of May 30, 2013.This information is current as Vitro Exposure to IL-15

Lymphoid/NK Cell Differentiation After In Cells Capable of−CD45+Lineage

−CD7−CD133−Human Cord Blood CD34Identification of a Novel Subpopulation of

PierelliScambia, Salvatore Mancuso, Giuseppe Leone and Lucade Ritis, Andrea Mariotti, Maria Teresa Voso, Giovanni Sergio Rutella, Giuseppina Bonanno, Maria Marone, Daniela

http://www.jimmunol.org/content/171/6/29772003; 171:2977-2988; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/171/6/2977.full#ref-list-1

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Identification of a Novel Subpopulation of Human Cord BloodCD34�CD133�CD7�CD45�Lineage� Cells Capable ofLymphoid/NK Cell Differentiation After In Vitro Exposure toIL-151

Sergio Rutella,2*† Giuseppina Bonanno,‡ Maria Marone,‡ Daniela de Ritis,‡ Andrea Mariotti,‡

Maria Teresa Voso,* Giovanni Scambia,‡ Salvatore Mancuso,‡ Giuseppe Leone,* andLuca Pierelli*†

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmentalpotential. Numerous CD34� cell subsets that might reside at an earlier stage of differentiation than CD34� HSCs have beendescribed and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation ofCD34�CD133�CD7�CD45dimlineage (lin)� HSCs contained within human UCB that were endowed with low but measurableextended long-term culture-initiating cell activity. Exposure of CD34�CD133�CD7�CD45dimlin� HSCs to stem cell factor pre-served cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 andCD133, 2) generation of CFU-granulocyte-macrophage, burst-forming unit erythroid, and megakaryocytic aggregates, 3) signif-icant extended long-term culture-initiating cell activity, and 4) up-regulation of mRNA signals for myeloperoxidase. At variancewith CD34�lin� cells, CD34�CD133�CD7�CD45dimlin� HSCs maintained with IL-15, but not with IL-2 or IL-7, proliferatedvigorously and differentiated into a homogeneous population of CD7�CD45brightCD25�CD44� lymphoid progenitors with highexpression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCR� genes, IL-15-primed CD34�CD133�CD7�CD45dimlin� HSCs failed to achieve full maturation, as manifested in their CD3�TCR������ pheno-type. Conversely, culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functionalfeatures of NK cells. Collectively, CD34�CD133�CD7�CD45dimlin� HSCs from human UCB displayed an exquisite sensitivity to IL-15and differentiated into lymphoid/NK cells. Whether the transplantation of CD34�lin� HSCs possessing T/NK cell differentiation po-tential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmuneor malignant diseases remains to be determined. The Journal of Immunology, 2003, 171: 2977–2988.

H emopoiesis can be defined as a hierarchical process sus-tained by pluripotent stem cells capable of extensiveself-renewal and differentiation (1). An orderly sequence

of changes in gene expression occurs, leading to lineage restrictionand, ultimately, production of mature blood cells. Hemopoieticstem cells (HSCs)3 possess enormous clinical relevance, becausethey can reconstitute both the lymphoid and the myeloid compart-ment in the recipients of autologous, allogeneic, or xenogeneic

transplants (1). Furthermore, HSCs can be targets for ex vivo ex-pansion protocols and for gene therapy strategies.

CD34 Ag is a syalomucin that has been considered to be ahallmark of human transplantable HSCs (2–4); accordingly, ex-perimental and clinical protocols have been designed exclusivelyfor CD34� subsets. Recently, several investigators have identifiedvery primitive CD34�lineage (lin)� HSCs capable of long-term re-population of nonobese diabetic/SCID mice and fetal sheep (5–9).The isolation of highly purified subsets of HSCs not expressing Agsassociated with lineage commitment (or lin� cells) has demonstrateda previously unrecognized heterogeneity within the human HSC com-partment (10). In particular, CD133�CD34�CD38�lin� cells iso-lated from human umbilical cord blood (UCB) possess in vitro growthfactor requirements and progenitor capacity equivalent to the CD34�

stem cell fraction (10). Because CD34� HSCs can be obscured bymature CD34� cells, functional assays have been developed to isolateHSCs, including Hoechst 33342 dye efflux (11). Using these ap-proaches, a rare side population of primitive CD34� HSCs has beenidentified in human UCB; CD34�lin� side-population cells expressCD7 Ag and contain lymphoid progenitors capable of rapid expansionand differentiation into functionally active NK cells, if plated onstroma supplemented with cytokines (11). In the study by Storms etal. (11), CD34�CD7�lin� cells were detected at very low frequencyand were excluded from analysis; thus, whether CD34�CD7�lin�

cells contain HSCs endowed with progenitor or stem cell activity hasnot been determined.

*Department of Hematology, †Laboratory of Immunology, and ‡Department of Gy-necology, Catholic University Medical School, Rome, Italy

Received for publication January 21, 2003. Accepted for publication July 16, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Ministero dell’Universita e della Ricerca Scientifica eTecnologica (Italy) and by the Stem Cell Project (Fondazione Cassa di Risparmio diRoma, Rome, Italy).2 Address correspondence and reprint requests to Dr. Sergio Rutella, Department ofHematology, Laboratory of Immunology, Catholic University Medical School, LargoA. Gemelli 8, 00168 Rome, Italy. E-mail addresses: sergiorutella@tin.it orsrutella@rm.unicatt.it3 Abbreviations used in this paper: HSC, hemopoietic stem cell; lin, lineage; UCB,umbilical cord blood; Flt3-L, Flt3 ligand; TPO, thrombopoietin; SCF, stem cell factor;CFU-GM, CFU-granulocyte-macrophage; BFU-E, burst-forming unit-erythroid;Meg-aggregate, megakaryocytic aggregate; ELTC-IC, extended long-term culture-initiating cell; LDA, limiting-dilution analysis; 7-AAD, 7-amino actinomycin-D.

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The molecular events occurring during lineage restriction in-volve activation or silencing of genes encoding cytokine receptors,signal transduction molecules, and transcription factors (12–14). Inthis respect, the isolation of progenitor populations from normal invivo sources can greatly contribute to the understanding of theprocess of differentiation and lineage plasticity, by providing ac-cess to intermediates that can be characterized by functional assaysand molecular approaches (15–17). In the present study, we iden-tified a novel, rare, and primitive subset of human UCBCD34�CD133�CD7�lin� progenitors that generate T cell precur-sors and functionally active NK cells after culture with stromalcells supplemented with the IL-2R �c-signaling cytokine IL-15.The CD34�CD133�CD7�CD45dimlin� lymphoid progenitorsthat we differentiated in vitro harbored rearrangement at the TCR�locus, expressed the T cell-specific transcription factor GATA-3,and could be hierarchically correlated to the recently describedlymphoid-oriented CD34�CD7�lin� cells and to the transitionalmyeloid CD34�CD133�CD7�lin� UCB cells.

Materials and MethodsUCB processing and isolation of CD34�CD133�CD7�lin�

cells

UCB samples were obtained after full-term cesarean delivery from healthydonors after written informed consent. The local human research commit-tee approved all investigations. UCB samples were collected in sterile bagscontaining citrate-phosphate dextrose and adenine anticoagulant and werediluted 1/3 in PBS. Low-density mononuclear cells were separated overFicoll-Hypaque (density, 1077 g/dl; Pharmacia, Uppsala, Sweden). Mono-nuclear cells were subsequently subjected to removal of lineage-committedcells by incubation with a mixture of mAb directed against CD2, CD3,CD14, CD16, CD19, CD24, CD56, CD66b, CD41, and glycophorin A(CD235a). The manufacturer (StemCell Technologies, Vancouver, BC,Canada) provided all mAb. After incubation, a secondary Ab conjugated toa metal colloid was added, and cells were depleted of lineage marker-positive elements by a magnetic device (StemSep; StemCell Technolo-gies). CD34�lin� cells were then removed and separately collected fromremaining lin� cells by binding with PE- or FITC-conjugated anti-CD34mAb (QBEND/10 clone; Serotec, Oxford, U.K.), followed by sorting on aFACStarPlus flow cytometer (BD Biosciences, San Jose, CA). After exten-sive washings with PBS/1% BSA, isolated CD34�lin� cells were stainedwith PE-conjugated anti-CD133 mAb (Miltenyi Biotec, Bergisch-Glad-bach, Germany) and FITC-conjugated anti-CD7 mAb (BD Biosciences).CD7� and CD133� cells were removed from the CD34�lin� starting pop-ulation by sorting on a FACStarPlus cytometer (BD Biosciences). The re-maining cells were designated as CD34�CD133�CD7�CD45dimlin� cells,in accordance with flow-cytometric reanalysis of surface markerexpression.

Culture of purified CD34�CD133�CD7�CD45dimlin� cells

In a preliminary set of experiments, we tested the ability of an array ofhuman growth factors to support viability, growth, and/or differentiation offreshly isolated CD34�CD133�CD7�CD45dimlin� cells in liquid cultures,with or without animal serum. IL-3 at 20 ng/ml, IL-6 at 20 ng/ml, IL-7 at40 ng/ml, IL-2 at 1000 IU/ml, Flt3 ligand (Flt3-L) at 20 ng/ml, platelet-derived growth factor at 3 ng/ml, epidermal growth factor at 0.4 ng/ml,thrombopoietin (TPO) at 10 ng/ml, GM-CSF at 20 ng/ml, stem cell factor(SCF) at 10 ng/ml, or IL-15 at 50 ng/ml (all purchased from R&D Systems,Oxon, Cambridge, U.K.) were tested individually in 72-h cultures for theircapacity to sustain viability and growth (as revealed by cell counts, exclu-sion of propidium iodide, and FACS analysis of light scatter characteris-tics) or to promote differentiation, which was monitored through the de-tection of CD34, CD133, and CD7 expression, as will be detailed.Cytokine concentrations were selected based on previously published stud-ies (18). Cultures were performed in �-MEM culture medium supple-mented with 10% horse serum, 10% bovine serum, and 10�6 M hydrocor-tisone, in the presence or absence of the indicated cytokine combinations(19). When the adequate culture conditions were established and standard-ized based on preliminary experiments, culture length was prolonged to 7days, using the optimal growth factor supplementation.

CFU assays, long-term culture-initiating cell assays, andlymphoid cell cultures

Colony-forming cells (CFU-granulocyte-macrophage (CFU-GM) andburst-forming unit-erythroid (BFU-E)) were generated from purified UCBcells (CD34�CD133�CD7�CD45dimlin� and CD34�lin� cells; the latterused as reference control) before and after cytokine treatments by 14-daymethylcellulose semisolid cultures in the presence of 25% serum substituteto avoid interference from serum factors (Bit 9500; StemCell Technolo-gies) and SCF, IL-3, GM-CSF, G-CSF, Flt3-L, and erythropoietin, as re-ported (18, 20). Replicates of cloning assays were established under thesame culture conditions but in the presence of SCF, TPO, and IL-6 toidentify megakaryocytic aggregates (Meg-aggregates), as previously de-scribed (18). Cloning activity was expressed as cumulative colony effi-ciency (CFU-GM plus BFU-E plus Meg-aggregates) both in terms of ab-solute number of colonies per number of seeded cells and in terms ofpercentage of seeded cells. Extended (12-wk) long-term culture-initiatingcell (ELTC-IC) activity was assessed by limiting-dilution analysis (LDA)and 12-wk long-term culture on a murine stromal cell line engineered toproduce human G-CSF and IL-3 (M2-10B4 cell line; a gift from Dr. C.Eaves (Terry Fox Laboratory, Vancouver, BC, Canada)) (18, 21). At wk 6,cultures were trypsinized, and cells were reseeded on a new irradiated layerof M2-10B4 cells to circumvent cell senescence. Freshly isolatedCD34�CD133�CD7�CD45dimlin� cells were tested in NK culture assays,which consisted of 21-day stroma-based culture on MS-5 murine cells in�-MEM supplemented with 10% horse serum, in the presence or absenceof IL-15 at 50 ng/ml. The starting population of CD34�CD133�

CD7�CD45dimlin� cells was seeded at 1 � 105 per well in 24-well plates,where a confluent stromal layer of MS-5 cells had been grown previously.

Limiting-dilution assays

Suspension cultures containing IL-15 were set up with purifiedCD34�CD133�CD7�CD45dimlin� cells in 20 �l of medium in the indi-vidual wells of a 60-well Terasaki microwell plate. After 7 days of culture,each well was examined for the presence of viable cells and then assayedindividually for the presence of lymphoid progenitors. Wells were scoredas positive for IL-15 responsiveness when �20 viable cells could be iden-tified on day 7 and when cells expressed detectable levels of the lymphoid-associated Ag CD7 by flow cytometry. All other wells were scored asnegative and usually contained no detectable cells. The frequency of IL-15-responsive cells in the CD34�CD133�CD7�CD45dimlin� cells wascalculated from the proportion of negative wells based on Poisson statis-tics, as previously reported (18).

Immunological markers

Aliquots of cells were incubated for 30 min at 4°C with pretitrated satu-rating dilutions of the following FITC-, PE-, PerCP-, or PE-Cy5 (Tricolor)-conjugated mAb: CD16 (NKP15 clone, IgG1), CD19 (4G7 clone, IgG1),CD22 (S-HCL-1 clone, IgG2b), CD34 (8G12 clone, IgG1), CD38 (HB7clone, IgG1), CD56 (My31 clone, IgG1), CD45RA (L48 clone, IgG1),CD45RO (UCHL-1 clone, IgG1), CD25 (2A3 clone, IgG1), CD44 (L178clone, IgG1), glycophorin-A (CD235a; GAR-2 clone, IgG2b), CD132(IL-2R �-chain; AG184 clone, IgG1), TCR�� (WT31 clone, IgG1),TCR�� (11F2 clone, IgG1; BD Biosciences), CD94 (HP-3D9 clone, IgG1),CD158a (HP-3E4 clone, IgM), CD158b (CH-L clone, IgG2b; BD PharM-ingen, San Diego, CA), CD3 (S4.1 clone, IgG1), CD4 (S3.5 clone, IgG2a),CD8 (3B5 clone, IgG2a), CD7 (CD7-6B7 clone, IgG2a), CD90 (F15-42-1clone, IgG1), CD64 (10.1 clone, IgG1), CD65 (VIM2 clone, IgG1),HLA-DR (TU36 clone, IgG2b), CD11b (CR3 Bear-1 clone, IgG1), CD13(TUK1 clone, IgG1), CD45 (HI30 clone, IgG1), Sca-1 (Ly-6A.2 clone,IgG2a; Caltag Laboratories, Burlingame, CA), CD105 (SN6 clone, IgG1;Serotec), and CD133 (AC133 clone, IgG1; Miltenyi Biotec). Appropriatefluorochrome-conjugated isotype-matched mAb purchased from the differ-ent manufacturers were used as control for background staining. After ex-tensive washings with PBS/1% BSA, cells were kept on ice until flow-cytometric analysis.

Assay for NK cell function

NK cell function was evaluated by flow cytometry, following a previouslydescribed protocol, which measures target cell death through the uptake ofmembrane-impermeable dyes (10). Briefly, 1 � 106 NK-sensitive K562cells were used as target cells and were labeled with 2.5 �M CFSE (Mo-lecular Probes, Eugene, OR) in PBS for 10 min at room temperature. Forlysis assay, K562 cells were plated in duplicate in RPMI 1640 culturemedium supplemented with Bit H9500 serum substitute at the various E:Tratios described in the figures. The cocultures were then incubated for 24 hat 37°C with 5% CO2. Cells were pelleted, washed with PBS supplemented

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with 1% BSA, and further incubated with 20 �g/ml 7-amino actinomy-cin-D (7-AAD; Molecular Probes) for an additional 30 min, before flowcytometry analysis.

Measurement of lymphocyte divisions by CFSE

For cell-doubling experiments, cells were resuspended in PBS containing2.5 �M CFSE (Molecular Probes) for 10 min at room temperature (22). Toquench the labeling process, an equal volume of FCS was added; afterwashings with PBS supplemented with 3% FCS, cells were incubated over-night with medium alone and then exposed to the various cytokine com-binations, as detailed in the figures.

Semiquantitative RT-PCR

mRNA expression levels were evaluated by RT-PCR, normalizing the lev-els of test RNA to that of the internal control, aldolase A. Total RNAextractions were conducted with the RNeasy mini-kit (Qiagen, Hilden,Germany), and RNA obtained from 2 � 104 cells was reverse transcribedwith 25 U of Moloney murine leukemia virus reverse transcriptase (PEApplied Biosystems, Foster City, CA) at 42°C for 30 min in the presenceof random hexamers. Two microliters of these cDNA products were am-plified with 1 U of AmpliTaq Gold (PE Applied Biosystems) in the pres-ence of the specific primers for the mRNA of interest, together with thealdolase-A primer (23). Reactions were conducted in the GeneAmp PCRSystem 9600 (PE Applied Biosystems). Conditions were chosen so thatnone of the amplification products obtained from the RNAs of interestreached a plateau, and that the two primer sets did not compete with eachother. Amplification of GATA-3 mRNA was achieved by 30 cycles of 45 sat 95°C, 45 s at 55°C, and 1 min at 72°C in the presence of 3 mM MgCl2.Amplification conditions and primer sequences for the other markers stud-ied in the present report were described elsewhere (24–28). The PCR prod-ucts were loaded onto 1.5% agarose gels and stained with ethidium bro-mide. Each set of reactions included a no-sample negative control. Theratio between the sample RNA to be determined and aldolase-A was cal-culated to normalize for initial variations in sample concentration and as acontrol for reaction efficiency. The following primers were used for am-plification of GATA-3 mRNA: 5�-GTCCTGTGCGAACTGTCAGA-3�and 5�-TAAACGAGCTGTTCTTGGGG-3�. All oligonucleotide primerswere synthesized by M-Medical (Florence, Italy).

DNA extraction and amplification

DNA was extracted using DNAzol (Life Technologies, Eggenstein, Ger-many), following the manufacturers’ instructions. TCR� rearrangementwas studied using a nested PCR technique (29). Briefly, DNA was ampli-fied using the following primers: 5�-AGGGTTGTGTTGGAATCAGG-3�(TCR-11) and 5�-CGTCGACAACAAGTGTTGTTCCAC-3� for the pri-mary PCR, and the TCR-11 primer and 5�-GGATCCACTGCCAAAGAGTTTCTT-3� for the nested PCR. For both amplifications, PCR con-sisted of 30 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 1 minand resulted in a fragment ranging between 150 and 180 bp following thenested PCR. As control, PCR of the housekeeping gene Bcl-2 was per-formed, using 5�-GCAATTCCGCATTTAATTCATGG-3� and 5�-GAAACAGGCCACGTAAAGCAAC-3� as primers. PCR was performed for 35cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, andextension at 72°C for 1 min. This resulted in a fragment of 154 bp. PCRswere conducted in a 25-�l mixture containing 200 ng of genomic DNA,and a master mix of PCR buffer, 1.5 mM MgCl2, 250 nM dNTPs, 1.25 Uof Taq polymerase (Taq Platinum; Life Technologies), and 0.8 mM eacholigonucleotide. A positive control and a negative control, containing waterinstead of DNA, were included in all PCR. PCR products were analyzed ona 3% agarose gel, stained with ethidium bromide.

Flow cytometry and immunofluorescence analysis

Cells were run through a FACScan flow cytometer (BD Biosciences),equipped with an argon laser emitting at 488 nm. Details on instrumentrequirements and settings were published elsewhere (30). FITC, PE, andPerCP, or PE-Cy5 (Tricolor) signals were collected at 530, 575, and 670nm, respectively; spectral overlap was minimized by electronic compen-sation with Calibrite beads (BD Biosciences) before each determinationseries. A minimum of 10,000 events was collected and acquired in listmode using the CellQuest software (BD Biosciences).

Statistical methods

The approximation of population distribution to normality was tested pre-liminarily using statistics for kurtosis and symmetry. Data were presentedas mean � SD. Consequently, all comparisons were performed with Stu-

dent’s t test for paired or unpaired data or with the ANOVA, as appropriate.The criterion for statistical significance was defined as p � 0.05.

ResultsPurification of CD34�CD133�CD7�lin� cells

UCB mononuclear cells were enriched in CD34� cells and lin�

cells by magnetic depletion of cells labeled with a mixture of mAbdirected against lymphoid, myeloid, and erythroid Ags, and byflow-cytometric removal of cells reactive with anti-CD34 mAb.The purity of sorted CD34�lin� cells always exceeded 98%, asassessed by FACS analysis (data not shown). CD133 and CD7were found on 1.6–22.3% and on 8.5–39% of CD34�lin� cells,respectively, in the different UCB samples tested. Furthermore,freshly isolated CD34�lin� cells were reactive, albeit at low fre-quency, with the anti-CD44 (11 � 5%) and anti-CD25 mAb (5 �2%). Next, we depleted CD34�lin� cells from CD7� and CD133�

cells using a flow cytometry-based cell-sorting strategy. Gating win-dows for cell-sorting experiments were constructed as depicted in Fig.1A. Coselection for absence of CD7 and CD133 resulted in a highlypurified CD34�CD133�CD7�lin� cell population (Fig. 1B). Con-tamination by residual CD7-expressing lymphoid progenitor cellscould be excluded based on FACS reanalysis (Fig. 1B) and on PCRanalysis of TCR� rearrangement status in CD34�CD133�CD7�lin�

cells (Fig. 1C). CD34�CD133�CD7�lin� cells comprised 0.16% onaverage (range, 0.03–0.28%) of UCB mononuclear cells collectedafter full-term deliveries from 10 normal donors.

Phenotypic and molecular features of highly purifiedCD34�CD133�CD7�lin� cells

The expression of informative differentiation Ags on highly puri-fied CD34�CD133�CD7�lin� UCB cells was investigated by

FIGURE 1. Isolation and characterization of CD34�CD133�CD7�lin�

cells. UCB CD34�lin� cells were depleted from CD7� and CD133� cellsthrough cell sorting on a FACStarPlus flow cytometer (BD Biosciences). A,Sorting windows (R1) were established to include �95% of theCD7�CD133� events. B and C, CD34�CD133�CD7�lin� cells were vir-tually devoid of contaminating residual T cells, as shown by FACS reanal-ysis and by molecular studies on TCR� rearrangement status. Lane 1, Pu-rified CD34� cells; lane 2, CD34�CD133�CD7�lin� cells; lane 3,CD34�CD133�CD7�lin� cells; lane 4, Jurkat cells; lane 5, monoclonalcontrol; lane 6, polyclonal control; and lane 7, dH2O. As control, PCR ofthe housekeeping gene Bcl-2 was performed and is shown in C (bottom).

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multiparameter flow cytometry. The T cell and B cell lineage-associated Ags CD3 and CD22 as well as CD235a and CD11b,assigned to erythroid and granulocytic cells, respectively, wereundetectable on CD34�CD133�CD7�lin� UCB cells (Fig. 2).Accordingly, TCR�� and TCR�� as well as CD4 and CD8 Agswere absent. A readily discernible subset of CD34�CD133�

CD7�lin� UCB cells was reactive with anti-CD105 (10 � 3%)and anti-CD90 mAb (12 � 4%); similarly, expression of HLA-DRand Sca-1 was shown on a small subset of cells (�10%), in ac-cordance with previous reports on putative stem/progenitor cellscapable of regenerating human myocardium (31). Of interest,CD34�CD133�CD7�lin� UCB cells uniformly expressed lowlevels of CD45 (mean fluorescence intensity equal to 27 � 6 com-pared with 130 � 15 in peripheral blood CD3� T cells used as acontrol) and were virtually negative for Ags assigned to the gran-ulocytic/monocytic differentiation pathway, e.g., CD33, CD13,CD64, CD65, as well as for the NK cell-associated Ags CD16 andCD56 (Fig. 2).

Next, we performed molecular studies to measure mRNA levelsfor the survival gene Bcl-2, cell cycle modulators (p27Kip1,p21Cip1, and TGF-�1), the lymphoid-associated transcription fac-tor GATA-3, and differentiation proteins (CD34 and myeloperox-idase). Relative to the TF-1 cell line and to CD34�lin� cells,CD34�CD133�CD7�CD45dimlin� cells displayed low Bcl-2 ex-pression, high p21Cip1 and TGF-�1 and intermediate p27Kip1 ex-pression, absence of CD34 and intermediate levels of myeloper-oxidase (Fig. 3). The differential expression of GATA-3 inCD34�CD133�CD7�CD45dimlin� compared with CD34�lin�

cells was of interest, given the undisputed role of GATA-3 in thepromotion of T cell differentiation (32). Collectively, the observedmolecular profile consisted of high expression levels of prototypicnegative modulators of the cell cycle and low expression levels ofsurvival-promoting proteins.

Growth factor requirements and functional activity of highlypurified CD34�CD133�CD7�CD45dimlin� cells

Next, we tested numerous growth factors for the ability to supportsurvival and/or differentiation of human CD34�CD133�CD7�

CD45dimlin� cells in liquid cultures performed in the presence ofhorse/bovine serum and 10�6 M hydrocortisone (19). Based ondata from preliminary experiments (not shown), corticosteroidsand animal serum were included in all growth experiments, becausetheir concomitant presence during the 48-h culture period reducedspontaneous cell death by 20% on average in any experimental con-dition tested as compared with medium supplementation with serumsubstitute alone. When used alone or in combinations, IL-3, IL-6,TPO, Flt3-L, IL-7, GM-CSF, IL-2, platelet-derived growth factor, andepidermal growth factor exerted no appreciable effects on growth,viability, and differentiation of CD34�CD133�CD7�CD45dimlin�

cells following 72 h of culture, as measured by viable cellcounts and CD133 and CD7 expression. Of interest, SCF pre-served cell viability and promoted the appearance of a popula-tion of cells that averaged 12% and expressed low levels ofCD133. At variance with the TCR� chain-signaling cytokinesIL-2 and IL-7, IL-15 was capable of promoting CD34�CD133�

CD7�CD45dimlin� cell growth, with an average 2.4-fold in-crease in cell number, maintained cell viability, and inducedexpression of the T cell-associated Ag CD7 on an average of15% of cultured cells on day 3.

Eight individual samples of purified CD34�CD133�CD7�

CD45dimlin� cells isolated from eight different UCB donors wereassayed for direct cloning efficiency in semisolid methylcelluloseculture in the presence of a combination of cytokines, as alreadydetailed. Following 14 days of serum-free culture, no colonies ofany lineage were scored when CD34�CD133�CD7�CD45dimlin�

cells were seeded at the concentration of either 1,000 or 5,000 cells

FIGURE 2. Phenotypic features of freshly iso-lated CD34�CD133�CD7�lin� cells. CD34�

CD133�CD7�lin� cells were further analyzed forthe expression levels of an array of differentiation/maturation Ags. Cells were gated in R1 based onforward-scatter (FSC)/side-scatter (SSC) character-istics. One representative experiment of six withsimilar results is shown. Markers were set accord-ing to the proper isotypic control. The percentage ofcells staining positively for a given Ag is indicatedin each bidimensional cytogram.

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per plate (Table I). Conversely, after increasing cells to 10,000 perplate, five small nonhemoglobinized colonies on average wereidentified in replicate plates containing SCF, erythropoietin plusG-CSF, GM-CSF, IL-3, and Flt3-L (cloning efficiency, 0.05%). Onthe contrary, the CD34�lin� counterpart isolated from the sameUCB samples and used as a control cell population produced con-fluent colonies when seeded at 5,000 and 10,000 cells per plate,and hemoglobinized and nonhemoglobinized colonies averaged100 at a starting concentration of 1,000 cells per plate (cloningefficiency, 10%; Table I). To assay for long-term functional activ-ity of CD34�CD133�CD7�CD45dimlin� cells, we performedELTC-IC assays through LDA and 12-wk culture on the murinestromal cell line M2-10B4, engineered to produce human G-CSFand IL-3. From these assays, we learned that human CD34�

CD133�CD7�CD45dimlin� cells were endowed with low butmeasurable long-term activity (average ELTC-IC frequency of1:12,000 in five independent experiments). For purposes of com-parison, the CD34�lin� counterpart from the same UCB samples

showed an average frequency of 1:600 ELTC-IC, in accordancewith previously published reports (18, 24).

CD34�CD133�CD7�CD45dimlin� cells cultured with SCFdifferentiate into CD34�CD133� progenitors

Based on the results obtained with SCF and IL-15 after 72-h liquidculture, we first performed a more in-depth analysis of the effectsproduced by SCF on survival and expansion of CD34�CD133�CD7�CD45dimlin� cells maintained in culture for up to 7 days.This experimental condition was established in three replicate ex-periments with UCB cells from three different donors. In the ab-sence of exogenous cytokines, cell count declined by 40% on av-erage as compared with the starting cell population, and cellviability averaged 40% (Table II). Conversely, cultures maintainedin the presence of SCF showed significant increases of cell counts,associated with cell viability scores that were consistently �80%.The onset of cell proliferation after exposure to SCF was alsoconfirmed by cell-doubling experiments with the fluorescent dye

Table I. Comparison of cloning activities of freshly isolated CD34�CD133�CD7�CD45dimlin� andCD34�lin� cellsa

Cell Subset Cells per Plate CFU-GM BFU-E Meg-Aggregates

CD34�CD133�CD7�CD45dimlin� 1,000 ND ND ND5,000 ND ND ND

10,000 5 � 1 ND NDCD34�lin� 1,000 35 � 19 58 � 26 19 � 10

5,000 Confluent Confluent 110 � 3010,000 Confluent Confluent 200 � 50

a CFU-GM, BFU-E, and Meg-aggregates were generated from freshly isolated CD34�CD133�CD7�CD45dimlin� cells, asdetailed in Materials and Methods. ND, Not detected. Results are expressed as the mean � SD observed in eight independentexperiments.

FIGURE 3. Molecular profile of freshly isolated CD34�CD133�CD7�lin� cells. mRNA levels for survival genes, cell cycle modulators, transcriptionfactors, and differentiation proteins were measured with semiquantitative RT-PCR. The TF-1 leukemic cell line and CD34�lin� cells were used as referencecontrol for gene expression levels. Absorbance relative to the housekeeping gene (aldolase A) was plotted in A. Data represent mean � SD from 10independent experiments performed in duplicate. One experiment of 10 with similar results is shown in B. Lane 1, CD34� cells; lane 2,CD34�CD133�CD7�CD45dimlin� cells; and lane 3, TF-1 cell line.

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CFSE. Whereas cells kept in the absence of SCF preserved theirCD34�CD133� phenotype throughout the culture period, cellsmaintained with SCF acquired the CD133 marker by day 4 in theabsence of significant CD34 expression. Notably, the majority ofSCF-treated cells coexpressed both CD133 and CD34 by day 7(Table II; Fig. 4).

Cells were then assayed for ELTC-IC activity by LDA and byculture on a stromal support. Twelve-week ELTC-IC assays re-vealed that long-term hemopoietic stem/progenitor cells were ab-sent after culturing CD34�CD133�CD7�CD45dimlin� cells with-out cytokine supplementation; conversely, an average frequency of1:4000 ELTC-IC was found after culturing in the presence of SCF.Finally, direct cloning assays did not reveal any colony inCD34�CD133�CD7�CD45dimlin� cells cultured in the absenceof SCF, whereas 55 colonies on average were scored after cultur-ing 5000 CD34�CD133�CD7�CD45dimlin� cells per plate withSCF (cloning efficiency, 1.1%; Table II).

Molecular analyses confirmed the commitment of SCF-treatedCD34�CD133�CD7�CD45dimlin� cells toward the myeloid lin-

eage, as indicated by high levels of transcripts for myeloperoxi-dase, when compared with cells cultured in the absence of exog-enous cytokines or with TF-1 erythroleukemia cells, used as acontrol cell population (Fig. 5). Also, reactivation of Bcl-2 genetranscription, lack of mRNA signals for TGF-�1, and significantdown-regulation of p21Cip1 and p27Kip1 mRNA levels occurred inSCF-treated CD34�CD133�CD7�CD45dimlin� cells, comparedwith cells maintained without SCF (Fig. 5) and with the startingcell population (Fig. 3). Conversely, no changes were observed inGATA-3 mRNA levels. As anticipated by the FACS analysis ofCD34 Ag expression, mRNA signals for CD34 were consistentlydetected after priming with SCF, indicating active gene transcrip-tion (Fig. 5).

CD34�CD133�CD7�CD45dimlin� cells cultured with IL-15differentiate into lymphoid progenitors

Following the observation that IL-15 promoted cell survival andinduced CD7 expression in liquid culture, CD34�CD133�CD7�

CD45dimlin� cells were maintained for 7 days in the presence ofexogenous IL-15 at 50 ng/ml. Under these experimental condi-tions, a population of putative lymphoid progenitors emerged thathomogeneously expressed the T cell-associated Ag CD7, togetherwith markers conventionally assigned to naive T cells, e.g.,CD45RA (Table III). Accordingly, minute fractions of cells ex-pressed the memory T cell-associated Ag CD45RO. Interestinglyenough, high percentages of cells differentiated with IL-15 stainedpositively with both the anti-CD25 and the anti-CD44 mAb. Thesefeatures were reminiscent of the early stages of normal T cell thy-mic development (12). In line with the exquisite sensitivity to IL-15, CD34�CD133�CD7�CD45dimlin� cells expressed high levelsof the IL-2/IL-15R� chain (Table III). However, cells completelylacked expression of CD3, as well as of CD4 and CD8 Ag; con-sistently, either surface �� or �� chains of the TCR complex wereundetectable. CD45 expression levels were significantly increasedafter culture, and IL-15-primed cells homogeneously acquired a

Table II. Culture of CD34�CD133�CD7�CD45dimlin� cells for 7 daysin the presence or absence of SCFa

Parameter SCF No Cytokine

Cell count (fold increase) 1.01 � 0.3 0.6 � 0.1Viability (%) 88 � 6 40 � 10CD133� (%) 98 � 5 5 � 3CD34� (%) 78 � 6 3 � 2CFU-GMb 35 � 7 NDBFU-Eb 16 � 5 NDMeg-Aggregatesb 6 � 2 ND

a CD34�CD133�CD7�CD45dimlin� cells were cultured for 7 days with 10%horse serum, 10% bovine serum, and 10�6 M hydrocortisone in the presence orabsence of 10 ng/ml SCF. ND, Not detected. Results are expressed as the mean � SDobserved in four independent experiments.

b Expressed as the absolute number of colonies per 5000 seeded cells.

FIGURE 4. Phenotypic features of SCF-treated CD34�CD133�CD7�CD45dimlin� cells. The expression of the stem cell-related Ag CD34 and CD133was evaluated by flow cytometry after culturing for 7 days in the presence (left panels) or in the absence (right panels) of SCF at 10 ng/ml. All cultureswere performed in �-MEM culture medium supplemented with 10% horse serum, 10% bovine serum, and 10�6 M hydrocortisone (19). The percentageof cells reactive with the anti-CD34 and/or the anti-CD133 mAb at the different time points is indicated in each bidimensional cytogram. Markers forquadrant analysis were set according to the proper isotypic controls. FSC, Forward scatter; SSC, side scatter. One representative experiment of four withsimilar results is shown.

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CD45bright staining pattern. Conversely, most CD34�CD133�

CD7�CD45dimlin� cells cultured without IL-15 retained aCD45dim phenotype (Table III). As shown in Table III, spontane-ous cell differentiation occurred and cells maintained in the ab-sence of exogenous IL-15 up-regulated the expression of the lym-phoid-cell associated Ag CD7. Furthermore, 24 � 5% of CD34�

CD133�CD7�CD45dimlin� cells cultured with medium alone dis-played a CD44�CD25� phenotype as compared with 5 � 2% offreshly isolated CD34�CD133�CD7�CD45dimlin� cells. Data onphenotype and viability of IL-15-primed CD34�CD133�CD7�

CD45dimlin� cells are summarized in Table III.When TCR gene rearrangement was investigated, the genes en-

coding the TCR� chain were nonclonally rearranged, at variancewith freshly isolated CD34�CD133�CD7�CD45dimlin� cells,which failed to rearrange the TCR� locus, as determined by a PCRanalysis of amplified genomic DNA (Fig. 6). As expected, TCR�chain gene rearrangements could be detected in CD34�lin� cellsas well as in CD34�CD133�CD7�CD45dimlin� cells, used ascontrol cell fractions (Fig. 6).

Notably, CD34�CD133�CD7�CD45dimlin� cells primed withIL-15 up-regulated mRNA transcripts for GATA-3 (Fig. 7); an an-ticipated finding of the molecular studies was the enhanced Bcl-2 andTGF-�1 expression levels after cytokine exposure, in line with pre-vious studies on the survival-promoting effects of IL-15 (33). Myelo-peroxidase mRNA was virtually undetectable, consistent with ab-sence of myeloid commitment after IL-15-priming (Fig. 7). Themolecular profile identified in this study indicates thatCD34�CD133�CD7�CD45dimlin� cells exposed to IL-15 differen-tiate into T cell precursors and selectively up-regulate survival genes

as well as transcription factors associated with T cell development.The specificity of our findings was confirmed by the use of a blockingAb directed against the IL-2/IL-15R� chain during culture in the pres-ence of IL-15; under these conditions, the generation of lymphoidprecursors from CD34�CD133�CD7� CD45dimlin� cells was sig-nificantly and specifically inhibited (data not shown).

Priming of CD34�CD133�CD7�CD45dimlin� cells with IL-15was also associated with preserved survival and with significant

Table III. Culture of CD34�CD133�CD7�CD45dimlin� cells for 7days in the presence or absence of IL-15a

Parameter IL-15 No Cytokine

Cell count (fold increase) 2.5 � 0.4* 0.5 � 0.2Viability (%) 98 � 5* 38 � 9CD133� (%) 2 � 1 3 � 1CD34� (%) 4 � 1 2 � 1CD7� (%) 80 � 2* 15 � 3CD25�CD44� (%) 81 � 3* 24 � 5CD45bright (%)b 65 � 7* 3 � 0.2CD45RO� (%) 9 � 4 6 � 3CD45RA� (%) 75 � 7* 22 � 4CD132� (%) 95 � 3* 2 � 1

a CD34�CD133�CD7�CD45dimlin� cells were cultured for 7 days with 10%horse serum, 10% bovine serum, and 10�6 M hydrocortisone in the presence orabsence of 50 ng/ml IL-15. ND, Not detected. Results are expressed as the mean �SD observed in four independent experiments performed in triplicate.

b Defined as cells showing a CD45-related fluorescence beyond the second decadeof a four-decade logarithmic scale.

�, p � 0.05 compared with cultures performed in the absence of exogenouslyadded IL-15.

FIGURE 5. Molecular profile of SCF-treated CD34�CD133�CD7�CD45dimlin� cells. mRNA levels for survival genes, cell cycle modulators, tran-scription factors, and differentiation proteins were measured with semiquantitative RT-PCR after 7 days of culture with SCF at 10 ng/ml. The TF-1 cellline was used as reference control for gene expression levels. Data on cells cultured without exogenous cytokines were presented for purposes ofcomparison. Absorbance relative to the housekeeping gene (aldolase A) was plotted in A. Data represent mean � SD from four independent experimentsperformed in duplicate. One representative experiment is depicted in B. Lane 1, CD34�CD133�CD7�CD45dimlin� cells (no cytokine); lane 2, SCF-treatedCD34�CD133�CD7�CD45dimlin� cells; and lane 3, TF-1 cell line.

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proliferation, compared with cultures maintained in the absence ofIL-15 (Table III). Experiments with the fluorescent probe CFSEallowed the identification and quantification of up to six genera-

tions of progeny in cultures conditioned with IL-15; conversely,cells cultured in the absence of IL-15 were incapable of cell cycleprogression beyond the second generation. Collectively, this ki-netic analysis suggested that IL-15’s effects on the development ofT cell precursors from CD34�CD133�CD7�CD45dimlin� cellswere partly accounted for by induction of proliferation.

LDAs performed with CD34�CD133�CD7�CD45dimlin� cellsplated at the concentration of one cell per well in the presence ofIL-15 supplementation indicated that the average frequency of IL-15-responsive CD34�CD133�CD7�CD45dimlin� cells was 1:8(range, 1:6–1:11) in three independent experiments performed intriplicate with three individual UCB samples. Finally, we wantedto clarify whether CD105� mesenchymal stem cells containedwithin the starting population of CD34�CD133�CD7�CD45dim

lin� cells (Fig. 2) possessed the ability to sustain the lymphoiddifferentiation process of CD34�CD133�CD7�CD45dimlin� cellsby providing soluble factors or accessory signals. To accomplishthis goal, we depleted CD105� cells from CD34�CD133�CD7�

CD45dimlin� cells before culturing in the presence of IL-15 (Fig.8A). These experiments indicated that CD105�CD34�CD133�

CD7�CD45dimlin� cells maintained the ability to differentiatealong the lymphoid lineage, as shown by immunophenotypic (Fig.8B) and molecular studies (not shown).

CD34�CD133�CD7�CD45dimlin� cells cultured on stromasupplemented with IL-15 differentiate into functional NK cells

To detect whether CD34�CD133�CD7�CD45dimlin� cells couldgive origin to terminally differentiated NK cells, a stroma-based21-day culture system was established in the presence or absenceof IL-15. On day 21, the expression of informative NK cell-asso-ciated Ags was investigated on cells recovered from the stromal

FIGURE 7. Molecular profile of IL-15-treated CD34�CD133�CD7�CD45dimlin� cells. mRNA levels for survival genes, cell cycle modulators, tran-scription factors, and differentiation proteins were measured with semiquantitative RT-PCR. The TF-1 cell line was used as reference control for geneexpression levels. Data on cells cultured without IL-15 were presented for purposes of comparison. Absorbance relative to the housekeeping gene (aldolaseA) was plotted in A. Data represent mean � SD from four independent experiments performed in duplicate. One representative experiment is shown in B.Lane 1, CD34�CD133�CD7�CD45dimlin� cells (no cytokine); lane 2, IL-15-treated CD34�CD133�CD7�CD45dimlin� cells; and lane 3, TF-1 cell line.

FIGURE 6. TCR� chain rearrangement after priming with IL-15. TCRrearrangement status was investigated using a nested PCR technique, asalready reported (29). A positive control and a negative control, containingwater instead of DNA, were included in all PCR. PCR products wereanalyzed on a 3% agarose gel, stained with ethidium bromide.CD34�CD133�CD7�lin� cells (lane 2) represented bona fide committedlymphoid precursors and were used as positive controls. Lane 1, MWmarker; lane 2, CD34�CD133�CD7�lin� cells; lane 3, IL-15-primedCD34�CD133�CD7�lin� cells; lane 4, monoclonal control; lane 5, poly-clonal control; and lane 6, dH2O.

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cell support. CD94 Ag was detected on 30% of lymphoid progen-itors on average, whereas the human neural-cell adhesion moleculeCD56 was expressed at high levels by the majority of cells ex-posed to IL-15 (Fig. 9A). In particular, �90% CD56-expressingcells resided in the CD56bright fraction, whereas CD16, the low-affinity Fc�RIII, could be detected on 10% of cells on average(Fig. 9A). In addition, the NK cell-associated killer Ig-like recep-tors CD158a and CD158b were expressed on 10% of cells (datanot shown). Collectively, these phenotypic features along with thelarge granular lymphocyte morphology (data not shown) werereminiscent of NK cells. In contrast, CD34�lin� cells were inca-pable of giving rise to NK cells when cultured on stroma cellssupplemented with IL-15, as shown in Fig. 9B. In addition, stromalcells not supplemented with IL-15 induced no measurable NK celldifferentiation from the CD34�CD133�CD7�CD45dimlin� cells,as shown by FACS analysis of NK cell-associated surface Ags(Fig. 9C). CD34�CD133�CD7�CD45dimlin� cells cultured onstroma supplemented with a combination of IL-2 (1000 IU/ml) andIL-7 (40 ng/ml) developed no phenotypic features indicative of

NK cell differentiation, further suggesting the specificity of IL-15in the determinism of the reported phenomena (Fig. 9D).

Finally, we attempted to test the NK cell activity of IL-15/stro-ma-primed CD34�CD133�CD7�CD45dimlin� cells. To this pur-pose, the K562 tumor cells were loaded with the fluorescent probeCFSE and then incubated with cytokine-primed CD34�CD133�

CD7�CD45dimlin� cells at graded E:T ratios. Results clearlydocumented that cells differentiated with IL-15 in the presenceof a stromal support acquired functional features of NK cells,including the ability to lyse tumor cells in an MHC-unrestrictedmanner (Fig. 10).

DiscussionThe human HSC compartment comprises primitive cells with dif-ferent multipotentiality, proliferative capacity, and ability to gen-erate committed progenitor cells through an orderly sequence ofchanges in gene expression and cell phenotype (1, 32–34). Al-though CD34 Ag expression has been conventionally assigned tohuman hemopoietic cells, recent studies have suggested thatCD34� cells might reside at earlier differentiation stages thanCD34� cells (1, 35–37). In particular, CD34�lin� HSCs express-ing the CD7 Ag possess limited differentiation potential within themyeloid lineage, but retain the ability to expand into NK cells ifcultured on stroma supplemented with cytokines (11, 38). Studiesby Gallacher et al. (10) identified complementary HSCs in humanUCB, which coexist with CD34�CD133� and CD34�CD7�lin�

cells, and possess a CD34�CD133�CD7�lin� phenotype and my-eloid potential, giving rise to CD34�CD133� cells after cytokinepriming. However, whether there exist other HSC subsets that areendowed with lymphoid or myeloid/lymphoid differentiation po-tential under the instructive action of individual cytokines or cy-tokine combinations remains to be thoroughly investigated.

The objectives of the present investigation were the following:1) to identify a relationship between the novel CD34�CD133�

CD7�CD45dimlin� HSC subset and the previously describedCD34�CD133�CD7�lin� and CD34�CD7�lin� cells and 2) to

FIGURE 8. Lymphoid differentiation potential of CD34�CD133�

CD7�CD45dimlin� after depletion of CD105� cells. CD34�CD133�

CD7�CD45dimlin� cells were initially depleted of contaminating CD105�

cells and then cultured with exogenous IL-15 as already described. Cellswere virtually devoid of contaminating CD105� as well as CD7� cells, asshown by FACS reanalysis (A). The expression of the lymphoid-associatedAg CD7 on CD105�CD34�CD133�CD7�CD45dimlin� cells culturedwith IL-15 was evaluated by multiparameter flow cytometry; results fromone representative experiment of three with similar results is shown in B.

FIGURE 9. Phenotypic features of CD34�

CD133�CD7�CD45dimlin� cells cultured on astromal cell support supplemented with IL-15.The expression of informative differentiationAgs on CD34�CD133�CD7�CD45dimlin�

cells was evaluated by multiparameter flowcytometry after culturing for 21 days on stro-mal cells in the presence (A) or absence (B)of IL-15 at 50 ng/ml. Parallel cultures were es-tablished with CD34�CD133�CD7�CD45dim

lin� cells in the presence of IL-2 at 1000 IU/mland IL-7 at 40 ng/ml (C). As a control,CD34�lin� cells cultured in the presence ofIL-15 and stromal cell support (D) showedonly modest induction of NK cell Ags. Mark-ers for quadrant analysis were set according tothe proper isotypic controls (not shown). Thepercentage of cells expressing a given Ag isindicated in each bidimensional cytogram. Onerepresentative experiment of four with similarresults is shown.

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evaluate the instructive action of individual cytokines on pheno-type, function, and molecular profile of human CD34�CD133�

CD7�CD45dimlin� HSCs. To this purpose, suitable methodolog-ical approaches to investigate T cell differentiation potential werecytokine-sustained, serum-containing liquid cultures and the cul-tivation on established stromal cell layers in adjunct to cytokinesupplementation, as previously reported by Storms et al. (11) forthe assessment of the differentiation repertoire of CD34�CD7�

lin� UCB cells.In this study, we isolated and characterized a previously unde-

scribed subset of CD34�CD133�CD7�CD45dimlin� HSCs fromhuman UCB. These cells significantly differed from those studiedby other investigators (10, 11), because they were selected basedon the lack of CD133 and CD7 expression. At variance with theCD34�CD7� lymphoid progenitors described elsewhere (11),CD34�CD133�CD7�CD45dimlin� HSCs did not express measur-able amounts of CD11b, but rather they displayed a CD105low

CD90lowCD117low phenotype.Preliminarily, CD34�CD133�CD7�CD45dimlin� HSCs were

plated in suspension cultures with the objective to determinegrowth factor requirements. Among the various cytokines that weused, SCF and IL-15 were the only growth factors capable of sus-taining cell viability. In line with these findings, both IL-2/IL-15R� chain (CD132) and SCFR (c-kit or CD117) were found on

freshly isolated CD34�CD133�CD7�CD45dimlin� cells. Thesedata prompted us to evaluate the effects of SCF and IL-15 on thedevelopmental fate of CD34�CD133�CD7�CD45dimlin� HSCs,as prototypical myeloid-supporting and lymphoid-supporting cyto-kines, respectively. When cultured in the presence of SCF,CD34�CD133�CD7�CD45dimlin� cells acquired a CD34�CD133�

phenotype, generated CFU-GM, BFU-E, and Meg-aggregates, man-ifested ELTC-IC activity, and up-regulated mRNA levels for myelo-peroxidase. Furthermore, SCF-primed HSCs down-regulated mRNAsignals for cell-cycle inhibitors, up-regulated Bcl-2 levels, and prolif-erated, as evaluated through cell-doubling experiments with CFSE.Particularly, differentiation of CD34�CD133�CD7�CD45dimlin�

cells toward the myeloid lineage occurred with sequential and coor-dinated appearance of CD133 and CD34 Ags. This interpretation ofthe data implies that CD34�CD133�CD7�CD45dimlin� cells repre-sent a more primitive HSC population than the previously describedCD34�CD133�CD7�lin� HSCs.

Interestingly, IL-15 promoted growth and differentiation ofCD34�CD133�CD7�CD45dimlin� HSCs into lymphoid progen-itors with TCR rearrangement and expression of CD45RA and theT cell-associated Ag CD7. Of interest, these cells lacked markerstypically found on mature lymphoid cells, e.g., surface membrane/intracellular TCR ���� chains, CD3, CD4, or CD8 Ags, suggest-ing absence of terminal T cell differentiation. mRNA signals for

FIGURE 10. Cytotoxicity of NK progenitors against K562 target cells. K562 cells were loaded with CFSE, as detailed in Materials and Methods.CD34�CD133�CD7�CD45dimlin� cells primed with IL-15 (putative NK progenitor cells) and stromal cells were cocultured with graded numbers ofNK-sensitive K562 cells or with NK-resistant (NK-res) Raji cells for 24 h, as indicated. A, The percentage of specific lysis was calculated by subtractingthe fraction of 7-AAD�CFSE� cells in control cultures not containing NK progenitors from the fraction of 7-AAD�CFSE� cells in the coculturescontaining NK progenitors (mean � SD recorded in four independent experiments performed in duplicate). �, p � 0.05 compared with cultures performedin the absence of IL-15-primed progenitor cells and with cultures containing IL-15 primed HSCs and K562 cells at a 1:1000 E:T ratio. B, One representativeexperiment of four with similar results is shown. The percentage of lysed K562 cells (7-AAD�CFSE� cells) is indicated in the upper right quadrant ofeach bidimensional cytogram.

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the T cell-associated transcription factor GATA-3, one of the cru-cial molecules implicated in the early phases of T lymphopoiesis(38, 39), as well as gene activity for the survival/antiapoptoticfactors TGF-�1 and Bcl-2 were strongly up-regulated after prim-ing with IL-15. Similar to our findings, high GATA-3 mRNA lev-els were recently detected in common lymphoid progenitors dif-ferentiated from UCB CD34�CD38�CD7� HSCs (40). CD34�

CD133�CD7�CD45dimlin� cells primed with IL-15 acquired aCD44�CD25� phenotype that closely resembled triple-negativethymic progenitors (41). Also, CD34�CD133�CD7�CD45dim

lin� HSCs displayed a modest ability to differentiate into cellswith a CD44�CD25�CD7� phenotype in cultures maintained inthe absence of exogenous IL-15, although cell viability was repro-ducibly poor. These observations imply that the effects of IL-15 onlymphoid differentiation of CD34�CD133�CD7�CD45dimlin�

HSCs might be, at least in part, accounted for by the promotion ofcell survival.

Experiments documenting lack of terminal T cell maturationafter priming with IL-15 alone prompted us to cultureCD34�CD133�CD7�CD45dimlin� cells on a stromal support inthe presence or the absence of IL-15 supplementation. Consistentwith the known actions of IL-15 on NK cell development andfunction (42), CD34�CD133�CD7�CD45dimlin� cells culturedwith stromal cells and IL-15 differentiated into a homogeneouspopulation of CD3�CD56�CD16�/�CD94�/� cells, reminiscentof immature NK cells (43, 44). Killer Ig-like receptor acquisitionwas polyclonal, because cells coexpressed different receptors, e.g.,CD158a and CD158b (data not shown). In our system model, stro-mal support was not dispensable for IL-15 to promote NK celldifferentiation, because no phenotypic features of NK cells weredetected after priming of CD34�CD133�CD7�CD45dimlin� cellswith IL-15 in the absence of stromal cells. Other cytokines be-longing to the family of IL-2/IL-15R� chain-binding cytokines,e.g., IL-2 and IL-7, were ineffective at promoting T/NK cell dif-ferentiation, when used either alone or in combination, as alsofound by others (45). In contrast, stromal cells were incapable ofsustaining NK cell development in the absence of IL-15 supple-mentation. Notably, the CD34�lin� counterpart exhibited minimalNK cell differentiation potential in the presence of a stromal layerand IL-15, when compared with CD34�CD133�CD7�CD45dim

lin� cells. When tested for their ability to lyse sensitive leukemictargets, cells differentiated with IL-15 and stromal cells exhibitedmeasurable lytic responses, even at low E:T ratio, indicating theacquisition of the functional features of NK cells.

It must be emphasized that the failure to detect CD7� lymphoidprogenitors by flow cytometry within the starting population ofCD34�CD133�CD7�CD45dimlin� HSCs as well as the absenceof mRNA signals for the TCR and the high frequency of IL-15-responsive progenitor cells measured with LDA argue against thepossibility that the functional properties here assigned toCD34�CD133�CD7�CD45dimlin� HSCs reflect the cytokine-driven expansion of contaminating T cells.

Collectively, CD34�CD133�CD7�CD45dimlin� UCB cellswere superimposable in terms of differentiation potential to theputative T cell/NK cell common progenitor that has been recentlycloned from fetal liver and from mouse fetal thymus (46, 47).Conceivably, UCB CD34�CD133�CD7�CD45dimlin� cells rep-resent a primitive HSC population with an inherent NK/T celldifferentiation capability. Coupled with previous descriptions ofthe coexistence of lymphoid-oriented CD34�CD7�lin� cells andmyeloid-oriented CD34�CD133�CD7�lin� HSCs in UCB, ourfindings support a hierarchical view of the stem cell compartment,in which the very primitive CD34�CD133�CD7�CD45dimlin� cellsthat we characterized in this study for the first time might reside in

dynamic equilibrium with lymphoid-restricted (CD34�CD7�lin�),transitional myeloid (CD34�CD133�CD7�lin�), and myeloid-ori-ented (CD34�CD133�) stem cells. Studies aimed at evaluatingwhether CD34�CD133�CD7�CD45dimlin� cells possess multilin-eage in vivo reconstitution potential are underway in animal trans-plantation models.

A final implication of our findings pertains to the transplantationof highly purified CD34� HSCs, which has enormously expandedin recent years for the treatment of malignant and nonmalignantdisease (48). Based on the data presented in this study, it can beassumed that depletion of CD34� HSC subsets with lymphoiddifferentiation potential contained within the unmanipulated HSCgraft is expected to contribute to the delayed immunological re-constitution that is routinely observed after CD34-selected HSCtransplantation (48). It remains to be evaluated whether ex vivocytokine-driven expansion of CD34� HSC subsets endowed withlymphoid developmental potential might be exploited to acceleratelymphoid recovery and boost antitumor responses after HSCtransplantation.

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