative bandsare indicated by arrows. Clones were present in primary (1°) mice alone (open arrows), in secondary (2°) or tertiary (3°) mice alone (black arrows) or in both primaryand secondary mice (gray arrows). The number before each tissue indicates the unique mouse identification numbers as follows: ‘Primary mouse number, secondarymouse number, tertiary mouse number’. Mice 6 and 7 were transplanted with cells from patient 2, whereas donors for other mice are described in Figure 3. (c) Cumulative number of clones with different self-renewal capacities over all serial transplants done for each patient tested. Numbers in intersecting circlesrepresent clones present in both or all three recipients. For patient 2, one experiment (right) is shown separately, as clonal analysis for the primary mouse wasinconclusive. (d) Kinetic analysis of SL-IC clones in secondary NOD-SCID recipients. Arrowhead shading is as described in Figure 3.©

2004

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

A RT I C L E S

742 VOLUME 5 NUMBER 7 JULY 2004 NATURE IMMUNOLOGY

gave rise to both SL-IC subclasses. The fact that both LSC and normalHSC compartments are structured as a hierarchy provides strong sup-port for the hypothesis that in AML the initial target cell for transforma-tion lies within the HSC compartment8,13,28. LSCs, like normal HSCs,have self-renewal capacity, although the clonal marking data as well asprior quantitative analysis by limiting dilution13 indicate that self-renewal is dysregulated, as LSCs have substantially higher self-renewalcapacity than do normal SRCs. LSCs still retain the ability to regulate self-renewal, resulting in a hierarchically organized stem cell pool withnotable similarities to the normal HSC compartment. Therefore, thecommitment and/or differentiation and renewal ‘decisions’ of LSCs arenot completely uncoupled, suggesting that the leukemogenic processesdo not abolish all the pathways that normally regulate stem cell develop-mental programs. The finding that the stem cell–specific gene Bmi1 is keyin the self-renewal of both normal and leukemic murine stem cells sup-ports this idea18,29. The model we favor, because fewer mutagenic eventsare required, ‘predicts’ that the intrinsic self-renewal capacity as well asthe means to regulate self-renewal of the HSCs targeted by leukemogenicevents continue to function in the resultant LSC. These studies supportthe idea that leukemia represents normal hematopoietic developmentgone slightly awry, with the leukemic clone retaining aspects of normaldevelopmental programs, perhaps assembled in aberrant ways, ratherthan the view that leukemia represents the generation of an entirely aber-rant cell type with little resemblance to normal hematopoiesis. Thismodel can accommodate the idea that for some forms of leukemia, theinitiating leukemogenic events occur in HSCs, but subsequent alterationscould occur in ‘downstream’ progenitors from which the LSCs emerge.Our data could also fit an alternative model in which normal committedprogenitors that have lost self-renewal capacity could reacquire both self-renewal capacity as well as the ability to regulate this process such that acomplex LSC hierarchy is re-established. However, this scenario requiresthat a progenitor accumulate many more mutations to attain the cellularproperties that are intrinsic to HSCs. The origin of at least some forms ofacute lymphoblastic leukemia might differ from AML because even somemature lymphocytes, such as memory cells, retain self-renewal poten-tial30. Progress to address the leukemogenic process in acute lymphoblas-tic leukemia must await identification of the acute lymphoblasticleukemia LSCs, a prospect that should be possible using NOD-SCIDrepopulation6,31.

The discovery of functional complexity in the LSC compartmenthas implications for the investigation of cancer-specific signalingpathways and the development of stem cell–targeted AML therapies.As cancer pathways may function differently in each LSC subclassthan in the bulk leukemic blasts, different responses to a given therapymay result. Effective therapy must target the highly self-renewinglong-term SL-ICs in a functionally heterogeneous SL-IC pool that isresponsible for aggressively driving the growth and relapse of AML.At present, AML therapies typically target proliferating cells; however,SL-ICs are quiescent, making them poorly responsive to such agents.More troubling from the standpoint of developing stem cell–targetedtherapeutics is the discovery of rare quiescent long-term SL-ICs thatself-renew in primary recipients but divide infrequently and/or donot produce daughter cells committed to leukemic cell populationexpansion. Such persistently quiescent and/or highly self-renewingLSCs could be difficult to eradicate, although recent purging strate-gies that inhibit the transcription factor NF-κB seem to target thisclass32. Additionally, the discovery of LSC subclasses may have clinicalcorrelates. AML patients that relapse within 1 year of first remissiontypically relapse with chemoresistant disease, suggesting tumor pro-gression. However, those who relapse later respond to the chemother-apeutic agents used to achieve first remission, suggesting reappearance

of LSCs that had been initially quiescent and unaffected by chemother-apy; quiescent long-term SL-ICs could be representative of this clinicalsituation. As the insights into the biology of cancer stem cells gainedfrom leukemia have been shown to be relevant to other cancers, includ-ing solid tumors such as brain and breast cancer1,33–36, our results inleukemia ‘predict’ that it will be important to explore the functionalheterogeneity of such tumor-initiating cells using the paradigm wehave established. Indeed the clonal dynamics of leukemic progressionwe noted here after serial transplantation could have implications forunderstanding of the mechanism that underlies tumor relapse, pro-gression and metastasis37.

METHODSPatient samples. After informed consent was obtained, peripheral blood cellswere obtained from patients with newly diagnosed AML according to proceduresapproved by the Human Experimentation Committee. Patients were diagnosedand classified based on the criteria of the French-American-British group: patients1 and 3 were M4; patients 4 and 5 were M5; and patient 2 was M2. Each of the sam-ples used for clonal analysis had normal cytogenetics. Mononuclear blood cellswere isolated by Ficoll Hypaque (Pharmacia) density gradient centrifugation andthen were cryopreserved in FCS containing 10% dimethyl sulfoxide.

Lentivirus production. A third-generation self-inactivating lentivirus vectorwith a gene encoding EGFP expressed from the human phosphoglyceratekinase promoter was used38. This vector also contains the central polypurinetrack as well as the woodchuck hepatitis virus post-regulatory element. Viralsupernatant was generated by transient transfection of 293T cells with packag-ing plasmids and was pseudotyped with the vesicular stomatitis virus G proteinas described25. High titer stocks were prepared by ultracentrifugation. Thefunctional titers of viral vectors, determined by infection of HeLa cells, weremore than 1 × 109 particle-forming units/ml.

Transduction of AML cells. Transduction was done in 10-cm dishes. FrozenAML mononuclear blood cells were thawed gradually at 37 °C and were washedin X-VIVO 10 (BioWhittaker) supplemented with 50% FCS and 100 µg/ml ofDNAse 1 (Roche). By trypan blue exclusion, the viability of thawed AML cellswas always more than 90%. AML cells were then incubated with viral super-natant at a multiplicity of infection of 30 in serum-free medium supplementedwith 15% BIT (BSA-insulin-transferrin) (Stem Cell Technologies) and 2 mM L-glutamine (Gibco BRL) in the absence of cytokines. The cells were infected for16–24 h at 37 °C and in 5% CO2. After infection, cells were collected and washedfor removal of virus.

In vitro assays. Immediately after infection, transduced AML cells were placed inserum-free suspension cultures with 15% BSA-insulin-transferrin and a cytokinemixture containing 100 ng/ml of stem cell factor, 20 ng/ml of interleukin 6 (IL-6),20 ng/ml of granulocyte colony-stimulating factor, 20 ng/ml of IL-3, 100 ng/ml ofFlt-3 ligand (each provided by Amgen), 20 ng/ml of granulocyte-monocytecolony-stimulating factor (R&D Systems) and 50 ng/ml of thrombopoietin(Kirin Brewery). The gene transfer efficiency into AML cells was estimated by theflow cytometric analysis of EGFP expression after 7, 14, 21 and 28 d of suspensionculture. For the AML-CFU assay, AML cells collected from suspension cultureswere plated at density of 0.1 × 105 to 2 × 105 cells/ml of in 0.9% methylcellulosecontaining 15% FCS, 15% pretested human plasma, 50 µM β-mercaptoethanol,and cytokines at concentrations of 100 ng/ml of stem cell factor, 100 ng/ml of Flt-3 ligand, 20 ng/ml of IL-6, 20 ng/ml of granulocyte-monocyte colony-stimulatingfactor, 20 ng/ml of IL-3 and 3 U/ml of erythropoietin (Amgen). After the cultureswere incubated for 12–14 d at 37 °C, duplicate plates were assigned scores for thepresence of AML-CFU (>50 cells). AML colonies expressing EGFP were viewedwith a fluorescent microscope for assignment of scores.

Analysis of SL-ICs by NOD-SCID mouse repopulation. NOD-SCID micewere bred at the University Health Network/Princess Margaret Hospital.Animal experimentation followed protocols approved by the UniversityHealth Network/Princess Margaret Hospital Animal Care Committee. NOD-SCID mice 6–8 weeks old were irradiated with 3.6 Gy before being injected in

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urei

mm

unol

ogy

A RT I C L E S

NATURE IMMUNOLOGY VOLUME 5 NUMBER 7 JULY 2004 743

the tail vein with transduced AML cells at a dose of 5 × 106 to 10 × 106 cells permouse. Some mice received stem cell factor, granulocyte-monocyte colony-stimulating factor and IL-3 by intraperitoneal injection at doses of 10 µg, 7 µgand 7 µg per mouse, respectively, three times a week for 2 weeks after trans-plant. A cohort of mice transplanted with certain AML samples did not receivecytokines, as cells from these samples could highly engraft mice in the absenceof cytokines. At 4-week intervals after transplantation, bone marrow sampleswere aspirated from the femur as described39. Mice were killed at 8 or 12 weeks(2 d after the last aspiration) and total bone marrow from both femurs andboth tibias, as well as spleen, thymus, liver, lung and kidney, were collected forflow cytometry and DNA extraction.

Flow cytometry. A FACScalibur (Becton Dickinson) was used for flow cytome-try. Isotypic controls were mouse immunoglobulin G conjugated to fluoresceinisothiocyanate (FITC), phycoerythrin (both from Becton Dickinson) or phyco-erythrin–cyanine 5 (Coulter). Human leukemia cells from transplanted micewere assessed with antibody to CD45 (anti-CD45)–phycoerythrin–cyanine 5,anti-CD15–phycoerythrin–cyanine 5 (Beckman-Coulter), anti-CD45–phyco-erythrin, anti-CD15–FITC, anti-CD14–phycoerythrin, anti-CD33–phycoery-thrin and anti-CD19–phycoerythrin (Becton-Dickinson). EGFP fluorescencewas detected with detector channel FL1 calibrated to the FITC emission profile.

Integration site analysis of lentivirus vector–transduced SL-ICs. GenomicDNA from the bone marrow aspirates, whole bone marrow, spleen, liver, kidney,lungs and thymus of the engrafted NOD-SCID mice was extracted and 15 µg wasdigested with BamHI, which cuts once within the proviral DNA upstream of thegene encoding EGFP. From those aspirates containing low cell numbers, less than15 µg of DNA was digested. After electrophoresis and transfer to a nylon mem-brane (Hybond N+; Amersham), DNA was hybridized to an EGFP probe.

Estimation of self-renewal potential of SL-IC clones. Bone marrow and spleencells from mice highly engrafted with transduced AML cells were obtained 8 or12 weeks after transplantation. Equal amounts of cells collected from each pri-mary mouse were injected through the tail vein into two sublethally irradiatedsecondary NOD-SCID recipients. Bone marrow cells were sampled by aspira-tion and were analyzed at 4, 8 and 12 weeks after transplantation. Bone marrowand spleen cells were collected from repopulated secondary recipients killed at12 weeks after transplantation, and tertiary transplantation was done as out-lined for secondary transplantation.

Statistical analysis. Data are presented as the mean ± standard error of the mean.

ACKNOWLEDGMENTSWe thank M. Minden (Princess Margaret Hospital, Toronto, Ontario, Canada) andE. Warren (Hutchinson Cancer Center, Seattle, Washington, USA) for providingAML samples; and members of the Dick lab, M. Minden, N. Iscove and C. Jordanfor critical comments on the manuscript. Supported by the Leukemia ResearchFund of Canada (L.J.) and Canadian Institutes for Health Research (L.J. and K.H.);and The Stem Cell Network of the National Centres of Excellence, National CancerInstitute of Canada and Canadian Cancer Society, Canadian Genetic DiseasesNetwork of the National Centres of Excellence, Canadian Institutes for HealthResearch, and Canada Research (J.E.D.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 31 December 2003; accepted 6 April 2004Published online at http://www.nature.com/natureimmunology/

1. Dick, J.E. Breast cancer stem cells revealed. Proc. Natl. Acad. Sci. USA 100,3547–3549 (2003).

2. McCulloch, E. Stem cells in normal and leukemic hemopoiesis (Henry StrattonLecture). Blood 62, 1–13 (1983).

3. Griffin, J. & Löwenberg, B. Clonogenic cells in acute myeloblastic leukemia. Blood68, 1185–1195 (1986).

4. Larochelle, A. et al. Identification of primitive human hematopoietic cells capable ofrepopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat.Med. 2, 1329–1337 (1996).

5. Kamel-Reid, S. & Dick, J.E. Engraftment of immune-deficient mice with humanhematopoietic stem cells. Science 242, 1706–1709 (1988).

6. Kamel-Reid, S. et al. A model of human acute lymphoblastic leukemia in

immune-deficient SCID mice. Science 246, 1597–1600 (1989).7. Lapidot, T. et al. Cytokine stimulation of multilineage hematopoiesis from immature

human cells engrafted in scid mice. Science 255, 1137–1141 (1992).8. Lapidot, T. et al. A cell initiating human acute myeloid leukaemia after transplanta-

tion into SCID mice. Nature 367, 645–648 (1994).9. Ailles, L.E., Gerhard, B., Kawagoe, H. & Hogge, D.E. Growth characteristics of acute

myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobesediabetic/severe combined immunodeficient mice. Blood 94, 1761–1772 (1999).

10. Rombouts, W.J., Martens, A.C. & Ploemacher, R.E. Identification of variables deter-mining the engraftment potential of human acute myeloid leukemia in the immun-odeficient NOD/SCID human chimera model. Leukemia 14, 889–897 (2000).

11. Bhatia, M., Wang, J.C.Y., Kapp, U., Bonnet, D. & Dick, J.E. Purification of primitivehuman hematopoietic cells capable of repopulating immune-deficient mice. Proc.Natl. Acad. Sci. USA 94, 5320–5325 (1997).

12. Conneally, E., Cashman, J., Petzer, A. & Eaves, C. Expansion in vitro of trans-plantable human cord blood stem cells demonstrated using a quantitative assay oftheir lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.Proc. Natl. Acad. Sci. USA 94, 9836–9841 (1997).

13. Bonnet, D. & Dick, J.E. Human acute myeloid leukemia is organized as a hierarchythat originates from a primitive hematopoietic cell. Nat. Med. 3, 730–737 (1997).

14. Blair, A., Hogge, D.E., Ailles, L.E., Lansdorp, P.M. & Sutherland, H.J. Lack ofexpression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term prolifer-ative ability in vitro and in vivo. Blood 89, 3104–3112 (1997).

15. Jordan, C.T. et al. The interleukin-3 receptor α chain is a unique marker for humanacute myelogenous leukemia stem cells. Leukemia 14, 1777–1784 (2000).

16. Blair, A., Hogge, D.E. & Sutherland, H.J. Most acute myeloid leukemia progenitorcells with long-term proliferative ability in vitro and in vivo have the phenotypeCD34+/CD71–/HLA-DR. Blood 92, 4325–4335 (1998).

17. Brendel, C. et al. Detection of cytogenetic aberrations both in CD90 (Thy-1)-positiveand (Thy-1)-negative stem cell (CD34) subfractions of patients with acute andchronic myeloid leukemias. Leukemia 13, 1770–1775 (1999).

18. Dick, J.E. Stem cells: Self-renewal writ in blood. Nature 423, 231–233 (2003).19. Guenechea, G., Gan, O.I., Dorrell, C. & Dick, J.E. Distinct classes of human stem cells

that differ in proliferative and self-renewal potential. Nat. Immunol. 2, 75–82 (2001).20. Ailles, L.E., Humphries, R.K., Thomas, T.E. & Hogge, D.E. Retroviral marking of

acute myelogenous leukemia progenitors that initiate long-term culture and growthin immunodeficient mice. Exp. Hematol. 27, 1609–1620 (1999).

21. Guzman, M.L. et al. Nuclear factor-κB is constitutively activated in primitive humanacute myelogenous leukemia cells. Blood 98, 2301–2307 (2001).

22. Terpstra, W. et al. Fluorouracil selectively spares acute myeloid leukemia cells withlong-term growth abilities in immunodeficient mice and in culture. Blood 88,1944–1950 (1996).

23. Mazurier, F., Gan, O., McKenzie, J., Doedens, M. & Dick, J. Lentivector-mediated clonaltracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compart-ment and culture-induced stem cell impairment. Blood 103, 545–552 (2004).

24. Miyoshi, H., Smith, K.A., Mosier, D.E., Verma, I.M. & Torbett, B.E. Transduction ofhuman CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIVvectors. Science 283, 682–686 (1999).

25. Guenechea, G. et al. Transduction of human CD34+ CD38– bone marrow and cordblood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol.Ther. 1, 566–573 (2000).

26. Terpstra, W. et al. Conditions for engraftment of human acute myeloid leukemia(AML) in SCID mice. Leukemia 9, 1573–1577 (1995).

27. Guan, Y., Gerhard, B. & Hogge, D.E. Detection, isolation, and stimulation of quies-cent primitive leukemic progenitor cells from patients with acute myeloid leukemia(AML). Blood 101, 3142–3149 (2003).

28. Till, J.E. & McCulloch, E.A. Hemopoietic stem cell differentiation. Biochim.Biophys. Acta 605, 431–459 (1980).

29. Lessard, J. & Sauvageau, G. Bmi-1 determines the proliferative capacity of normaland leukemic stem cells. Nature 423, 455–460 (2003).

30. Greaves, M. Differentiation-linked leukemogenesis in lymphocytes. Science 234,697–704 (1986).

31. Kamel-Reid, S. et al. Bone marrow from children in relapse with pre-B acute lym-phoblastic leukemia proliferates and disseminates rapidly in scid mice. Blood 78,2973–2981 (1991).

32. Guzman, M.L. et al. Preferential induction of apoptosis for primary human leukemicstem cells. Proc. Natl. Acad. Sci. USA 99, 16220–16225 (2002).

33. Al-Hajj, M., Wicha, M., Morrison, S.J. & Clarke, M.F. Prospective identification oftumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100, 3983–3988(2003).

34. Reya, T., Morrison, S.J., Clarke, M.F. & Weissman, I.L. Stem cells, cancer, and can-cer stem cells. Nature 414, 105–111 (2001).

35. Singh, S. et al. Identification of a cancer stem cell in human brain tumours. CancerRes. 63, 5281–5288 (2003).

36. Marx, J. Cancer research. Mutant stem cells may seed cancer. Science 301,1308–1310 (2003).

37. Bernards, R. & Weinberg, R.A. A progression puzzle. Nature 418, 823 (2002).38. Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by

lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 polsequences. Nat. Genet. 25, 217–222 (2000).

39. Guenechea, G. et al. Delayed engraftment of nonobese diabetic/severe combinedimmunodeficient mice transplanted with ex vivo-expanded human CD34+ cordblood cells. Blood 93, 1097–1105 (1999).

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urei

mm

unol

ogy