Identification of a Small Subpopulation of Candidate Leukemia-Initiating Cells in the Side Population of Patients with Acute Myeloid Leukemia

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In acute myeloid leukemia (AML), apart from the CD34+CD38− compartment, the side population (SP) compartment contains leukemic stem cells (LSCs). We have previously shown that CD34+CD38− LSCs can be identified using stem cell-associated cell surface
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  Identification of a Small Subpopulation of CandidateLeukemia-Initiating Cells in the Side Population of Patientswith Acute Myeloid Leukemia B IJAN  M OSHAVER , A NNA VAN  R HENEN , A NG ` ELE  K ELDER , M ARJOLEIN VAN DER  P OL , M ONIQUE  T ERWIJN ,C OSTA  B ACHAS , A UGUST  H. W ESTRA , G ERT  J. O SSENKOPPELE , S ONJA  Z WEEGMAN , G ERRIT  J AN  S CHUURHUIS Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands Key Words.  Acute myeloid leukemia • Stem cell • Side population • Flow cytometry A BSTRACT In acute myeloid leukemia (AML), apart from theCD34  CD38  compartment, the side population (SP) com-partment contains leukemic stem cells (LSCs). We havepreviously shown that CD34  CD38  LSCs can be identifiedusing stem cell-associated cell surface markers, includingC-type lectin-like molecule-1 (CLL-1), and lineage markers,such as CD7, CD19, and CD56. A similar study was per-formed for AML SP to further characterize the SP cells withthe aim of narrowing down the putatively very low stem cellfraction. Fluorescence-activated cell sorting (FACS) analysisof 48 bone marrow and peripheral blood samples at diag-nosis showed SP cells in 41 of 48 cases that were partly orcompletely positive for the markers, including CD123. SPcells in normal bone marrow (NBM) were completely neg-ative for markers, except CD123. Further analysis revealedthattheSPfractioncontainsdifferentsubpopulations:(a)threesmall lymphoid subpopulations (with T-, B-, or natural killer-cellmarkers);(b)adifferentiatedmyeloidpopulationwithhighforward scatter (FSC high ) and high sideward scatter (SSC high ),high CD38 expression, and usually with aberrant marker ex-pression; (c) a more primitive FSC low  /SSC low , CD38 low , mark-er-negative myeloid fraction; and (d) a more primitive FSC low  / SSC low , CD38 low , marker-positive myeloid fraction. NBMcontained the first three populations, although the aberrantmarkers were absent in the second population. Suspensionculture assay showed that FSC low  /SSC low SP cells were highlyenriched for primitive cells. Fluorescence in situ hybridization(FISH) analyses showed that cytogenetically abnormal coloniessrcinated from sorted marker positive cells, whereas the cy-togenetically normal colonies srcinated from sorted marker-negative cells. In conclusion, AML SP cells could be discrimi-nated from normal SP cells at diagnosis on the basis of expression of CLL-1 and lineage markers. This reveals thepresence of a low-frequency (median, 0.0016%) SP subfractionas a likely candidate to be enriched for leukemia stem cells. S TEM  C ELLS  2008;26:3059–3067  Disclosure of potential conflicts of interest is found at the end of this article. I NTRODUCTION Acute myeloid leukemia (AML) is generally regarded as adisease likely to originate from the hematopoietic stem cell(HSC) [1]. There is an increasing body of evidence pointingtoward the importance of the leukemic stem cells (LSCs) for theoccurrence of minimal residual disease (MRD) and relapse. Thismight well be explained by properties LSCs share with normalstem cells, such as relative quiescence and resistance to apopto-sis. Accordingly, we have previously described that a high stemcell frequency in AML predicts MRD cell frequencies afterchemotherapeutic treatment, resulting in poor prognosis [2]. Toboth determine the number of remaining LSCs after therapy forprognostic purposes, as well as to apply targeted therapy, im-munophenotypic molecular and functional characterization of LSCs is of utmost importance. However, there is a need for newmarkers, preferably on the cell surface, to discriminate betweennormal CD34  CD38  cells and malignant CD34  CD38  cells,as there is considerable overlap in expression of currently avail-able markers. Recently, we found that C-type lectin-like mole-cule-1 (CLL-1) and leukemia-associated lineage markers pro-vide the opportunity to discriminate between HSCs and LSCs,as they were found to be solely expressed on leukemicCD34  CD38  cells [3, 4]. In addition, new definitions of LSCsshould be generated, as not all AML cases have one or more of the aberrant markers present, and moreover, not all LSCs mayhave the CD34  CD38  immunophenotype (e.g., in true CD34-negative AML). Moreover, the real number of stem cells islikely to be much lower than the number present in the so-calledstem cell compartment [1, 5], suggesting that the definition of the stem cell compartment should be refined. An alternativestem cell compartment may be the so-called side population(SP). SP cells are defined by their ability to efficiently effluxHoechst 33342 dye. In normal bone marrow (NBM), the SP wasindeed found to be enriched for stem cells [6, 7]. Accordingly, Author contributions: B.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing;A.V.R., A.K., M.V.D.P., M.T., C.B., and A.H.W.: collection and/or assembly of data; G.J.O.: provision of study material or patients, finalapproval of manuscript; S.Z.: provision of study material or patients, manuscript writing, final approval of manuscript; G.J.S.: conception anddesign, data analysis and interpretation, manuscript writing, final approval of manuscript.Correspondence: G.J. Schuurhuis, Ph.D., VU University Medical Center, Department of Hematology, CCA building, Room 4.24, DeBoelelaan 1117, 1081 HV Amsterdam, The Netherlands. Telephone: 0031-20-4443838; Fax: 0031-20-4442277; e-mail: gj.schuurhuis@vumc.nl Received October 12, 2007; accepted for publication September 18, 2008; first published online in S TEM C ELLS  E   XPRESS   October2, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0861 C  ANCER   S TEM  C ELLS S TEM C ELLS 2008;26:3059–3067 www.StemCells.com   a  t   Vr i   j   e  Uni   v e r  s i   t   e i   t  Bi   b l  i   o t  h  e  e k  onD e  c  e m b  e r 1  9  ,2  0  0  8  w w w . S  t   e m C  e l  l   s  . c  omD o wnl   o a  d  e  d f  r  om   in AML, the SP compartment is able to initiate leukemia inNOD/SCID mice, whereas the non-side population (NSP) com-partment is not [8]. It is tempting to speculate that the frequencyof real LSCs within the SP compartment is higher than withinthe CD34  CD38  compartment, as the SP frequency is muchlower than that of the CD34  CD38  cells [8]. Since the SPcompartment has previously been shown by fluorescence in situhybridization (FISH) analysis to contain both malignant andnormal cells [9], we sought characteristics/markers with theability to discriminate between these malignant and normal SPcells. These would allow the primitive AML SP cells to betraced at diagnosis and during/after treatment. Moreover, bothnormal and AML SP cells could then be studied separately fortherapeutic target finding. This would provide functional andmolecular biological differences between AML and normalstem cells under the most clinically relevant conditions: bothtypes of stem cells present in the same bone marrow. Lastly, itwould enable the definition of AML SP stem cells to be fine-tuned. M ATERIALS AND  M ETHODS Leukemic and Normal Bone Marrow Cells Bone marrow (BM) samples were collected at diagnosis afterinformed consent from 48 AML patients (20 females, 28 males)with a median age of 49 years (range, 19–75). In four cases, BMwas not available at diagnosis, and peripheral blood (PB) wasused. NBM was obtained after informed consent from patientsundergoing cardiac surgery. The majority of the samples wereanalyzed immediately. Mononuclear cells were isolated by Ficollgradient (1.077 g/ml; Amersham Biosciences, Freiburg, Ger-many, http://www.amersham.com). Red blood cells were lysedafterward by 10 minutes of incubation on ice, using 10 ml of asolution containing 155 mM NH 4 Cl, 10 mM KHCO 3 , and 0.1mM Na 2  EDTA, pH 7.4, added directly to the cell pellet. Afterwashing, cells were frozen in RPMI (Gibco, Paisley, U.K.,http://www.invitrogen.com) with 20% heat-inactivated fetal bo-vine serum (FBS; Greiner Bio-One, Alphen a/d Rijn, The Neth-erlands, http://www.gbo.com/en) and 10% dimethyl sulfoxide(Riedel-de Haen, Seelze, Germany, http://www.riedeldehaen.de)in isopropanol-filled containers and subsequently stored in liquidnitrogen. When needed for analysis, cells were thawed andsuspended in prewarmed RPMI with 40% FBS at 37°C. Cellswere washed and allowed to recover for 45 minutes in the samemedium at 37°C. Cells were washed again and resuspended inphosphate-buffered saline with 0.1% bovine serum albumin (ICNBiomedicals, Aurora, OH, http://www.icn.biomed.com). Flow Cytometry and Cell Sorting Primary AML cells (1  10 6 cells per milliliter) were stained with5   g/ml Hoechst 33342 dye (Molecular Probes, Eugene, OR, http:// probes.invitrogen.com) with or without breast cancer resistanceprotein (BCRP) inhibitor KO143 (200 nM; Sigma-Aldrich, Stein-heim, Germany, http://www.sigmaaldrich.com) and incubated at37°C for 2 hours according to Goodell et al. [6]. After Hoechststaining, cells were washed and resuspended into 100   l of cold(4°C) Hanks’ balanced salt solution (HBSS; Cambrex, Verviers,Belgium, http://www.cambrex.com)    2% fetal calf serum (FCS)and incubated for 30 minutes on ice with combinations of fluores-cein isothiocyanate- (FITC), phycoerythrin- (PE), allophycocyanin(APC)-labeled monoclonal antibodies (MoAbs). Anti-CD45 APC,anti-CD45 PE, anti-CD38 APC, anti-CD34 FITC, anti-CD7 PE,anti-CD19 PE, anti-CD56 PE, anti-CD48 PE [10], and anti-CD123PE MoAbs were all from BD Biosciences (San Jose, CA, http:// www.bdbiosciences.com). To define the leukemic stem cells, weused CD7, CD19, and CD56, which are frequently used in AMLMRD detection using leukemia-associated phenotype; anti-CLL-1and isotype control were used as previously described [4, 11]. Afterantibody staining, cells were washed with cold HBSS  (HBSS  2% FCS), resuspended in 1 ml of cold HBSS   and stained for 5minutes with 2   g/ml propidium iodide (Sigma-Aldrich), enablingexclusion of dead cells. Cells were kept on ice until fluorescence-activated cell sorting (FACS) analysis. Data acquisition was per-formed using either a FACSVantage (equipped with red, blue andultra violet lasers) or a FACSCanto II (with red, blue and violetsolid-state lasers), both from BD Biosciences; analysis was per-formed using CellQuest and FACSDiva software (BD Biosciences).The Hoechst dye was excited with a 350-nm UV (FACSVantage) or405-nm violet (FACSCanto II) laser and detected with 450/BP20and 450/BP50 optical filters, respectively. Gates were set to detectthe viable SP cells as shown in Figure 1A. Cells were sorted usinga FACSAria (with red, blue, and violet solid-state lasers; BDBiosciences). Cells were kept on ice during the whole procedure.For further culturing, cells were sorted directly into cold culturemedium. Purity of sorted populations was  98%. Suspension Culture of AML SP Cells The suspension culture was performed essentially as has beenpreviously described [12]. The sorted SP (sub)populations (3,000–5,000 cells) were mixed with 1  10 5 NSP cells and resuspended in250   l per well of CellGro medium (Cellgenix, Vancouver, BC,Canada, http://www.cellgenix.com) containing 20 ng/ml interleukin(IL)-3, 100 ng/ml Flt-3 ligand, and 100 ng/ml stem cell factor (SCF)(all from Peprotech, Basel, Switzerland, http://www.peprotech.com)prior to plating in 96-well round-bottomed plates (Greiner Bio-One). In addition, 1    10 5 and 1    10 6 NSP cells were plated ascontrol for the mixed SP    NSP. Suspension cultures were incu-bated at 37°C in 5% CO 2  and received weekly half-mediumchanges. Usually in this assay these weekly half-medium changesare accompanied by demipopulation of cells; however, since thenumbers of SP cells were very low, we chose to harvest all cells atone time point only (i.e., 5 weeks). Subsequently, all harvested cellswere cultured in a 14-day colony-forming unit (CFU) assay. CFU Assay Assays for leukemic CFUs were performed by plating cells inmethylcellulose medium (H4434; StemCell Technologies, Vancou-ver, BC, Canada, http://www.stemcell.com). Cultures were scoredafter 14 days for the presence of clusters (4–20 cells) and colonies(more than 20 cells). The number of colonies from the sorted SP  NSP or NSP cells was calculated as previously described [12]. Finalclonogenic output was expressed as number of colonies per millioninput cells (i.e., cells immediately following sorting at the start of the [5  2 weeks] experiment). FISH Analysis of FACS-Sorted SP Cells For interphase FISH, the FACS-sorted SPs were washed three timeswith 3 ml of 3:1 methanol/acetic acid fixative and suspended in 100  l of fixative. Subsequently, one droplet was gently placed on anobject slide and air-dried. Dual-color (spectrum green and spectrumorange fluorophores) labeled LSI DNA probes (Vysis, DownersGrove, IL, http://www.vysis.com) were applied to the denaturatedcells and incubated as previously described [13]. The followingprobes were used: the LSI AML1/ETO dual color for t(8;21) and theLSI TEL/AML1 ES dual color for del 12(p13). Hybridization anddeletion signals were scored in 50 interphase nuclei with an Ax-ioscop 20 (Carl Zeiss, Jena, Germany, http://www.zeiss.com) fluo-rescence microscope with three single-band-pass filters and onetriple-band-pass filter. Nuclei were scored positive for the fusiongene, when a green spot and an orange spot were less than one spotdiameter apart. Nuclei were scored positive for deletion 12, whenone green spot was absent. The images were captured with a digitalcamera using CytoVision 4.1 software (Applied Imaging Corp.,Newcastle, U.K., http://www.appliedimaging.com). Statistical Analysis Statistical analysis was performed using SPSS 9.0 software package(SPSS, Chicago, http://www.spss.com). The Wilcoxon signed-rank test was used to determine differences between paired samples.Statistical significance was evaluated at  p    .05. Average valueswere expressed as mean  SEM.3060  Stem Cell Identification in AML Side Population   a  t   Vr i   j   e  Uni   v e r  s i   t   e i   t  Bi   b l  i   o t  h  e  e k  onD e  c  e m b  e r 1  9  ,2  0  0  8  w w w . S  t   e m C  e l  l   s  . c  omD o wnl   o a  d  e  d f  r  om   Figure 1.  Side population (SP) cell immu-nophenotyping: gating strategy and SP sub-populations. Cells were stained for SP,CD45, lineage markers (CD7, CD19,CD56), and CD48, as outlined in Materialsand Methods.  (A1–A4):  One representativeexample of acute myeloid leukemia (AML)SP immunophenotyping with differentmarker expression patterns and populationswith different FSC and SSC.  (A1):  FSC ver-sus PI of an AML sample (R1). Gating onPI-negative cells identified viable cells (R1).Gate R1 was used in  (A2) .  (A2):  Hoechst redversus Hoechst blue identified the viableside population cells (R2). Gates R1    R2were used in  (A3) .  (A3):  FSC/SSC was usedto select a cluster or clusters of cells (R3).Nonspecific events are excluded here. Notethe presence of two clusters differing in SSCbut especially in FSC  (A3) . The large clusterwas located in the whole blast region (notshown). Gates R1  R2  R3 were used in (A4) .  (A4):  This shows the final populationof viable SP cells containing as few as pos-sible nonspecific events (R4).  (A5–A8): Representative example of lymphoid SPsubpopulations. Three small lymphoid sub-populations were detected within SP cells. (A5):  Gate R5 shows CD45 high  /CD56  SPcells.  (A6):  Gate R6 shows CD45 high  / CD19  SP cells.  (A7):  Gate R7 showsCD45 high  /CD7  SP cells. The CD45 high cells with low expression of CD7 are theCD56  cells from A5.  (A8):  Location of cells from gates R5  R6  R7 in FSC/SSC. (B1–B4):  One representative example of CD48 expression on SP and non-SP sub-populations of an AML patient.  (B1):  GateR6 shows CD48   /CD45 dim myeloid SP.Lymphocytes (CD45 high ) were CD48  . (B2):  CD45 high Lymphocytes (R1) andCD45 dim myeloid cells (R2) from non-SPwere selected and used in  (B3, B4) .  (B3): Lymphocytes (CD45 high ) from non-SP wereessentially CD48-positive.  (B4):  Myeloidcells (CD45 dim ) from non-SP were CD48negative.  (B5–B8):  One representative ex-ample of CD48 expression on SP andnon-SP subpopulations of a normal bonemarrow. The results were similar to  (B1–B4) ; that is, the lymphoid population wasCD48-positive and the myeloid populationwas CD48-negative. Abbreviations: APC,allophycocyanin; FITC, fluorescein isothio-cyanate; FSC, forward scatter; PI, propidiumiodide; SSC, sideward scatter. 3061 Moshaver, van Rhenen, Kelder et al. www.StemCells.com   a  t   Vr i   j   e  Uni   v e r  s i   t   e i   t  Bi   b l  i   o t  h  e  e k  onD e  c  e m b  e r 1  9  ,2  0  0  8  w w w . S  t   e m C  e l  l   s  . c  omD o wnl   o a  d  e  d f  r  om   R ESULTS To distinguish AML SP cells from normal SP cells at diagnosis,we attempted to find leukemia stem cell-associated immunophe-notypic markers (i.e., those not staining the normal SP stemcells). We used CLL-1 and IL-3 receptor   -chain CD123, pre-viously reported to be leukemic stem cell markers [4, 14], andthe lineage markers CD7, CD19, and CD56, used to define blastcell aberrancies suitable for immunophenotypic MRD detection[15] and staining of AML CD34  CD38  LSCs [3]. Relationship Between CD34 and CD38 Expressionand the SP Phenotype To define the immunophenotype of AML SP cells in relation toNBM SP cells, 44 bone marrow samples and 4 PB samples of AMLdiagnosispatientswereinvestigated.SPcellsweredetectablein 41 of 48 AML patients (85%), with a median frequency of 0.07% (expressed as percentage of whole blast cells; range,0.002%–7.6%). In all individual cases with both CD34  CD38  and SP stem cell compartments present ( n    36), theCD34  CD38  compartment had a higher frequency than the SP inthis subset of samples: 0.47% (range, 0.01%–26.6%) versus 0.03%(range, 0.002%–7.6%;  p    .002). The median frequency of CD34  CD38  cells within the SP compartment was 2.5% (range,0–49%). SP cells were detected in all 12 NBM samples, with amedian frequency of 0.12% (range, 0.008%–4.1%). The SP Compartment in AML Contains NormalLymphocytic Cells During the immunophenotyping of AML SP cells we detected asmall SP with a low forward scatter (FSC) and a slightly lowersideward scatter (SSC); one representative example is shown inFigure 1A3. Some of these cells showed expression of themarkers CD7 or CD19 or showed CD56 expression, character-istics shared by blast cells in some AML cases [15]. However,as FSC/SSC was lower than for blast cells and similar to normallymphocytes, further analysis was performed using antibodiesagainst CD45, which enables discrimination between differenttypes of white blood cells (WBC), and CD48 [10], a glyco-sylphosphatidylinositol (GPI)-anchored protein that is expressedon mature lymphocytes. Figures 1A5–1A8 identifies three smalllymphoid subpopulations (all CD34-negative and CD33-nega-tive) within the SP compartment, which also had low FSC: (a)natural killer-like cells with CD45 high  /CD56   /CD7 low expres-sion, with a median frequency of 4% (percentage of whole SPcompartment; range, 2%–8%;  n  8; example in Fig. 1A5); (b)B lymphocytes with CD45 high  /CD19  phenotype, and a medianfrequency of 2% (range, 0%–6%;  n  8; example in Fig. 1A6);and (c) T lymphocytes with CD45 high  /CD56   /CD7 high expres-sion, with a median frequency of 7% (range, 2%–14%;  n  8;example in Fig. 1A7). The latter resembled the previouslydescribed CD34  CD7  SP cells [16]. All three CD45 high pop-ulations cluster together in the low FSC area (Fig. 1A8; comparewith Fig. 1A3). Similar to the AML samples, NBM samples alsoshowed these three lymphoid SP subpopulations: NK-like, Band T cells, with median frequencies of 5%, 2%, and 8%, respec-tively ( n    3). Therefore, it is highly likely that the AML SPcompartment contains a subset of normal lymphocyte cells. In themalignant samples, all three lymphoid SP subpopulations(CD45 high , FSC low ) were CD48-positive (Fig. 1B1). In contrast,myeloid SP cells (CD45 dim ) were CD48-negative (Fig. 1B1, R6).For comparison, the CD45 high lymphoid cells within the non-SP(Fig. 1B2, R2) were also CD48-positive (Fig. 1B3), whereas themyeloid cells were not (Fig. 1B4). The NBM samples showedsimilar results (Fig. 1B5–1B8). Thus, staining with CD48 enablesthe nonmyeloid cells to be excluded from further analyses of stemcell activity in the myeloid SP subcompartment. Leukemic Cells with Aberrant Marker ExpressionAre Present in the SP Compartment Marker expression on the CD45 dim AML SP cells was deter-mined after exclusion of the CD45 high lymphocytic cells. In 39of 41 cases with SP present, the SP cells were partly or com-pletely positive (defined as  10% expression) for CLL-1 (in all41 samples: median, 53%; range, 2%–100%) and in 27 of 41cases partly or completely positive for CD123 (median, 30%;range, 0%–100%;  n    41). In 25 of 41 cases SP cells werepartly or completely positive for one or more of the three lineagemarkers previously shown to mark AML CD34  CD38  stemcells [3], that is, CD7, CD19, and CD56. Median expression was35% for CD7 (range, 18%–80%;  n    13), 55% for CD19(range, 20%–95%;  n    5), and 50% for CD56 (range, 25%–100%;  n  17). As CLL-1 and aberrant lineage marker expres-sion have never been found on normal CD34  CD38  previ-ously by our laboratory [3, 4], this strongly suggest thatmalignant myeloid cells in the SP cell compartment in AML atdiagnosis can be identified using aberrant expression of mark-ers. This was confirmed by comparison with surface markerexpression on SP cells in 12 normal bone marrow samples.These SP cells were negative for CLL-1 (median, 0%; range,0%–4%), CD7 (median, 0%; range, 0%–3%), CD19 (median,0%; range, 0%–3%), and CD56 (median, 0%; range, 0%–4%),although not for CD123 (median, 27%; range, 1%–82%).Therefore, both CLL-1 and lineage markers, but not CD123,are suitable to discriminate between malignant and normal SPstem cells at diagnosis. FISH analysis for three t(8;21) andone del(12) AML patients (in Table 1, patients 2, 10, 41, and22, respectively) showed that the majority of cells wereindeed malignant (88%, 76%, 78%, and 80%, respectively).However, both marker patterns and FISH analyses indicatedthat even after correction for the lymphoid compartment (notshown), some of the myeloid SP cells should still be of normal srcin. The Myeloid SP Compartment Is Heterogeneous inScatter Properties and CD34 and CD38 Expression On closer inspection, in 39 of 41 samples heterogeneity forsideward scatter was observed in the myeloid SP compartment(example in Fig. 2A1), showing two different subpopulations: (a)high sideward scatter (HSSC) cells with a median frequency of 56% (percentage of whole SP compartment; range, 4%–91%); (b)low sideward scatter (LSSC) myeloid SP cells with a medianfrequency of 44% (percentage of whole SP compartment; range,9%–96%). In 2 of 41 cases only the LSSC SP cells presented as aseparate population (an example is shown in Fig. 1A3).Backgating of HSSC and LSSC cells in Hoechst bivariateplots showed their presence throughout the SP, with no signif-icant difference in Hoechst staining between LSSC and HSSCSP cells (Fig. 2B1–2B3). HSSC and LSSC populations werealso seen in 9 of 12 NBM SP cells (one representative exampleis shown in Fig. 2C1), with only LSSC SP present in theremaining three cases (not shown).HSSC AML SP cells had high CD38 expression, with amedian frequency of 84% (range, 0%–100%;  n  39; examplein Fig. 2A4). LSSC SP cells had significantly (  p    .04) lowerCD38 expression (example in Fig. 2A4), with a median fre-quency of 43% (range, 1%–100%;  n    41;  p    .04). Themedian CD34 expression on HSSC and LSSC SP cells was19% (range, 0%–99%) and 41% (range, 1%–99%), respec-tively. Apparently, compared with HSSC cells, LSSC cells 3062  Stem Cell Identification in AML Side Population   a  t   Vr i   j   e  Uni   v e r  s i   t   e i   t  Bi   b l  i   o t  h  e  e k  onD e  c  e m b  e r 1  9  ,2  0  0  8  w w w . S  t   e m C  e l  l   s  . c  omD o wnl   o a  d  e  d f  r  om   have characteristics indicative of their primitive nature:higher CD34 expression, lower CD38 expression, and lowerSSC. Combining Scatter Properties and MarkerExpression Defines the Presence of at Least ThreeDifferent Myeloid SP Subpopulations Subsequently, aberrant marker expression was studied sepa-rately in HSSC and LSSC (data provided per patient in Table 1).CD123 was not included since, as reported earlier, it is ex-pressed on normal bone marrow SP cells as well. HSSC showedheterogeneous expression patterns for the markers CD7, CD19,CD56, and CLL-1 (e.g., patient 10: 62% CLL-1  , 3% CD7  ,6% CD19  , and 65% CD56  ). However, the HSSC populationusually showed homogeneous expression (either high or low orabsent) for all of these markers. In contrast, within the LSSC SPcells in general, two clearly discernable myeloid subpopula-tions could be identified: one negative for aberrant markersand the other with aberrant markers present. As a particularexample, Figure 2A2 shows CD56 expression on the majorityof HSSC SP cells but only on part of the LSSC fraction. Ingeneral, both HSSC and LSSC compartments showed heter-ogeneous expression patterns of CLL-1 and aberrant markers,with often large intrapatient differences in expression be- Table 1.  Marker expression on myeloid, scatter-defined SP subpopulations in acute myeloid leukemia patients As % of LSSC and HSSC SPCLL-1 CD7 CD19 CD56Patient LSSC HSSC LSSC HSSC LSSC HSSC LSSC HSSC 1 46 78 — a — — — — —2 25 — b — — b 95 — b 85 — b 3 72 80 — — — — 92 904 43 89 — — — — — —5 57 75 — — — — 60 586 8 54 36 24 — — 26 437 20 93 78 37 — — — —8 100 100 — — — — 100 1009 57 87 — — 11 5 — —10 22 62 26 3 16 6 22 6511 6 48 57 23 — — — —12 46 — b — — b — — b 39 — b 13 34 86 37 2 — — 21 7514 50 86 41 5 — — — —15 20 60 — — — — 28 7016 33 75 — — 52 33 — —17 18 45 — — — — — —18 10 84 — — — — — —19 77 100 50 42 — — — —20 3 20 — — — — — —21 53 63 29 7 — — — —22 2 15 — — — — 43 2123 65 77 — — — — — —24 62 83 — — — — — —25 48 83 — — — — — —26 10 75 35 4 — — — —27 15 78 21 11 — — — —28 78 87 — — — — — —29 50 99 — — — — — —30 20 72 25 5 — — — —31 34 95 40 0 — — — —32 1 0 — — — — — —33 28 57 — — — — 36 3834 50 48 — — — — — —35 23 92 — — — — — 4936 86 99 — — — — — —37 34 54 — — — — — —38 20 65 — — — — 54 2939 63 92 45 20 — — — —40 66 92 — — 60 29 — —41 30 99 — — 56 22 19 35Median 34 (1–100) c,d 78 (0–100) c 37 (21–78) c,d 7 (2–42) c 54 (11–95) c,d 22 (5–33) c 39 (19–100) c,d 53 (21–100) c NBM median e 0 (0–0) 0 (0–4) 0 (0–3) 0 (0–0) 0 (0–3) 0 (0–0) 0 (0–0) 0 (0–4) a Not measured. b —, HSSC population not present. c Differences in intrapatient marker expression between HSSC and LSSC (HSSC  LSSC for CLL-1 and CD56; HSSC  LSSC for CD7 andCD19) were significant for CLL-1 (  p  .0001), CD7 (  p  .001), and CD19 (  p  .04), but not for CD56. d No significant differences were found in interpatient marker expression on LSSC cells between CLL-1, CD7, CD19, and CD56. e NBM:  n  12, for all markers.Abbreviations: CLL-1, C-type lectin-like molecule-1; HSSC, high sideward scatter; LSSC, low sideward scatter; NBM, normal bone marrow;SP, side population. 3063 Moshaver, van Rhenen, Kelder et al. www.StemCells.com   a  t   Vr i   j   e  Uni   v e r  s i   t   e i   t  Bi   b l  i   o t  h  e  e k  onD e  c  e m b  e r 1  9  ,2  0  0  8  w w w . S  t   e m C  e l  l   s  . c  omD o wnl   o a  d  e  d f  r  om 
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